[Title 40 CFR ]
[Code of Federal Regulations (annual edition) - July 1, 1998 Edition]
[From the U.S. Government Printing Office]
[[Page i]]
40
Protection of Environment
PART 790 TO END
Revised as of July 1, 1998
CONTAINING
A CODIFICATION OF DOCUMENTS
OF GENERAL APPLICABILITY
AND FUTURE EFFECT
AS OF JULY 1, 1998
With Ancillaries
Published by
the Office of the Federal Register
National Archives and Records
Administration
as a Special Edition of
the Federal Register
[[Page ii]]
U.S. GOVERNMENT PRINTING OFFICE
WASHINGTON : 1998
For sale by U.S. Government Printing Office
Superintendent of Documents, Mail Stop: SSOP, Washington, DC 20402-9328
[[Page iii]]
Table of Contents
Page
Explanation................................................. v
Title 40:
Chapter I--Environmental Protection Agency (Continued).... 3
Chapter V--Council on Environmental Quality............... 345
Finding Aids:
Material Approved for Incorporation by Reference.......... 391
Table of CFR Titles and Chapters.......................... 393
Alphabetical List of Agencies Appearing in the CFR........ 411
Table of OMB Control Numbers.............................. 421
Toxic Substances CAS Number--Chemical Index............... 441
List of CFR Sections Affected............................. 463
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Cite this Code: CFR
To cite the regulations in this volume use title, part and
section number. Thus, 40 CFR 790.1 refers to title 40, part
790, section 1.
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[[Page v]]
EXPLANATION
The Code of Federal Regulations is a codification of the general and
permanent rules published in the Federal Register by the Executive
departments and agencies of the Federal Government. The Code is divided
into 50 titles which represent broad areas subject to Federal
regulation. Each title is divided into chapters which usually bear the
name of the issuing agency. Each chapter is further subdivided into
parts covering specific regulatory areas.
Each volume of the Code is revised at least once each calendar year
and issued on a quarterly basis approximately as follows:
Title 1 through Title 16.................................as of January 1
Title 17 through Title 27..................................as of April 1
Title 28 through Title 41...................................as of July 1
Title 42 through Title 50................................as of October 1
The appropriate revision date is printed on the cover of each
volume.
LEGAL STATUS
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HOW TO USE THE CODE OF FEDERAL REGULATIONS
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To determine whether a Code volume has been amended since its
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EFFECTIVE AND EXPIRATION DATES
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OMB CONTROL NUMBERS
The Paperwork Reduction Act of 1980 (Pub. L. 96-511) requires
Federal agencies to display an OMB control number with their information
collection request.
[[Page vi]]
Many agencies have begun publishing numerous OMB control numbers as
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OBSOLETE PROVISIONS
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INCORPORATION BY REFERENCE
What is incorporation by reference? Incorporation by reference was
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This material, like any other properly issued regulation, has the force
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What is a proper incorporation by reference? The Director of the
Federal Register will approve an incorporation by reference only when
the requirements of 1 CFR part 51 are met. Some of the elements on which
approval is based are:
(a) The incorporation will substantially reduce the volume of
material published in the Federal Register.
(b) The matter incorporated is in fact available to the extent
necessary to afford fairness and uniformity in the administrative
process.
(c) The incorporating document is drafted and submitted for
publication in accordance with 1 CFR part 51.
Properly approved incorporations by reference in this volume are
listed in the Finding Aids at the end of this volume.
What if the material incorporated by reference cannot be found? If
you have any problem locating or obtaining a copy of material listed in
the Finding Aids of this volume as an approved incorporation by
reference, please contact the agency that issued the regulation
containing that incorporation. If, after contacting the agency, you find
the material is not available, please notify the Director of the Federal
Register, National Archives and Records Administration, Washington DC
20408, or call (202) 523-4534.
CFR INDEXES AND TABULAR GUIDES
A subject index to the Code of Federal Regulations is contained in a
separate volume, revised annually as of January 1, entitled CFR Index
and Finding Aids. This volume contains the Parallel Table of Statutory
Authorities and Agency Rules (Table I), and Acts Requiring Publication
in the Federal Register (Table II). A list of CFR titles, chapters, and
parts and an alphabetical list of agencies publishing in the CFR are
also included in this volume.
An index to the text of ``Title 3--The President'' is carried within
that volume.
The Federal Register Index is issued monthly in cumulative form.
This index is based on a consolidation of the ``Contents'' entries in
the daily Federal Register.
[[Page vii]]
A List of CFR Sections Affected (LSA) is published monthly, keyed to
the revision dates of the 50 CFR titles.
REPUBLICATION OF MATERIAL
There are no restrictions on the republication of material appearing
in the Code of Federal Regulations.
INQUIRIES
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Raymond A. Mosley,
Director,
Office of the Federal Register.
July 1, 1998.
[[Page ix]]
THIS TITLE
Title 40--Protection of Environment is composed of twenty-three
volumes. The parts in these volumes are arranged in the following order:
parts 1-49, parts 50-51, parts 52.01-52.1018, part 52.1019-end, parts
53-59, part 60, parts 61-62, part 63, parts 64-71, parts 72-80, parts
81-85, part 86, parts 87-135, parts 136-149, parts 150-189, parts 190-
259, parts 260-265, parts 266-299, parts 300-399, parts 400-424, parts
425-699, parts 700-789, and part 790 to end. The contents of these
volumes represent all current regulations codified under this title of
the CFR as of July 1, 1998.
Chapter I--Environmental Protection Agency appears in all twenty-two
volumes. A Pesticide Tolerance Commodity/Chemical Index appears in parts
150-189. A Toxic Substances Chemical--CAS Number Index appears in parts
700-789 and part 790 to end. Redesignation Tables appear in the volumes
containing parts 50-51, parts 150-189, and parts 700-789. Regulations
issued by the Council on Environmental Quality appear in the volume
containing part 790 to end.
The OMB control numbers for title 40 appear in Sec. 9.1 of this
chapter. For the convenience of the user, Sec. 9.1 appears in the
Finding Aids section of the volumes containing part 50 to the end.
For this volume, Gregory R. Walton was Chief Editor. The Code of
Federal Regulations publication program is under the direction of
Frances D. McDonald, assisted by Alomha S. Morris.
[[Page x]]
[[Page 1]]
TITLE 40--PROTECTION OF ENVIRONMENT
(This book contains part 790 to End)
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Part
Chapter i--Environmental Protection Agency (Continued)...... 790
Chapter v--Council on Environmental Quality................. 1500
[[Page 3]]
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
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SUBCHAPTER R--TOXIC SUBSTANCES CONTROL ACT (CONTINUED)
Part Page
790 Procedures governing testing consent
agreements and test rules............... 5
791 Data reimbursement.......................... 26
792 Good laboratory practice standards.......... 33
795 Provisional test guidelines................. 46
796 Chemical fate testing guidelines............ 80
797 Environmental effects testing guidelines.... 101
798 Health effects testing guidelines........... 140
799 Identification of specific chemical
substance and mixture testing
requirements............................ 225
Editorial Note: Nomenclature changes to Chapter I appear at 57 FR
28087, June 24, 1992.
[[Page 5]]
SUBCHAPTER R--TOXIC SUBSTANCES CONTROL ACT (Continued)
PART 790--PROCEDURES GOVERNING TESTING CONSENT AGREEMENTS AND TEST RULES--Table of Contents
Subpart A--General Provisions
Sec.
790.1 Scope, purpose, and authority.
790.2 Applicability.
790.3 Definitions.
790.5 Submission of information.
790.7 Confidentiality.
Subpart B--Procedures for Developing Consent Agreements and Test Rules
790.20 Recommendation and designation of testing candidates by the ITC.
790.22 Procedures for gathering information and negotiating consent
agreements on chemicals which the ITC has recommended for
testing with an intent to designate.
790.24 Criteria for determining whether a consensus exists concerning
the provisions of a draft consent agreement.
790.26 Initiation and completion of rulemaking proceedings on ITC-
designated chemicals.
790.28 Procedures for developing consent agreements and/or test rules
for chemicals that have not been designated or recommended
with intent to designate by the ITC.
Subpart C--Implementation, Enforcement, and Modification of Test Rules
790.40 Promulgation of test rules.
790.42 Persons subject to a test rule.
790.45 Submission of letter of intent to conduct testing or exemption
application.
790.48 Procedure if no one submits a letter of intent to conduct
testing.
790.50 Submission of study plans.
790.52 Phase II test rule.
790.55 Modification of test standards or schedules during conduct of
test.
790.59 Failure to comply with a test rule.
Subpart D--Implementation, Enforcement and Modification of Consent
Agreements
790.60 Contents of consent agreements.
790.62 Submission of study plans and conduct of testing.
790.65 Failure to comply with a consent agreement.
790.68 Modification of consent agreements.
Subpart E--Exemptions From Test Rules
790.80 Submission of exemption applications.
790.82 Content of exemption application.
790.85 Submission of equivalence data.
790.87 Approval of exemption applications.
790.88 Denial of exemption application.
790.90 Appeal of denial of exemption application.
790.93 Termination of conditional exemption.
790.97 Hearing procedures.
790.99 Statement of financial responsibility.
Appendix A to Subpart E--Schedule for Developing Consent Agreements and
Test Rules
Authority: 15 U.S.C. 2603.
Subpart A--General Provisions
Sec. 790.1 Scope, purpose, and authority.
(a) This part establishes procedures for gathering information,
conducting negotiations, and developing and implementing test rules or
consent agreements on chemical substances and mixtures under section 4
of TSCA.
(b) Section 4 of the Act authorizes EPA to require manufacturers and
processors of chemical substances and mixtures to test these chemicals
to determine whether they have adverse health or environmental effects.
Section 4 (a) empowers the Agency to promulgate rules which require such
testing. In addition, EPA has implied authority to enter into
enforceable consent agreements requiring testing where they provide
procedural safeguards equivalent to those that apply where testing is
conducted by rule.
(c) EPA intends to use enforceable consent agreements to accomplish
testing where a consensus exists among EPA, affected manufacturers and/
or processors, and interested members of the public concerning the need
for and scope of testing. If such a consensus does not exist and the
Agency believes that it can make the findings specified in section 4(a),
EPA will initiate proceedings to promulgate test rules which will be
codified in part 799 of this chapter.
[[Page 6]]
(d) Appendix A to this part presents timetables for various steps in
the evaluation of chemicals under consideration for testing, the
initiation and completion of negotiations to develop consent agreements,
and the proposal and promulgation of test rules. All deadlines which are
imposed by the Act are binding on EPA and will be observed by the
Agency. The remaining deadlines represent target dates that EPA intends
to meet.
[51 FR 23712, June 30, 1986]
Sec. 790.2 Applicability.
This part is applicable to manufacturers and processors of chemical
substances or mixtures who are subject to the testing requirements of a
consent agreement or a rule under section 4(a) of the Act. The
procedures for test rules are applicable to each test rule in part 799
or this chapter unless otherwise stated in specific test rules in part
799 of this chapter.
[51 FR 23712, June 30, 1986]
Sec. 790.3 Definitions.
Terms defined in the Act and not explicitly defined herein are used
with the meaning given in the Act. For the purpose of this part:
Act means the Toxic Substances Control Act, 15 U.S.C. 2601 et seq.
Additive means a chemical substance that is intentionally added to
another chemical substance to improve its stability or impart some other
desirable quality.
Chemical means a chemical substance or mixture.
Consortium means an association of manufacturers and/or processors
who have made an agreement to jointly sponsor testing.
EPA means the U.S. Environmental Protection Agency.
Equivalence data means chemical data or biological test data
intended to show that two substances or mixtures are equivalent.
Equivalent means that a chemical substance or mixture is able to
represent or substitute for another in a test or series of tests, and
that the data from one substance can be used to make scientific and
regulatory decisions concerning the other substance.
Exemption means an exemption from a testing requirement of a test
rule promulgated under section 4 of the Act and part 799 of this
chapter.
Impurity means a chemical substance which is uninitentionally
present with another chemical substance.
Joint sponsor means a person who sponsors testing pursuant to
section 4(b)(3)(A) of the Act.
Joint sponsorship means the sponsorship of testing by two or more
persons in accordance with section
4(b)(3)(A) of the Act.
Person means an individual, partnership, corporation, association,
scientific or academic establishment, or organizational unit thereof,
and any other legal entity.
Principal sponsor means an individual sponsor or the joint sponsor
who assumes primary responsibility for the direction of a study and for
oral and written communication with EPA.
Protocol means the plan and procedures which are to be followed in
conducting a test.
Reimbursement period refers to a period that begins when the data
from the last non-duplicative test to be completed under a test rule are
submitted to EPA and ends after an amount of time equal to that which
had been required to develop data or after five years, whichever is
later.
Sponsor means the person or persons who design, direct and finance
the testing of a substance or mixture.
Test substance means the form of chemical substance or mixture that
is specified for use in testing.
[49 FR 39782, Oct. 10, 1984, as amended at 51 FR 23712, June 30, 1986]
Sec. 790.5 Submission of information.
(a) All submissions to EPA under this part must bear the Code of
Federal Regulations (CFR) section number of the subject chemical test
rule, or indicate the identity of the consent agreement. For all
submissions under this part, six copies must be provided to EPA.
(b) Submissions containing both confidential business information or
non-confidential business information must be addressed to the Document
Control
[[Page 7]]
Office (7407), Office of Pollution Prevention and Toxics, U.S.
Environmental Protection Agency, Room G-099, 401 M St., SW., Washington,
DC 20460, ATTN: TSCA Section 4.
[50 FR 20656, May 17, 1985, as amended at 51 FR 23712, June 30, 1986; 58
FR 34205, June 23, 1993; 60 FR 31922, June 19, 1995; 60 FR 34466, July
3, 1995]
Sec. 790.7 Confidentiality.
(a) Any person subject to the requirements of a consent agreement or
a test rule under section 4 of the Act may assert a claim of
confidentiality for certain information submitted to EPA in response to
the consent agreement or the test rule. Any information claimed as
confidential will be treated in accordance with the procedures in part 2
of this title and section 14 of the Act. Failure to assert a claim of
confidentiality at the time the information is submitted will result in
the information being made available to the public without further
notice to the submitter.
(b) A claim of confidentiality must be asserted by circling or
otherwise marking the specific information claimed as confidential and
designating it with the words ``confidential business information,''
``trade secret,'' or another appropriate phrase indicating its
confidential character.
(c) If a person asserts a claim of confidentiality for study plan
information described in Secs. 790.50(c)(1)(iii)(D), (iv), (v), and (vi)
and 790.62(b)(6), (7), (8), (9), and (10), the person must provide a
detailed written substantiation of the claim by answering the questions
in this paragraph. Failure to provide written substantiation at the time
the study plan information is submitted will be considered a waiver of
the claim of confidentiality, and the study plan information will be
disclosed to the public without further notice.
(1) Would disclosure of the study plan information disclose
processes used in the manufacture or processing of a chemical substance
or mixture? Describe how this would occur.
(2) Would disclosure of the study plan information disclose the
portion of a mixture comprised by any of the substances in the mixture?
Describe how this would occur.
(3) What harmful effects to your competitive position, if any, do
you think would result from disclosure of this information? How would a
competitor use such information? How substantial would the harmful
effects be? What is the causal relationship between disclosure and the
harmful effects?
(4) For what period of time should confidential treatment be given?
Until a specific date, the occurrence of a specific event, or
permanently? Why?
(5) What measures have you taken to guard against disclosure of this
information to others?
(6) To what extent has this information been disclosed to others?
What precautions have been taken in connection with such disclosures?
(7) Has this information been disclosed to the public in any forms?
Describe the circumstances.
(8) Has the information been disclosed in a patent?
(9) Has EPA, another Federal agency, or any Federal court made any
pertinent confidentiality determination regarding this information? If
so, copies of such determinations must be included in the
substantiation.
(d) If the substantiation provided under paragraph (c) of this
section contains information which the submitter considers confidential,
the submitter must assert a separate claim of confidentiality for that
information at the time of submission in accordance with paragraph (b)
of this section.
[49 FR 39782, Oct. 10, 1984, as amended at 51 FR 23713, June 30, 1986]
Subpart B--Procedures for Developing Consent Agreements and Test Rules
Source: 51 FR 23713, June 30, 1986, unless otherwise noted.
Sec. 790.20 Recommendation and designation of testing candidates by the ITC.
(a) Recommendations with intent to designate. The ITC has advised
EPA that it will discharge its responsibilities under section 4(e) of
the Act in the following manner:
[[Page 8]]
(1) When the ITC identifies a chemical substance or mixture that it
believes should receive expedited consideration by EPA for testing, the
ITC may add the substance or mixture to its list of chemicals
recommended for testing and include a statement that the ITC intends to
designate the substance or mixture for action by EPA in accordance with
section 4(e)(1)(B) of the Act.
(2) Chemical substances or mixtures selected for expedited review
under paragraph (a)(1) of this section may, at a later time, be
designated for EPA action within 12 months of such designation. The
ITC's subsequent decision would be based on the ITC's review of TSCA
sections 8(a) and 8(d) data and other relevant information.
(3) Where the ITC concludes that a substance or mixture warrants
testing consideration but that expedited EPA review of testing needs is
not justified, the ITC will add the substance or mixture to its list of
testing recommendations without expressing an intent to designate the
substance or mixture for EPA action in accordance with section
4(e)(1)(B) of the Act.
(4) The ITC reserves its right to designate any chemical that it
determines the Agency should, within 12 months of the date first
designated, initiate a proceeding under section 4(a) of the Act.
(b) EPA consideration of ITC recommendations. (1) Where a substance
or mixture is designated for EPA action under section 4(e)(1)(B) of the
Act, the Agency will take either one of the following actions within 12
months after receiving the ITC designation:
(i) Initiate rulemaking proceedings under section 4(a) of the Act.
(ii) Publish a Federal Register notice explaining the Agency's
reasons for not initiating such rulemaking proceedings. EPA may conclude
that rulemaking proceedings under section 4(a) of the Act are
unnecessary if it determines that the findings specified in section 4(a)
of the Act cannot be made or if the Agency has entered into a consent
agreement requiring testing in accordance with the provisions of this
subpart.
(2) Where a substance or mixture has been recommended for testing by
the ITC without an intent to designate, EPA will use its best efforts to
act on the ITC's recommendations as rapidly as possible consistent with
its other priorities and responsiblities. EPA may respond to the ITC's
recommendations either by:
(i) Initiating rulemaking proceedings under section 4(a) of the Act.
(ii) Publishing a Federal Register notice explaining the Agency's
reasons for concluding that testing is unnecessary.
(iii) Entering into a consent agreement in accordance with this
subpart.
Sec. 790.22 Procedures for gathering information and negotiating consent agreements on chemicals which the ITC has recommended for testing with an intent to
designate.
(a) Preliminary EPA evaluation. Following receipt of an ITC report
containing a recommendation with an intent to designate, EPA will use
the following procedure for completing a preliminary evaluation of
testing needs. Appendix A \1\ to this part presents the schedule that
EPA intends to follow for this purpose.
---------------------------------------------------------------------------
\1\ Editorial Note: Appendix A appears at the end of subpart E.
---------------------------------------------------------------------------
(1) EPA will publish the ITC report in the Federal Register and
announce that interested persons have 30 days to submit comments on the
ITC's testing recommendations.
(2) EPA will publish a Federal Register notice adding all ITC-
recommended chemicals to the automatic reporting provisions of its rules
under sections 8(a) and 8(d) of the Act (40 CFR parts 712 and 716).
(3) EPA will hold a public ``focus meeting'' to discuss the ITC's
testing recommendations and obtain comments and information from
interested parties.
(4) EPA will evaluate submissions received under the sections 8(a)
and 8(d) reporting requirements, comments filed on the ITC's
recommendations, and other information and data compiled by the Agency.
(5) EPA will make a preliminary staff determination of the need for
testing and, where testing appears warranted, will tentatively select
the studies to be performed.
[[Page 9]]
(6) EPA will hold a public meeting to announce its preliminary
testing determinations.
(b) Negotiation procedures for consent agreements. Where EPA
believes that testing is necessary, the Agency will explore whether a
consent agreement can be negotiated that satisfies the testing needs
identified by the Agency. EPA will use the following procedures for
negotiating, formulating and accepting consent agreements. Appendix A
\1\ to this part presents the schedule that EPA intends to follow for
this purpose.
(1) In the Federal Register notice described in paragraph (a)(1) of
this section, EPA will explain its procedures and timetable for
negotiating consent agreements and invite persons interested in
participating in or monitoring negotiations to contact the Agency in
writing.
(2) Persons who respond to EPA's notice by the announced date of the
Agency's course-setting meeting will be deemed ``interested parties''
for purposes of any negotiations that EPA conducts.
(3) Following the course-setting meeting announcing EPA's
preliminary testing determinations, the Agency will meet with
manufacturers, processors and other interested parties for the purpose
of attempting to negotiate a consent agreement. To facilitate attendance
at these meetings, EPA will contact all interested parties who have
expressed a desire to participate in or monitor negotiations under
paragraph (b)(2) of this section and advise them of meeting dates.
(4) All negotiating meetings will be open to members of the public.
The minutes of each meeting will be prepared by EPA. Meeting minutes,
testing proposals, background documents and other materials exchanged at
or prepared for negotiating meetings will be included in the public file
established by EPA on each ITC-recommended chemical. Materials in this
file will be made available for inspection in the OPPTS Reading Room
during EPA working hours.
(5) While negotiations are underway, EPA will promptly circulate
meeting minutes, testing proposals, correspondence and other relevant
materials to interested parties who expressed a desire to participate in
or monitor negotiations pursuant to paragraph (b)(2) of this section.
(6) As negotiations progress, EPA will make a tentative decision
either to proceed with formulation of a consent agreement or to initiate
rulemaking. EPA will terminate negotiations after 10 weeks and proceed
with rulemaking unless negotiations are likely to result in a draft
consent agreement within 4 additional weeks. By the end of this 4-week
period, EPA either will have prepared a draft consent agreement
reflecting the apparent consensus of the parties or will terminate
negotiations and proceed with rulemaking. If EPA decides to proceed with
rulemaking, no further opportunity for negotiations will be provided.
EPA will promptly send written notice to all interested parties of the
termination of negotiations.
(7) Where EPA prepares a draft consent agreement, it will be
circulated for comment to all interested parties who expressed a desire
to participate in or monitor negotiations under paragraph (b)(2) of this
section. A period of 4 weeks will be provided for submitting comments or
written objections under Sec. 790.24(a).
(8) If necessary, EPA will hold a public meeting to discuss comments
on the draft consent agreement and to determine whether revisions in the
agreement are appropriate.
(9) Where a consensus exists concerning the contents of a draft
consent agreement, it will be circulated to EPA management and
interested parties for final approval and signature.
(10) Upon final approval of a consent agreement, EPA will publish a
Federal Register notice that summarizes the agreement, describes the ITC
recommendations for the test substance, outlines the chemical's use and
exposure characteristics, and explains the background, objectives and
rationale of the testing to be conducted, and codifies in subpart C of
part 799 the name of the substance(s) to be tested and the citation to
the Federal Register notice of the agreement.
[[Page 10]]
Sec. 790.24 Criteria for determining whether a consensus exists concerning the provisions of a draft consent agreement.
(a) EPA will enter into consent agreements only where there is a
consensus among the Agency, one or more manufacturers and/or processors
who agree to conduct or sponsor the testing, and all other interested
parties who identify themselves in accordance with Sec. 790.22(b)(2).
EPA will not enter into a consent agreement in either of the following
circumstances:
(1) EPA and affected manufacturers and/or processors cannot reach a
consensus on the testing requirements or other provisions to be included
in the consent agreement.
(2) A draft consent agreement is considered inadequate by other
interested parties who, pursuant to Sec. 790.22(b)(2), have asked to
participate in or monitor negotiations; and these parties have submitted
timely written objections to the draft consent agreement which provide a
specific explanation of the grounds on which the draft agreement is
objectionable.
(b) EPA may reject objections described in paragraph (a)(2) of this
section only where the Agency concludes the objections are either:
(1) Not made in good faith.
(2) Untimely.
(3) Do not involve the adequacy of the proposed testing program or
other features of the agreement that may affect EPA's ability to fulfill
the goals and purposes of the Act.
(4) Not accompanied by a specific explanation of the grounds on
which the draft agreement is considered objectionable.
(c) The unwillingness of some manufacturers and/or processors of a
prospective test chemical to sign the draft consent agreement does not,
in itself, establish a lack of consensus if EPA concludes that those
manufacturers and/or processors who are prepared to sign the agreement
are capable of accomplishing the testing to be required and that the
draft agreement will achieve the purposes of the Act in all other
respects.
Sec. 790.26 Initiation and completion of rulemaking proceedings on ITC-designated chemicals.
(a) Where EPA concludes that a consensus does not exist concerning
the provisions of a draft consent agreement and that the findings
specified by section 4(a) can be made, the Agency will proceed with
rulemaking under section 4(a) of TSCA.
(b) When EPA decides to proceed with rulemaking under paragraph (a)
of this section, the Agency intends to publish a rulemaking proposal and
a final rule or a notice terminating the rulemaking proceeding in
accordance with the schedule specified in Appendix A \1\ to this part.
---------------------------------------------------------------------------
\1\ Editorial Note: Appendix A appears at the end of subpart E.
---------------------------------------------------------------------------
(c) Where the testing recommendations of the ITC raise unusually
complex and novel issues that require additional Agency review and
opportunity for public comment, the Agency may publish an Advance Notice
of Proposed Rulemaking (ANPR). The schedule that EPA intends to follow
for rulemaking proceedings initiated by publication of an ANPR is
presented in appendix A \1\ to this part.
Sec. 790.28 Procedures for developing consent agreements and/or test rules for chemicals that have not been designated or recommended with intent to designate
by the ITC.
(a) Where EPA believes that testing is needed, it may also develop
consent agreements and/or test rules on chemical substances or mixtures
that either:
(1) Have been recommended but not ``recommended with intent to
designate'' by the ITC.
(2) Have been selected for testing consideration by EPA on its own
initiative.
(b) When EPA wishes to initiate negotiations concerning chemicals
described in paragraph (a) of this section, it will publish a Federal
Register notice describing its tentative evaluation of testing needs,
announcing a date for a public course-setting meeting, and inviting
persons interested in participating in or monitoring negotiations to
[[Page 11]]
contact the Agency in writing. Any negotiations that EPA conducts will
conform to the procedures specified in Sec. 790.22(b) and, to the extent
feasible, will follow the schedules presented in appendix A \1\ to this
part.
(c) EPA will enter into consent agreements on chemicals described in
paragraph (a) of this section only if there is a consensus among EPA,
affected manufacturers and/or processors, and any other persons who have
asked to participate in or monitor negotiations. In determining whether
such a consensus exists, EPA will employ the criteria specified in
Sec. 790.24. In the absence of consensus, EPA will initiate rulemaking
if it concludes that the findings specified in section 4(a) of the Act
can be made. The schedule for initiating and completing such rulemaking
proceedings will, to the extent feasible, follow the schedule specified
in appendix A \1\ to this part.
---------------------------------------------------------------------------
\1\ Editorial Note: Appendix A appears at the end of subpart E.
---------------------------------------------------------------------------
Subpart C--Implementation, Enforcement, and Modification of Test Rules
Source: 50 FR 20657, May 17, 1985, unless otherwise noted.
Redesignated at 51 FR 23713, June 30, 1986.
Sec. 790.40 Promulgation of test rules.
(a) If EPA determines that it is necessary to test a chemical
substance or mixture by rule under section 4 of the Act, it will
promulgate a test rule in part 799 of this chapter.
(b) EPA will promulgate specific test rules in part 799 of this
chapter either by a single-phase rulemaking procedure or by a two-phase
rulemaking procedure.
(1) Under single-phase test rule development, EPA will promulgate a
test rule in part 799 of this chapter through a notice and comment
rulemaking which specifies the following:
(i) Identification of the chemical for which testing is required
under the rule.
(ii) The health or environmental effect or effects or other
characteristics for which testing is being required.
(iii) Which test substance(s) must be tested.
(iv) Standards for the development of test data.
(v) The EPA Good Laboratory Practice requirements for the required
testing.
(vi) Schedule for submission of interim reports and/or final reports
to EPA.
(vii) Who must submit either letters of intent to conduct testing or
exemption applications.
(viii) What types of data EPA will examine in determining
equivalence if more than one test substance is to be tested.
(2) Under two-phase test rule development, EPA will promulgate a
Phase I test rule in part 799 of this chapter through a notice and
comment rulemaking which specifies the following:
(i) Identification of the chemical for which testing is required
under the rule.
(ii) The health or environmental effect or effects or other
characteristics for which testing is being required.
(iii) Which test substance(s) must be tested.
(iv) A reference to appropriate guidelines for the development of
test data.
(v) The EPA Good Laboratory Practice requirements for the required
testing.
(vi) Who must submit either letters of intent to conduct testing and
study plans, or exemption applications.
(vii) What types of data EPA will examine in determining equivalence
if more than one test substance is to be tested.
(3) Under two-phase test rule development, test standards and
schedules will be developed in a second phase of rulemaking as described
in Secs. 790.50 and 790.52.
[50 FR 20657, May 17, 1985. Redesignated and amended at 51 FR 23713,
June 30, 1986; 54 FR 36313, Sept. 1, 1989]
Sec. 790.42 Persons subject to a test rule.
(a) Each test rule described in Sec. 790.40 will specify whether
manufacturers, processors, or both are subject to the requirement for
testing of the subject chemical under section 4(b)(3)(B) of the
[[Page 12]]
Act and will indicate who will be required to submit letters of intent
to conduct testing.
(1) If testing is being required to allow evaluation of risks:
(i) Primarily associated with manufacture of the chemical, or
(ii) Associated with both manufacturer and processing of the
chemical, or
(iii) Associated with distribution in commerce, use, and/or disposal
activities concerning the chemical, each manufacturer of the chemical
will be subject and must comply with the requirements of the test rule.
(2) While legally subject to the test rule in circumstances
described in paragraphs (a)(1) (ii) and (iii) of this section,
processors of the chemical must comply with the requirements of the test
rule only if processors are directed to do so in a subsequent notice as
set forth in Sec. 790.48(b).
(3) If testing is being required to allow evaluation of risks
associated solely with processing of the chemical, processors will be
subject and must comply with the requirements of the test rule.
(4) While legally subject to the test rule in circumstances
described in paragraph (a)(1) of this section, persons who manufacture
less than 500 kg (1,100 lb) of the chemical annually during the period
from the effective date of the test rule to the end of the reimbursement
period, must comply with the requirements of the test rule only if such
manufacturers are directed to do so in a subsequent notice as set forth
in Sec. 790.48, or if directed to do so in a particular test rule.
(5) While legally subject to the test rule in circumstances
described in paragraph (a)(1) of this section, persons who manufacture
small quantities of the chemical solely for research and development
(meaning quantities that are not greater than those necessary for
purposes of scientific experimentation or analysis or chemical research
on, or analysis of, such chemical or another chemical, including such
research or analysis for development of a product) from the effective
date of the test rule to the end of the reimbursement period, must
comply with the requirements of the test rule only if such manufacturers
are directed to do so in subsequent notice set forth in Sec. 790.48, or
if directed to do so in a particular test rule.
(6) If testing is being required to allow evaluation of risks
associated primarily with manufacture of a chemical for research and
development (R & D) purposes, manufacturers of the chemical for R & D
will be subject and must comply with the requirements of the test rule.
(b) [Reserved]
[50 FR 20657, May 17, 1985. Redesignated at 51 FR 23713, June 30, 1986,
and amended at 55 FR 18884, May 7, 1990]
Sec. 790.45 Submission of letter of intent to conduct testing or exemption application.
(a) No later than 30 days after the effective date of a test rule
described in Sec. 790.40, each person subject to that rule and required
to comply with the requirements of that rule as provided in
Sec. 790.42(a) must, for each test required, either notify EPA by letter
of his or her intent to conduct testing or submit to EPA an application
for an exemption from testing requirements for the test.
(b) EPA will consider letters of intent to test as commitments to
sponsor the tests for which they are submitted unless EPA agrees to the
substitution of an exemption application in instances where more than
one person indicates an intent to sponsor equivalent tests.
(c) Each letter of intent to conduct testing must include:
(1) Identification of test rule.
(2) Name, address, and telephone number of the firm(s) which will be
sponsoring the tests.
(3) Name, address, and telephone number of the appropriate
individual to contact for further information.
(4) For sponsors participating in a testing consortium--a list of
all members of the consortium, the signature of an authorized
representative of each member, and a designation of who is to serve as
principal sponsor.
(5) A list of the testing requirements for which the sponsor(s)
intends to conduct tests.
[[Page 13]]
(6) If EPA is requiring testing of more than one representative
substance--which test substance the sponsor(s) intends to use in each of
the tests.
(d)(1) Any person not manufacturing or processing the subject
chemical as of the effective date of the test rule describing in
Sec. 790.40 or by 30 days after the effective date of the rule who,
before the end of the reimbursement period, manufacturers or processes
the test chemical and who is subject to and required to comply with the
requirements of the test rule must submit the letter of intent to test
or an exemption application required by paragraph (a) of this section by
the date manufacture or processing begins, or
(2) When both manufacturers and processors are subject to the rule,
any person not processing the subject chemical as of the effective date
of the test rule described in Sec. 790.40 or by 30 days after
publication of the Federal Register notice described in
Sec. 790.48(b)(2) who, before the end of the reimbursement period,
processes the test chemical and who is required to comply with the
requirements of the rule must submit the letter of intent to test or an
exemption application required by Sec. 790.48(b)(3) of the date
processing begins.
(e) Manufacturers subject to a test rule described in Sec. 790.40
who do not submit to EPA either a letter of their intent to conduct
tests or a request for an exemption from testing for each test for which
testing is required in the test rule will be considered in violation of
that rule beginning on the 31st day after the effective date of the test
rule described in Sec. 790.40 or on the date manufacture begins as
described in paragraph (d) of this section.
(f) Processors subject to a test rule described in Sec. 790.40 and
required to comply with the requirements of test rule pursuant to
Sec. 790.42(a)(2) or a Federal Register notice as described in
Sec. 790.48(b)(2) who do not submit to EPA either a letter of their
intent to conduct tests or a request for an exemption for each test for
which testing is required in the test rule will be considered in
violation of that rule beginning on the 31st day after the effective
date of the test rule described in Sec. 790.40 or 31 days after
publication of the Federal Register notice described in
Sec. 790.48(b)(2) or on the date processing begins as described in
paragraph (d) of this section, as appropriate.
Sec. 790.48 Procedure if no one submits a letter of intent to conduct testing.
(a) If only manufacturers are subject to the rule. (1) This
paragraph applies if testing is being required solely to allow
evaluation of risks associated with manufacturing and the test rule
described in Sec. 790.40 states that manufacturers only are responsible
for testing.
(2) If no manufacturer subject to the test rule has notified EPA of
its intent to conduct one or more of the required tests within 30 days
after the effective date of the test rule described in Sec. 790.40, EPA
will notify all manufacturers, including those described in
Sec. 790.42(a)(4) and (a)(5), by certified mail or by publishing a
notice of this fact in the Federal Register specifying the tests for
which no letter of intent has been submitted and will give such
manufacturers an opportunity to take corrective action.
(3) If no manufacturer submits a letter of intent to conduct one or
more of the required tests within 30 days after receipt of the certified
letter or publication of the Federal Register notice described in
paragraph (a)(2) of this section, all manufacturers subject to the rule
will be in violation of the test rule from the 31st day after receipt of
the certified letter or publication of the Federal Register notice
described in this paragraph.
(b) If manufacturers and processors are subject to the rule. (1)
This paragraph applies if testing is being required to allow evaluation
of risks associated with manufacturing and processing or with
distribution in commerce, use, or disposal of the chemical and the test
rule described in Sec. 790.40 states that manufacturers and processors
are responsible for testing.
(2) If no manufacturer subject to the rule has notified EPA of its
intent to conduct testing for one or more of the required tests within
30 days after the effective date of the test rule described in
Sec. 790.40, EPA will publish a notice in the Federal Register of this
fact
[[Page 14]]
specifying the tests for which no letter of intent has been submitted.
(3) No later than 30 days after the date of publication of the
Federal Register notice described in paragraph (b)(2) of this section,
each person described in Sec. 790.40(a)(4) and (5) and each person
processing the subject chemical as of the effective date of the test
rule described in Sec. 790.40 or by 30 days after the date of
publication of the Federal Register notice described in paragraph (b)(2)
of this section must, for each test specified in the Federal Register
notice, either notify EPA by letter of his or her intent to conduct
testing or submit to EPA an application for an exemption from testing
requirements for the test.
(4) If no manufacturer or processor of the test chemical has
submitted a letter of intent to conduct one or more of the required
tests within 30 days after the date of publication of the Federal
Register notice described in paragraph (b)(2) of this section, EPA will
notify all manufacturers and processors by certified letter or publish a
Federal Register notice of this fact specifying the tests for which no
letter of intent has been submitted. This letter or Federal Register
notice will give the manufacturers and processors an opportunity to take
corrective action.
(5) If no manufacturer or processor submits a letter of intent to
conduct one or more of the required tests within 30 days after receipt
of the certified letter or publication of the Federal Register notice
described in paragraph (b)(4) of this section, all manufacturers and
processors subject to the rule will be in violation of the test rule
from the 31st day after receipt of the certified letter or publication
of the Federal Register notice described in paragraph (b)(4) of this
section.
(c) Only processors are subject to the rule. (1) This paragraph
applies if testing is being required solely to allow evaluation of risks
associated with processing and the test rule described in Sec. 790.40
states that only processors are responsible for testing.
(2) If no processor subject to the rule has notified EPA of its
intent to conduct one or more of the required tests within 30 days after
the effective date of the test rule described in Sec. 790.40, EPA will
notify all the processors by certified mail or publish a notice in the
Federal Register of this fact, specifying the tests for which no letter
of intent has been submitted and give the processors an opportunity to
take corrective action.
(3) If no processor submits a letter of intent to conduct one or
more of the required tests within 30 days after receipt of the certified
letter or publication of the Federal Register notice described in
paragraph (c)(2) of this section, all processors subject to the rule
will be in violation of the test rule from the 31st day after receipt of
the certified letter or publication of the Federal Register notice
described in this paragraph.
[50 FR 20657, May 17, 1985. Redesignated at 51 FR 23713, June 30, 1986,
and amended at 55 FR 18884, May 7, 1990]
Sec. 790.50 Submission of study plans.
(a) Who must submit study plans. (1) Persons who notify EPA of their
intent to conduct tests in compliance with the requirements of a single
phase test rule as described in Sec. 790.40(b)(1) must submit study
plans for those tests prior to the initiation of each of these tests,
unless directed by a particular test rule or consent agreement to submit
study plans at a specific time.
(2) Persons who notify EPA of their intent to conduct tests in
compliance with the requirements of a Phase I test rule as described in
Sec. 790.40(b)(2) must submit the proposed study plans for those tests
on or before 90 days after the effective date of the Phase I rule; or,
for processors complying with the notice described in Sec. 790.48(b)(2),
90 days after the publication date of that notice; or 60 days after the
date manufacture or processing begins as described in Sec. 790.45(d), as
appropriate, to the address in Sec. 790.5(b).
(3) Study plans must be prepared according to the requirements of
this subpart B and part 792 of this chapter. Only one set of study plans
should be prepared and submitted by persons who are jointly sponsoring
testing.
(4) Any person subject to a test rule may submit a study plan for
any test
[[Page 15]]
required by the rule at any time, regardless of whether the person
previously submitted an application for exemption from testing for that
test.
(5) Unless EPA has granted an extension of time for submission of
proposed study plans, manufacturers who notify EPA that they intend to
conduct testing in compliance with the requirements of a Phase I test
rule as described in Sec. 790.40(b)(2) and who do not submit proposed
study plans for those tests on or before 90 days after the effective
date of the Phase I test rule or 60 days after the date manufacture
begins as described in Sec. 790.45(d) will be considered in violation of
the test rule as if no letter of intent to test had been submitted.
(6) Unless EPA has granted an extension of time for submission of
proposed study plans, processors who notify EPA that they intend to
conduct testing in compliance with the requirements of a Phase I test
rule as described in Sec. 790.40(b)(2) and who do not submit proposed
study plans for those tests on or before 90 days after the effective
date of the Phase I test rule or 90 days after the publication date of
the notice described in Sec. 790.48(b)(2), or 60 days after the date
processing begins as described in Sec. 790.45(d), as appropriate, will
be considered in violation of the test rule as if no letter of intent to
test had been submitted.
(b) Extensions of time for submission of study plans. (1) EPA may
grant requests for additional time for the development of study plans on
a case-by-case basis. Requests for additional time for study plan
development must be made in writing to EPA at the address in
Sec. 790.5(b). Each extension request must state why EPA should grant
the extension.
(2) Under two-phase rulemaking, extension requests must be submitted
to EPA within 60 days after the effective date of the Phase I test rule
as described in Sec. 790.40(b)(2); or for processors complying with the
notice described in Sec. 790.48(b)(2), 60 days after the publication
date of that notice; or 30 days after the date manufacture or processing
begins as described in Sec. 790.45(d), as appropriate.
(3) EPA will notify the submitter by certified mail of EPA's
decision to grant or deny an extension request.
(4) Persons who have been granted an extension of time for
submission of study plans as described in paragraph (b)(1) of this
section and who do not submit proposed study plans in compliance with
the requirements of a Phase I test rule in accordance with the new
deadline granted by EPA will be considered in violation of the test rule
as if no letter of intent to test had been submitted as described in
Sec. 790.45(e) and (f).
(c) Content of study plans. (1) All study plans are required to
contain the following information:
(i) Identity of the test rule.
(ii) The specific test requirements of that rule to be covered by
the study plan.
(iii)(A) The names and addresses of the test sponsors.
(B) The names, addresses, and telephone numbers of the responsible
administrative officials and project manager(s) in the principal
sponsor's organization.
(C) The name, address, and telephone number of the appropriate
individual to contact for oral and written communications with EPA.
(D)(1) The names and addresses of the testing facilities and the
names, addresses, and telephone numbers of the testing facilities'
administrative officials and project manager(s) responsible for the
testing.
(2) Brief summaries of the training and experience of each
professional involved in the study, including study director,
veterinarian(s), toxicologist(s), pathologist(s), chemist(s),
microbiologist(s), and laboratory assistants.
(iv) Identity and data on the chemical substance(s) being tested,
including physical constants, spectral data, chemical analysis, and
stability under test and storage conditions, as appropriate.
(v) Study protocol, including the rationale for any combination of
test protocols; the rationale for species/strain selection; dose
selection (and supporting data); route(s) or method(s) of exposure;
description of diet to be used and its source; including nutrients
[[Page 16]]
and contaminants and their concentrations; for in vitro test systems, a
description of culture medium and its source; and a summary of expected
spontaneous chronic diseases (including tumors), genealogy, and life
span.
(vi) Schedule for initiation and completion of each short-term test
and of each major phase of long-term tests; dates for submission of
interim progress and final reports to EPA that are within the reporting
deadlines specified by EPA In the final test rule.
(2) Information required in paragraph (c)(1)(iii)(D) of this section
is not required in proposed study plans submitted in compliance with the
requirements of a Phase I test rule if the information is not available
at the time of study plan submission; however, the information must be
submitted before the initiation of testing.
(d) Incomplete study plans. (1) Upon receipt of a study plan, EPA
will review the study plan to determine whether it complies with
paragraph (c) of this section. If EPA determines that the study plan
does not comply with paragraph (c) of this section, EPA will notify the
submitter that the submission is incomplete and will identify the
deficiencies and the steps necessary to complete the submission.
(2) The submitter will have 15 days after the day it receives this
notice to submit appropriate information to make the study plan
complete.
(3) If the submitter fails to provide appropriate information to
complete a proposed study plan submitted in compliance with the
requirements of a Phase I test rule on or before 15 days after receipt
of the notice, the submitter will be considered in violation of the test
rule as if no letter of intent to conduct the test had been submitted as
described in Sec. 790.45(e) and (f).
(e) Amendments to study plans. Test sponsors shall submit all
amendments to study plans to the Director, Office of Compliance
Monitoring at the address in Sec. 790.5(d).
[50 FR 20657, May 17, 1985. Redesignated and amended at 51 FR 23713,
June 30, 1986; 52 FR 36569, Sept. 30, 1987; 54 FR 36313, Sept. 1, 1989;
55 FR 18884, May 7, 1990; 58 FR 34205, June 23, 1993; 60 FR 34466, July
3, 1995]
Sec. 790.52 Phase II test rule.
(a) If EPA determines that the proposed study plan described in
Sec. 790.50(a)(2) complies with Sec. 790.50(c), EPA will publish a
proposed Phase II test rule in the Federal Register requesting comments
on the ability of the proposed study plan to ensure that data from the
test will be reliable and adequate.
(b) EPA will provide a 45-day comment period and will provide an
opportunity for an oral presentation upon the request of any person. EPA
may extend the comment period if it appears from the nature of the
issues raised by EPA's review or from public comments that further
comment is warranted.
(c) After receiving and considering public comments on the study
plan, EPA will adopt, as proposed or as modified in response to EPA
review and public comments, the study protocol section of the study
plan, as defined by Sec. 790.50(c)(1)(v) of this chapter, as the test
standard for the required testing, and the schedule section of the study
plan, as defined by Sec. 790.50(c)(1)(vi) of this chapter, as the
schedule for the required testing in a final Phase II test rule.
[50 FR 20657, May 17, 1985. Redesignated at 51 FR 23713, June 30, 1986,
and amended at 52 FR 36569, Sept. 30, 1987]
Sec. 790.55 Modification of test standards or schedules during conduct of test.
(a) Application. Any test sponsor who wishes to modify the test
schedule for the mandatory testing conditions or requirements (i.e.,
``shall statements'') in the test standard for any test required by a
test rule must submit an application in accordance with this paragraph.
Application for modification must be made in writing to EPA at the
address in Sec. 790.5(b), or by phone with written confirmation to
follow within 10 working days. Applications must include an appropriate
explanation and rationale for the modification. Where a test sponsor
requests EPA to provide guidance or to clarify a non-mandatory testing
requirement (i.e., ``should statements'') in a test standard, the test
sponsor should submit these requests to EPA at the address in
Sec. 790.5(b).
[[Page 17]]
(b) Adoption. (1) Where EPA concludes that the requested
modification of a test standard or schedule for a test required under a
test rule is appropriate, EPA will proceed in accordance with this
paragraph (b).
(2) Where, in EPA's judgment, the requested modification of the test
standard or schedule would not alter the scope of the test or
significantly change the schedule for completing the test, EPA will not
ask for public comment before approving the modification. EPA will
notify the test sponsor by letter of EPA's approval. EPA will place
copies of each application and EPA approval letter in the rulemaking
record for the test rule in question. EPA will publish a notice annually
in the Federal Register indicating the test standards or schedules for
tests required in test rules which have been modified under this
paragraph (b)(2) and describing the nature of the modifications. Until
the Federal Register notice is published, any modification approved by
EPA under this paragraph (b)(2) shall apply only to the test sponsor who
applied for the modification under this paragraph (a) of this section.
(3) Where, in EPA's judgment, the requested modification of a test
standard or schedule would significantly alter the scope of the test or
significantly change the schedule for completing the test, EPA will
publish a notice in the Federal Register requesting comment on the
proposed modification. However, EPA will approve a requested
modification of a test standard under paragraph (b)(3) of this section
without first seeking public comment if EPA believes that an immediate
modification to the test standard is necessary to preserve the accuracy
or validity of an ongoing test. EPA may also modify a testing
requirement or test condition in a test standard if EPA determines that
the completion or achievement of this requirement or condition is not
technically feasible. EPA may approve a test schedule extension under
paragraph (b)(3) of this section without first seeking public comment if
EPA determines, on a case-by-case basis, that a delay of over 12 months
is not the fault of the test sponsor and is the result of unforeseen
circumstances such as a lack of laboratory availability, lack of
availability of suitable test substance (e.g., 14-C labelled test
substance), lack of availability of healthy test organisms, or the
unexpected failure of a long-term test. EPA will publish an annual
notice in the Federal Register announcing the approval of any test
standard modifications and test schedule extensions under paragraph
(b)(3) of this section and provide a brief rationale of why the
modification was granted.
(4) For purposes of this paragraph (b), a requested modification of
a test standard or schedule for a test required under a test rule would
alter the scope of the test or significantly change the schedule for
completing the test if the modification would:
(i) Change the test species.
(ii) Change the route of administration of the test chemical.
(iii) Change the period of time during which the test species is
exposed to the test chemical.
(iv) Except as provided in paragraph (b)(3) of this section, extend
the final reporting deadline more than 12 months from the date specified
in the final rule.
(c) Disapproval. Where EPA concludes that the requested modification
of a test standard or schedule for a test required under a test rule is
not appropriate, EPA will so notify the test sponsor in writing.
(d) Timing. (1) Test sponsors should submit all applications for
test schedule modifications at least 60 days before the reporting
deadline for the test in question.
(2) EPA will not normally approve any test schedule extensions
submitted less than 30 days before the reporting deadline for the test
in question.
(3) Except as provided in paragraph (b)(3) of this section, EPA may
grant extensions for up to 1 year but will normally limit extensions to
a period of time equal to the in-life portion of the test plus 60 days.
(4) EPA will normally approve only one deadline extension for each
test.
(5) Test sponsors should submit requests for test standard
modifications as soon as they determine that the test
[[Page 18]]
cannot be successfully completed according to the test standard
specified in the rule.
[50 FR 20657, May 17, 1985. Redesignated at 51 FR 23713, June 30, 1986,
and amended at 52 FR 36571, Sept. 30, 1987; 54 FR 36314, Sept. 1, 1989;
60 FR 34466, July 3, 1995]
Sec. 790.59 Failure to comply with a test rule.
(a) Persons who notified EPA of their intent to conduct a test
required in a test rule in part 799 of this chapter and who fail to
conduct the test in accordance with the test standards and schedules
adopted in the test rule, or as modified in accordance with Sec. 790.55,
will be in violation of the rule.
(b) Any person who fails or refuses to comply with any aspect of
this part or a test rule under part 799 of this chapter is in violation
of section 15 of the Act. EPA will treat violations of the Good
Laboratory Practice standards as indicated in Sec. 792.17 of this
chapter.
Subpart D--Implementation, Enforcement and Modification of Consent
Agreements
Source: 51 FR 23715, June 30, 1986, unless otherwise noted.
Sec. 790.60 Contents of consent agreements.
(a) Standard provisions. All consent agreements will contain the
following provisions:
(1) Identification of the chemical(s) to be tested.
(2) The health effects, environmental effects and/or other
characteristics for which testing will be required.
(3) The names and addresses of each manufacturer and/or processor
who will sign the agreement.
(4) The name and address of the manufacturer, processor or other
entity who has agreed to act as the principal test sponsor.
(5) The technical or commercial grade, level of purity or other
characteristics of the test substances(s) or mixture(s).
(6) Standards for the development of test data.
(7) A requirement that testing will be conducted in accordance with
the EPA Good Laboratory Practice (GLP) regulations (40 CFR part 792).
(8) Schedules with reasonable deadlines for submitting interim
progress and/or final reports to EPA.
(9) A requirement that the principal sponsor will submit a study
plan to EPA in accordance with Sec. 790.62.
(10) A statement that the results of testing conducted pursuant to
the consent agreement will be announced to the public in accordance with
the procedures specified in section 4(d) of the Act and that the
disclosure of data generated by such testing will be governed by section
14(b) of the Act.
(11) A requirement that the manufacturers and/or processors signing
the consent agreement will comply with the notification requirements of
section 12(b)(1) of the Act and part 707 of this chapter if they export
or intend to export the substance or mixture for which the submission of
data is required under the agreement and a statement that any other
person who exports or intends to export such substance or mixture is
subject to the above cited export notification requirements.
(12) A requirement that, in the event EPA promulgates a significant
new use rule applicable to the test chemical under section 5(a)(2), the
consent agreement will have the status of a test rule for purposes of
section 5(b)(1)(A) and manufacturers and/or processors signing the
agreement will comply with the data submission requirements imposed by
that provision.
(13) A statement that each manufacturer and/or processor signing the
agreement agrees that violation of its requirements will constitute a
``prohibited act'' under section 15(1) of the Act and will trigger all
provisions of TSCA applicable to a violation of section 15.
(14) A statement that, in the event one or more provisions of the
agreement are determined to be unenforceable by a court, the remainder
of the agreement would not be presumed to be valid and EPA will then
either initiate a rulemaking proceeding or publish in the Federal
Register the Administrator's reason for not initiating such a
proceeding.
(15) A statement that the Agency may conduct laboratory inspections
[[Page 19]]
and/or study audits of the testing being conducted pursuant to the
consent agreement in accordance with the authority and procedures
contained in section 11 of the Act.
(16) A statement that EPA acceptance of a consent agreement
constitutes ``final agency action'' for purposes of 5 U.S.C. 704.
(17) Any other requirements that the parties agree are necessary to
achieve the purposes of the Act.
(b) Contents of standards for the development of data. The standards
for the development of the data included in consent agreements will be
based on the TSCA test guidelines in 40 CFR parts 796, 797, and 798, the
Organization for Economic Cooperation and Development (OECD) test
guidelines, the EPA pesticide assessment guidelines published by The
National Technical Information Service (NTIS), or other suitable test
methodologies. During the negotiation of consent agreements, EPA will
initially propose suitable test guidelines as the required test
standards; manufacturers and processors or other interested parties may
then suggest alternative methodologies or modifications to the Agency's
proposed guidelines. These alternative methodologies or modifications
will be adopted only where, in the judgment of EPA, they will develop at
least equally reliable and adequate data on the chemical substance or
mixture subject to the agreement.
(c) Statement of rationale for consent agreement. EPA will prepare a
written explanation of the basis for each consent agreement. This
document will summarize the agreement, describe any ITC testing
recommendations for the chemical involved, outline the chemical's use
and exposure characteristics, and explain the objectives of the testing
to be conducted and the rationale for the specific studies selected.
This document will be published in the Federal Register and, for ITC-
designated chemicals, will constitute the statement of EPA's reasons for
not initiating rulemaking required by section 4(e)(1)(B) of the Act.
[51 FR 23715, June 30, 1986, as amended at 54 FR 36314, Sept. 1, 1989]
Sec. 790.62 Submission of study plans and conduct of testing.
(a) Timing of submission. The principal sponsor of testing conducted
pursuant to a consent agreement shall submit a study plan no later than
45 days prior to the initiation of testing.
(b) Content of study plans. All study plans are required to contain
the following information:
(1) Identity of the consent agreement under which testing will be
performed.
(2) The specific test requirements to be covered by the study plan.
(3) The name and address of the principal test sponsor.
(4) The names, addresses, and telephone numbers of the responsible
administrative official[s] and project manager[s] in the principal
sponsor's organization.
(5) The names, addresses, and telephone numbers of the technical
contacts at each manufacturer and/or processor subject to the agreement.
(6) The names and addresses of the testing facilities responsible
for the testing and the names, addresses, and telephone numbers of the
administrative officials[s] and project manager[s] assigned to oversee
the testing program at these facilities.
(7) Brief summaries of the training and experience of each
professional involved in the study, including study director,
veterinarian[s], toxicologist[s], pathologist[s], chemist[s],
microbiologist[s], and laboratory assistants.
(8) Identity and supporting data on the chemical substance[s] being
tested, including physical constants, spectral data, chemical analysis,
and stability under test and storage conditions, as appropriate.
(9) Study protocol, including the rationale for any combination of
test protocols; the rationale for species/strain selection; dose
selection (and supporting data); route(s) or method(s) of exposure;
description of diet to be used and its source, including nutrients and
contaminants and their concentrations; for in vitro test systems, a
description of culture medium and its source; and a summary of expected
spontaneous chronic diseases (including tumors), genealogy, and life
span.
[[Page 20]]
(10) A schedule, with reasonable timeables and deadlines, for
initiation and completion of each short-term test and of each major
phases of long-term tests, and submission of interim progress and/or
final reports to EPA.
(c) Review and modification. (1) Upon receipt of a study plan, EPA
will review it to determine whether it complies with paragraph (b) of
this section. If EPA determines that the study plan does not comply with
paragraph (b) of this section, EPA will notify the submitter that the
plan is incomplete and will identify the deficiencies and the steps
necessary to complete the plan. It is the responsibility of the test
sponsor to review the study protocols to determine if they comply with
all the mandatory testing conditions and requirements in the test
standards (i.e., ``shall statements'').
(2) The submitter will have 15 days after the day it receives a
notice under paragraph (c)(1) of this section to submit appropriate
information to make the study plan complete.
(3) If the submitter fails to provide appropriate information to
complete a study plan within 15 days after having received a notice
under paragraph (c)(1) of this section, the submitter will be considered
to be in violation of the consent agreement and subject to enforcement
proceedings pursuant to Sec. 790.65 (c) and (d).
(4) The test sponsor shall submit any amendments to study plans to
EPA at the address specified in Sec. 790.5(b).
(d) Functions of the principal test sponsor. When testing is being
conducted pursuant to a consent agreement, the principal test sponsor
will be responsible for submitting interim progress and final reports to
EPA, informing the Agency of any proposed changes in standards for the
development of data, study plans or testing schedules, and communicating
with the Agency about laboratory inspections and other matters affecting
the progress of testing.
[51 FR 23715, June 30, 1986, as amended at 54 FR 36314, Sept. 1, 1989;
60 FR 34466, July 3, 1995]
Sec. 790.65 Failure to comply with a consent agreement.
(a) Manufacturers and/or processors who have signed a consent
agreement and who fail to comply with the test requirements, test
standards, GLP regulations, schedules, or other provisions contained in
the consent agreement, or in modifications to the agreement adopted
pursuant to Sec. 790.68, will be in violation of the consent agreement.
(b) The Agency considers failure to comply with any aspect of a
consent agreement to be a ``prohibited act'' under section 15 of TSCA,
subject to all of the provisions of the Act applicable to violations of
section 15. Section 15(1) of TSCA makes it unlawful for any person to
fail or refuse to comply with any rule or order issued under section 4.
Consent agreements adopted pursuant to this part are ``orders issued
under section 4'' for purposes of section 15(1) of TSCA.
(c) Manufacturers and/or processors who violate consent agreements
are subject to criminal and/or civil liability. Under the penalty
provisions of section 16 of TSCA, such firms could be subject to a civil
penalty of up to $25,000 per violation with each day in violation
constituting a separate violation of section 15. Intentional violations
could lead to the imposition of criminal penalties of up to $25,000 for
each day of violation and imprisonment for up to one year. In addition,
EPA could invoke the remedies available under section 17 of TSCA,
including seeking an injunction to compel adherence to the requirements
of the consent agreement.
(d) Noncompliance with a consent agreement will constitute conduct
``in violation of this Act'' under section 20(a)(1) of TSCA. Thus,
failure to comply with the requirements of a consent agreement could
result in a citizens' civil action under section 20(a)(1) of TSCA.
Sec. 790.68 Modification of consent agreements.
(a) Changes in the scope of testing. (1) Manufacturers or processors
subject to a consent agreement, other persons or EPA may seek
modifications in the scope of testing performed under the consent
agreement. If, upon receiving a request for modification, EPA determines
that new issues have been raised that warrant reconsideration of the
scope of testing, or if EPA determines
[[Page 21]]
on its own that such reconsideration is appropriate, EPA will publish a
Federal Register notice describing the proposed modification and
soliciting public comment. If, based on the comments received, EPA
concludes that differences of opinion may exist about the proposed
modification, EPA will establish a schedule for conducting negotiations
and invite parties who wish to participate in or monitor these
negotiations to contact the Agency in writing. Any negotiations that EPA
conducts will conform to the procedures specified in Sec. 790.22(b).
(2) The scope of testing required by a consent agreement will be
modified only where there is a consensus concerning the modified testing
requirements among EPA, affected manufacturers and/or processors, and
other persons who have asked to participate in or monitor negotiations
under paragraph (a)(1) of this section. In determining whether a
consensus exists, EPA will employ the criteria specified in Sec. 790.24.
In the absence of consensus, EPA may initiate rulemaking under section
4(a) of the Act if it concludes that any testing beyond that required by
the consent agreement is necessary and that the other statutory findings
required by section 4(a) can be made. While such rulemaking proceedings
are underway, the consent agreement will remain in effect unless EPA
finds that the testing required by the agreement is or may be
unnecessary in view of the testing requirements included in EPA's
proposed rule.
(b) Changes in test standards or schedules. (1) Any test sponsor who
wishes to modify the test schedule for any test required under a consent
order must submit an application in accordance with this paragraph.
Application for modification must be made in writing to EPA at the
address in Sec. 790.5(b), or by phone with written confirmation to
follow within 10 working days. Applications must include an appropriate
explanation and rationale for the modification. EPA will consider only
those applications that request modifications to mandatory testing
conditions or requirements (``shall statements'' in the consent order).
Where a test sponsor requests EPA to provide guidance or to clarify a
non-mandatory testing requirement (i.e., ``should statements''), the
test sponsor should submit these requests to EPA at the address in
section 790.5(b).
(2)(i) Where EPA concludes that the requested modification of a test
standard or schedule for a test required under a consent agreement is
appropriate, EPA will proceed in accordance with this paragraph (b)(2).
(ii) Where, in EPA's judgment, the requested modification of a test
standard or schedule would not alter the scope of the test or
significantly change the schedule for completing the test, EPA will not
ask for public comment before approving the modification. EPA will
notify the test sponsor, and any other persons who have signed the
consent agreement, by letter of EPA's approval. EPA will place copies of
each application and EPA approval letter in the administrative record
maintained for the consent agreement in question. EPA will publish a
notice annually in the Federal Register indicating the test standards or
schedules for test required in consent agreements which have been
modified under this paragraph (b)(2)(ii) and describing the nature of
the modifications.
(iii) Where, in EPA's judgment, the requested modification of a test
standard or schedule would significantly alter the scope of the test or
significantly change the schedule for completing the test, EPA will
publish a notice in the Federal Register requesting comment on the
proposed modification. However, EPA will approve a requested
modification of a test standard under paragraph (b)(2)(iii) of this
section without first seeking public comment if EPA believes that an
immediate modification to the test standard is necessary to preserve the
accuracy or validity of an ongoing test. EPA also may modify a testing
requirement or test condition in a test standard if EPA determines that
the completion or achievement of this requirement or condition is not
technically feasible. EPA may approve a requested modification of a test
schedule under paragraph (b)(2)(iii) of this section without first
seeking public comment if EPA determines, on a case-by-case basis, that
a delay of over 12 months is not the fault of the test sponsor and is
due
[[Page 22]]
to unforeseen circumstances such as a lack of laboratory availability,
lack of availability of suitable test substance (e.g., 14-C labelled
test substance), lack of availability of healthy test organisms, or the
unexpected failure of a long-term test. EPA will publish an annual
notice in the Federal Register announcing the approval of any test
standard modifications and test scheduled extensions under paragraph
(b)(2)(iii) of this section, and provide a brief rationale of why the
modification was granted.
(iv) For purposes of this paragraph (b)(2), a requested modification
of a test standard of schedule for a test required under a consent
agreement would alter the scope of the test or significantly change the
schedule for completing the test if the modification would:
(A) Change the test species.
(B) Change the route of administration of the test chemical.
(C) Change the period of time during which the test species is
exposed to the test chemical.
(D) Except as provided in paragraph (b)(2)(iii) of this section,
extend the final reporting deadline more than 12 months from the date
specified in the consent order.
(3) Where EPA concludes that the requested modification of a test
standard or schedule for a test requirement under a consent agreement is
not appropriate, EPA will so notify the test sponsor in writing.
(c) Timing. (1) Test sponsors should submit all applications for
test schedule modifications at least 60 days before the reporting
deadline for the test in question.
(2) EPA will not normally approve any test schedule extensions
submitted less than 30 days before the reporting deadline for the test
in question.
(3) Except as provided in paragraph (b)(2)(iii) of this section, EPA
may grant extensions as shown necessary for up to 1 year but will
normally limit extensions to a period of time equal to the in-life
portion of the test plus 60 days.
(4) EPA will normally approve only one deadline extension for each
test.
(5) Test sponsors should submit requests for test standard
modifications as soon as they determine that the test cannot be
successfully completed according to the test standard specified in the
consent order.
[51 FR 23715, June 30, 1986, as amended at 52 FR 36571, Sept. 30, 1987;
54 FR 36314, Sept. 1, 1989; 60 FR 34466, July 3, 1995]
Subpart E--Exemptions From Test Rules
Source: 50 FR 20660, May 17, 1985, unless otherwise noted.
Sec. 790.80 Submission of exemption applications.
(a) Who should file applications. (1) Any manufacturer or processor
subject to a test rule in part 799 of this chapter may submit an
application to EPA for an exemption from performing any or all of the
tests required under the test rule.
(2) Processors will not be required to apply for an exemption or
conduct testing unless EPA so specifies in a test rule or in a special
Federal Register notice as described in Sec. 790.48(b)(2) under the
following circumstances:
(i) If testing is being required to allow evaluation of risks
associated with manufacturing and processing or with distribution in
commerce, use, or disposal of the chemical and manufacturers do not
submit notice(s) of intent to conduct the required testing; or
(ii) If testing is being required solely to allow evaluation of
risks associated with processing of the chemical.
(b) When applications must be filed. (1) Exemption applications must
be filed within 30 days after the effective date of the test rule
described in Sec. 790.40 or, if being submitted in compliance with the
Federal Register notice described in Sec. 790.48(b)(2), within 30 days
after the publication of that notice.
(2) Exemption applications must be filed by the date manufacture or
processing begins by any person not manufacturing or processing the
subject chemical as of the effective date of the test rule described in
Sec. 790.40 or by 30 days after the effective date of the test rule
described in Sec. 790.40, who, before the end of the reimbursement
period, manufactures or processes the test substance and who is subject
to the requirement to submit either a letter of
[[Page 23]]
intent to test or an exemption application.
(3) When both manufacturers and processors are subject to the rule,
exemption applications must be filed by the date processing begins by
any person not processing as of the effective date of the test rule
described in Sec. 790.40 or by 30 days after publication of the Federal
Register notice described in Sec. 790.48(b)(2) who, before the end of
the reimbursement period, processes the test substance and who is
subject to the requirement to submit either a letter of intent to test
or an exemption application.
(c) Scope of application. A person may apply for an exemption from
all, or one or more, specific testing requirements in a test rule in
part 799 of this chapter.
[50 FR 20660, May 17, 1985, as amended at 58 FR 34205, June 23, 1993]
Sec. 790.82 Content of exemption application.
The exemption application must contain:
(a) The identity of the test rule, the chemical identity, and the
CAS No. of the test substance on which the application is based.
(b) The specific testing requirement(s) from which an exemption is
sought and the basis for the exemption request.
(c) Name, address, and telephone number of applicant.
(d) Name, address, and telephone number of appropriate individual to
contact for further information.
(e)(1) If required in the test rule to establish equivalence:
(i) The chemical identity of the test substance on which the
application is based.
(ii) Equivalence data specified in Sec. 790.85.
(2) If a test rule requires testing of a single representative
substance, EPA will consider all forms of the chemical subject to that
rule to be equivalent and will not require the submission of equivalence
data as described in Sec. 790.85.
[50 FR 20660, May 17, 1985, as amended at 54 FR 36315, Sept. 1, 1989]
Sec. 790.85 Submission of equivalence data.
If EPA requires in a test rule promulgated under section 4 of the
Act the testing of two or more test substances which are forms of the
same chemical, each exemption applicant must submit the following data:
(a) The chemical identity of each technical-grade chemical substance
or mixture manufactured and/or processed by the applicant for which the
exemption is sought. The exact type of identifying data required will be
specified in the test rule, but may include all characteristics and
properties of the applicant's substance or mixture, such as boiling
point, melting point, chemical analysis (including identification and
amount of impurities), additives, spectral data, and other physical or
chemical information that may be relevant in determining whether the
applicant's substance or mixture is equivalent to the specific test
substance.
(b) The basis for the applicant's belief that the substance or
mixture is equivalent to the test substance or mixture.
(c) Any other data which exemption applicants are directed to submit
in the test rule which may bear on a determination of equivalence. This
may include a description of the process by which each technical-grade
chemical substance or mixture for which an exemption is sought is
manufactured or processed prior to use or distribution in commerce by
the applicant.
Sec. 790.87 Approval of exemption applications.
(a) EPA will conditionally approve exemption applications if:
(1)(i) For single-phase test rules, EPA has received a letter of
intent to conduct the testing from which exemption is sought;
(ii) For two-phase test rules, EPA has received a complete proposed
study plan for the testing from which exemption is sought and has
adopted the study plan, as proposed or modified, as test standards and
schedules in a final Phase II test rule; and
(2) The chemical substance or mixture with respect to which the
application was submitted is equivalent to a test substance or mixture
for which the
[[Page 24]]
required data have been or are being submitted in accordance with a test
rule; and
(3) Submission of the required test data concerning that chemical
substance or mixture would be duplicative of data which have been or are
being submitted to EPA in accordance with a test rule.
(b)(1) If a single representative substance is to be tested under a
test rule, EPA will consider all forms of the chemical subject to that
rule to be equivalent and will contact the exemption applicant only if
information is missing or unclear.
(2) If two or more representative substances are to be tested under
a test rule, EPA will evaluate equivalence claims made in each exemption
application according to the criteria discussed in the test rule.
(i) If EPA finds an equivalence claim to be in error or inadequately
supported, the applicant will be notified by certified mail. The
applicant will be given 15 days to provide clarifying information.
(ii) Exemption applicants will be notified that equivalence has been
accepted or rejected.
(c) The final Phase II test rule which adopts the study plans in
two-phase rulemaking, a separate Federal Register notice in single-phase
rulemaking, or a letter by certified mail will give exemption applicants
final notice that they have received a conditional exemption. All
conditional exemptions thus granted are contingent upon the test
sponsors' successful completion of testing according to the
specifications in the test rule.
Sec. 790.88 Denial of exemption application.
(a) EPA may deny any exemption application if:
(1) EPA determines that the applicant has failed to demonstrate that
the applicant's chemical is equivalent to the test substance; or
(2) The exemption applicant fails to submit any of the information
specified in Sec. 790.82; or
(3) The exemption applicant fails to submit any of the information
specified in Sec. 790.85 if required in the test rule; or
(4)(i) For single-phase test rules, EPA has not received a letter of
intent to conduct the test for which exemption is sought; or
(ii) For two-phase test rules, EPA has not received an adequate
study plan for the test for which exemption is sought; or
(5) The study sponsor(s) fails to initiate the required testing by
the deadlines adopted in the test rule; or
(6) The study sponsor(s) fails to submit data as required in the
test standard and deadlines for submission of test data as adopted in
the test rule or as modified in accordance with Sec. 790.55.
(b) EPA will notify the exemption applicant by certified mail or
Federal Register notice of EPA's determination that the exemption
application is denied.
Sec. 790.90 Appeal of denial of exemption application.
(a) Within 30 days after receipt of notification that EPA has denied
an application for exemption, the applicant may file an appeal with EPA.
(b) The appeal shall indicate the basis for the applicant's request
for reconsideration.
(c)(1) The applicant may also include a request for a hearing.
Hearings will be held according to the procedures described in
Sec. 790.97.
(2) Hearing requests must be in writing and must be received by EPA
within 30 days of receipt of the letter or publication of the Federal
Register notice described in Sec. 790.88(b). Hearing requests must
provide reasons why a hearing is necessary.
(d) If EPA determines that there are material issues of fact, then
the request for a hearing will be granted. If EPA denies a hearing
request, EPA will base its decision on the written submission.
(e) EPA will notify the applicant of its decision within 60 days
after EPA receives the appeal described in paragraph (a) of this section
or within 60 days after completion of a hearing described in paragraph
(c) of this section.
(f) The filing of an appeal from the denial of an exemption shall
not act to stay the applicant's legal obligations under a test rule
promulgated under section 4 of the Act.
[[Page 25]]
Sec. 790.93 Termination of conditional exemption.
(a) EPA shall terminate a conditional exemption if it determines
that:
(1) The test which provided the basis for approval of the exemption
application has not been started by the deadlines for initiation of
testing adopted in the test rule or modified in accordance with
Sec. 790.55; or
(2) Data required by the test rule have not been generated in
accordance with the test standards or submitted in accordance with the
deadlines for submission of test data that were adopted in the test rule
or modified in accordance with Sec. 790.55; or
(3) The testing has not been conducted or the data have not been
generated in accordance with the Good Laboratory Practice requirements
in part 792 of this chapter.
(b) If EPA determines that one or more of the criteria listed in
paragraph (a) of this section has been met, EPA will notify each holder
of an affected conditional exemption by certified mail or Federal
Register notice of EPA's intent to terminate that conditional exemption.
(c) Within 30 days after receipt of a letter of notification or
publication of a notice in the Federal Register that EPA intends to
terminate a conditional exemption, the exemption holder may submit
information to rebut EPA's preliminary decision or notify EPA by letter
of its intent to conduct the required test pursuant to the test standard
established in the final test rule. Such a letter of intent shall
contain all of the information required by Sec. 790.45(c).
(d)(1) The exemption holder may also include a request for a
hearing. Hearings will be held in accordance with the procedures set
forth in Sec. 790.97.
(2) Hearing requests must be in writing and must be received by EPA
within 30 days after receipt of the letter or publication in the Federal
Register notice described in paragraph (b) of this section.
(e) EPA will notify the exemption holder by certified letter or by
Federal Register notice of EPA's final decision concerning termination
of conditional exemptions and will give instructions as to what actions
the former exemption holder must take to avoid being found in violation
of the test rule.
Sec. 790.97 Hearing procedures.
(a) Hearing requests must be in writing to EPA and must include the
applicant's basis for appealing EPA's decision.
(b) If more than one applicant has requested a hearing on similar
grounds, all of those appeals will be considered at the same hearing
unless confidentiality claims preclude a joint hearing.
(c) EPA will notify each applicant of EPA's decision within 60 days
after the hearing.
Sec. 790.99 Statement of financial responsibility.
Each applicant for an exemption shall submit the following sworn
statement with his or her application:
I understand that if this application is granted before the
reimbursement period described in section 4(c)(3)(B) of TSCA expires, I
must pay fair and equitable reimbursement to the person or persons who
incurred or shared in the costs of complying with the requirement to
submit data and upon whose data the granting of my application was
based.
Appendix A to Subpart E--Schedule for Developing Consent Agreements and
Test Rules
EPA intends to follow the schedule set forth in this Appendix to
evaluate testing candidates, conduct negotiations, develop consent
agreements where appropriate, and propose and promulate test rules in
those instances where testing can be required under section 4(a) of TSCA
but agreement cannot be reached in timely manner on a consent agreement.
Where deadlines are imposed by the statute, they are binding on EPA and
will be observed by the Agency. The remaining dates represent targets
that EPA intends to meet.
This schedule is based on what EPA currently believes are reasonable
target dates. As EPA gains experience with the process and determines
the feasibility of these schedules, it may adjust the schedule
accordingly. EPA will solicit public comment before implementing any
changes in the schedule.
------------------------------------------------------------------------
Week \1\ Event
------------------------------------------------------------------------
0............................. Receive ITC report, recommendation.
2............................. Publish ITC report, 8(a) and 8(d)
notices, and invitation for public
participation in negotiations.
[[Page 26]]
3-6........................... Comment period on ITC report.
6............................. Public focus meeting.
7-14.......................... 8(a) and 8(d) reporting period.
22............................ Public meeting on course-setting
decision and deadline for requests to
participate in negotiations.
22-30......................... Negotiations.
32............................ EPA decision point: consent agreement or
test rule.
------------------------------------------------------------------------
\1\ The dates contained in the left-hand column are calculated from the
date EPA receives the ITC report recommending a chemical for testing.
------------------------------------------------------------------------
Week Consent Agreement Week Test Rule
------------------------------------------------------------------------
36-40.......... Comment period on 32-60 Rule preparation,
consent agreement. agency review and
sign-off.
42............. Comment resolution 62 Publish proposed rule
meeting if necessary. in Federal
Register.\1\
48............. Sign-off consent 70-106 Agency reviews
agreement and Federal comments;
Register notice. preparation of final
rule or no-test
decision, agency
review and sign-
off.\1\
50............. Publish Federal 108 Publish final rule or
Register notice. no-test decision in
Federal Register.\1\
------------------------------------------------------------------------
\1\ As stated in Sec. 790.26, EPA may publish an Advance Notice of
Proposed Rulemaking (ANPR) where the testing recommendations of the
ITC raise unusually novel and complex issues that require additional
Agency review and opportunity for public comment. EPA intends to
publish such ANPRs by Week 62 following receipt of the initial ITC
report; to publish a proposed rule or decision-not-to-test by Week
108; and to publish a final rule or notice terminating the rulemaking
process by Week 154.
[51 FR 23717, June 30, 1986]
PART 791--DATA REIMBURSEMENT--Table of Contents
Subpart A--General Provisions
Sec.
791.1 Scope and authority.
791.2 Applicability.
791.3 Definitions.
Subpart B--Hearing Procedures
791.20 Initiation of reimbursement proceeding.
791.22 Consolidation of hearings.
791.27 Pre-hearing preparation.
791.29 Appointment of hearing officer.
791.30 Hearing procedures.
791.31 Expedited procedures.
791.34 Serving of notice.
791.37 The award.
791.39 Fees and expenses.
Subpart C--Basis for Proposed Order
791.40 Basis for the proposed order.
791.45 Processors.
791.48 Production volume.
791.50 Costs.
791.52 Multiple tests.
Subpart D--Review
791.60 Review.
Subpart E--Final Order
791.85 Availability of final Agency order.
Subpart F--Prohibited Acts
791.105 Prohibited acts.
Authority: 15 U.S.C. 2603 and 2607.
Source: 48 FR 31791, July 11, 1983, unless otherwise noted.
Subpart A--General Provisions
Sec. 791.1 Scope and authority.
(a) This part establishes procedures and criteria to be used in
determining fair amounts of reimbursement for testing costs incurred
under section 4(a) of the Toxic Substances Control Act (TSCA) (15 U.S.C.
2603(a)).
(b) Section 4(c) of TSCA requires EPA to develop rules for the
determination of fair and equitable reimbursement (15 U.S.C. 2603 (c)).
Sec. 791.2 Applicability.
(a) This rule is potentially applicable to all manufacturers,
importers and processors who may be required by a specific test rule
promulgated under section 4(a) of TSCA to conduct tests and submit data,
and who seek the assistance of the Administrator in determining the
amount or method of reimbursement. Persons subject to a test rule have
an obligation from the date the test rule becomes effective until the
end of the reimbursement period, either to test or to obtain an
exemption and pay reimbursement.
(b) The provisions of this rule will take effect only when private
efforts to resolve a dispute have failed and a manufacturer or processor
requests EPA's assistance.
Sec. 791.3 Definitions.
Terms defined in the Act, and not explicitly defined herein, are
used with the meanings given in the Act.
[[Page 27]]
(a) The Act refers to the Toxic Substances Control Act (TSCA) (15
U.S.C. 2601 et seq.).
(b) The Agency or EPA refers to the Environmental Protection Agency.
(c) Byproduct refers to a chemical substance produced without a
separate commercial intent during the manufacture, processing, use or
disposal of another chemical substance or mixture.
(d) Dispute refers to a present controversy between parties subject
to a test rule over the amount or method of reimbursement for the cost
of developing health and environmental data on the test chemical.
(e) Exemption holder refers to a manufacturer or processor, subject
to a test rule, that has received an exemption under sections 4(c)(1) or
4(c)(2) of TSCA from the requirement to conduct a test and submit data.
(f) Impurity refers to a chemical substance unintentionally present
with another chemical substance or mixture.
(g) A party refers to a person subject to a section 4 test rule,
who:
(1) Seeks reimbursement from another person under these rules, or
(2) From whom reimbursement is sought under these rules.
(h) Reimbursement period refers to a period that begins when the
data from the last non-duplicative test to be completed under a test
rule is submitted to EPA and ends after an amount of time equal to that
which had been required to develop that data or after 5 years, whichever
is later.
(i) Small business refers to a manufacturer or importer whose annual
sales, when combined with those of its parent company (if any) are less
than $30 million.
(j) Test rule refers to a regulation ordering the development of
data on health or environmental effects or chemical fate for a chemical
substance or mixture pursuant to TSCA section 4(a).
Subpart B--Hearing Procedures
Sec. 791.20 Initiation of reimbursement proceeding.
(a) When persons subject to a test rule are unable to reach an
agreement on the amount or method of reimbursement for test data
development as described in TSCA section 4(c)(3)(A), any of them may
initiate a proceeding by filing two signed copies of a request for a
hearing with a regional office of the American Arbitration Association
and mailing a copy of the request to EPA, and to each person from whom
they seek reimbursement, or who seeks reimbursement from them.
(b) The request for hearing must contain the following:
(1) The names and addresses of the filing party and its counsel, if
any.
(2) Identification of the test rule under which the dispute arose.
(3) A list of the parties from whom reimbursement is sought or who
are seeking reimbursement, a brief description of the attempts to reach
agreement and a concise explanation of the issues on which the parties
are unable to agree.
(c) The request for a hearing shall be accompanied by the
appropriate administrative fee, as provided in a current Fee Schedule of
the American Arbitration Association.
Sec. 791.22 Consolidation of hearings.
(a) Promptly upon receipt of the request for a hearing, the
Administrator will publish a notice in the Federal Register, advising
those subject to the test rule that a request for a hearing has been
made.
(b) Any other person wishing to participate in the hearing shall so
notify EPA within 45 days of the Federal Register notice. EPA will
promptly inform the regional office of the American Arbitration
Association where the request has been filed of the additional parties.
Sec. 791.27 Pre-hearing preparation.
(a) Responses to requests for hearings. After filing of the request
for hearing, if any other party desires to file an answer it shall be
made in writing and filed with the American Arbitration Association, and
a copy thereof shall be mailed to the other parties within a period of
fourteen days from the date of receiving the complete list of parties.
After the hearing officer is appointed, however, no new or different
claim may be submitted except with the hearing officer's consent.
[[Page 28]]
(b) Pre-hearing conference. At the request of the parties or at the
discretion of the American Arbitration Association, a pre-hearing
conference with a representative of the American Arbitration Association
and the parties or their counsel will be scheduled in appropriate cases
to arrange for an exchange of information and the stipulation of
uncontested facts so as to expedite the proceedings.
(c) Fixing of locale. The parties may mutually agree on the locale
where the hearing is to be held. If the locale is not designated within
45 days from the time the complete list of parties is received, the
American Arbitration Association shall have power to determine the
locale. Its decision shall be final and binding. If any party requests,
and informs the other parties of its request, that the hearing be held
in a specific locale and the other parties file no objection thereto
within 14 days of the request, the locale shall be the one requested.
(d) Time and place. The hearing officer shall fix the time and place
for each hearing. The American Arbitration Association will mail notice
to each party at least 14 days in advance.
Sec. 791.29 Appointment of hearing officer.
(a) Qualifications of hearing officer. All hearing officers shall be
neutral, subject to disqualification for the reasons specified in
paragraph (f) of this section.
(b) Appointment from panel. Promptly after receiving the complete
list of parties at the close of the notice period described in
Sec. 791.22, the American Arbitration Association shall submit
simultaneously to each party to the dispute an identical list of names.
Each party to the dispute shall have thirty days from the mailing date
in which to cross off any names objected to, number the remaining names
to indicate the order of preference, and return the list to the American
Arbitration Association. If a party does not return the list within the
time specified, all persons named therein shall be deemed acceptable to
that party. From among the persons who have been approved on all lists,
and in accordance with the designated order of mutual preference, the
American Arbitration Association shall invite the acceptance of a
hearing officer to serve. If the parties fail to agree upon any of the
persons named, or if acceptable hearing officers are unable to act, or
if for any other reason the appointment cannot be made from the
submitted lists, the American Arbitration Association shall have the
power to make the appointment without the submission of any additional
list.
(c) Nationality of hearing officer in international dispute. If one
of the parties is a national or resident of a country other than the
United States, the hearing officer shall upon the request of any party,
be appointed from among the nationals of a country other than that of
the parties.
(d) Number of hearing officers. The dispute shall be heard and
determined by one hearing officer unless the American Arbitration
Association, in its discretion, directs that a greater number of hearing
officers be appointed.
(e) Notice of appointment. Notice of the appointment of the hearing
officer, together with a copy of these rules, and the signed acceptance
of the hearing officer shall be filed prior to the opening of the first
hearing.
(f) Disclosure and challenge procedure. A person appointed as
hearing officer shall disclose to the American Arbitration Association
any circumstances likely to affect impartiality, including any bias or
any financial or personal interest in the result of the hearing or any
past or present relationship with the parties or their counsel. Upon
receipt of such information from such hearing officer or other source,
the American Arbitration Association shall communicate such information
to the parties, and, if it deems it appropriate to do so, to the hearing
officer and others. Thereafter, the American Arbitration Association
shall determine whether the hearing officer should be disqualified and
shall inform the parties of its decision, which shall be conclusive.
(g) Vacancies. If any hearing officer should resign, die, withdraw,
refuse, be disqualified or be unable to perform the duties of the
office, the American Arbitration Association may, on proof
[[Page 29]]
satisfactory to it, declare the office vacant. Vacancies shall be filled
in accordance with the applicable provisions of these rules and the
matter shall be reheard unless the parties shall agree otherwise.
Sec. 791.30 Hearing procedures.
(a) Representation by counsel. Any party may be represented by
counsel. A party intending to be so represented shall notify the other
parties and the American Arbitration Association of the name and address
of counsel at least 5 days prior to the date set for the hearing at
which counsel is first to appear. When a hearing is initiated by
counsel, or where an attorney replies for the other party, such notice
is deemed to have been given.
(b) Stenographic record. The American Arbitration Association shall
make the necessary arrangements for the taking of a stenographic record.
The parties shall share the cost of such record.
(c) Attendance at hearings. The hearing officer shall have the power
to require the exclusion of anyone, including a party or other essential
person, during the testimony of any witness to protect confidential
business information. It shall be discretionary with the hearing officer
to determine the propriety of the attendance of any other person.
(d) Oaths. Hearing officers shall swear or affirm their neutrality
and their dedication to a fair and equitable resolution. Witnesses shall
swear or affirm that they are telling the truth.
(e) Order of proceedings. (1) A hearing shall be opened by the
filing of the oath of the hearing officer and by the recording of the
place, time and date of the hearing, the presence of the hearing officer
and parties, and counsel, if any, and by the receipt by the hearing
officer of the request for hearing and answer, if any.
(2) The hearing officer may, at the beginning of the hearing, ask
for statements clarifying the issues involved.
(3) The party or parties seeking reimbursement shall then present a
claim and proofs and witnesses, who shall submit to questions or other
examination. The party or parties from whom reimbursement is sought
shall then present a defense and proofs and witnesses, who shall submit
to questions or other examination. The hearing officer has discretion to
vary this procedure but shall afford full and equal opportunity to all
parties for the presentation of any material or relevant proofs.
(4) Exhibits, when offered by any party, shall be received in
evidence by the hearing officer. The names and addresses of all
witnesses and exhibits in order received shall be made a part of the
record.
(f) Hearing in the absence of a party. A hearing may proceed in the
absence of any party which, after due notice, fails to be present or
fails to obtain an adjournment. An award shall not be made solely on the
default of a party. The hearing officer shall require the parties who
are present to submit such evidence as the hearing officer may require
for the making of an award.
(g) Evidence. (1) The parties may offer such evidence as they desire
and shall produce such additional evidence as the hearing officer may
deem necessary to an understanding and determination of the dispute. The
hearing officer shall be the judge of the relevancy and materiality of
the evidence offered and conformity to legal rules of evidence shall not
be necessary. All evidence shall be taken in the presence of all the
hearing officers and of all the parties, except where any of the parties
is absent in default, has waived the right to be present, or has been
excluded by the hearing officer to protect confidential business
information.
(2) All documents not filed with the hearing officer at the hearing,
but arranged for by agreement of the parties, shall be filed with the
American Arbitration Association for transmission to the hearing
officer, according to the agreed-upon schedule. All parties shall be
afforded opportunity to examine such documents.
(h) Evidence by affidavit and filing of documents. The hearing
officer shall receive and consider the evidence of witnesses by
affidavit, but shall give it only such weight as the hearing officer
deems it entitled to after consideration of any objections made to its
admission.
(i) Closing of hearings. The hearing officer shall specifically
inquire of all
[[Page 30]]
parties whether they have any further proofs to offer or witnesses to be
heard. Upon receiving negative replies, the hearing officer shall
declare the hearings closed and record the time of closing of the
hearing. If briefs are to be filed, the hearings shall be declared
closed as of the final date set by the hearing officer for the receipt
of briefs. If documents are to be filed as provided for in paragraph
(g)(2) of this section and the date set for their receipt is later than
that set for the receipt of briefs, the later date shall be the date of
closing the hearings.
(j) Reopening of hearings. The hearings may be reopened on the
hearing officer's own motion, or upon application of a party at any time
before the award is made. If the reopening of the hearings would prevent
the making of the award within the specified time the matter may not be
reopened, unless the parties agree upon the extension of the time limit.
(k) Waiver of oral hearings. The parties may provide, by written
agreement, for the waiver of oral hearings. If the parties are unable to
agree as to the procedure, the American Arbitration Association shall
specify a fair and equitable procedure.
(l) Waiver of rules. Any party who proceeds with the hearing after
knowledge that any provision or requirement of these rules has not been
complied with and who fails to state objection thereto in writing, shall
be deemed to have waived the right to object.
(m) Extensions of time. The parties may modify any period of time by
mutual agreement. The American Arbitration Association for good cause
may extend any period of time established by these rules, except the
time for making the award. (Sec. 791.37(a)) The American Arbitration
Association shall notify the parties of any such extension of time and
its reason therefor.
(n) Communication with hearing officer. There shall be no direct
communication between the parties and a hearing officer other than at
oral hearings. Any other oral or written communications from the parties
to the hearing officer shall be directed to the American Arbitration
Association for transmittal to the hearing officer.
Sec. 791.31 Expedited procedures.
Unless the American Arbitration Association in its discretion
determines otherwise, the Expedited Procedures described in this section
shall be applied in any case where the total claim of any party does not
exceed $5,000, exclusive of interest and hearing costs, and may be
applied in other cases if the parties agree.
(a) Application of rules. The expedited hearings will be conducted
according to the same procedures as the regular ones, except for those
specifically changed by the expedited rules in this section,
Sec. 791.31.
(b) Notice by telephone. The parties shall accept all notices from
the American Arbitration Association by telephone. Such notices by the
American Arbitration Association shall subsequently be confirmed in
writing to the parties. Notwithstanding the failure to confirm in
writing any notice or objection hereunder, the proceeding shall
nonetheless be valid if notice or obligation has, in fact, been given by
telephone.
(c) Appointment and qualifications of hearing officers. The American
Arbitration Association shall submit simultaneously to each party to the
dispute an identical list of five persons from which one hearing officer
shall be appointed. Each party shall have the right to strike two names
from the list on a peremptory basis. The list is returnable to the
American Arbitration Association within 10 days from the date of
mailing. If for any reasons the appointment cannot be made from the
list, the American Arbitration Association shall have the authority to
make the appointment without the submission of additional lists. Such
appointment shall be subject to disqualification for the reasons
specified in Sec. 791.29(f). The parties shall be given notice by
telephone by the American Arbitration Association of the appointment of
the hearing officer. The parties shall notify the American Arbitration
Association, by telephone, within 7 days of any objections to the
hearing officer(s) appointed. Any objection by a party to such hearing
officer shall be confirmed in writing to the American Arbitration
Association with a copy to the other parties.
[[Page 31]]
(d) Time and place of hearing. The hearing officer shall fix the
date, time and place of the hearing. The American Arbitration
Association will notify the parties by telephone, 7 days in advance of
the hearing date. Formal notice of hearing will be sent by the American
Arbitration Association to the parties.
(e) The hearing. Generally, the hearing shall be completed within 1
day. The hearing officer, for good cause shown, may schedule an
additional hearing to be held within 5 days.
(f) Time of award. Unless otherwise agreed to by the parties, the
Award shall be rendered not later than 15 business days from the date of
the closing of the hearing.
Sec. 791.34 Serving of notice.
(a) Each party shall be deemed to have consented that any papers,
notices or process necessary or proper for the initiation or
continuation of a hearing under these rules and for any appeal to EPA or
any court action in connection therewith may be served upon such party
by mail addressed to such party or its attorney at its last known
address or by personal service, within or without the state wherein the
arbitration is to be held (whether such party be within or without the
United States of America), provided that reasonable opportunity to be
heard with regard thereto has been granted such party.
(b) The American Arbitration Association shall, upon the written
request of a party, furnish to such party, at its expense, certified
facsimiles of any papers in the American Arbitration Association's
possession that may be required in appeal to EPA or judicial proceedings
relating to the hearing.
Sec. 791.37 The award.
(a) Time of award. The award shall be made promptly by the hearing
officer and, unless otherwise agreed by the parties, no later than 30
days from the date of closing the hearings, or if oral hearings have
been waived, from the date of transmitting the final statements and
proofs to the hearing officer.
(b) Form of award. The award shall be in writing and shall be signed
either by the sole hearing officer or by at least a majority if there is
more than one. It shall contain a concise statement of its basis and
rationale, and a timetable for payment of any ordered reimbursement.
(c) Delivery of award to parties. Parties shall accept as legal
delivery of the award the delivery of the award or a true copy thereof
by certified mail to the party at its last known address or to its
attorney, or by personal service.
Sec. 791.39 Fees and expenses.
(a) Administrative fees. (1) As a not-for-profit organization, the
American Arbitration Association shall prescribe an Administrative Fee
Schedule and a Refund Schedule to compensate it for the cost of
providing administrative services. The schedule in effect at the time of
filing or the time of refund shall be applicable.
(2) The administrative fees shall be advanced by the initiating
party or parties, subject to final apportionment by the hearing officer
in the award. The administrative fee is increased by 10 percent of the
original for each additional party.
(3) Fees and expenses in excess of the limit contained in section
26(b) of TSCA ($2,500 per person, or $100 per small business) will be
paid by EPA.
(b) Expenses. Subject to paragraph (a)(3) of this section, all
expenses of the hearing, including the cost of recording (though not
transcribing) the hearing and required traveling and other expenses of
the hearing officer and of American Arbitration Association
representatives, and the expenses of any witness or the cost of any
proofs produced at the direct request of the hearing officer, shall be
borne equally by the parties, unless they agree otherwise, or unless the
hearing officer, in the award, assesses such expenses or any part
thereof against any specified party or parties.
(c) Hearing officer's fee. Hearing officers will normally serve
without a fee. In prolonged or special cases the American Arbitration
Association in consultation with the Administrator may determine that
payment of a fee by the parties is appropriate and may establish a
reasonable amount, taking into account the extent of service by the
[[Page 32]]
hearing officer and other relevant circumstances of the case. Any
arrangements for compensation shall be made through the American
Arbitration Association and not directly between the parties and the
hearing officer.
Subpart C--Basis for Proposed Order
Sec. 791.40 Basis for the proposed order.
(a) The hearing officer shall propose a fair and equitable amount of
reimbursement. The formula in paragraph (b) of this section shall be
presumed to be fair and equitable as applied to all persons subject to a
test rule. However, the hearing officer has the discretion to modify the
formula, or to use some other basis for allocation if necessary.
Additional factors that may be taken into account include, but are not
limited to, relative amounts of exposure attributable to each person and
the effect of the reimbursement share on competitive position.
(b) In general, each person's share of the test cost shall be in
proportion to its share of the total production volume of the test
chemical:
[GRAPHIC] [TIFF OMITTED] TC15NO91.044
Where:
R=the reimbursement share owed by company X.
C=the total cost of the testing required by the test rule.
Vx=the volume of the test chemical produced or imported by
company X over the period defined by Sec. 791.48.
Vt=the total volume of the test chemical produced or imported
over the period defined by Sec. 791.48.
(c) The burden of proposing modifications to the formula shall lie
with the party requesting the modification.
Sec. 791.45 Processors.
(a) Generally, processors will be deemed to have fulfilled their
testing and reimbursement responsibilities indirectly, through higher
prices passed on by those directly responsible, the manufacturers. There
are three circumstances in which processors will have a responsibility
to provide reimbursement directly to those paying for the testing:
(1) When a test rule or subsequent Federal Register notice
pertaining to a test rule expressly obligates processors as well as
manufacturers to assume direct testing and data reimbursement
responsibilities.
(2) When one or more manufacturers demonstrate to the hearing
officer that it is necessary to include processors in order to provide
fair and equitable reimbursement in a specific case.
(3) When one or more processors voluntarily agree to reimburse
manufacturers for a portion of test costs. Only those processors who
volunteer will incur the obligation.
(b) A hearing including processors shall be initiated in the same
way as those including only manufacturers. Voluntary negotiations must
be attempted in good faith first, and the request for a hearing must
contain the names of the parties and a description of the unsuccessful
negotiations.
(c) When processors as well as manufacturers are required to provide
reimbursement, the hearing officer will decide for each case how the
reimbursement should be allocated among the participating parties. When
a test rule is applicable solely to processors, the hearing officer will
apply the formula to the amount of the test chemical purchased or
processed.
Sec. 791.48 Production volume.
(a) Production volume will be measured over a period that begins one
calendar year before publication of the final test rule in the Federal
Register and continues up to the latest data available upon resolution
of a dispute.
(b) For the purpose of determining fair reimbursement shares,
production volume shall include amounts of the test chemical imported in
bulk form and mixtures, and the total domestic production of the
chemical including that produced as a byproduct. Impurities will not be
included unless the test rule specifically includes them.
(c) Amounts of the test chemical manufactured for export will not be
included unless covered by a finding under TSCA section 12(a)(2).
[[Page 33]]
(d) Chemicals excluded from the jurisdiction of TSCA by section
3(2)(B) need not be included in the computation of production volume.
(Chemicals used as intermediates to produce pesticides are covered by
TSCA.)
(e) The burden of establishing the fact that particular amounts of
the test chemical are produced for exempt purposes lies with the party
seeking to exclude those amounts from the calculation of his production
volume.
Sec. 791.50 Costs.
(a) All costs reasonable and necessary to comply with the test rule,
taking into account the practices of other laboratories in conducting
similar tests, are eligible for reimbursement. Necessary costs include:
(1) Direct and indirect costs of planning, conducting, analyzing and
submitting the test results to EPA.
(2) A reasonable profit, and a reasonable rate of interest and
depreciation on the tester's initial capital investment.
(3) The cost of repeating or repairing tests where failure was
demonstrably due to some cause other than negligence of the tester.
(b) Costs attributable to tests beyond those specified by EPA shall
not be eligible for reimbursement under this rule.
Sec. 791.52 Multiple tests.
When more than one of a particular kind of test required by the test
rule is performed, the additional costs will be shared among all those
holding exemptions. The costs of all the tests will be added together
and each exemption holder shall be responsible for a share of the total
which is equal to its share of the total production of the test
chemical. The exemption holders shall divide their shares between test
sponsors in proportion to the costs of their respective tests. Those
sponsoring a particular test do not have to obtain exemptions for that
test and therefore do not have reimbursement responsibilities for the
same tests done by others.
Subpart D--Review
Sec. 791.60 Review.
(a) The hearing officer's proposed order shall become the final
Agency order 30 days after issuance unless within the 30-day period one
of the parties requests Agency review or the Administrator of his own
initiative decides to review the proposed order.
(b) The proposed order may be reviewed upon the record of the
hearing and the petitions for review. If necesary, the Administrator may
order the transcription of the stenographic record of the hearing,
written briefs, oral arguments or any other reasonable aids to making an
equitable decision.
(c) The final Agency order may be reviewed in federal court as
provided by 26 U.S.C. 2603(c).
Subpart E--Final Order
Sec. 791.85 Availablity of final Agency order.
The final Agency order shall be available to the public for
inspection and copying pursuant to 5 U.S.C. 552(a)(2), subject to
necessary confidentiality restrictions.
Subpart F--Prohibited Acts
Sec. 791.105 Prohibited acts.
Failure to provide information required by the Agency or to pay the
amounts awarded under this rule within time alloted in the final order
shall constitute a violation of 15 U.S.C. 2614(1) or 2614(3).
PART 792--GOOD LABORATORY PRACTICE STANDARDS--Table of Contents
Subpart A--General Provisions
Sec.
792.1 Scope.
792.3 Definitions.
792.10 Applicability to studies performed under grants and contracts.
792.12 Statement of compliance or non-compliance.
792.15 Inspection of a testing facility.
792.17 Effects of non-compliance.
[[Page 34]]
Subpart B--Organization and Personnel
792.29 Personnel.
792.31 Testing facility management.
792.33 Study director.
792.35 Quality assurance unit.
Subpart C--Facilities
792.41 General.
792.43 Test system care facilities.
792.45 Test system supply facilities.
792.47 Facilities for handling test, control, and reference substances.
792.49 Laboratory operation areas.
792.51 Specimen and data storage facilities.
Subpart D--Equipment
792.61 Equipment design.
792.63 Maintenance and calibration of equipment.
Subpart E--Testing Facilities Operation
792.81 Standard operating procedures.
792.83 Reagents and solutions.
792.90 Animal and other test system care.
Subpart F--Test, Control, and Reference Substances
792.105 Test, control, and reference substance characterization.
792.107 Test, control, and reference substance handling.
792.113 Mixtures of substances with carriers.
Subpart G--Protocol for and Conduct of A Study
792.120 Protocol.
792.130 Conduct of a study.
792.135 Physical and chemical characterization studies.
Subparts H-I [Reserved]
Subpart J--Records and Reports
792.185 Reporting of study results.
792.190 Storage and retrieval of records and data.
792.195 Retention of records.
Authority: 15 U.S.C. 2603.
Source: 54 FR 34043, Aug. 17, 1989, unless otherwise noted.
Subpart A--General Provisions
Sec. 792.1 Scope.
(a) This part prescribes good laboratory practices for conducting
studies relating to health effects, environmental effects, and chemical
fate testing. This part is intended to ensure the quality and integrity
of data submitted pursuant to testing consent agreements and test rules
issued under section 4 of the Toxic Substances Control Act (TSCA) (Pub.
L. 94-469, 90 Stat. 2006, 15 U.S.C. 2603 et seq.).
(b) This part applies to any study described by paragraph (a) of
this section which any person conducts, initiates, or supports on or
after September 18, 1989.
(c) It is EPA's policy that all data developed under section 5 of
TSCA be in accordance with provisions of this part. If data are not
developed in accordance with the provisions of this part, EPA will
consider such data insufficient to evaluate the health and environmental
effects of the chemical substances unless the submitter provides
additional information demonstrating that the data are reliable and
adequate.
Sec. 792.3 Definitions.
As used in this part the following terms shall have the meanings
specified:
Batch means a specific quantity or lot of a test, control, or
reference substance that has been characterized according to
Sec. 792.105(a).
Carrier means any material, including but not limited to, feed,
water, soil, and nutrient media, with which the test substance is
combined for administration to a test system.
Control substance means any chemical substance or mixture, or any
other material other than a test substance, feed, or water, that is
administered to the test system in the course of a study for the purpose
of establishing a basis for comparison with the test substance for
chemical or biologicaI measurements.
EPA means the U.S. Environmental Protection Agency.
Experimental start date means the first date the test substance is
applied to the test system.
Experimental termination date means the last date on which data are
collected directly from the study.
FDA means the U.S. Food and Drug Administration.
Person includes an individual, partnership, corporation,
association, scientific or academic establishment, government agency, or
organizational unit thereof, and any other legal entity.
[[Page 35]]
Quality assurance unit means any person or organizational element,
except the study director, designated by testing facility management to
perform the duties relating to quality assurance of the studies.
Raw data means any laboratory worksheets, records, memoranda, notes,
or exact copies thereof, that are the result of original observations
and activities of a study and are necessary for the reconstruction and
evaluation of the report of that study. In the event that exact
transcripts of raw data have been prepared (e.g., tapes which have been
transcribed verbatim, dated, and verified accurate by signature), the
exact copy or exact transcript may be substituted for the original
source as raw data. ``Raw data'' may include photographs, microfilm or
microfiche copies, computer printouts, magnetic media, including
dictated observations, and recorded data from automated instruments.
Reference substance means any chemical substance or mixture, or
analytical standard, or material other than a test substance, feed, or
water, that is administered to or used in analyzing the test system in
the course of a study for the purposes of establishing a basis for
comparison with the test substance for known chemical or biological
measurements.
Specimen means any material derived from a test system for
examination or analysis.
Sponsor means:
(1) A person who initiates and supports, by provision of financial
or other resources, a study;
(2) A person who submits a study to the EPA in response to a TSCA
section 4(a) test rule and/or a person who submits a study under a TSCA
section 4 testing consent agreement or a TSCA section 5 rule or order to
the extent the agreement, rule or order references this part; or
(3) A testing facility, if it both initiates and actually conducts
the study.
Study means any experiment at one or more test sites, in which a
test substance is studied in a test system under laboratory conditions
or in the environment to determine or help predict its effects,
metabolism, environmental and chemical fate, persistence, or other
characteristics in humans, other living organisms, or media. The term
``study'' does not include basic exploratory studies carried out to
determine whether a test substance or a test method has any potential
utility.
Study completion date means the date the final report is signed by
the study director.
Study director means the individual responsible for the overall
conduct of a study.
Study initiation date means the date the protocol is signed by the
study director.
Test substance means a substance or mixture administered or added to
a test system in a study, which substance or mixture is used to develop
data to meet the requirements of a TSCA section 4(a) test rule and/or is
developed under a TSCA section 4 testing consent agreement or section 5
rule or order to the extent the agreement, rule or order references this
part.
Test system means any animal, plant, microorganism, chemical or
physical matrix, including but not limited to, soil or water, or
components thereof, to which the test, control, or reference substance
is administered or added for study. ``Test system'' also includes
appropriate groups or components of the system not treated with the
test, control, or reference substance.
Testing facility means a person who actually conducts a study, i.e.,
actually uses the test substance in a test system. ``Testing facility''
encompasses only those operational units that are being or have been
used to conduct studies.
TSCA means the Toxic Substances Control Act (15 U.S.C, 2601 et seq.)
Vehicle means any agent which facilitates the mixture, dispersion,
or solubilization of a test substance with a carrier.
Sec. 792.10 Applicability to studies performed under grants and contracts.
When a sponsor or other person utilizes the services of a consulting
laboratory, contractor, or grantee to perform all or a part of a study
to which this part applies, it shall notify the consulting laboratory,
contractor, or grantee that the service is, or is part of, a study that
must be conducted in
[[Page 36]]
compliance with the provisions of this part.
Sec. 792.12 Statement of compliance or non-compliance.
Any person who submits to EPA a test required by a testing consent
agreement or a test rule issued under section 4 of TSCA shall include in
the submission a true and correct statement, signed by the sponsor and
the study director, of one of the following types:
(a) A statement that the study was conducted in accordance with this
part; or
(b) A statement describing in detail all differences between the
practices used in the study and those required by this part; or
(c) A statement that the person was not a sponsor of the study, did
not conduct the study, and does not know whether the study was conducted
in accordance with this part.
Sec. 792.15 Inspection of a testing facility.
(a) A testing facility shall permit an authorized employee or duly
designated representative of EPA or FDA, at reasonable times and in a
reasonable manner, to inspect the facility and to inspect (and in the
case of records also to copy) all records and specimens required to be
maintained regarding studies to which this part applies. The records
inspection and copying requirements shall not apply to quality assurance
unit records of findings and problems, or to actions recommended and
taken, except the EPA may seek production of these records in litigation
or formal adjudicatory hearings.
(b) EPA will not consider reliable for purposes of showing that a
chemical substance or mixture does not present a risk of injury to
health or the environment any data developed by a testing facility or
sponsor that refuses to permit inspection in accordance with this part.
The determination that a study will not be considered reliable does not,
however, relieve the sponsor of a required test of any obligation under
any applicable statute or regulation to submit the results of the study
to EPA.
(c) Since a testing facility is a place where chemicals are stored
or held, it is subject to inspection under section 11 of TSCA.
Sec. 792.17 Effects of non-compliance.
(a) The sponsor or any other person who is conducting or has
conducted a test to fulfill the requirements of a testing consent
agreement or a test rule issued under section 4 of TSCA will be in
violation of section 15 of TSCA if:
(1) The test is not being or was not conducted in accordance with
any requirement of this part;
(2) Data or information submitted to EPA under this part (including
the statement required by Sec. 792.12) include information or data that
are false or misleading, contain significant omissions, or otherwise do
not fulfill the requirements of this part; or
(3) Entry in accordance with Sec. 792.15 for the purpose of auditing
test data or inspecting test facilities is denied. Persons who violate
the provisions of this part may be subject to civil or criminal
penalties under section 16 of TSCA, legal action in United States
district court under section 17 of TSCA, or criminal prosecution under
18 U.S.C. 2 or 1001.
(b) EPA, at its discretion, may not consider reliable for purposes
of showing that a chemical substance or mixture does not present a risk
of injury to health or the environment any study which was not conducted
in accordance with this part. EPA, at its discretion, may rely upon such
studies for purposes of showing adverse effects. The determination that
a study will not be considered reliable does not, however, relieve the
sponsor of a required test of the obligation under any applicable
statute or regulation to submit the results of the study to EPA.
(c) If data submitted to fulfill a requirement of a testing consent
agreement or a test rule issued under section 4 of TSCA are not
developed in accordance with this part, EPA may determine that the
sponsor has not fulfilled its obligations under section 4 of TSCA and
may require the sponsor to develop data in accordance with the
requirements of this part in order to satisfy such obligations.
[[Page 37]]
Subpart B--Organization and Personnel
Sec. 792.29 Personnel.
(a) Each individual engaged in the conduct of or responsible for the
supervision of a study shall have education, training, and experience,
or combination thereof, to enable that individual to perform the
assigned functions.
(b) Each testing facility shall maintain a current summary of
training and experience and job description for each individual engaged
in or supervising the conduct of a study.
(c) There shall be a sufficient number of personnel for the timely
and proper conduct of the study according to the protocol.
(d) Personnel shall take necessary personal sanitation and health
precautions designed to avoid contamination of test, control, and
reference substances and test systems.
(e) Personnel engaged in a study shall wear clothing appropriate for
the duties they perform. Such clothing shall be changed as often as
necessary to prevent microbiological, radiological, or chemical
contamination of test systems and test, control, and reference
substances.
(f) Any individual found at any time to have an illness that may
adversely affect the quality and integrity of the study shall be
excluded from direct contact with test systems, test, control, and
reference substances and any other operation or function that may
adversely affect the study until the condition is corrected. All
personnel shall be instructed to report to their immediate supervisors
any health or medical conditions that may reasonably be considered to
have an adverse effect on a study.
Sec. 792.31 Testing facility management.
For each study, testing facility management shall:
(a) Designate a study director as described in Sec. 792.33 before
the study is initiated.
(b) Replace the study director promptly if it becomes necessary to
do so during the conduct of a study.
(c) Assure that there is a quality assurance unit as described in
Sec. 792.35.
(d) Assure that test, control, and reference substances or mixtures
have been appropriately tested for identity, strength, purity,
stability, and uniformity, as applicable.
(e) Assure that personnel, resources, facilities, equipment,
materials and methodologies are available as scheduled.
(f) Assure that personnel clearly understand the functions they are
to perform.
(g) Assure that any deviations from these regulations reported by
the quality assurance unit are communicated to the study director and
corrective actions are taken and documented.
Sec. 792.33 Study director.
For each study, a scientist or other professional of appropriate
education, training, and experience, or combination thereof, shall be
identified as the study director. The study director has overall
responsibility for the technical conduct of the study, as well as for
the interpretation, analysis, documentation, and reporting of results,
and represents the single point of study control. The study director
shall assure that:
(a) The protocol, including any change, is approved as provided by
Sec. 792.120 and is followed.
(b) All experimental data, including observations of unanticipated
responses of the test system are accurately recorded and verified.
(c) Unforeseen circumstances that may affect the quality and
integrity of the study are noted when they occur, and corrective action
is taken and documented.
(d) Test systems are as specified in the protocol.
(e) All applicable good laboratory practice regulations are
followed.
(f) All raw data, documentation, protocols, specimens, and final
reports are transferred to the archives during or at the close of the
study.
Sec. 792.35 Quality assurance unit.
(a) A testing facility shall have a quality assurance unit which
shall be responsible for monitoring each study to assure management that
the facilities, equipment, personnel, methods, practices, records, and
controls are in
[[Page 38]]
conformance with the regulations in this part. For any given study, the
quality assurance unit shall be entirely separate from and independent
of the personnel engaged in the direction and conduct of that study. The
quality assurance unit shall conduct inspections and maintain records
appropriate to the study.
(b) The quality assurance unit shall:
(1) Maintain a copy of a master schedule sheet of all studies
conducted at the testing facility indexed by test substance and
containing the test system, nature of study, date study was initiated,
current status of each study, identity of the sponsor, and name of the
study director.
(2) Maintain copies of all protocols pertaining to all studies for
which the unit is responsible.
(3) Inspect each study at intervals adequate to ensure the integrity
of the study and maintain written and properly signed records of each
periodic inspection showing the date of the inspection, the study
inspected, the phase or segment of the study inspected, the person
performing the inspection, findings and problems, action recommended and
taken to resolve existing problems, and any scheduled date for re-
inspection. Any problems which are likely to affect study integrity
found during the course of an inspection shall be brought to the
attention of the study director and management immediately.
(4) Periodically submit to management and the study director written
status reports on each study, noting any problems and the corrective
actions taken.
(5) Determine that no deviations from approved protocols or standard
operating procedures were made without proper authorization and
documentation.
(6) Review the final study report to assure that such report
accurately describes the methods and standard operating procedures, and
that the reported results accurately reflect the raw data of the study.
(7) Prepare and sign a statement to be included with the final study
report which shall specify the dates inspections were made and findings
reported to management and to the study director.
(c) The responsibilities and procedures applicable to the quality
assurance unit, the records maintained by the quality assurance unit,
and the method of indexing such records shall be in writing and shall be
maintained. These items including inspection dates, the study inspected,
the phase or segment of the study inspected, and the name of the
individual performing the inspection shall be made available for
inspection to authorized employees or duly designated representatives of
EPA or FDA.
(d) An authorized employee or a duly designated representative of
EPA or FDA shall have access to the written procedures established for
the inspection and may request testing facility management to certify
that inspections are being implemented, performed, documented, and
followed up in accordance with this paragraph.
Subpart C--Facilities
Sec. 792.41 General.
Each testing facility shall be of suitable size and construction to
facilitate the proper conduct of studies. Testing facilities which are
not located within an indoor controlled environment shall be of suitable
location to facilitate the proper conduct of studies. Testing facilities
shall be designed so that there is a degree of separation that will
prevent any function or activity from having an adverse effect on the
study.
Sec. 792.43 Test system care facilities.
(a) A testing facility shall have a sufficient number of animal
rooms or other test system areas, as needed, to ensure: proper
separation of species or test systems, isolation of individual projects,
quarantine or isolation of animals or other test systems, and routine or
specialized housing of animals or other test systems.
(1) In tests with plants or aquatic animals, proper separation of
species can be accomplished within a room or area by housing them
separately in different chambers or aquaria. Separation of species is
unnecessary where the protocol specifies the simultaneous exposure of
two or more species in the
[[Page 39]]
same chamber, aquarium, or housing unit.
(2) Aquatic toxicity tests for individual projects shall be isolated
to the extent necessary to prevent cross-contamination of different
chemicals used in different tests.
(b) A testing facility shall have a number of animal rooms or other
test system areas separate from those described in paragraph (a) of this
section to ensure isolation of studies being done with test systems or
test, control, and reference substances known to be biohazardous,
including volatile substances, aerosols, radioactive materials, and
infectious agents.
(c) Separate areas shall be provided, as appropriate, for the
diagnosis, treatment, and control of laboratory test system diseases.
These areas shall provide effective isolation for the housing of test
systems either known or suspected of being diseased, or of being
carriers of disease, from other test systems.
(d) Facilities shall have proper provisions for collection and
disposal of contaminated water, soil, or other spent materials. When
animals are housed, facilities shall exist for the collection and
disposal of all animal waste and refuse or for safe sanitary storage of
waste before removal from the testing facility. Disposal facilities
shall be so provided and operated as to minimize vermin infestation,
odors, disease hazards, and environmental contamination.
(e) Facilities shall have provisions to regulate environmental
conditions (e.g., temperature, humidity, photoperiod) as specified in
the protocol.
(f) For marine test organisms, an adequate supply of clean sea water
or artificial sea water (prepared from deionized or distilled water and
sea salt mixture) shall be available. The ranges of composition shall be
as specified in the protocol.
(g) For freshwater organisms, an adequate supply of clean water of
the appropriate hardness, pH, and temperature, and which is free of
contaminants capable of interfering with the study shall be available as
specified in the protocol.
(h) For plants, an adequate supply of soil of the appropriate
composition, as specified in the protocol, shall be available as needed.
Sec. 792.45 Test system supply facilities.
(a) There shall be storage areas, as needed, for feed, nutrients,
soils, bedding, supplies, and equipment. Storage areas for feed,
nutrients, soils, and bedding shall be separated from areas where the
test systems are located and shall be protected against infestation or
contamination. Perishable supplies shall be preserved by appropriate
means.
(b) When appropriate, plant supply facilities shall be provided.
These include:
(1) Facilities, as specified in the protocol, for holding,
culturing, and maintaining algae and aquatic plants.
(2) Facilities, as specified in the protocol, for plant growth,
including but not limited to, greenhouses, growth chambers, light banks,
and fields.
(c) When appropriate, facilities for aquatic animal tests shall be
provided. These include but are not limited to aquaria, holding tanks,
ponds, and ancillary equipment, as specified in the protocol.
Sec. 792.47 Facilities for handling test, control, and reference substances.
(a) As necessary to prevent contamination or mixups, there shall be
separate areas for:
(1) Receipt and storage of the test, control, and reference
substances.
(2) Mixing of the test, control, and reference substances with a
carrier, e.g., feed.
(3) Storage of the test, control, and reference substance mixtures.
(b) Storage areas for test, control, and/or reference substance and
for test, control, and/or reference mixtures shall be separate from
areas housing the test systems and shall be adequate to preserve the
identity, strength, purity, and stability of the substances and
mixtures.
Sec. 792.49 Laboratory operation areas.
Separate laboratory space and other space shall be provided, as
needed, for the performance of the routine and specialized procedures
required by studies.
[[Page 40]]
Sec. 792.51 Specimen and data storage facilities.
Space shall be provided for archives, limited to access by
authorized personnel only, for the storage and retrieval of all raw data
and specimens from completed studies.
Subpart D--Equipment
Sec. 792.61 Equipment design.
Equipment used in the generation, measurement, or assessment of data
and equipment used for facility environmental control shall be of
appropriate design and adequate capacity to function according to the
protocol and shall be suitably located for operation, inspection,
cleaning, and maintenance.
Sec. 792.63 Maintenance and calibration of equipment.
(a) Equipment shall be adequately inspected, cleaned, and
maintained. Equipment used for the generation, measurement, or
assessment of data shall be adequately tested, calibrated, and/or
standardized.
(b) The written standard operating procedures required under
Sec. 792.81(b)(11) shall set forth in sufficient detail the methods,
materials, and schedules to be used in the routine inspection, cleaning,
maintenance, testing, calibration, and/or standardization of equipment,
and shall specify, when appropriate, remedial action to be taken in the
event of failure or malfunction of equipment. The written standard
operating procedures shall designate the person responsible for the
performance of each operation.
(c) Written records shall be maintained of all inspection,
maintenance, testing, calibrating, and/or standardizing operations.
These records, containing the date of the operation, shall describe
whether the maintenance operations were routine and followed the written
standard operating procedures. Written records shall be kept of
nonroutine repairs performed on equipment as a result of failure and
malfunction. Such records shall document the nature of the defect, how
and when the defect was discovered, and any remedial action taken in
response to the defect.
Subpart E--Testing Facilities Operation
Sec. 792.81 Standard operating procedures.
(a) A testing facility shall have standard operating procedures in
writing, setting forth study methods that management is satisfied are
adequate to insure the quality and integrity of the data generated in
the course of a study. All deviations in a study from standard operating
procedures shall be authorized by the study director and shall be
documented in the raw data. Significant changes in established standard
operating procedures shall be properly authorized in writing by
management.
(b) Standard operating procedures shall be established for, but not
limited to, the following:
(1) Test system room preparation.
(2) Test system care.
(3) Receipt, identification, storage, handling, mixing, and method
of sampling of the test, control, and reference substances.
(4) Test system observations.
(5) Laboratory or other tests.
(6) Handling of test systems found moribund or dead during study.
(7) Necropsy of test systems or postmortem examination of test
systems.
(8) Collection and identification of specimens.
(9) Histopathology.
(10) Data handling, storage and retrieval.
(11) Maintenance and calibration of equipment.
(12) Transfer, proper placement, and identification of test systems.
(c) Each laboratory or other study area shall have immediately
available manuals and standard operating procedures relative to the
laboratory or field procedures being performed. Published literature may
be used as a supplement to standard operating procedures.
(d) A historical file of standard operating procedures, and all
revisions thereof, including the dates of such revisions, shall be
maintained.
Sec. 792.83 Reagents and solutions.
All reagents and solutions in the laboratory areas shall be labeled
to indicate identity, titer or concentration,
[[Page 41]]
storage requirements, and expiration date. Deteriorated or outdated
reagents and solutions shall not be used.
Sec. 792.90 Animal and other test system care.
(a) There shall be standard operating procedures for the housing,
feeding, handling, and care of animals and other test systems.
(b) All newly received test systems from outside sources shall be
isolated and their health status or appropriateness for the study shall
be evaluated. This evaluation shall be in accordance with acceptable
veterinary medical practice or scientific methods.
(c) At the initiation of a study, test systems shall be free of any
disease or condition that might interfere with the purpose or conduct of
the study. If during the course of the study, the test systems contract
such a disease or condition, the diseased test systems should be
isolated, if necessary. These test systems may be treated for disease or
signs of disease provided that such treatment does not interfere with
the study. The diagnosis, authorization of treatment, description of
treatment, and each date of treatment shall be documented and shall be
retained.
(d) Warm-blooded animals, adult reptiles, and adult terrestrial
amphibians used in laboratory procedures that require manipulations and
observations over an extended period of time, or in studies that require
these test systems to be removed from and returned to their test system-
housing units for any reason (e.g., cage cleaning, treatment, etc.),
shall receive appropriate identification (e.g., tattoo, color code, ear
tag, ear punch, etc.). All information needed to specifically identify
each test system within the test system-housing unit shall appear on the
outside of that unit. Suckling mammals and juvenile birds are excluded
from the requirement of individual identification unless otherwise
specified in the protocol.
(e) Except as specified in paragraph (e)(1) of this section, test
systems of different species shall be housed in separate rooms when
necessary. Test systems of the same species, but used in different
studies, should not ordinarily be housed in the same room when
inadvertent exposure to test, control, or reference substances or test
system mixup could affect the outcome of either study. If such mixed
housing is necessary, adequate differentiation by space and
identification shall be made.
(1) Plants, invertebrate animals, aquatic vertebrate animals, and
organisms that may be used in multispecies tests need not be housed in
separate rooms, provided that they are adequately segregated to avoid
mixup and cross contamination.
(2) [Reserved]
(f) Cages, racks, pens, enclosures, aquaria, holding tanks, ponds,
growth chambers, and other holding, rearing, and breeding areas, and
accessory equipment, shall be cleaned and sanitized at appropriate
intervals.
(g) Feed, soil, and water used for the test systems shall be
analyzed periodically to ensure that contaminants known to be capable of
interfering with the study and reasonably expected to be present in such
feed, soil, or water are not present at levels above those specified in
the protocol. Documentation of such analyses shall be maintained as raw
data.
(h) Bedding used in animal cages or pens shall not interfere with
the purpose or conduct of the study and shall be changed as often as
necessary to keep the animals dry and clean.
(i) If any pest control materials are used, the use shall be
documented. Cleaning and pest control materials that interfere with the
study shall not be used.
(j) All plant and animal test systems shall be acclimatized to the
environmental conditions of the test, prior to their use in a study.
Subpart F--Test, Control, and Reference Substances
Sec. 792.105 Test, control, and reference substance characterization.
(a) The identity, strength, purity, and composition, or other
characteristics which will appropriately define the test, control, or
reference substance shall be determined for each batch and shall be
documented before its use in a study. Methods of synthesis, fabrication,
or derivation of the test, control,
[[Page 42]]
or reference substance shall be documented by the sponsor or the testing
facility, and such location of documentation shall be specified.
(b) When relevant to the conduct of the study the solubility of each
test, control, or reference substance shall be determined by the testing
facility or the sponsor before the experimental start date. The
stability of the test, control or reference substance shall be
determined before the experimental start date or concomitantly according
to written standard operating procedures, which provide for periodic
analysis of each batch.
(c) Each storage container for a test, control, or reference
substance shall be labeled by name, chemical abstracts service number
(CAS) or code number, batch number, expiration date, if any, and, where
appropriate, storage conditions necessary to maintain the identity,
strength, purity, and composition of the test, control, or reference
substance. Storage containers shall be assigned to a particular test
substance for the duration of the study.
(d) For studies of more than 4 weeks experimental duration, reserve
samples from each batch of test, control, and reference substances shall
be retained for the period of time provided by Sec. 792.195.
(e) The stability of test, control, and reference substances under
storage conditions at the test site shall be known for all studies.
Sec. 792.107 Test, control, and reference substance handling.
Procedures shall be established for a system for the handling of the
test, control, and reference substances to ensure that:
(a) There is proper storage.
(b) Distribution is made in a manner designed to preclude the
possibility of contamination, deterioration, or damage.
(c) Proper identification is maintained throughout the distribution
process.
(d) The receipt and distribution of each batch is documented. Such
documentation shall include the date and quantity of each batch
distributed or returned.
Sec. 792.113 Mixtures of substances with carriers.
(a) For each test, control, or reference substance that is mixed
with a carrier, tests by appropriate analytical methods shall be
conducted:
(1) To determine the uniformity of the mixture and to determine,
periodically, the concentration of the test, control, or reference
substance in the mixture.
(2) When relevant to the conduct of the experiment, to determine the
solubility of each test, control, or reference substance in the mixture
by the testing facility or the sponsor before the experimental start
date.
(3) To determine the stability of the test, control or reference
substance in the mixture before the experimental start date or
concomitantly according to written standard operating procedures, which
provide for periodic analysis of each batch.
(b) Where any of the components of the test, control, or reference
substance carrier mixture has an expiration date, that date shall be
clearly shown on the container. If more than one component has an
expiration date, the earliest date shall be shown.
(c) If a vehicle is used to facilitate the mixing of a test
substance with a carrier, assurance shall be provided that the vehicle
does not interfere with the integrity of the test.
Subpart G--Protocol for and Conduct of A Study
Sec. 792.120 Protocol.
(a) Each study shall have an approved written protocol that clearly
indicates the objectives and all methods for the conduct of the study.
The protocol shall contain but shall not necessarily be limited to the
following information:
(1) A descriptive title and statement of the purpose of the study.
(2) Identification of the test, control, and reference substance by
name, chemical abstracts service (CAS) number or code number.
(3) The name and address of the sponsor and the name and address of
the testing facility at which the study is being conducted.
[[Page 43]]
(4) The proposed experimental start and termination dates.
(5) Justification for selection of the test system.
(6) Where applicable, the number, body weight, sex, source of
supply, species, strain, substrain, and age of the test system.
(7) The procedure for identification of the test system.
(8) A description of the experimental design, including methods for
the control of bias.
(9) Where applicable, a description and/or identification of the
diet used in the study as well as solvents, emulsifiers and/or other
materials used to solubilize or suspend the test, control, or reference
substances before mixing with the carrier. The description shall include
specifications for acceptable levels of contaminants that are reasonably
expected to be present in the dietary materials and are known to be
capable of interfering with the purpose or conduct of the study if
present at levels greater than established by the specifications.
(10) The route of administration and the reason for its choice.
(11) Each dosage level, expressed in milligrams per kilogram of body
or test system weight or other appropriate units, of the test, control,
or reference substance to be administered and the method and frequency
of administration.
(12) The type and frequency of tests, analyses, and measurements to
be made.
(13) The records to be maintained.
(14) The date of approval of the protocol by the sponsor and the
dated signature of the study director.
(15) A statement of the proposed statistical method.
(b) All changes in or revisions of an approved protocol and the
reasons therefor shall be documented, signed by the study director,
dated, and maintained with the protocol.
Sec. 792.130 Conduct of a study.
(a) The study shall be conducted in accordance with the protocol.
(b) The test systems shall be monitored in conformity with the
protocol.
(c) Specimens shall be identified by test system, study, nature, and
date of collection. This information shall be located on the specimen
container or shall accompany the specimen in a manner that precludes
error in the recording and storage of data.
(d) In animal studies where histopathology is required, records of
gross findings for a specimen from postmortem observations shall be
available to a pathologist when examining that specimen
histopathologically.
(e) All data generated during the conduct of a study, except those
that are generated by automated data collection systems, shall be
recorded directly, promptly, and legibly in ink. All data entries shall
be dated on the day of entry and signed or initialed by the person
entering the data. Any change in entries shall be made so as not to
obscure the original entry, shall indicate the reason for such change,
and shall be dated and signed or identified at the time of the change.
In automated data collection systems, the individual responsible for
direct data input shall be identified at the time of data input. Any
change in automated data entries shall be made so as not to obscure the
original entry, shall indicate the reason for change, shall be dated,
and the responsible individual shall be identified.
Sec. 792.135 Physical and chemical characterization studies.
(a) All provisions of the GLPs shall apply to physical and chemical
characterization studies designed to determine stability, solubility,
octanol water partition coefficient, volatility, and persistence (such
as biodegradation, photodegradation, and chemical degradation studies).
(b) The following GLP standards shall not apply to studies designed
to determine physical and chemical characteristics of a test, control,
or reference substance:
Section 792.31 (c), (d), and (g)
Section 792.35 (b) and (c)
Section 792.43
Section 792.45
Section 792.47
Section 792.49
Section 792.81(b) (1), (2), (6) through (9), and (12)
Section 792.90
Section 792.105 (a) through (d)
[[Page 44]]
Section 792.113
Section 792.120(a) (5) through (12), and (15)
Section 792.185(a) (5) through (8), (10), (12), and (14)
Section 792.195 (c) and (d)
Subparts H-I [Reserved]
Subpart J--Records and Reports
Sec. 792.185 Reporting of study results.
(a) A final report shall be prepared for each study and shall
include, but not necessarily be limited to, the following:
(1) Name and address of the facility performing the study and the
dates on which the study was initiated and was completed, terminated, or
discontinued.
(2) Objectives and procedures stated in the approved protocol,
including any changes in the original protocol.
(3) Statistical methods employed for analyzing the data.
(4) The test, control, and reference substances identified by name,
chemical abstracts service (CAS) number or code number, strength,
purity, and composition, or other appropriate characteristics.
(5) Stability, and when relevant to the conduct of the study, the
solubility of the test, control, and reference substances under the
conditions of administration.
(6) A description of the methods used.
(7) A description of the test system used. Where applicable, the
final report shall include the number of animals or other test organisms
used, sex, body weight range, source of supply, species, strain and
substrain, age, and procedure used for identification.
(8) A description of the dosage, dosage regimen, route of
administration, and duration.
(9) A description of all circumstances that may have affected the
quality or integrity of the data.
(10) The name of the study director, the names of other scientists
or professionals and the names of all supervisory personnel, involved in
the study.
(11) A description of the transformations, calculations, or
operations performed on the data, a summary and analysis of the data,
and a statement of the conclusions drawn from the analysis.
(12) The signed and dated reports of each of the individual
scientists or other professionals involved in the study, including each
person who, at the request or direction of the testing facility or
sponsor, conducted an analysis or evaluation of data or specimens from
the study after data generation was completed.
(13) The locations where all specimens, raw data, and the final
report are to be stored.
(14) The statement prepared and signed by the quality assurance unit
as described in Sec. 792.35(b)(7).
(b) The final report shall be signed and dated by the study
director.
(c) Corrections or additions to a final report shall be in the form
of an amendment by the study director. The amendment shall clearly
identify that part of the final report that is being added to or
corrected and the reasons for the correction or addition, and shall be
signed and dated by the person responsible. Modification of a final
report to comply with the submission requirements of EPA does not
constitute a correction, addition, or amendment to a final report.
(d) A copy of the final report and of any amendment to it shall be
maintained by the sponsor and the test facility.
Sec. 792.190 Storage and retrieval of records and data.
(a) All raw data, documentation, records, protocols, specimens, and
final reports generated as a result of a study shall be retained.
Specimens obtained from mutagenicity tests, specimens of soil, water,
and plants, and wet specimens of blood, urine, feces, and biological
fluids, do not need to be retained after quality assurance verification.
Correspondence and other documents relating to interpretation and
evaluation of data, other than those documents contained in the final
report, also shall be retained.
(b) There shall be archives for orderly storage and expedient
retrieval of all raw data, documentation, protocols, specimens, and
interim and final reports. Conditions of storage shall minimize
deterioration of the documents or
[[Page 45]]
specimens in accordance with the requirements for the time period of
their retention and the nature of the documents of specimens. A testing
facility may contract with commercial archives to provide a repository
for all material to be retained. Raw data and specimens may be retained
elsewhere provided that the archives have specific reference to those
other locations.
(c) An individual shall be identified as responsible for the
archives.
(d) Only authorized personnel shall enter the archives.
(e) Material retained or referred to in the archives shall be
indexed to permit expedient retrieval.
Sec. 792.195 Retention of records.
(a) Record retention requirements set forth in this section do not
supersede the record retention requirements of any other regulations in
this subchapter.
(b)(1) Except as provided in paragraph (c) of this section,
documentation records, raw data, and specimens pertaining to a study and
required to be retained by this part shall be retained in the archive(s)
for a period of at least ten years following the effective date of the
applicable final test rule.
(2) In the case of negotiated testing agreements, each agreement
will contain a provision that, except as provided in paragraph (c) of
this section, documentation records, raw data, and specimens pertaining
to a study and required to be retained by this part shall be retained in
the archive(s) for a period of at least ten years following the
publication date of the acceptance of a negotiated test agreement.
(3) In the case of testing submitted under section 5, except for
those items listed in paragraph (c) of this section, documentation
records, raw data, and specimens pertaining to a study and required to
be retained by this part shall be retained in the archive(s) for a
period of at least five years following the date on which the results of
the study are submitted to the agency.
(c) Wet specimens, samples of test, control, or reference
substances, and specially prepared material which are relatively fragile
and differ markedly in stability and quality during storage, shall be
retained only as long as the quality of the preparation affords
evaluation. Specimens obtained from mutagenicity tests, specimens of
soil, water, and plants, and wet specimens of blood, urine, feces,
biological fluids, do not need to be retained after quality assurance
verification. In no case shall retention be required for longer periods
than those set forth in paragraph (b) of this section.
(d) The master schedule sheet, copies of protocols, and records of
quality assurance inspections, as required by Sec. 792.35(c) shall be
maintained by the quality assurance unit as an easily accessible system
of records for the period of time specified in paragraph (b) of this
section.
(e) Summaries of training and experience and job descriptions
required to be maintained by Sec. 792.29(b) may be retained along with
all other testing facility employment records for the length of time
specified in paragraph (b) of this section.
(f) Records and reports of the maintenance and calibration and
inspection of equipment, as required by Sec. 792.63 (b) and (c), shall
be retained for the length of time specified in paragraph (b) of this
section.
(g) If a facility conducting testing or an archive contracting
facility goes out of business, all raw data, documentation, and other
material specified in this section shall be transferred to the archives
of the sponsor of the study. The EPA shall be notified in writing of
such a transfer.
(h) Specimens, samples, or other non-documentary materials need not
be retained after EPA has notified in writing the sponsor or testing
facility holding the materials that retention is no longer required by
EPA. Such notification normally will be furnished upon request after EPA
or FDA has completed an audit of the particular study to which the
materials relate and EPA has concluded that the study was conducted in
accordance with this part.
(i) Records required by this part may be retained either as original
records or as true copies such as photocopies, microfilm, microfiche, or
other accurate reproductions of the original records.
[[Page 46]]
PART 795--PROVISIONAL TEST GUIDELINES--Table of Contents
Subpart A [Reserved]
Subpart B--Provisional Chemical Fate Guidelines
Sec.
795.70 Indirect photolysis screening test: Sunlight photolysis in
waters containing dissolved humic substances.
Subpart C--Provisional Environmental Effects Guidelines
795.120 Gammarid acute toxicity test.
Subpart D--Provisional Health Effects Guidelines
795.225 Dermal pharmacokinetics of DGBE and DGBA.
795.228 Oral/dermal pharmacokinetics.
795.231 Pharmacokinetics of isopropanal.
795.232 Inhalation and dermal pharmacokinetics of commercial hexane.
795.250 Developmental neurotoxicity screen.
Authority: 15 U.S.C. 2603.
Subpart A [Reserved]
Subpart B--Provisional Chemical Fate Guidelines
Sec. 795.70 Indirect photolysis screening test: Sunlight photolysis in waters containing dissolved humic substances.
(a) Introduction. (1) Chemicals dissolved in natural waters are
subject to two types of photoreaction. In the first case, the chemical
of interest absorbs sunlight directly and is transformed to products
when unstable excited states of the molecule decompose. In the second
case, reaction of dissolved chemical is the result of chemical or
electronic excitation transfer from light-absorbing humic species in the
natural water. In contrast to direct photolysis, this photoreaction is
governed initially by the spectroscopic properties of the natural water.
(2) In general, both indirect and direct processes can proceed
simultaneously. Under favorable conditions the measurement of a
photoreaction rate constant in sunlight (KpE) in a natural
water body will yield a net value that is the sum of two first-order
reaction rate constants for the direct (kDE) and indirect
(kIE) pathways which can be expressed by the relationship
Equation 1
kpE=kDE+kIE.
This relationship is obtained when the reaction volume is optically thin
so that a negligible fraction of the incident light is absorbed and is
sufficiently dilute in test chemical; thus the direct and indirect
photoreaction processes become first-order.
(3) In pure water only, direct photoreaction is possible, although
hydrolysis, biotransformation, sorption, and volatilization also can
decrease the concentraton of a test chemical. By measuring
kpE in a natural water and kDE in pure water,
kIE can be calculated.
(4) Two protocols have been written that measure kDE in
sunlight or predict kDE in sunlight from laboratory
measurements with monochromatic light (USEPA (1984) under paragraph
(f)(14) and (15) of this section; Mill et al. (1981) under paragraph
(f)(9) of this section; Mill et al. (1982) under paragraph (f)(10) of
this section; Mill et al. (1983) under paragraphs (f)(11) of this
section). As a preface to the use of the present protocol, it is not
necessary to know kDE; it will be determined under conditions
that definitively establish whether kIE is significant with
respect to kDE.
(5) This protocol provides a cost effective test method for
measuring kIE for test chemicals in a natural water
(synthetic humic water, SHW) derived from commercial humic material. It
describes the preparation and standardization of SHW. To implement the
method, a test chemical is exposed to sunlight in round tubes containing
SHW and tubes containing pure water for defined periods of time based on
a screening test.
(6) To correct for variations in solar irradiance during the
reaction period, an actinometer is simultaneously insolated. From these
data, an indirect photoreaction rate constant is calculated that is
applicable to clear-sky, near-surface, conditions in fresh water bodies.
(7) In contrast to kDE, which, once measured, can be
calculated for different seasons and latitudes, kIE only
[[Page 47]]
applies to the season and latitude for which it is determined. This
condition exists because the solar action spectrum for indirect
photoreaction in humic-containing waters is not generally known and
would be expected to change for different test chemicals. For this
reason, kpE, which contains kIE, is likewise valid
only for the experimental data and latitude.
(8) The value of kpE represents an atypical quantity
because kIE will change somewhat from water body to water
body as the amount and quality of dissolved aquatic humic substances
change. Studies have shown, however, that for optically-matched natural
waters, these differences are usually within a factor of two (Zepp et
al. (1981) under paragraph (f)(17) of this section).
(9) This protocol consists of three separate phases that should be
completed in the following order: In Phase 1, SHW is prepared and
adjusted; in Phase 2, the test chemical is irradiated in SHW and pure
water (PW) to obtain approximate sunlight photoreaction rate constants
and to determine whether direct and indirect photoprocesses are
important; in Phase 3, the test chemical is again irradiated in PW and
SHW. To correct for photobleaching of SHW and also solar irradiance
variations, tubes containing SHW and actinometer solutions are exposed
simultaneously. From these data kpE is calculated that is the
sum of kIE and kDE (Equation 1) (Winterle and Mill
(1985) under paragraph (f)(12) of this section).
(b) Phase 1--Preparation and standardization of synthetic natural
water--(1) Approach. (i) Recent studies have demonstrated that natural
waters can promote the indirect (or sensitized) photoreaction of
dissolved organic chemicals. This reactivity is imparted by dissolved
organic material (DOM) in the form of humic substances. These materials
absorb sunlight and produce reactive intermediates that include singlet
oxygen (102) (Zepp et al. (1977) under paragraph
(f)(20) of this section, Zepp et al. (1981) under paragraph (f)(17) of
this section, Zepp et al. (1981) under paragraph (f)(18) of this
section, Wolff et al. (1981) under paragraph (f)(16) of this section,
Haag et al. (1984) under paragraph (f)(6) of this section, Haag et al.
(1984) under paragraph (f)(7) of this section); peroxy radicals
(RO2-) (Mill et al. (1981) under paragraph (f)(9) of this
section; Mill et al. (1983) under paragraph (f)(8) of this section);
hydroxyl radicals (HO-) (Mill et al. (1981) under paragraph (f)(9) of
this section, Draper and Crosby (1981, 1984) under paragraphs (f)(3) and
(4) of this section); superoxide anion (02--) and
hydroperoxy radicals (HO-). (Cooper and Zika (1983) under paragraph
(f)(1) of this section, Draper and Crosby (1983) under paragraph (f)(2)
of this section); and triplet excited states of the humic substances
(Zepp et al. (1981) under paragraph (f)(17) of this section, Zepp et al.
(1985) under paragraph (f)(21) of this section). Synthetic humic waters,
prepared by extracting commercial humic or fulvic materials with water,
photoreact similarly to natural waters when optically matched (Zepp et
al. (1981) under paragraphs (f)(17) and (18) of this section).
(ii) The indirect photoreactivity of a chemical in a natural water
will depend on its response to these reactive intermediates, and
possibly others yet unknown, as well as the ability of the water to
generate such species. This latter feature will vary from water-to-water
in an unpredictable way, judged by the complexity of the situation.
(iii) The approach to standardizing a test for indirect
photoreactivity is to use a synthetic humic water (SHW) prepared by
water-extracting commercial humic material. This material is
inexpensive, and available to any laboratory, in contrast to a specific
natural water. The SHW can be diluted to a dissolved organic carbon
(DOC) content and uv-visible absorbance typical of most surface fresh
waters.
(iv) In recent studies it has been found that the reactivity of SHW
mixtures depends on pH, and also the history of sunlight exposure (Mill
et al. (1983) under paragraph (f)(11) of this section). The SHW
solutions initially photobleach with a time-dependent rate constant. As
such, an SHW test system has been designed that is buffered to maintain
pH and is pre-aged in sunlight to produce, subsequently, a predictable
bleaching behavior.
(v) The purpose of Phase 1 is to prepare, pre-age, and dilute SHW to
a
[[Page 48]]
standard mixture under defined, reproducible conditions.
(2) Procedure. (i) Twenty grams of Aldrich humic acid are added to a
clean 2-liter Pyrex Erlenmeyer flask. The flask is filled with 2 liters
of 0.1 percent NaOH solution. A stir bar is added to the flask, the
flask is capped, and the solution is stirred for 1 hour at room
temperature. At the end of this time the dark brown supernatant is
decanted off and either filtered through coarse filter paper or
centrifuged and then filtered through 0.4 )m microfilter. The pH is
adjusted to 7.0 with dilute H2SO4 and filter
sterilized through a 0.2 )m filter into a rigorously cleaned 2-liter
Erlenmeyer flask. This mixture contains roughly 60 ppm DOC and the
absorbance (in a 1 cm path length cell) is approximately 1.7 at 313 nm
and 0.7 at 370 nm.
(ii) Pre-aging is accomplished by exposing the concentrated solution
in the 2-liter flask to direct sunlight for 4 days in early spring or
late fall; 3 days in late spring, summer, or early fall. At this time
the absorbance of the solution is measured at 370 nm, and a dilution
factor is calculated to decrease the absorbance to 0.50 in a 1 cm path
length cell. If necessary, the pH is re-adjusted to 7.0. Finally, the
mixture is brought to exact dilution with a precalculated volume of
reagent-grade water to give a final absorbance of 0.500 in a 1-cm path
length cell at 370 nm. It is tightly capped and refrigerated.
(iii) This mixture is SHW stock solution. Before use it is diluted
10-fold with 0.010 M phosphate buffer to produce a pH 7.0 mixture with
an absorbance of 5.00 x 10-2 at 370 nm, and a dissolved
organic carbon of about 5 ppm. Such values are characteristic of many
surface fresh waters.
(3) Rationale. The foregoing procedure is designed to produce a
standard humic-containing solution that is pH controlled, and
sufficiently aged that its photobleaching first-order rate constant is
not time dependent. It has been demonstrated that after 7 days of winter
sunlight exposure, SHW solutions photobleached with a nearly constant
rate constant (Mill et al. (1983) under paragraph (f)(11) of this
section).
(c) Phase 2--Screening test--(1) Introduction and purpose. (i) Phase
2 measurements provide approximate solar photolysis rate constants and
half-lives of test chemicals in PW and SHW. If the photoreaction rate in
SHW is significantly larger than in PW (factor of > 2X) then the test
chemical is subject to indirect photoreaction and Phase 3 is necessary.
Phase 2 data are needed for more accurate Phase 3 measurements, which
require parallel solar irradiation of actinometer and test chemical
solutions. The actinometer composition is adjusted according to the
results of Phase 2 for each chemical, to equalize as much as possible
photoreaction rate constants of chemical in SHW and actinometer.
(ii) In Phase 2, sunlight photoreaction rate constants are measured
in round tubes containing SHW and then mathematically corrected to a
flat water surface geometry. These rate constants are not corrected to
clear-sky conditions.
(2) Procedure. (i) Solutions of test chemicals should be prepared
using sterile, air-saturated, 0.010 M, pH 7.0 phosphate buffer and
reagent-grade (or purer) chemicals.\1\ Reaction mixtures should be
prepared with chemicals at concentrations at less than one-half their
solubility in pure water and at concentrations such that, at any
wavelengths above 290 nm, the absorbance in a standard quartz sample
cell with a 1-cm path length is less than 0.05. If the chemicals are too
insoluble in water to permit reasonable handling or analytical
procedures, 1-volume percent acetonitrile may be added to the buffer as
a cosolvent.
---------------------------------------------------------------------------
\1\ The water should be ASTM Type IIA, or an equivalent grade.
---------------------------------------------------------------------------
(ii) This solution should be mixed 9.00:1.00 by volume with PW or
SHW stock solution to provide working solutions. In the case of SHW, it
gives a ten-fold dilution of SHW stock solution. Six mL aliquots of each
working solution should then be transferred to separate 12 x 100 mm
quartz tubes with screw tops and tightly sealed with Mininert valves.\2\
Twenty four tubes are required for each chemical solution
[[Page 49]]
(12 samples and 12 dark controls), to give a total of 48 tubes.
---------------------------------------------------------------------------
\2\ Mininert Teflon sampling vials are available from Alltech
Associates, Inc., 202 Campus Dr., Arlington Heights, IL 60004.
---------------------------------------------------------------------------
(iii) The sample tubes are mounted in a photolysis rack with the
tops facing geographically north and inclined 30 deg. from the
horizontal. The rack should be placed outdoors over a black background
in a location free of shadows and excessive reflection.
(iv) Reaction progress should be measured with an analytical
technique that provides a precision of at least 5 percent.
High pressure liquid chromatography (HPLC) or gas chromatograph (GC)
have proven to be the most general and precise analytical techniques.
(v) Sample and control solution concentrations are calculated by
averaging analytical measurements for each solution. Control solutions
should be analyzed at least twice at zero time and at other times to
determine whether any loss of chemical in controls or samples has
occurred by some adventitious process during the experiment.
(vi) Whenever possible the following procedures should be completed
in clear, warm, weather so that solutions will photolyze more quickly
and not freeze.
(A) Starting at noon on day zero, expose to sunlight 24 sample tubes
mounted on the rack described above. Tape 24 foil-wrapped controls to
the bottom of the rack.
(B) Analyze two sample tubes and two unexposed controls in PW and
SHW for chemical at 24 hours. Calculate the round tube photolysis rate
constants (kp)SHW and (kp)W
if the percent conversions are J 20 percent but F 80 percent. The rate
constants (kp)SHW and (kp)W
are calculated, respectively, from Equations 2 and 3:
Equation 2
(kp)SHW=(1/t)Pn(Co/
Ct)SHW (in d-1)
Equation 3
(kp)W=(1/t)Pn(Co/
Ct)W (in d-1),
where the subscript identifies a reaction in SHW or PW; t is the
photolysis time in calendar days; Co is the initial molar
concentration; and Ct is the molar concentration in the
irradiated tube at t. In this case t=1 day.
(C) If less than 20 percent conversion occurs in SHW in 1 day,
repeat the procedure for SHW and PW at 2 days, 4 days, 8 days, or 16
days, or until 20 percent conversion is reached. Do not extend the
experiment past 16 days. If less than 20 percent photoreaction occurs in
SHW at the end of 16 days the chemical is ``photoinert''. Phase 3 is not
applicable.
(D) If more than 80 percent photoreaction occurs at the end of day 1
in SHW, repeat the experiment with eight each of the remaining foil-
wrapped PW and SHW controls. Divide these sets into four sample tubes
each, leaving four foil-wrapped controls taped to the bottom of the
rack.
(1) Expose tubes of chemical in SHW and PW to sunlight starting at
0900 hours and remove one tube and one control at 1, 2, 4, and 8 hours.
Analyze all tubes the next day.
(2) Extimate (kp)SHW for the first tube in
which photoreaction is J 20 percent but F 80 percent. If more than 80
percent conversion occurs in the first SHW tube, report: ``The half-life
is less than one hour'' and end all testing. The chemical is
``photolabile.'' Phase 3 is not applicable.
(3) The rate constants (kp)SHW and
(kp)W are calculated from equations 2 and 3 but
the time of irradiation must be adjusted to reflect the fact that day-
averaged rate constants are approximately one-third of rate constants
averaged over only 8 daylight hours. For 1 hour of insolation enter
t=0.125 day into equation 2. For reaction times of 2, 4, and 8 hours
enter 0.25, 0.50 and 1.0 days, respectively. Proceed to Phase 3 testing.
(4) Once (kp)SHW and
(kp)W are measured, determine the ratio R from
equation 4:
Equation 4
R=(kp)SHW/(kp)W.
The coefficient R, defined by Equation 4, is equal to
[(kI+kD)/kD]. If R is in the range 0 to
1, the photoreaction is inhibited by the synthetic humic water and Phase
3 does not apply. If R is in the range 1 to 2, the test chemical is
marginally susceptable to indirect photolysis. In this case, Phase 3
studies are optional. If R is greater than 2, Phase 3 measurements are
necessary to measure kpE and to evaluate kIE.
(vii) Since the rate of photolysis in tubes is faster than the rate
in natural
[[Page 50]]
water bodies, values of near-surface photolysis rate constants in
natural and pure water bodies, kpE and kDE,
respectively, can be obtained from (kp)SHW and
(kp)W from Equations 5 and 6:
Equation 5
kpE=0.45(kp)SHW
Equation 6
kDE=0.45(kp)W.
The factor 0.45 is an approximate geometric correction for scattered
light in tubes versus horizontal surfaces. A rough value of
kIE, the rate constant for indirect photolysis in natural
waters or SHW, can be estimated from the difference between
kpE and kDE using Equation 7:
Equation 7
kIE=kpE-kDE.
(3) Criteria for Phase 2. (i) If no loss of chemical is found in
dark control solutions compared with the analysis in tubes at zero time
(within experimental error), any loss of chemical in sunlight is assumed
to be due to photolysis, and the procedure provides a valid estimate of
kpE and kDE. Any loss of chemical in the dark-
control solutions may indicate the intervention of some other loss
process such as hydrolysis, microbial degradation, or volatilization. In
this case, more detailed experiments are needed to trace the problem and
if possible eliminate or minimize the source of loss.
(ii) Rate constants determined by the Phase 2 protocol depend upon
latitude, season, and weather conditions. Note that
(kp)SHW and kD values apply to round
tubes and kpE and kDE values apply to a natural
water body. Because both (kp)SHW and kD
are measured under the same conditions the ratio
((kp)SHW/kD) is a valid measure of the
susceptibility of a chemical to indirect photolysis. However, since SHW
is subject to photobleaching, (kp)SHW will
decrease with time because the indirect rate will diminish. Therefore, R
>2 is considered to be a conservative limit because
(kp)SHW will become systematically smaller with
time.
(4) Rationale. The Phase 2 protocol is a simple procedure for
evaluating direct and indirect sunlight photolysis rate constants of a
chemical at a specific time of year and latitude. It provides a rough
rate constant for the chemical in SHW that is necessary for Phase 3
testing. By comparison with the direct photoreaction rate constant, it
can be seen whether the chemical is subject to indirect photoreaction
and whether Phase 3 tests are necessary.
(5) Scope and limitations. (i) Phase 2 testing separates test
chemicals into three convenient categories: ``Photolabile'',
``photoinert'', and those chemicals having sunlight half-lives in round
tubes in the range of 1 hour to 50 days. Chemicals in the first two
categories fall outside the practical limits of the test, and cannot be
used in Phase 3. All other chemicals are suitable for Phase 3 testing.
(ii) The test procedure is simple and inexpensive, but does require
that the chemical dissolve in water at sufficient concentrations to be
measured by some analytical technique but not have appreciable
absorbance in the range 290 to 825 nm. Phase 2 tests should be done
during a clear-sky period to obtain the best results. Testing will be
less accurate for chemicals with half-lives of less than 1 day because
dramatic fluctuations in sunlight intensity can arise from transient
weather conditions and the difficulty of assigning equivalent reaction
times. Normal diurnal variations also affect the photolysis rate
constant. Phase 3 tests should be started as soon as possible after the
Phase 2 tests to ensure that the (kp)SHW estimate
remains valid.
(6) Illustrative Example. (i) Chemical A was dissolved in 0.010 M pH
7.0 buffer. The solution was filtered through a 0.2 )m filter, air
saturated, and analyzed. It contained 1.7 x 10 -5 M A, five-
fold less than its water solubility of 8.5 x 10 -5 M at
25 deg.C. A uv spectrum (1-cm path length) versus buffer blank showed no
absorbance greater than 0.05 in the wavelength interval 290 to 825 nm, a
condition required for the Phase 2 protocol. The 180 mL mixture was
diluted by the addition of 20 mL of SHW stock solution.
(ii) The SHW solution of A was photolyzed in sealed quartz tubes
(12 x 100 mm) in the fall season starting on October 1. At the end of 1
and 2
[[Page 51]]
days, respectively, the concentration of A was found to be 1.13 x 10
-5 M and 0.92 x 10 -5 M compared to unchanged dark
controls (1.53 x 10 -5 M).
(iii) The tube photolysis rate constant of chemical A was calculated
from Equation 2 under paragraph (c)(2)(vi)(B) of this section. The first
time point at day 1 was used because the fraction of A remaining was in
the range 20 to 80 percent:
(kp)SHW=(1/1d)Pn(1.53 x 10 -5/1.13
x 10 -5) (kp)SHW=0.30
d-1.
(iv) From this value, kpE was found to be 0.14
d-1 using equation 5 under paragraph (c)(2)(vii) of this
section:
kpE=0.45(0.30 d-1)=0.14d-1.
(v) From measurements in pure water, kD for chemical A
was found to be 0.085 d-1. Because the ratio of
(kp)SHW/kD(=3.5) is greater than 2,
Phase 3 experiments were started.
(d) Phase 3--Indirect photoreaction with actinometer: Calculation of
kIE and kpE--(1) Introduction and purpose.
(i) The purpose of Phase 3 is to measure kIo, the
indirect photolysis rate constant in tubes, and then to calculate
kpE for the test chemical in a natural water. If the
approximate (kp)SHW determined in Phase 2 is not
significantly greater than kD measured for the experiment
date of Phase 2, then Phase 3 is unnecessary because the test chemical
is not subject to indirect photoreaction.
(ii) In the case (kp)SHW is significantly
larger than kD, Phase 3 is necessary. The rate constant
(kp)SHW is used to choose an actinometer
composition that matches the actinometer rate to the test chemical rate.
Test chemical solutions in SHW and in pure water buffer are then
irradiated in sunlight in parallel with actinometer solutions, all in
tubes.
(iii) The actinometer used is the p-nitroacetophenone-pyridine
(PNAP/PYR) system developed by Dulin and Mill (1982) under paragraph
(f)(5) of this section and is used in two EPA test guidelines (USEPA
(1984) under paragraphs (f) (14) and (15) of this section). By varying
the pyridine concentration, the PNAP photolysis half-life can be
adjusted over a range of several hours to several weeks. The starting
PNAP concentration is held constant.
(iv) SHW is subject to photobleaching that decreases its ability to
promote indirect photolysis based on its ability to absorb sunlight.
This effect will be significant when the test period exceeds a few days.
To correct for photobleaching, tubes containing SHW are irradiated in
action to the other tubes above.
(v) At any time, the loss of test chemical is given by Equation 8
assuming actinometric correction to constant light flux:
Equation 8
-(d[C]/dt)=kI[C]+kD[C].
(vi) The indirect photolysis rate constant, kI, is
actually time dependent because SHW photobleaches; the rate constant
kI, after pre-aging, obeys the formula:
Equation 9
kI=kIo exp(-kt),
in which kIo is the initial indirect photoreaction rate
constant and k is the SHW photobleaching rate constant. After
substituting equation 9 for kI in Equation 8 under paragraph
(d)(1)(v) of this section, and rearranging, one obtains
-(d[C]/[C]=kIo[exp(-kt)]dt+kD dt.
This expression is integrated to give Equation 10:
Equation 10
Pn(Co/C)SHW=(kIo/
k)[1-exp(-kt)]+kD t.
The term (kIo/k) can now be evaluated. Since in pure water,
Pn(Co/C)W=kD t, then subtracting this
equation from Equation 10 gives
Equation 11
Pn(Co/C)SHW-Pn(co/
C)W=(kIo/k)[1-exp(-kt)].
The photobleaching fraction, [1-exp(-kt)], is equivalent to the
expression [1-(A370/A deg.370)], where
A deg.370 and A370 are the absorbances at 370 nm,
and are proportional to humic sensitizer content at times zero and t.
Therefore, (kIo/k) is derived from the slope of a linear
regression using [Pn(Co/C)SHW-Pn(Co/
C)W] as the dependent variable and [1-(A370/
A deg.370)SHW] as the independent variable.
(vii) To evaluate kIo, the parameter k has to be
evaluated under standard
[[Page 52]]
sunlight conditions. Therefore, the photolysis rate constant for the
PNAP/PYR actinometer (kA) is used to evaluate k by linear
regression on Equation 12:
Equation 12
Pn(A deg.370/A370)=(k/
kA)Pn(Co/C)PNAP,
where the slope is (k/kA) and the value of kA is
calculated from the concentration of pyridine and the absorption of
light by PNAP: kA=2.2(0.0169)[PYR]ka. Values of
ka are listed in the following Table 1.
Table 1--Day Averaged Rate Constant (ka) \1\ For Sunlight Absorption by
PNAP as a Function of Season and Decadic Latitude \2\
------------------------------------------------------------------------
Season
Latitude ------------------------------
Spring Summer Fall Winter
------------------------------------------------------------------------
20 deg.N................................. 515 551 409 327
30 deg.N................................. 483 551 333 232
40 deg.N................................. 431 532 245 139
50 deg.N................................. 362 496 154 64
------------------------------------------------------------------------
\1\ ka=@ ega Lg in the units of day -\1\, (Mill et al. (1982) under
paragraph (f)(10) of this section).
\2\ For use in Equation 15 under paragraph (d)(2)(i) of this section.
The value of kIo is then given by Equation 13:
Equation 13
kIo=(kIo/k)(k/kA)kA.
(viii) To obtain kD, determine the ratio (kD/
kA) from a linear regression of Pn(Co/
C)W versus Pn(Co/C)PNAP according to
Equation 13a:
Equation 13a
Pn(Co/C)W=(kD/
kA)Pn(Co/C)PNAP.
The slope is (kD/kA), and kD is
obtained by multiplication of this slope with the known value of
kA: i.e., kD=(kD/
kA)kA.
(ix) Then, (kp)SHW values in SHW are
determined by summing kD and KIo as follows:
Equation 14
(kp)SHW=kIo+kD.
(x) Finally, kpE is calculated from the precise
relationship, Equation 5a:
Equation 5a
kpE=0.455(kp)SHW.
(2) Procedure. (i) Using the test chemical photoreaction rate
constant in round tubes, (kp) SHW, determined in
Phase 2 under paragraph (c) of this section, and the absorption rate
constant, k found in Table 1, under paragraph (d)(1)(vii) of
this section, calculate the molar pyridine concentration required by the
PNAP/PYR actinometer using Equation 15:
Equation 15
[PYR]/M=26.9[(kp) SHW/ka].
This pyridine concentration makes the actinometer rate constant match
the test chemical rate constant.
(A) The variable ka (= @ e ga Lg)
is equal to the day-averaged rate constant for sunlight absorption by
PNAP (USEPA (1984) under paragraph (f)(14) of this section; Mill et al.
(1982) under paragraph (f)(10) of this section, Zepp and Cline (1977)
under paragraph (f)(19) of this section) which changes with season and
latitude.
(B) The variable ka is selected from Table 1 under
paragraph (d)(1)(vii) of this section for the season nearest the mid-
experiment date of Phase 2 studies and the decadic latitude nearest the
experimental site.
(ii) Once [PYR] is determined, an actinometer solution is prepared
by adding 1.00 mL of 1.0 x 10-2 M (0.165 gms/100 mL) PNAP
stock solution (in CH3 CN solvent) and the required volume,
V, of PYR to a 1 liter volumetric flask. The flask is then filled with
distilled water to give 1 liter of solution. The volume V can be
calculated from Equation 16:
Equation 16
V/mL=[PYR]/0.0124.
The PNAP/PYR solutions should be wrapped with aluminum foil and kept out
of bright light after preparation.
(iii) The following solutions should be prepared and individually
added in 6.00 mL aliquots to 12/100 mm quartz sample tubes; 8 tubes
should be filled with each solution:
(A) PNAP/PYR actinometer solution.
(B) Test chemical in pH 7.0, 0.010 M phosphate buffer.
(C) Test chemcial in pH 7.0, 0.010 M phosphate buffer/SHW.
[[Page 53]]
(D) pH 7.0, 0.010 M phosphate buffer/SHW. Four tubes of each set are
wrapped in foil and used as controls.
(iv) The tubes are placed in the photolysis rack (Phase 2,
Procedure) at 0900 hours on day zero, with the controls taped to the
bottom of the rack. One tube of each composition is removed, along with
their respective controls, according to a schedule found in Table 2,
which categorizes sampling times on the basis of
(kp)SHW determined in Phase 1.
Table 2--Category and Sampling Procedure for Test and Actinometry
Solutions
------------------------------------------------------------------------
Category kp (d-1)SHW Sampling procedure
------------------------------------------------------------------------
A.............................. 5.5 J Kp J 0.69 Sample at 0, 1, 2,
4, and 8h.
B.............................. 0.69> kp J 0.017 Sample at 0, 1, 2,
4, and 8d.
C.............................. 0.17> kp J 0.043 Sample at 0, 4, 8,
16, and 32d.
------------------------------------------------------------------------
(v) The tubes containing PNAP, test chemical, and their controls are
analyzed for residual concentrations soon after the end of the
experiment. PNAP is conveniently analyzed by HPLC, using a 30 cm
C18 reverse phase column and a uv detector set at 280 nm. The
mobile phase is 2 percent acetic acid, 50 percent acetonitrile and 48
percent water (2 mL/min flow rate). Tubes containing only SHW (solution
D) should be analyzed by absorption spectroscopy at 370 nm after storage
at 4 deg.C in the dark. The absorbance range to be measured is 0.05 to
0.01 AU (1 cm).
(vi) If controls are well-behaved and show no significant loss of
chemical or absorbance change, then kI can be calculated. In
tabular form (see Table 4 under paragraph (d)(6)(iii)(A) of this
section) arrange the quantities Pn(Co/Ct)
SHW, Pn(Co/Ct)SHW,
[1-(A370/Ao370)],
Pn(Ao370/A370), and Pn(Co/
C)PNAP in order of increasing time. According to Equation 11
under paragraph (d)(1)(vi) of this section in the form of Equation 17,
Equation 17
Pn(Co/C)SHW-Pn(Co/
C)W=(kIo/k)[1-(A370/
Ao370)],
plot the quantities [Pn(Co/
Ct)SHW-Pn(Co/
Ct)W] versus the independent variable
[1-(A370/Ao370)]. Obtain the slope (S1)
by least square linear regression. Under the assumptions of the
protocol, S1=(kIo/k).
(vii) According to Equation 12 under paragraph (d)(1)(vii) of this
section, plot the quantities Pn(Ao370/
A370) versus the independent variable Pn(Co/
Ct)PNAP. Obtain the slope (S2) by least squares
linear regression on Equation 12 under paragraph (d)(1)(vii) of this
section. Under the assumptions of the protocol, S2=(k/kA).
(viii) Then, using Equation 13a under paragraph (d)(1)(vii) of this
section, determine the slope (S3) by least squares linear regression.
Under the assumptions of the protocol, S3 is equal to (kD/
kA).
(ix) From Equation 18
Equation 18
kA=0.0372[PYR]ka,
calculate kA using ka values found in Table 1
under paragraph (d)(1)(vii) of this section. The value of ka
chosen must correspond to the date closest to the mid-experiment date
and latitude closest to that of the experimental site.
(x) The indirect photoreaction rate constant, kIo, is
determined using Equation 19,
Equation 19
kIo=(S1)(kA)(S2),
by incorporating the quantities kA, S1, and S2 determined as
described in paragraphs (d)(2) (ix), (vi), and (vii) of this section,
respectively.
(xi) The rate constant kD is calculated from Equation 20,
Equation 20
kD=(S3)(kA),
using the quantities S3 and kA determined as described above.
(xii) Then, (kp)SHW is obtained by summing
kD and kIo, as described by Equation 14 in
paragraph (d)(1)(ix) of this section:
Equation 14
(kp)SHW=kIo+kD.
(xiii) Finally, kpE is obtained by multiplying
(kp) SNW by the factor 0.455, as described by
Equation 5a in paragraph (d)(1)(x) of this section:
[[Page 54]]
Equation 5a
kpE=0.455 (kp)SHW
As determined, kpE is the net environmental photoreaction
rate constant. It applies to clear sky conditions and is valid for
predicting surface photoreaction rates in an average humic containing
freshwater body. It is strictly valid only for the experimental latitude
and season.
(3) Criteria for Phase 3. As in Phase 2, Phase 3 tests are assumed
valid if the dark controls are well behaved and show no significant loss
of chemical. In such a case, loss of test chemical in irradiated samples
is due to photoreaction.
(4) Rationale. Simultaneous irradiation of a test chemical and
actinometer provide a means of evaluating sunlight intensities during
the reaction period. Parallel irradiation of SHW solutions allows
evaluation of the extent of photobleaching and loss of sensitizing
ability of the natural water.
(5) Scope and limitations of Phase 3 protocol. Test chemicals that
are classified as having half-lives in SHW in the range of 1 hour to 50
days in Phase 2 listing are suitable for use in Phase 3 testing. Such
chemicals have photoreaction half-lives in a range accommodated by the
PNAP/PYR actinometry in sunlight and also accommodate the persistence of
SHW in sunlight.
(6) Illustrative example. (i) From Phase 2 testing, under paragraph
(c)(6)(iii) of this section, chemical A was found to have a photolysis
rate constant, (kp)SHW' of 0.30 d-1 in
fall in round tubes at latitude 33 deg. N. Using Table 1 under paragraph
(d)(1)(vii) of this section for 30 deg. N, the nearest decadic latitude,
a fall value of ka equal to 333 d-1 is found for
PNAP. Substitution of (kp)SHW and ka
into Equation 15 under paragraph (d)(2)(i) of this section gives [PYR] =
0.0242 M. This is the concentration of pyridine that gives an
actinometer rate constant of 0.30 d-1 in round tubes in fall
at this latitude.
(ii) The actinometer solution was made up by adding a volume of
pyridine (1.95 mL) calculated from equation 16 under paragraph
(d)(2)(ii) of this section to a 1 liter volumetric flask containing 1.00
mL of 1.00 x 10-2 M PNAP in acetonitrile. The flask was
filled to the mark with distilled water to give final concentrations of
[PYR]=0.0242 M and [PNAP]=1.00 x 10-5 M. Ten tubes of each of
the following solutions were placed in the photolysis rack at 1,200
hours on day zero:
(A) Chemical A (1.53 x 10-5 M) in standard SHW (0.010 M,
pH 7 phosphate buffer).
(B) Chemical A (1.53 x 10-5), in 0.010 M, pH 7 phosphate
buffer.
(C) SHW standard solution diluted with water 0.90 to 1.00 to match
solution A.
(D) PNAP/PYR actinometer solution. Ten additional foil-wrapped
controls of each mixture were taped to the bottom of the rack.
(iii) The test chemical had been placed in category B, Table 2 under
the paragraph (d)(2)(iv) of this section, on the basis of its Phase 2
rate constant under paragraph (c) of this section. Accordingly, two
tubes of each irradiated solution and two tubes of each blank solution
were removed at 0, 1, 2, 4, and 8 days at 1,200 hours. The averaged
analytical results obtained at the end of the experiment are shown in
the following Table 3.
Table 3--Chemical Analytical Results for Illustrative Example, Phase 3
----------------------------------------------------------------------------------------------------------------
Day 10\5\[C]SHW, M 10\5\[C]W, M ASHW370 105 [PNAP], M
----------------------------------------------------------------------------------------------------------------
0............................................... 1.53 1.53 0.0500 1.00
1............................................... 1.03 1.40 0.0470 0.810
2............................................... 0.760 1.30 0.0440 0.690
4............................................... 0.300 1.01 0.0370 0.380
8............................................... 0.130 0.800 0.0320 0.220
----------------------------------------------------------------------------------------------------------------
Data for solutions A through D are given in column 2 through 5,
respectively. No significant chemical loss was found in the dark
controls.
[[Page 55]]
(A) From these items the functions Pn(Co/C)
SNW' Pn(Co/C)W' [1--(A370/
Ao370)SNW],
Pn(Ao370/A370), and Pn(Co/
C)PNAP were calculated, as shown in the following Table 4
which was derived from Table 3 under paragraph (d)(6)(iii) of this
section:
Table 4--Photoreaction Function for Illustrative Examples, Phase 3, Derived From Table 3
----------------------------------------------------------------------------------------------------------------
1-(A 370 / Pn(Ao370 /
Day Pn(Co/C)SHW Pn(Co/C)W Ao370) A370) Pn(Co /C) PNAP
----------------------------------------------------------------------------------------------------------------
0............................... 0 0 0 0 0
1............................... 0.396 0.0888 0.0600 0.0618 0.211
2............................... 0.700 0.163 0.120 0.128 0.371
4............................... 1.629 0.415 0.260 0.301 0.968
8............................... 2.465 0.648 0.360 0.446 1.514
----------------------------------------------------------------------------------------------------------------
(B) Slope S1=(kIo/k) was calculated according to Equation
17 under paragraph (d)(2)(vi) of this section and was found to be 4.96
by a least squares regression with a correlation coefficient equal to
0.9980. The following Figure 1 shows a plot of Equation 17 under
paragraph (d)(2)(vi) of this section and its best-fit line.
[GRAPHIC] [TIFF OMITTED] TC01AP92.034
Figure 1--Graphic determination of S1=(kIo/k) based on
Equation 17 under paragraph (d)(2)(vi) of this section.
(C) Slope S2=(k/ka) was also derived from Table 4 under
paragraph (d)(6)(iii)(A) of this section by a fit of
Pn(Ao370 /A370) SHW and
Pn(Co /C)PNAP to Equation 12 under paragraph
(d)(l)(vii) of this section. This plot is displayed in the following
Figure 2; the slope S2 was found to be 0.295 and the correlation
coefficient was equal to 0.9986.
[GRAPHIC] [TIFF OMITTED] TC01AP92.035
Figure 2--Graphic determination of S2=(k/kA) based on
Equation 12 under paragraph (d)(1)(vii) of this section.
(D) Using the data in columns 3 and 6 in Table 4 under paragraph
(d)(6)(iii)(A) of this section, slope S3 was calculated by regression
from Equation 13a under paragraph (d)(1)(viii) of this section and was
found to be 0.428 with correlation coefficient euqal to 0.99997.
(E) Using Equation 18 under paragraph (d)(2)(ix) of this section,
kA was found to be =0.300 d-1.
(F) The values of S1, S2, and kA were then combined in
Equation 19 under paragraph (d)(2)(x) of this section to give
kIo as follows:
Equation 19
kIo=(4.96)(0.300)(0.295)=0.439 d-1.
(G) The rate constant kD was calculated from the product
of S3 and kA
[[Page 56]]
as expressed in Equation 20 under paragraph (d)(2)(xi) of this section
as follows:
Equation 20
kD=(0.428)(0.300)=0.128d-1.
(H) The sum of kD and kIo was multiplied by
0.455 to obtain kpE as follows:
Equation 21
kpE=(0.455)(0.439+0.128)d-1=0.258 d-1.
(I) Since kpE is a first-order rate constant, the half-
life, t1/2E, is given by Equation 22:
Equation 22
t1/2E=0.693/kpE.
Substituting the value of kpE from Equation 21 under
paragraph (d)(6)(iii)(H) of this section in Equation 22 yielded
Equation 23
t1/2E=0.693/0.258d-1=2.7 d.
(e) Data and reporting--(1) Test conditions--(i) Specific analytical
and recovery procedures. (A) Provide a detailed description or reference
for the analytical procedures used, including the calibration data and
precision.
(B) If extraction methods were used to separate the solute from the
aqueous solution, provide a description of the extraction method as well
as the recovery data.
(ii) Other test conditions. (A) Report the site and latitude where
the photolysis experiments were carried out.
(B) Report the dates of photolysis, weather conditions, times of
exposure, and the duration of exposure.
(C) If acetonitrile was used to solubilize the test chemical, report
the volume percent.
(D) If a significant loss of test chemical occurred in the control
solutions for pure water and SHW, indicate the causes and how they were
eliminated or minimized.
(2) Test data report--(i) Phase 2 Screening Test under paragraph (c)
of this section. (A) Report the initial molar concentration of test
chemical, Co, in pure water and SHW for each replicate and
the mean value.
(B) Report the molar concentration of test chemical, Ct,
in pure water and SHW for each replicate and the mean value for each
time point t.
(C) Report the molar concentration of test chemical for each
replicate control sample and the mean value for each time point.
(D) Report the values of (kp)SHW and
(kp)W for the time point t in which the fraction
of test chemical photoreacted is in the range 20 to 80 percent.
(E) If small losses of test chemical were observed in SHW and pure
water, report a first-order rate constant loss,
(kp)loss. Calculate and report
(kp)obs for SHW and/or pure water. Calculate and
report the corrected first-order rate constant for SHW and/or pure water
using the relationship expressed in Equation 24:
Equation 24
kp=(kp)obs-(kp)loss
.
(F) Report the value of R calculated from Equation 4 under paragraph
(c)(2)(vi)(D)(4) of this section.
(G) Report the values of kpE and kDE obtained
from Equations 5 and 6, respectively under paragraph (c)(2)(vii) of this
section; report the corresponding half-life calculated from Equation 22
under paragraph (d)(6)(iii)(I) of this section.
(ii) Phase 3--Indirect photoreaction with actinometer. (A) Report
the initial molar concentration of test chemical, Co, in pure
water and in SHW for each replicate and the mean value.
(B) Report the initial absorbance Ao370 of the
SNW solution.
(C) Report the initial molar concentration of PNAP of each replicate
and the mean value in the actinometer. Report the concentration of
pyridine used in the actinometer which was obtained from Equation 15
under paragraph (d)(2)(i) of this section.
(D) Report the time and date the photolysis experiments were
started, the time and date the experiments were completed, and the
elapsed photolysis time in days.
(E) For each time point t, report the separate values of the
absorbance of the SHW solution, and the mean values.
(F) For each time point for the controls, report the separate values
of the molar concentrations of test chemical
[[Page 57]]
in pure water and SHW, and the absorbance of the SHW solution, and the
mean values.
(G) Tabulate and report the following data: t, [C]SHW,
[C]W, ASNW370, [PNAP].
(H) From the data in (G), tabulate and report the following data: t,
Pn(Co/C)SNW, Pn(Co/C)W,
[1-(A370/Ao370)SNW],
Pn(Ao370/A370), Pn(Co/
C)PNAP.
(I) From the linear regression analysis of the appropriate data in
step (H) in Equation 17 under paragraph (d)(2)(vi) of this section,
report the slope S1 and the correlation coefficient.
(J) From the linear regression analysis of the appropriate data in
step (H) in Equation 12 under paragraph (d)(1)(vii) of this section,
report the slope S2 and the correlation coefficient.
(K) From the linear regression analysis of the appropriate data in
step (H) in Equation 13a under paragraph (d)(1)(viii) of this section,
report the slope S3 and the correlation coefficient.
(L) If loss of chemical was observed during photolysis in pure water
and SHW, then report the data Pn(Co/C)corr,
Pn(Co/C)obs, Pn(Co/C)loss as
described in paragraph (e)(2)(E) of this section. Repeat steps (H), (I),
(J), (K) where applicable and report S1, S2, S3 and the corresponding
correlation coefficients.
(M) Report the value of the actinometer rate constant obtained from
Equation 18 under paragraph (d)(2)(ix) of this section.
(N) Report the value of kIo obtained from Equation 19
under paragraph (d)(2)(x) of this section.
(O) Report the value of kD obtained from Equation 20
under paragraph (d)(2)(xi) of this section.
(P) Report the value of (kpE)SHW, obtained
from Equation 14 under paragraph (d)(1)(ix) of this section, and the
value of kpE obtained from Equation 5a under paragraph
(d)(1)(x) of this section.
(Q) Report the half-life, t1/2E, obtained from Equation
22 under paragraph (d)(6)(iii)(I) of this section.
(f) References. For additional background information on this test
guideline the following references should be consulted.
(1) Cooper W.J., Zika R.G. ``Photochemical formation of hydrogen
peroxide in surface and ground waters exposed to sunlight.'' Science,
220:711. (1983).
(2) Draper W.M., Crosby D.G. ``The photochemical generation of
hydrogen peroxide in natural waters.'' Archives of Environmental
Contamination and Toxicology, 12:121. (1983).
(3) Draper, W.M. and Crosby D.G. ``Solar photooxidation of
pesticides in dilute hydrogen peroxide.'' Journal of Agricultural and
Food Chemistry, 32:231. (1984).
(4) Draper W.M., Crosby D.G. ``Hydrogen peroxide and hydroxyl
radical: Intermediates in indirect photolysis reactions in water.''
Journal of Agricultural and Food Chemistry, 29:699. (1981).
(5) Dulin D., Mill T. ``Development and evaluation of sunlight
actinometers.'' Environmental Science and Technology, 6:815. (1982).
(6) Haag H.R., Hoigne J., Gassman E., Braun A.M. ``Singlet oxygen in
surface waters--Part I; Furfuryl alcohol as a trapping agent.''
Chemosphere, 13:631. (1984).
(7) Haag W.R., Hoigne J., Gassman E., Braun A.M. ``Singlet oxygen in
surface waters--Part II: Quantum yields of its production by some
natural humic materials as a function of wavelength.'' Chemosphere,
13:641. (1984).
(8) Mill T., Winterle J.S., Fischer A., Tse D., Mabey W.R., Drossman
H., Liu A., Davenport J.E. Toxic substances process data generation and
protocol development. Work assignment 12, test standard development.
``Section 3. Indirect photolysis.'' Draft final report. EPA Contract No.
68-03-2981. Environmental Research Laboratory, Office of Research and
Development, EPA, Athens, GA, and Office of Pollution Prevention and
Toxics, EPA, Washington, DC. (1984).
(9) Mill T., Mabey W.R., Bomberger D.C., Chou T.W., Hendry D.G.,
Smith J.H. ``Laboratory protocols for evaluating the fate of organic
chemicals in air and water. Chapter 3. Photolysis in water. Chapter 4.
Oxidation in water.'' EPA 600/3-82-022. Environmental Research
Laboratory, Office of Research and Development, EPA, Athens, GA. (1981).
[[Page 58]]
(10) Mill T., Mabey W.R., Winterle J.S., Davenport J.E., Barich
V.P., Dulin D.E., Tse D.S., Lee G. ``Design and validation of screening
and detailed methods for environmental processes. Apendix C. Lower-tier
direct photolysis protocol.'' Draft final report. EPA Contract No. 68-
01-6325. Office of Pollution Prevention and Toxics, EPA, Washington, DC.
(1982).
(11) Mill T., Davenport J.E., Winterle J.S., Mabey W.R., Dossman H.,
Tse D., Liu A. Toxic substances process data generation and protocol
development. Work assignment 12. ``Appendix B. Upper-tier protocol for
direct photolysis in water.'' Draft final report. EPA Contract No. 68-
03-2981. Environmental Research Laboratory, Office of Research and
Development, EPA, Athens, GA, and Office of Pollution Prevention and
Toxics, EPA, Washington, DC. (July 1983).
(12) Winterle J.S., Mill T. Toxic substances process data generation
and protocol development. Work assignment 18. ``Indirect photoreaction
protocol.'' Draft EPA special report. EPA Contract No. 68-03-2981.
Environmental Research Laboratory, Office of Research and Development,
EPA, Athens, GA and Office of Pollution Prevention and Toxics, EPA,
Washington, DC. (1985).
(13) Mill T., Hendry D.G., Richardson H. ``Free radical oxidants in
natural waters.'' Science, 207:886. (1980).
(14) U.S. Environmental Protection Agency (USEPA), Office of
Pollution Prevention and Toxics (OPPT). ``Chemical fate test guidelines.
Test guideline (CG, CS-6000). Photolysis in aqueous solution.'' EPA-560/
6-84-003. NTIS publication PB-84-233287. (1984).
(15) USEPA, OPPT. ``Chemical fate test guidelines. Test guildeline
(CG, CS-6010). Laboratory determination of the direct photolysis
reaction quantum yield in aqueous solution and sunlight photolysis.''
EPA-560/6-84-003. NTIS publication PB-84-233287. (1984).
(16) Wolff C.J.M., Halmans M.T.H., Van der Heijde H.B. ``The
formation of singlet oxygen in surface waters.'' Chemosphere, 10:59.
(1981).
(17) Zepp R.G., Baughman G.L., Schlotzhauer P.F. ``Comparison of
photochemical behavior of various humic substances in water: I. Sunlight
induced reactions of aquatic pollutants photosensitized by humic
substances.'' Chemosphere, 10:109. (1981).
(18) Zepp R.G., Baughman G.L., Schlozhauer P.F. ``Comparison of
photochemical behavior of various humic substances in water: II.
Photosensitized oxygenations.'' Chemosphere, 10:119. (1981).
(19) Zepp R.G., Cline D.M. ``Rates of direct photolysis in aquatic
environments.'' Environmental Science and Technology, 11:359. (1977).
(20) Zepp, R.G., Wolfe N.L., Baughman G.L., Hollis R.C. ``Singlet
oxygen in natural waters.'' Nature, 267:421. (1977).
(21) Zepp R.G., Schlotzhauer P.F., Merritt S.R. ``Photosensitized
transformations involving electronic energy transfer in natural waters:
role of humic substances.'' Environmental Science and Technology, 19:74.
(1985).
[53 FR 34522, Sept. 7, 1988; 53 FR 37393, Sept. 26, 1988]
Subpart C--Provisional Environmental Effects Guidelines
Sec. 795.120 Gammarid acute toxicity test.
(a) Purpose. This guideline is intended for use in developing data
on the acute toxicity of chemical substances and mixtures subject to
environmental effects test regulations under the Toxic Substances
Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003 (15 U.S.C. 2601 et
seq.)). This guideline describes a test to develop data on the acute
toxicity of chemicals to gammarids. The United States Environmental
Protection Agency (EPA) will use data from this test in assessing the
hazard of a chemical to aquatic organisms.
(b) Definitions. The definitions in section 3 of TSCA and in part
792 of this chapter, Good Laboratory Practice Standards, apply to this
test guideline. The following definitions also apply to this guideline:
Death means the lack of reaction of a test organism to gentle
prodding.
Flow-through means a continuous or an intermittent passage of test
solution or dilution water through a test chamber or a holding or
acclimation tank, with no recycling.
[[Page 59]]
LC50 means the median lethal concentration, i.e., that concentration
of a chemical in air or water killing 50 percent of the test batch of
organisms within a particular period of exposure (which shall be
stated).
Loading means the ratio of the biomass of gammarids (grams, wet
weight) to the volume (liters) of test solution in either a test chamber
or passing through it in a 24-hour period.
Solvent means a substance (e.g., acetone) which is combined with the
test substance to facilitate introduction of the test substance into the
dilution water.
Static system means a test chamber in which the test solution is not
renewed during the period of the test.
(c) Test procedures--(1) Summary of the test. In preparation for the
test, test chambers are filled with appropriate volumes of dilution
water. If a flow-through test is performed, the flow of dilution water
through each chamber is adjusted to the rate desired. In a static test,
the test substance is introduced into each test chamber. In a flow-
through test, the rate in which the test substance is added is adjusted
to establish and maintain the desired concentration of test substance in
each test chamber. The test is started by randomly introducing
gammarids, which have been acclimated to the test conditions, into the
test chambers. Gammarids in the test chambers are observed periodically
during the test; the dead gammarids are removed and the findings
recorded. Dissolved oxygen concentration, pH, temperature, and the
concentration of test substance in test chambers are measured at
specified intervals. Data collected during the test are used to develop
concentration--response curves and LC50 values for the test substance.
(2) [Reserved]
(3) Range-finding test. (i) A range-finding test should be conducted
to establish test substance concentrations to be used for the definitive
test.
(ii) The gammarids shall be exposed to a wide-range of
concentrations of the test substance (e.g., 1, 10, 100 mg/1, etc.),
usually under static conditions.
(iii) A minimum of five gammarids should be exposed to each
concentration of test substance for a period of 96 hours. The exposure
period may be shortened if data suitable for determining concentrations
in the definitive test can be obtained in less time. Nominal
concentrations of the test substance may be acceptable.
(4) Definitive test. (i) The purpose of the definitive test is to
determine the 24, 48, 72, and 96--hour LC50 values and the
concentration-response curves.
(ii) A minimum of 20 gammarids per concentration shall be exposed to
five or more concentrations of the test substance chosen in a geometric
series in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32,
64 mg/L). The range and number of concentrations to which the organisms
are exposed shall be such that in 96 hours there is at least one
concentration resulting in mortality greater than 50 and less than 100
percent, and one concentration causing greater than zero and less than
50 percent mortality. An equal number of gammarids may be placed in two
or more replicate test chambers. Solvents should be avoided, if
possible. If solvents have to be used, a solvent control, as well as a
dilution control, shall be tested at the highest solvent concentration
employed in the treatments. The solvent should not be toxic or have an
effect on the toxicity of the test substance. The concentration of
solvent should not exceed 0.1 ml/L.
(iii) Every test shall include a concurrent control using gammarids
from the same population or culture container. The control group shall
be exposed to the same dilution water, conditions and procedures, except
that none of the test substance shall be is added to the chamber.
(iv) The dissolved oxygen concentration, temperature and pH of the
test solution shall be measured at the beginning of the test and at 24,
48, 72 and 96 hours in at least one replicate each of the control, and
the highest, lowest and middle test concentrations.
(v) The test duration is 96 hours. The test is unacceptable if more
than 10 percent of the control organisms die during the test.
(vi) In addition to death, any abnormal behavior or appearance shall
also be reported.
(vii) Gammarids shall be randomly assigned to the test chambers.
Test
[[Page 60]]
chambers shall be positioned within the testing area in a random manner
or in a way in which appropriate statistical analyses can be used to
determine whether there is any variation due to placement.
(viii) Gammarids shall be introduced into the test chambers after
the test substance has been added.
(ix) Observations on compound solubility shall be recorded. The
investigator should record the appearance of surface slicks,
precipitates, or material adhering to the sides of the test chambers.
(5) [Reserved]
(6) Analytical measurements--(i) Water quality analysis. The
hardness, acidity, alkalinity, pH, conductivity, TOC or COD, and
particulate matter of the dilution water shall be measured at the
beginning of each definitive test.
(ii) Collection of samples for measurement of test substance. Each
sample to be analyzed for the test substance concentrations shall be
taken at a location midway between the top, bottom, and sides of the
test chamber. Samples should not include any surface scum or material
dislodged from the bottom or sides. Samples shall be analyzed
immediately or handled and stored in a manner which minimizes loss of
test substance through microbial degradation, photogradation, chemical
reaction, volatilization, or sorption.
(iii) Measurement of test substance. (A) For static tests, the
concentration of dissolved test substance (that which passes through a
0.45 micron filter) shall be measured in each test chamber at least at
the beginning (zero-hour, before gammarids are added) and at the end of
the test. During flow-through tests, the concentration of dissolved test
substance shall be measured in each test chamber at least at 0 and 96-
hours and in at least one chamber whenever a malfunction of the test
substance delivery system is observed.
(B) The analytical methods used to measure the amount of test
substance in a sample shall be validated before beginning the test. This
involves adding a known amount of the test substance to each of three
water samples taken from a chamber containing dilution water and the
same number of gammarids as are placed in each test chamber. The nominal
concentrations of the test substance in these samples should span the
concentration range to be used in the test. Validation of the analytical
method should be performed on at least two separate days prior to
starting the test.
(C) An analytical method is not acceptable if likely degradation
products of the test substance give positive or negative interferences,
unless it is shown that such degradation products are not present in the
test chambers during the test.
(D) Among replicate test chambers, the measured concentrations shall
not vary more than 20 percent. The measured concentration of the test
substance in any chamber during the test shall not vary more than plus
or minus 30 percent from the measured concentration in that chamber at
zero time.
(E) The mean measured concentration of dissolved test substance
shall be used to calculate all LC50's and to plot all concentration-
response curves.
(d) Test conditions for definitive test--(1) Test species--(i)
Selection. (A) The amphipods, Gammarus fasciatus, G. pseudolimnaeus, and
G. lacustris are specified for this test.
(B) Gammarids can be cultured in the laboratory or collected from
natural sources. If collected, they must be held in the laboratory for
at least 14 days prior to testing.
(C) Gammarids used in a particular test shall be of similar age and/
or size and from the same source or culture population.
(ii) Acclimation. If the holding water is from the same source as
the dilution water, acclimation to the dilution water shall be done
gradually over a 48-hour period. The gammarids then shall be held at
least 7 days in the dilution water prior to testing. Any changes in
water temperature should not exceed 2 deg.C per day. Gammarids should
be held for a minimum of 7 days at the test temperature prior to
testing.
(iii) Care and handling. Gammarids shall be cultured in dilution
water under similar environmental conditions to those used in the test.
Organisms shall be handled as little as possible. When handling is
necessary it should be done as gently, carefully and
[[Page 61]]
quickly as possible. During culturing and acclimation, gammarids shall
be observed carefully for signs of stress and mortality. Dead and
abnormal individuals shall be discarded.
(iv) Feeding. The organisms shall not be fed during testing. During
culturing, holding, and acclimation, a sufficient quantity of deciduous
leaves, such as maple, aspen, or birch, should be placed in the culture
and holding containers to cover the bottom with several layers. These
leaves should be aged for at least 30 days in a flow-through system
before putting them in aquaria. As these leaves are eaten, more aged
leaves should be added. Pelleted fish food may also be added.
(2) Facilities--(i) Apparatus--(A) Facilities needed to perform this
test include:
(1) Containers for culturing, acclimating and testing gammarids;
(2) Containers for aging leaves under flow-through conditions;
(3) A mechanism for controlling and maintaining the water
temperature during the culturing, acclimation and test periods;
(4) Apparatus for straining particulate matter, removing gas
bubbles, or aerating the dilution water, as necessary; and
(5) An apparatus for providing a 16-hour light and 8-hour dark
photoperiod with a 15- to 30-minute transition period.
(B) Facilities should be well ventilated and free of fumes and
disturbances that may affect the test organism.
(C) Test chambers shall be covered loosely to reduce the loss of
test solution or dilution water due to evaporation and to minimize the
entry of dust or other particulates into the solutions.
(ii) Construction materials. Construction materials and equipment
that may contact the stock solution, test solution or dilution water
should not contain substances that can be leached or dissolved into
aqueous solutions in quantities that can alter the test results.
Materials and equipment that contact stock or test solutions should be
chosen to minimize sorption of test substances. Glass, stainless steel,
and perfluorocarbon plastic should be used wherever possible. Concrete,
fiberglass, or plastic (e.g., PVC) may be used for holding tanks,
acclimation tanks, and water supply systems, but they should be aged
prior to use. Rubber, coopper, brass, galvanized metal, and lead should
not come in contact with the dilution water, stock solution, or test
solution.
(iii) Test substance delivery system. In flow-through tests,
diluters, metering pump systems or other suitable devices shall be used
to deliver the test substance to the test chambers. The system used
shall be calibrated before each test. The general operation of the test
substance delivery system shall be checked twice daily during a test.
The 24-hour flow shall be equal to at least five times the volume of the
test chamber. During a test, the flow rates should not vary more than 10
percent from one test chamber to another.
(iv) Test chambers. Test chambers shall contain at least one liter
of test solution. Test chambers made of stainless steel should be
welded, not soldered. Test chambers made of glass should be glued using
clear silicone adhesive. As little adhesive as possible should be left
exposed in the interior of the chamber. A substrate, such as a bent
piece of stainless steel screen, should be placed on the bottom of each
test chamber to provide cover for the gammarids.
(v) Cleaning of test system. Test substance delivery systems and
test chambers should be cleaned before each test. They should be washed
with detergent and then rinsed sequentially with clean water, pesticide-
free acetone, clean water, and 5-percent nitric acid, followed by two or
more changes of dilution water.
(vi) Dilution water. (A) Clean surface or ground water,
reconstituted water, or dechlorinated tap water is acceptable as
dilution water if gammarids will survive in it for the duration of the
culturing, acclimating, and testing periods without showing signs of
strees. The quality of the dilution water should be constant enough that
the month-to-month variation in hardness, acidity, alkalinity,
conductivity, TOC or COD, and particulate matter is
[[Page 62]]
not more than 10 percent. The pH should be constant within 0.4 unit. In
addition, the dilution water should meet the following specifications
measured at least twice a year:
------------------------------------------------------------------------
Substance Maximum concentration
------------------------------------------------------------------------
Particulate matter......................... 20 mg/L
Total organic carbon (TOC) or.............. 2 mg/L
chemical oxygen demand (COD)............. 5 mg/L
Boron, fluoride............................ 100 ug/L
Un-ionized ammonia......................... 1 ug/L
Aluminum, arsenic, chromium, cobalt, 1 ug/L
copper, iron, lead, nickel, zinc.
Residual chlorine.......................... 3 ug/L
Cadmium, mercury, silver................... 100 ng/L
Total organophosphorus pesticides.......... 50 ng/L
Total organochlorine pesticides plus:
polychlorinated biphenyls (PCBs) or...... 50 ng/L
organic chlorine......................... 25 ng/L
------------------------------------------------------------------------
(B) If the dilution water is from a ground or surface water source,
conductivity and total organic carbon (TOC) or chemical oxygen demand
(COD) shall be measured. Reconstituted water can be made by adding
specific amounts of reagent-grade chemicals to deionized or distilled
water. Glass-distilled or carbon-filtered deionized water with a
conductivity less than 1 micromho/cm is acceptable as the diluent for
making reconstituted water.
(C) The concentration of dissolved oxygen in the dilution water
shall be between 90 and 100 percent saturation. If necessary, the
dilution water can be aerated before the addition of the test substance.
All reconstituted water should be aerated before use.
(3) Test parameters. Environmental parameters during the test shall
be maintained as specified below:
(i) Water temperature of 18 1 deg.C.
(ii) Dissolved oxygen concentration between 60 and 105 percent
saturation.
(iii) The number of gammarids placed in a test chamber shall not be
so great as to affect the results of the test. Ten gammarids per liter
is the recommended level of loading for the static test. Loading
requirements for the flow-through test will vary depending on the flow
rate of dilution water. The loading should not cause the dissolved
oxygen concentration to fall below the recommended levels.
(iv) Photoperiod of 16 hours light and 8 hours darkness.
(e) Reporting. The sponsor shall submit to the EPA all data
developed by the test that are suggestive or predictive of toxicity. In
addition, the test report shall include, but not necessarily be limited
to, the following information:
(1) Name and address of the facility performing the study and the
dates on which the study was initiated and completed.
(2) Objectives and procedures stated in the approved protocol,
including any changes in the original protocol.
(3) Statistical methods employed for analyzing the data.
(4) The test substance identified by name, Chemical Abstracts (CAS)
number or code number, source, lot or batch number, strength, purity,
and composition, or other appropriate characteristics.
(5) Stability of the test substance under the conditions of the
test.
(6) A description of the methods used, including:
(i) The source of the dilution water, its chemical characteristics
(e.g., hardness, pH, etc.) and a description of any pretreatment.
(ii) A description of the test substance delivery system, test
chambers, the depth and volume of solution in the chamber, the way the
test was begun (e.g., test substance addition), the loading, the
lighting, and the flow rate.
(iii) Frequency and methods of measurements and observations.
(7) The scientific name, weight, length, source, and history of the
organisms used, and the acclimation procedures and food used.
(8) The concentrations tested, the number of gammarids and
replicates per test concentration. The reported results should include:
(i) The results of dissolved oxygen, pH and temperature
measurements.
(ii) If solvents are used, the name and source of the solvent, the
nominal concentration of the test substance in the stock solution, the
highest solvent concentration in the test solution and a description of
the solubility determination in water and solvents.
(iii) The measured concentration of the test substance in each test
chamber just before the start of the test and at all subsequent sampling
periods.
[[Page 63]]
(iv) In each test chamber at each observation period, the number of
dead and live test organisms, the percentage of organisms that died, and
the number of test organisms that showed any abnormal effects in each
test chamber at each observation period.
(v) The 48, 72 and 96-hour LC50's and their 95 percent confidence
limits. When sufficient data have been generated, the 24-hour LC50 value
also. These calculations should be made using the mean measured test
substance concentrations.
(vi) The observed no-effect concentration (the highest concentration
tested at which there were no mortalities or abnormal behavioral or
physiological effects), if any.
(vii) Methods and data for all chemical analyses of water quality
and test substance concentrations, including method validations and
reagent blanks.
(9) A description of all circumstances that may have affected the
quality or integrity of the data.
(10) The names of the sponsor, study director, principal
investigator, names of other scientists or professionals, and the names
of all supervisory personnel involved in the study.
(11) A description of the transformations, calculations, or
operations performed on the data, a summary and analysis of the data,
and a statement of the conclusions drawn from the analysis. Results of
the analysis of data should include the calculated LC50 value, 95
percent confidence limits, slope of the transformed concentration-
response line, and the results of a goodness-of-fit test (e.g., chi-
square test).
(12) The signed and dated reports prepared by any individual
scientist or other professional involved in the study, including each
person who, at the request or direction of the testing facility or
sponsor, conducted an analysis or evaluation of data or specimens from
the study after data generation was completed.
(13) The locations where all specimens, raw data, and the final
report are stored.
(14) The statement prepared and signed by the quality assurance
unit.
[52 FR 24462, July 1, 1987]
Subpart D--Provisional Health Effects Guidelines
Sec. 795.225 Dermal pharmacokinetics of DGBE and DGBA.
(a) Purpose. The purpose of these studies is to determine:
(1) The absorption of diethylene glycol butyl ether (DGBE) after
administration by the dermal route.
(2) The biotransformation of DGBE administered dermally.
(3) The dermal absorption of DGBE and diethylene glycol butyl ether
acetate (DGBA).
(b) Test procedures--(1) Animal selection--(i) Species. The species
utilized for investigating DGBE and DGBA shall be the rat, a species for
which historical data on the toxicity and carcinogenicity of many
compounds are available and which is used extensively in percutaneous
absorption studies.
(ii) Animals. Adult female Sprague Dawley rats shall be used. The
rats shall be 7 to 8 weeks old and weigh 180 to 220 grams. Prior to
testing, the animals shall be selected at random for each group. Animals
showing signs of ill health shall not be used.
(iii) Animal care. (A) The animals should be housed in
environmentally controlled rooms with 10 to 15 air changes per hour. The
rooms should be maintained at a temperature of 25 2 deg.C
and humidity of 50 10 percent with a 12-hour light/dark
cycle per day. The rats should be isolated for at least 7 days prior to
use.
(B) During the acclimatization period, the rats should be housed in
cages on hardwood chip bedding. All animals shall be provided with
conventional laboratory diets and water ad libitum.
(2) Administration of DGBE and DGBA--(i) Test substances. These
studies require the use of 14C-labeled DGBE and DGBA. The use
of 14C-DGBE and 14C-DGBA is required for the
determinations in paragraphs (a) (1), (2), and (3) of this section
because they will facilitate the work and improve the reliability of
quantitative determinations.
(ii) Dosage and treatment. (A) Two doses of DGBA shall be used in
the study, a ``low'' dose and a ``high'' dose. Three doses of DGBE shall
be used in the study, a neat ``low'' dose, an aqueous ``low'' dose, and
neat ``high'' dose.
[[Page 64]]
When administered dermally, the ``high'' dose level should ideally
induce some overt toxicity such as weight loss. The ``low'' dose level
should correspond to a no observed effect level.
(B) For dermal treatment, the doses shall be applied in a volume
adequate to deliver the prescribed doses. The backs of the rats should
be lightly shaved with an electric clipper shortly before treatment. The
dose shall be applied with a micropipette on a specific area (for
example, 2 cm2) on the freshly shaven skin.
(iii) Washing efficiency study. Before initiation of the dermal
absorption studies described in paragraph (b)(2)(iv)(A) of this section,
an initial washing efficiency experiment shall be performed to assess
the extent of removal of the applied DGBE and DGBA by washing with soap
and water. Groups of four rats should be lightly anesthetized with
sodium pentobarbital. These animals shall then be treated with dermal
doses of test substance at the low dose level. Soon after application (5
to 10 minutes) the treated animals shall be washed with soap and water
then housed in individual metabolism cages for excreta collection. Urine
and feces shall be collected at 8, 24, and 48 hours following dosing.
Collection of excreta shall continue every 24 hours if a significant
amounts of DGBE, DGBA, or metabolites continue to be eliminated.
(iv) Determination of absorption, biotransformation, and excretion.
(A) Eight animals shall be dosed once dermally with the low dose of
14C-DGBE.
(B) Eight animals shall be dosed once dermally with the high dose of
14C-DGBE.
(C) Eight animals shall be dosed once dermally with the low dose of
14C-DGBA.
(D) Eight animals shall be dosed once dermally with the high dose of
14C-DGBA.
(E) The high and low doses of 14C-DGBE and
14C-DGBA shall be kept on the skin for 24 hours. After
application, the animals shall be placed in metabolism cages for excreta
collection. After 24 hours, any test material remaining on the skin will
be washed off and the containment cell removed. Radiolabeled material in
the wash will be accounted for in the total recovery. Urine and feces
shall be collected at 8, 24, 48, 72, and 96 hours after dosing, and if
necessary, daily thereafter until at least 90 percent of the dose has
been excreted or until 7 days after dosing, whichever occurs first.
(3) Observation of animals--(i) Urinary and fecal excretion. The
quantities of total 14C excreted in urine and feces by rats
dosed as specified in paragraph (b)(2)(iv) of this section shall be
determined at 8, 24, 48, 72 and 96 hours after dosing, and if necessary,
daily thereafter until at least 90 percent of the dose has been excreted
or until 7 days after dosing (whichever occurs first). Four animals from
each group shall be used for this purpose.
(ii) Biotransformation after dermal dosing. Appropriate qualitative
and quantitative methods shall be used to assay urine specimens
collected from rats dosed with DGBE as specified in paragraph (b)(2)(iv)
of this section. Any metabolite which comprises greater than 10 percent
of the dose shall be identified.
(c) Data and reporting--(1) Treatment of results. Data shall be
summarized in tabular form.
(2) Evaluation of results. All observed results, quantitative or
incidental, shall be evaluated by an appropriate statistical method.
(3) Test report. In addition to the reporting requirements as
specified in the TSCA Good Laboratory Practice Standards, in part 792,
subpart J of this chapter, the following specific information shall be
reported:
(i) Species, strain, and supplier of laboratory animals.
(ii) Information on the degree (i.e., specific activity for a
radiolabel) and sites of labeling of the test substances.
(iii) A full description of the sensitivity and precision of all
procedures used to produce the data.
(iv) Relative percent absorption by the dermal route for rats
administered low and high doses of 14C-DGBE and
14C-DGBA.
(v) Quantity of isotope, together with percent recovery of the
administered dose, in feces and urine.
(vi) Biotransformation pathways and quantities of DGBE and
metabolites in
[[Page 65]]
urine collected after administering single high and low dermal doses to
rats.
[53 FR 5946, Feb. 26, 1988, as amended at 54 FR 41834, Oct. 12, 1989]
Sec. 795.228 Oral/dermal pharmacokinetics.
(a) Purpose. The purposes of these studies are to:
(1) Ascertain whether the pharmacokinetics and metabolism of a
chemical substance or mixture (``test substance'') are similar after
oral and dermal administration.
(2) Determine bioavailability of a test substance after oral and
dermal administration.
(3) Examine the effects of repeated dosing on the pharmacokinetics
and metabolism of the test substance.
(b) Definitions. (1) Bioavailability refers to the rate and relative
amount of administered test substance which reaches the systemic
circulation.
(2) Metabolism means the study of the sum of the processes by which
a particular substance is handled in the body and includes absorption,
tissue distribution, biotransformation, and excretion.
(3) Percent absorption means 100 times the ratio between total
excretion of radioactivity following oral or dermal administration and
total excretion following intravenous administration of test substance.
(4) Pharmacokinetics means the study of the rates of absorption,
tissue distribution, biotransformation, and excretion.
(c) Test procedures--(1) Animal selection--(i) Species. The rat
shall be used for pharmacokinetics testing because it has been used
extensively for metabolic and toxicological studies. For dermal
bioavailability studies, the rat and the mini-pig shall be used.
(ii) Test animals. For pharmacokinetics testing and dermal studies,
adult male and female Sprague-Dawley rats, 7 to 9 weeks of age, shall be
used. For dermal studies, young adult mini-pigs shall also be used. The
animals should be purchased from a reputable dealer and shall be
identified upon arrival at the testing laboratory. The animals shall be
selected at random for the test groups and any animal showing signs of
ill health shall not be used. In all studies, unless otherwise
specified, each test group shall contain at least 4 animals of each sex
for a total of at least 8 animals.
(iii) Animal care. (A) The animals shall be housed in
environmentally controlled rooms with at least 10 air changes per hour.
The rooms shall be maintained at a temperature of 24 2
deg.C and humidity of 50 20 percent with a 12-hour light/
dark cycle per day. The animals shall be kept in a quarantine facility
for at least 7 days prior to use and shall be acclimated to the
experimental environment for a minimum of 48 hours prior to
administration of the test substance.
(B) During the acclimatization period, the animals shall be housed
in suitable cages. All animals shall be provided with certified feed and
tap water ad libitum. The mini-pig diet shall be supplemented with
adequate amounts of ascorbic acid in the drinking water.
(2) Administration of test substance--(i) Test substance. The use of
a radioactive test substance is required for all studies. Ideally, the
purity, radioactive and nonradioactive, is greater than 99 percent. The
radioactive and nonradioactive test substances shall be chromatographed
separately and together to establish purity and identity. If the purity
is less than 99 percent or if the chromatograms differ significantly,
EPA should be consulted.
(ii) Dosage and treatment--(A) Intravenous. The low dose of test
substance, in an appropriate vehicle, shall be administered
intravenously to groups of rats and mini-pigs of each sex. If feasible,
the same low dose should be used for intravenous, oral, and dermal
studies.
(B) Oral. Two doses of text substance shall be used in the oral
study, a low dose and a high dose. The high dose should ideally induce
some overt toxicity, such as weight loss. The low dose should correspond
to a no-observed effect level. The oral dosing shall be accomplished by
gavage or by administering the encapsulated test substance. If feasible,
the same high and low doses should be used for oral and dermal studies.
[[Page 66]]
(C) Dermal. (1) Dermal treatment. For dermal treatment, two doses,
comparable to the low and high oral doses, shall be dissolved in a
suitable vehicle and applied in volumes adequate to deliver comparable
doses. The backs of the animals should be lightly shaved with an
electric clipper 24 hours before treatment. The test substance shall be
applied to the intact shaven skin (approximately 2 cm\2\ for rats, 5
cm\2\ for mini-pigs). The dosed areas shall be protected with a suitable
porous covering which is secured in place, and the animals shall be
housed separately.
(2) Washing efficacy study. Before initiation of the dermal
absorption studies, an initial washing efficacy experiment shall be
conducted to assess the removal of the applied low dose of the test
substance by washing the exposed skin area with soap and water and an
appropriate organic solvent. The low dose shall be applied to 4 rats and
4 mini-pigs in accordance with paragraph (c)(2)(ii)(C)(1) of this
section. After application (5 to 10 minutes), the treated areas of 2
rats and 2 mini-pigs shall be washed with soap and water and the treated
areas of the remaining rats and pigs shall be washed with an appropriate
solvent. The amounts of test substance recovered in the washings shall
be determined to assess efficacy of its removal by washing.
(iii) Dosing and sampling schedule--(A) Rat studies. After
administration of the test substance, each rat shall be placed in a
metabolic unit to facilitate collection of excreta. For the dermal
studies, excreta from the rats shall also be collected during the 6 hour
exposure periods. At the end of each collection period, the metabolic
units shall be cleaned to recover any excreta that might adhere to them.
All studies, except the repeated dosing study, shall be terminated at 7
days or after at least 90 percent of the radioactivity has been
recovered in the excreta, whichever occurs first.
(1) Intravenous study. Group A shall be dosed once intravenously at
the low dose of test substance.
(2) Oral study. (i) Group B shall be dosed once per os with the low
dose of test substance.
(ii) Group C shall be dosed once per os with the high dose of test
substance.
(3) Dermal studies. Unless precluded by corrosivity, the test
substance shall be applied and kept on the skin for a minimum of 6
hours. At the time of removal of the porous covering, the treated area
shall be washed with an appropriate solvent to remove any test substance
that may be on the skin surface. Both the covering and the washing shall
be assayed to recover residual radioactivity. At the termination of the
studies, each animal shall be sacrificed and the exposed skin area
removed. An appropriate section of the skin shall be solubilized and
assayed for radio-activity to ascertain if the skin acts as a reservoir
for the test substance. Studies on the dermal absorption of corrosive
test substances should be discussed with EPA prior to initiation.
(i) Group D shall be dosed once dermally with the low dose of test
compound.
(ii) Group E shall be dosed once dermally with the high dose of the
test substance.
(4) Repeated dosing study. Group F shall receive a series of single
daily oral low doses of nonradioactive test substance over a period of
at least 7 days. Twenty-four hours after the last nonradioactive dose, a
single oral low dose of radioactive test substance shall be
administered. Following dosing with the radioactive substance, the rats
shall be placed in individual metabolic units as described in paragraph
(c)(2)(iii) of this section. The study shall be terminated at 7 days
after the last dose, or after at least 90 percent of the radioactivity
has been recovered in the excreta, whichever occurs first.
(B) Mini-Pig studies. For all mini-pig studies, the test groups
shall consist of four young adult animals. After administration of the
test substance, each mini-pig shall be kept in a metabolic unit to
facilitate collection of excreta. At the end of each collection period,
the metabolic units are to be cleaned to recover any excreta that might
adhere to them. All studies shall be terminated at 7 days, or after at
least 90 percent of the radio-activity has been recovered in the
excreta, whichever occurs first.
[[Page 67]]
(1) Intravenous study. Group G is to be dosed once intravenously at
the low dose of the test substance.
(2) Dermal studies. Following the experimental guidance described in
(c)(2)(iii)(A)(3) of this section:
(i) Group H shall be dosed once dermally with the low dose of test
substance.
(ii) Group I shall be dosed once dermally with the high dose of the
test substance.
(3) Types of studies--(i) Pharmacokinetics studies--(A) Rat studies.
Groups A through F shall be used to determine the kinetics of absorption
of the test substance. In the group administered the test substance by
intravenous routes, (i.e., Group A), the concentration of radioactivity
in blood and excreta shall be measured following administration. In
groups administered the test substance by the oral and dermal route
(i.e., Groups B, C, D, E and F), the concentration of radioactivity in
blood and excreta shall be measured at selected time intervals during
and following the exposure period.
(B) Mini-Pig studies. Groups G, H, and I shall be used to determine
the extent of dermal absorption of the test substance. The amount of
radioactivity in excreta shall be determined at selected time intervals.
(ii) Metabolism studies--Rat studies. Groups A through F shall be
used to determine the metabolism of the test substance. Urine, feces,
and expired air shall be collected for identification and quantification
of the test substance and metabolites.
(4) Measurements--(i) Pharmacokinetics. Four animals from each group
shall be used for these purposes.
(A) Rat studies--(1) Bioavailability. The levels of radioactivity
shall be determined in whole blood, blood plasma or blood serum at 15
and 30 minutes and at 1, 2, 8, 24, 48, and 96 hours after initiation of
dosing.
(2) Extent of absorption. The total quantities of radioactivity
shall be determined for excerta collected daily for 7 days or until at
least 90 percent of the radioactivity has been recovered in the excreta.
(3) Excretion. The quantities of radioactivity eliminated in the
urine, feces, and expired air shall be determined separately at
appropriate time intervals. The collection of carbon dioxide may be
discontinued when less than one percent of the dose is found to be
exhaled as radioactive carbon dioxide in 24 hours.
(4) Tissue distribution. At the termination of each study, the
quantities of radioactivity in blood and in various tissues, including
bone, brain, fat, gastrointestinal tract, gonads, heart, kidney, liver,
lungs, muscle, skin, and residual carcass of each animal shall be
determined.
(5) Changes in pharmacokinetics. Results of pharmacokinetics
measurements (i.e., bioavailability and extent of absorption, tissue
distribution, and excretion) obtained in rats receiving the single low
oral dose of the test substance (Groups B and C) shall be compared to
the corresponding results obtained in rats receiving repeated oral doses
of the test substance (Group F).
(B) Mini-Pig studies--Extent of absorption. The total quantities of
radioactivity shall be determined for excreta daily for 7 days or until
at least 90 percent of the test substance has been excreted.
(ii) Metabolism. Four animals from each group shall be used for
these purposes.
(A) Rat studies--(1) Biotransformation. Appropriate qualitative and
quantitative methods shall be used to assay urine, feces, and expired
air collected from rats. Efforts shall be made to identify any
metabolite which comprises 5 percent or more of the administered dose
and the major radioactive components of blood.
(2) Changes in biotransformation. Appropriate qualitative and
quantitative assay methodology shall be used to compare the composition
of radioactive compounds in excreta from rats receiving a single oral
dose (Groups B and C) with those in the excreta from rats receiving
repeated oral doses (Group H).
(d) Data and reporting. The final test report shall include the
following:
(1) Presentation of results. Numerical data shall be summarized in
tabular form. Pharmacokinetic data shall also be presented in graphical
form. Qualitative observations shall also be reported.
[[Page 68]]
(2) Evaluation of results. All quantitative results shall be
evaluated by an appropriate statistical method.
(3) Reporting results. In addition to the reporting requirements as
specified in 40 CFR part 792, the following specific information shall
be reported:
(i) Species and strains of laboratory animals.
(ii) Chemical characterization of the test substance, including:
(A) For the radioactive test substances, information on the site(s)
and degree of radiolabeling, including type of label, specific activity,
chemical purity, and radiochemical purity.
(B) For the nonradioactive compound, information on chemical purity.
(C) Results of chromatography.
(iii) A full description of the sensitivity, precision, and accuracy
of all procedures used to generate the data.
(iv) Percent of absorption of test substance after oral and dermal
exposures to rats and dermal exposure to mini-pigs.
(v) Quantity and percent recovery of radioactivity in feces, urine,
expired air, and blood. In dermal studies on rats and mini-pigs, include
recovery data for skin, skin washings, and residual radioactivity in the
covering as well as results of the washing efficacy study.
(vi) Tissue distribution reported as quantity of radioactivity in
blood and in various tissues, including bone, brain, fat,
gastrointestinal tract, gonads, heart, kidney, liver, lung, muscle, skin
and in residual carcass of rats.
(vii) Materials balance developed from each study involving the
assay of body tissues and excreta.
(viii) Biotransformation pathways and quantities of test substance
and metabolites in excreta collected after administering single high and
low doses to rats.
(ix) Biotransformation pathways and quantities of the test substance
and metabolites in excreta collected after administering repeated low
doses to rats.
(x) Pharmacokinetics model(s) developed from the experimental data.
[54 FR 33411, Aug. 14, 1989; 54 FR 49844, Dec. 1, 1989; 55 FR 25392,
June 21, 1990]
Sec. 795.231 Pharmacokinetics of isopropanal.
(a) Purpose. The purposes of these studies are to:
(1) Ascertain whether the pharmacokinetics and metabolism of the
``test substance'' are similar after oral and inhalation administration.
(2) Determine bioavailability of the test substance after oral and
inhalation administration.
(3) Examine the effects of repeated dosing on the pharmacokinetics
and metabolism of the test substance.
(b) Definitions. (1) ``Bioavailability'' refers to the rate and
relative amount of administered test substance which reaches the
systemic circulation.
(2) ``Metabolism'' means the study of the sum of the processes by
which a particular substance is handled in the body, and includes
absorption, tissue distribution, biotransformation, and excretion.
(3) ``Pharmacokinetics'' means the study of the rates of absorption,
tissue distribution, biotransformation, and excretion.
(c) Test procedures--(1) Animal selection--(i) Species. The rat
shall be used because it has been used extensively for metabolic and
toxicological studies.
(ii) Test animals. For pharmacokinetics testing, adult male and
female rats (Fischer 344 or strain used for major toxicity testing), 7
to 9 weeks of age, shall be used. The animals should be purchased from a
reputable dealer and shall be identified upon arrival at the testing
laboratory. The animals shall be selected at random for the testing
groups and any animal showing signs of ill health shall not be used. In
all studies, unless otherwise specified, each test group shall contain
at least four animals of each sex for a total of at least eight animals.
(iii) Animal care. (A) Animal care and housing should be in
accordance with DHEW Publication No. (NIH)-85-23, 1985, entitled
``Guidelines for the Care and Use of Laboratory Animals.''
(B) The animals should be housed in environmentally controlled rooms
with at least 10 air changes per hour. The rooms shall be maintained at
a temperature of 222 deg.C and humidity of 5020
percent with a 12-hour light/dark cycle per day. The animals shall be
[[Page 69]]
kept in a quarantine facility for at least 7 days prior to use and shall
be acclimated to the experimental environment for a minimum of 48 hours
prior to treatment.
(C) During the acclimatization period, the animals should be housed
in suitable cages. All animals shall be provided with certified feed and
tap water ad libitum.
(2) Administration of test substance--(i) Test substance. The use of
radioactive test substance is required for all materials balance and
metabolite identification requirements of the study. Ideally, the purity
of both radioactive and nonradioactive test substance should be greater
than 99 percent. The radioactive and nonradioactive substances shall be
chromatographed separately and together to establish purity and
identity. If the purity is less than 99 percent or if the chromatograms
differ significantly, EPA should be consulted.
(ii) Dosage and treatment--(A) Intravenous. The low dose of test
substance, in an appropriate vehicle, shall be administered
intravenously to four rats of each sex.
(B) Oral. Two doses of test substance shall be used in the oral
portion of the study, a low dose and a high dose. The high dose should
ideally induce some overt toxicity, such as weight loss. The low dose
level should correspond to a no-observed effect level. The oral dosing
shall be accomplished by gavage or by administering an encapsulated test
substance. If feasible, the same high and low doses should be used for
oral and dermal studies.
(C) Inhalation. Two concentrations of the test substance shall be
used in this portion of the study, a low concentration and a high
concentration. The high concentration should ideally induce some overt
toxicity, while the low concentration should correspond to a no observed
level. Inhalation treatment should be conducted using a ``nose-cone'' or
``head only'' apparatus to prevent ingestion of the test substance
through ``grooming''.
(iii) Dosing and sampling schedule. After administration of the test
substance, each rat shall be placed in a separate metabolic unit to
facilitate collection of excreta. For the inhalation studies, excreta
from the rats shall also be collected during the exposure periods. At
the end of each collection period, the metabolic units shall be cleaned
to recover any excreta that might adhere to the cages. All studies,
except the repeated dose study, shall be terminated at 7 days, or after
at least 90 percent of the radioactivity has been recovered in the
excreta, whichever occurs first.
(A) Intravenous study. Group A shall be dosed once intravenousely at
the low dose of test substance.
(B) Oral studies. (1) Group B shall be dosed once per os with the
low dose of the test substance.
(2) Group C shall be dosed once per os with the high dose of the
test substance.
(C) Inhalation studies. A single 6-hour exposure period shall be
used for each group.
(1) Group D shall be exposed to a mixture of the test substance in
air at the low concentration.
(2) Group E shall be exposed to a mixture of test substance in air
at the high concentration.
(D) Repeated dosing study. Group F shall receive a series of single
daily oral low doses of nonradioactive test substance over a period of
at least 7 consecutive days. Twenty four hours after the last
nonradioactive dose, a single oral low dose of radioactive test
substance shall be administered. Following dosing with radioactive
substance, the rats shall be placed in individual metabolic units as
described in paragraph (c)(2)(iii) of this section. The study shall be
terminated 7 days after the last dose, or after at least 90 percent of
the radioactivity has been recovered in the excreta, whichever occurs
first.
(3) Types of studies--(i) Pharmacokinetics studies. Groups A through
F shall be used to determine the kinetics of absorption of the test
substance. In groups administered the substance by intravenous or oral
routes, (i.e., Groups A, B, C, F), the concentration of radioactivity in
blood and excreta including expired air shall be measured following
administration. In groups administered the substance by the inhalation
route (i.e., Groups D and E), the concentration of radioactivity in
blood shall be measured at selected time intervals
[[Page 70]]
during and following the exposure period. In the groups administered the
substance by inhalation (i.e., Groups D and E), the concentration of
radioactivity in excreta (including expired air) shall be measured at
selected time intervals following the exposure period. In addition, in
the groups administered the substance by inhalation, the concentration
of test substance in inspired air shall be measured at selected time
intervals during the exposure period.
(ii) Metabolism studies. Groups A through F shall be used to
determine the metabolism of the test substance. Excreta (urine, feces,
and expired air) shall be collected for identification and
quantification of test substance and metabolites.
(4) Measurements--(i) Pharmacokinetics. Four animals from each group
shall be used for these purposes.
(A) Bioavailability. The levels of radioactivity shall be determined
in whole blood, blood plasma or blood serum at 15 minutes, 30 minutes,
1, 2, 3, 6, 9, and 18 hours after dosing; and at 30 minutes, 3, 6, 6.5,
7, 8, 9, 12, and 18 hours after initation of inhalation exposure.
(B) Extent of absorption. The total quantities of radioactivity
shall be determined for excreta collected daily for 7 days, or after at
least 90 percent of the radioactivity has been recovered in the excreta,
whichever occurs first.
(C) Excretion. The quantities of radioactivity eliminated in the
urine, feces, and expired air shall be determined separately at
appropriate time intervals. The collection of the intact test substance
or its metabolites, including carbon dioxide, may be discontinued when
less than 1 percent of the administered dose is found to be exhaled as
radioactive carbon dioxide in 24 hours.
(D) Tissue distribution. At the termination of each study, the
quantities of radioactivity in blood and in various tissues, including
bone, brain, fat, gastrointestinal tract, gonads, heart, kidney, liver,
lungs, muscle, skin, spleen, and residual carcass of each animal shall
be determined.
(E) Changes in pharmacokinetics. Results of pharmacokinetics
measurements (i.e., biotransformation, extent of absorption, tissue
distribution, and excretion) obtained in rats receiving the single low
oral dose of test substance (Group B) shall be compared to the
corresponding results obtained in rats receiving repeated oral doses of
test substance (Group F).
(F) Biotransformation. Appropriate qualitative and quantitative
methods shall be used to assay urine, feces, and expired air collected
from rats. Efforts shall be made to identify any metabolite which
comprises 5 percent or more of the dose eliminated.
(G) Changes in biotransformation. Appropriate qualitative and
quantitative assay methodology shall be used to compare the composition
of radioactive substances in excreta from the rats receiving a single
oral dose (Groups B and C) with those in the excreta from rats receiving
repeated oral doses (Group F).
(ii) [Reserved]
(d) Data and reporting. The final test report shall include the
following:
(1) Presentation of results. Numerical data shall be summarized in
tabular form. Pharmacokinetics data shall also be presented in graphical
form. Qualitative observations shall also be reported.
(2) Evaluation of results. All quantitative results shall be
evaluated by an appropriate statistical method.
(3) Reporting results. In addition to the reporting requirements as
specified in the EPA Good Laboratory Practice Standards (40 CFR
792.185), the following specific information shall be reported:
(i) Species and strains of laboratory animals.
(ii) Chemical characterization of the test substance, including:
(A) For the radioactive test substance, information on the site(s)
and degree of radiolabeling, including type of label, specific activity,
chemical purity, and radiochemical purity.
(B) For the nonradioactive substance, information on chemical
purity.
(C) Results of chromatography.
(iii) A full description of the sensitivity, precision, and accuracy
of all procedures used to generate the data.
(iv) Extent of absorption of the test substance as indicated by:
percent absorption of the administered oral dose; and total body burden
after inhalation exposure.
[[Page 71]]
(v) Quantity and percent recovery of radioactivity in feces, urine,
expired air, and blood.
(vi) Tissue distribution reported as quantity of radioactivity in
blood and in various tissues, including bone, brain, fat,
gastrointestinal tract, gonads, heart, kidney, liver, lung, muscle,
skin, spleen and in residual carcass of each rat.
(vii) Biotransformation pathways and quantities of the test
substance and metabolites in excreta collected after administering
single high and low doses to rats.
(viii) Biotransformation pathways and quantities of the test
substance and metabolites in excreta collected after administering
repeated low doses to rats.
(ix) Pharmacokinetics model(s) developed from the experimental data.
[54 FR 43261, Oct. 23, 1989]
Sec. 795.232 Inhalation and dermal pharmacokinetics of commercial hexane.
(a) Purposes. The purposes of these studies are to:
(1) Determine the bioavailability of the test substances after
dermal and inhalation administration.
(2) Compare the pharmacokinetics and metabolism of the test
substances after intravenous, dermal, and inhalation administration.
(3) Examine the effects of repeated doses on the pharmacokinetics
and metabolism of the test substances.
(b) Definitions. (1) Bioavailability refers to the relative amount
of administered test substance which reaches the systemic circulation
and the rate at which this process occurs.
(2) Metabolism means the sum of the enzymatic and nonenzymatic
processes by which a particular substance is handled in the body.
(3) Pharmacokinetics means the study of the rates of absorption,
tissue distribution, biotransformation, and excretion.
(4) Low dose should correspond to 1 /10 of the high dose.
(5) High dose shall not exceed the lower explosive limit (LEL) and
ideally should induce minimal toxicity.
(6) Test substance refers to the unlabeled and both radiolabeled
mixtures (14C-n-hexane and 14C-methylcyclopentane)
of commercial hexane used in the testing.
(c) Test procedures--(1) Animal selection--(i) Species. The rat
shall be used for pharmacokinetics testing because it has been used
extensively for metabolic and toxicological studies.
(ii) Test animals. Adult male and female rats shall be used for
testing. The rats shall be 7 to 9 weeks old and their weight range
should be comparable from group to group. The animals shall be purchased
from a reputable dealer and shall be permanently identified upon
arrival. The animals shall be selected at random for the testing groups,
and any animal showing signs of ill health shall not be used.
(iii) Animal care. (A) Animal care and housing shall be in
accordance with DHHS/PHS NIH Publication No. 86-23, 1985, ``Guidelines
for the Care and Use of Laboratory Animals.''
(B) The animals shall be housed in environmentally controlled rooms
with at least 10 air changes per hour. The rooms shall be maintained at
a temperature of 18 to 26 degrees centigrade and humidity of 40 to 70
percent with a 12-hour light/dark cycle per day. The animal subjects
shall be kept in a quarantine facility for at least 7 days prior to use,
and shall be acclimated to the experimental environment for a minimum of
48 hours prior to treatment.
(C) During the acclimatization period, the rats shall be housed in
suitable cages. All animals shall be provided with certified feed and
tap water ad libitum.
(2) Administration of test substances--(i) Test substances. The
study will require the use of both radiolabeled and unlabeled test
substances. All unlabeled commercial hexane shall be from the same lot
number. Two kinds of radiolabeled test substances will be tested.
14C-n-hexane shall be the only radiolabeled component of one,
and 14C-MCP shall be the only radiolabeled component of the
other test substance. The use of both radiolabeled test substances is
required for all pharmacokinetics and metabolism studies described in
this rule, except for the bioavailability measurements required in
(c)(4)(i)(A) of this section. The bioavailability measurements need only
be conducted with the test substance
[[Page 72]]
containing 14C-n-hexane or an unlabeled test substance may be
used if it can be demonstrated that the analytical sensitivity of the
method used with the unlabeled test substance is equal to or greater
than the sensitivity which could be obtained with the radiolabeled test
substance. If an unlabeled test substance is used for bioavailability
measurements, these measurements shall be extended to include relevant
metabolites of n-hexane. These test substances shall contain at least 40
liquid volume percent but no more than 55 liquid volume percent n-hexane
and no less than 10 liquid volume percent methylcyclopentane (MCP) and
otherwise conform to the specifications prescribed in the American
Society for Testing and Materials Designation D 1836-83 (ASTM D 1836),
``Standard Specification for Commercial Hexanes'', published in the 1986
Annual Book of ASTM Standards: Petroleum Products and Lubricants, ASTM D
1836-83, pp. 966-967, 1986, which is incorporated by reference in
accordance with 5 U.S.C. 552(a). ASTM D 1863-83 is available for public
inspection at the Office of the Federal Register, Suite 700, 800 North
Capitol St., NW., Washington, DC, and copies may be obtained from the
Non-Confidential Information Center (NCIC) (7407), Office of Pollution
Prevention and Toxics, U.S. Environmental Protection Agency, Room B-607
NEM, 401 M Street, SW., Washington, DC 20460, between the hours of 12
p.m. and 4 p.m. weekdays excluding legal holidays. This incorporation by
reference was approved by the Director of the Office of the Federal
Register in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. This
material is incorporated as it exists on the date of approval, and a
notice of any change in this material will be published in the Federal
Register.
(ii) Dosage and treatment--(A) Intravenous. An appropriate dose of
the test substance shall be administered intravenously. The intravenous
data obtained in this portion of the study shall be suitable for the
determination of absorption, distribution, and excretion parameters of
the test substance. Factors that should be considered in the selection
of the intravenous doses are: The acute toxicity of the test substance,
the availability of a suitable vehicle (if saline is unsuitable) and the
solubility of the test substance in the vehicle.
(B) Inhalation. Two concentrations of each test substance shall be
used in this portion of the study, a low concentration and a high
concentration. The high concentration should induce minimal toxicity,
but shall not exceed the lower explosive limit (LEL). The low
concentration shall correspond to 1/10 of the high concentration.
Inhalation treatment shall be conducted using a ``nose-cone'' or ``head
only'' apparatus to reduce ingestion of the test substance through
``grooming'' or dermal absorption.
(C) Dermal. Dermal absorption studies should be conducted by the
methodology of Susten, A.S., Dames, B.L. and Niemeier, R.W., ``In vivo
percutaneous absorption studies of volatile solvents in hairless mice.
I. Description of a skin depot'', In: Journal of Applied Toxicology
6:43-46, (1986), or by some other suitable method because the test
substances have significant volatility. The high and low doses shall be
tested in rats.
(iii) Dosing and sampling schedule. Each experimental group shall
contain at least four animals of each sex. After administration of the
test substance, each rat shall be placed in an individual metabolic unit
for collection of urine, feces, and expired air. For the dermal studies,
excreta from the rats shall also be collected during the exposure
periods. At the end of each collection period, the metabolic units shall
be cleaned to recover any excreta that might adhere to the units. All
studies, except the repeated dose studies, shall be terminated at 7
days, or after at least 90 percent of the administered radioactivity has
been recovered in the excreta, whichever occurs first. All studies
described below shall be conducted separately with each radiolabeled
test substance.
(A) Intravenous study. Group A shall be given a single intravenous
dose of the radiolabeled test substance to result in a level of
commercial hexane in the blood that approximates the level from the
other routes of exposure so that the data can be used to determine
absorption and excretion parameters.
[[Page 73]]
(B) Inhalation studies. A single 6-hour exposure period shall be
used for each group.
(1) Group B shall be exposed to a mixture of the radiolabeled test
substance in air at the low concentration.
(2) Group C shall be exposed to a mixture of the radiolabeled test
substance in air at the high concentration.
(C) Dermal studies. The test substance shall be applied and kept on
the skin for a minimum of 6 hours. The covering apparatus components
shall be assayed to recover residual radioactivity. At the termination
of the studies, each animal shall be sacrificed and the exposed skin
area removed. An appropriate section of the skin shall be solubilized
and assayed for radioactivity to ascertain whether the skin acts as a
reservoir for the test substance.
(1) Group D shall be given one dermal, low dose of the radiolabeled
test substance.
(2) Group E shall be given one dermal, high dose of the radiolabeled
test substance.
(D) Repeated dosing study. Group F shall receive a series of single
daily 6-hour inhalation exposures to unlabeled test substance at the low
dose over a period of at least 7 days. A single 6-hour inhalation
exposure to the radiolabeled test substance at the low dose shall be
administered 24 hours after the last unlabeled exposure. Following
administration of the radiolabeled substance, the rats shall be placed
in individual metabolic units and excreta collected. The study shall be
terminated 7 days after the last exposure, or after at least 90 percent
of the radioactivity has been recovered in the excreta, whichever occurs
first.
(3) Types of studies--(i) Pharmacokinetics studies. Groups A through
F shall be used to determine the kinetics of absorption of the test
substance. In animal subjects administered the test substance
intravenously (i.e., Group A), the concentration of test substance in
blood and excreta shall be measured following administration. In animal
subjects administered the test substance by the inhalation and dermal
routes (i.e., Groups B through F), the concentration of test substance
in blood shall be measured at selected time intervals during and
following the exposure period. In animal subjects administered the test
substance by the inhalation route (i.e., Groups B, C, and F) the
concentration of test substance in excreta shall be measured following
exposure. In animal subjects administered the test substance by the
dermal route (i.e., Groups D and E) the concentration of test substance
in excreta shall be measured during and following exposure. These
measurements allow calculation of uptake, half lives, and clearance. In
addition, in the groups administered the test substance by inhalation
(i.e., Groups B, C, and F), the concentration of test substance in the
exposure chamber air shall be measured at selected time intervals during
the exposure period.
(ii) Metabolism studies. Groups A through F shall be used to
determine the metabolism of the test substance. Excreta (urine, feces,
and expired air) shall be collected for identification and measurement
of the quantities of test substance and metabolites.
(4) Measurements--(i) Pharmacokinetics. At least four animals from
each group shall be used for these purposes.
(A) Bioavailability. The levels of test substance and relevant
metabolites, as appropriate, shall be determined in whole blood, blood
plasma or blood serum at appropriate intervals after initiation of
intravenous, dermal, and inhalation exposure. The sampling intervals
should be compatible with the exposure route under study. The
determinations need only be done on animals administered the test
substance containing 14C-n-hexane or, if the analytical
sensitivity is equal or greater, unlabeled test substance may be used.
(B) Extent of absorption. The total quantities of radioactivity
shall be determined for excreta collected daily for 7 days, or until at
least 90 percent of theradioactivity has been recovered in the excreta,
whichever occurs first.
(C) Excretion. The quantities of radioactivity eliminated in the
urine, feces, and expired air shall be determined separately at time
intervals that provide accurate measurement of clearance and excretory
rates. The collection of carbon dioxide may be discontinued when less
than one percent of the dose is found to be exhaled as radioactive
carbon dioxide in 24 hours.
[[Page 74]]
(D) Tissue distribution. At the termination of each study, the
quantities of radioactivity shall be determined in blood and in various
tissues, including bone, brain, fat, gastrointestinal tract, gonads,
heart, kidney, liver, lungs, muscle, skin, spleen, thymus, and residual
carcass of each animal.
(E) Change in pharmacokinetics. Results of pharmacokinetics
measurements (i.e., biotransformation, extent of absorption, tissue
distribution, and excretion) obtained in rats receiving the single
inhalation exposure to the low dose of the test substance (Group B)
shall be compared to the corresponding results obtained in rats
receiving repeated inhalation exposures to the low dose of the test
substance (Group F).
(ii) Metabolism. At least four animals from each group shall be used
for these purposes.
(A) Biotransformation. Appropriate qualitative and quantitative
methods shall be used to assay urine, feces, and expired air collected
from rats. Efforts shall be made to identify any metabolite which
comprises 5 percent or more of the dose administered.
(B) Changes in biotransformation. Appropriate qualitative and
quantitative assay methods shall be used to compare the composition of
radioactive compounds in excreta from rats receiving a single inhalation
exposure (Groups B and C) with that from rats receiving repeated
inhalation exposures (Group F).
(d) Data and reporting. The final test report shall include the
following:
(1) Presentation of results. Numerical data shall be summarized in
tabular form. Pharmacokinetics data shall also be presented in graphical
form. Qualitative observations shall also be reported.
(2) Evaluation of results. All data shall be evaluated by
appropriate statistical methods.
(3) Reporting results. In addition to the reporting requirements as
specified in 40 CFR part 792, the following information shall be
reported.
(i) Strain of laboratory animals.
(ii) Chemical characterization of the test substances, including:
(A) For the radiolabeled test substances, information on the sites
and degree of radiolabeling, including type of label, specific activity,
chemical purity prior to mixing with the unlabeled hexane mixture, and
radiochemical purity.
(B) For the unlabeled test substance, information on lot number and
the percentage of MCP and n-hexane.
(C) Results of chromatography.
(iii) A full description of the sensitivity, precision, and accuracy
of all procedures used to obtain the data.
(iv) Percent and rate of absorption of the test substance after
inhalation and dermal exposures.
(v) Quantity and percent recovery of radioactivity in feces, urine,
expired air, and blood. For dermal studies, include recovery data for
skin and residual radioactivity in the covering apparatus.
(vi) Tissue distribution reported as quantity of radioactivity in
blood, in various tissues including bone, brain, fat, gastrointestinal
tract, gonads, heart, kidney, liver, lung, muscle, skin, spleen, thymus,
and in residual carcass.
(vii) Biotransformation pathways, to the extent possible, and
quantities of the test substances and metabolites in excreta collected
after administering single high and low doses.
(viii) Biotransformation pathways, to the extent possible, and
quantities of test substances and metabolites in excreta collected after
administering repeated low doses.
(ix) Pharmacokinetics models to the extent they can be developed
from the experimental data.
[55 FR 632, Jan. 8, 1990, as amended at 58 FR 34205, June 23, 1993; 60
FR 34466, July 3, 1995]
Sec. 795.250 Developmental neurotoxicity screen.
(a) Purpose. In the assessment and evaluation of the toxic
characteristics of a chemical, it is important to determine when
acceptable exposures in the adult may not be acceptable to a developing
organism. This test is designed to provide information on the potential
functional and morphologic hazards to the nervous system which may arise
in the offspring from exposure of the mother during pregnancy and
lactation.
[[Page 75]]
(b) Principle of the test method. The test substance is administered
to several groups of pregnant animals during gestation and lactation,
one dose level being used per group. Offspring are randomly selected
from within litters for neurotoxicity evaluation. The evaluation
includes observation to detect gross neurological and behavioral
abnormalities, determination of motor activity, neuropathological
evaluation, and brain weights. Measurements are carried out periodically
during both postnatal development and adulthood.
(c) Test procedures--(1) Animal selection--(i) Species and strain.
Testing should be performed in the Sprague Dawley rat.
(ii) Age. Young adult animals (nulliparous females) shall be used.
(iii) Sex. Pregnant females shall be used at each dose level.
(iv) Number of animals. The objective is for a sufficient number of
pregnant rats to be exposed to ensure that an adequate number of
offspring are produced for neurotoxicity evaluation. At least 20 litters
are recommended at each dose level. This number assumes a coefficient of
variation of 20 to 25 percent for most behavioral tests. If, based upon
experience with historical control data or data for positive controls in
a given laboratory, the coefficient of variation for a given task is
higher than 20 to 25 percent, then calculation of appropriate sample
sizes to detect a 20 percent change from control values with 80 percent
power would need to be done. For most designs, calculations can be made
according to Dixon and Massey (1957) under paragraph (e)(5) of this
section, Neter and Wasserman (1974) under paragraph (e)(10) of this
section, Sokal and Rohlf (1969) under paragraph (e)(11) of this section,
or Jensen (1972) under paragraph (e)(8) of this section.
(A) On day 4 after birth, the size of each litter should be adjusted
by eliminating extra pups by random selection to yield, as nearly as
possible, 4 males and 4 females per litter. Whenever the number of male
or female pups prevents having 4 of each sex per litter, partial
adjustment (for example, 5 males and 3 females) is permitted.
Adjustments are not appropriate for litters of less than 8 pups.
Elimination of runts only is not appropriate. Individual pups should be
identified uniquely after standardization of litters. A method that may
be used can be found in Adams et al. (1985) under paragraph (e)(1) of
this section.
(B) After standardization of litters, males and females shall be
randomly assigned to one of each of three behavioral tasks.
Alternatively, more than one of the behavioral tasks may be conducted in
the same animal. In the latter case, a minimum of 1 to 2 days should
separate the tests when conducted at about the same age.
(C) One male and one female shall be randomly selected from each
litter for sacrifice at weaning as specified in paragraph (c)(8) of this
section.
(2) Control group. A concurrent control group shall be used. This
group shall be a sham treated group, or, if a vehicle is used in
administering the test substance, a vehicle control group. Animals in
the control groups shall be handled in an identical manner to test group
animals. The vehicle shall neither be developmentally toxic nor have
effects on reproduction.
(3) Dose levels and dose selection. (i) At least 3 dose levels plus
a control (vehicle control, if a vehicle is used) shall be used.
(ii) If the substance has been shown to be developmentally toxic
either in a standard developmental toxicity study or a pilot study, the
highest dose level shall be the maximum dose which will not induce in
utero or neonatal deaths or malformations sufficient to preclude a
meaningful evaluation of neurotoxicity.
(iii) In the absence of standard developmental toxicity, unless
limited by the physicochemical nature or biologicial properties of the
substance, the highest dose level shall induce some overt maternal
toxicity but shall not result in a reduction in weight gain exceeding 20
percent during gestation and lactation.
(iv) The lowest dose should not produce any grossly observable
evidence of either maternal or developmental neurotoxicity.
(v) The intermediate dose(s) shall be equally spaced between the
highest and lowest dose.
[[Page 76]]
(4) Dosing period. Day 0 in the test is the day on which a vaginal
plug and/or sperm are observed. The dose period shall cover the period
from day 6 of gestation through weaning (21 days postnatally).
(5) Administration of test substance. The test substance or vehicle
should be administered orally by intubation. The test substance shall be
administered at the same time each day. The animals shall be weighed
periodically and the dosage based on the most recent weight
determination.
(6) Observation of dams. (i) A gross examination of the dams shall
be made at least once each day, before daily treatment. The animals
shall be observed by trained technicians who are blind with respect to
the animal's treatment, using standardized procedures to maximize inter-
observer reliability. Where possible, it is advisable that the same
observer be used to evaluate the animals in a given study. If this is
not possible, some demonstration of inter-observer reliability is
required.
(ii) During the treatment and observation periods, cage-side
observations shall include:
(A) Any responses with respect to body position, activity level,
coordination of movement, and gait.
(B) Any unusual or bizarre behavior including, but not limited to
headflicking, head searching, compulsive biting or licking, self-
mutilation, circling, and walking backwards.
(C) The presence of:
(1) Convulsions.
(2) Tremors.
(3) Increased levels of lacrimation and/or red-colored tears.
(4) Increased levels of salivation.
(5) Piloerection.
(6) Pupillary dilation or constriction.
(7) Unusual respiration (shallow, labored, dyspneic, gasping, and
retching) and/or mouth breathing.
(8) Diarrhea.
(9) Excessive or diminished urination.
(10) Vocalization.
(iii) Signs of toxicity shall be recorded as they are observed,
including the time of onset, the degree and duration.
(iv) Animals shall be weighed at least weekly.
(v) The day of delivery of litters shall be recorded.
(7) Study conduct--(i) Observation of offspring. (A) All offspring
shall be examined cage-side daily for gross signs of mortality and
morbidity.
(B) All offspring shall be examined outside the cage for gross signs
of toxicity whenever they are weighed or removed from their cages for
behavioral testing. The offspring shall be observed by trained
technicians, who are blind with respect to the animal's treatment using
standardized procedures to maximize inter-observer reliability. Where
possible, it is advisable that the same observer be used to evaluate the
animals in a given study. If this is not possible, some demonstration of
inter-observer reliability is required. At a minimum, the end points
outlined in paragraph (c)(6)(ii) of this section shall be monitored as
appropriate for the developmental stage being observed.
(C) Any gross signs of toxicity in the offspring shall be recorded
as they are observed, including the time of onset, the degree, and
duration.
(ii) Developmental landmarks. Live pups should be counted and
litters weighed by weighing each individual pup at birth, or soon
thereafter, and on days 4, 7, 13, 17, and 21, and biweekly thereafter.
The age of the pups at the time of the appearance of the following
developmental landmarks shall be determined:
(A) Vaginal opening. General procedure for this determination may be
found in Adams et al. (1985) under paragraph (e)(1) of this section.
(B) Testes descent. General procedure for this determination may be
found in Adams et al. (1985) under paragraph (e)(1) of this section.
(iii) Motor activity. (A) Motor activity shall be monitored
specifically on days 13, 17, 21, 45 (2 days), and 60
(2 days). Motor activity shall be monitored by an automated
activity recording apparatus. The device used shall be capable of
detecting both increases and decreases in activity, i.e., baseline
activity as measured by the device shall not be so low as to preclude
decreases nor so high as to preclude increases. Each
[[Page 77]]
device shall be tested by standard procedures to ensure, to the extent
possible, reliability of operation across devices and testing of animals
within dose groups shall be balanced across devices.
(B) Each animal shall be tested individually. The test session shall
be long enough to demonstrate habituation of motor activity in control
animals, i.e., to approach asymptotic levels by the last 20 percent of
the session. Animals' activity counts shall be collected in equal time
periods of no greater than 10 minutes duration. All sessions shall have
the same duration. Treatment groups shall be counter-balanced across
test times.
(C) Efforts shall be made to ensure that variations in the test
conditions are minimal and are not systematically related to treatment.
Among the variables which can affect motor activity are sound level,
size, and shape of the test cage, temperature, relative humidity,
lighting conditions, odors, use of home cage or novel test cage, and
environmental distractions.
(D) Additional information on the conduct of a motor activity study
may be obtained in the TSCA motor activity guideline, in Sec. 798.6200
of this chapter.
(iv) Auditory startle test. An auditory startle habituation test
shall be performed on the offspring on days 22 and 60. Details on the
conduct of this testing may be obtained in Adams et al. (1985) under
paragraph (e)(1) of this section. In performing the auditory startle
task, the mean response amplitude on each block of 10 trials (5 blocks
of 10 trials per session on each day of testing) shall be made. While
use of pre-pulse inhibition is not a requirement, it may be used at the
discretion of the investigator. Details on the conduct of this testing
may be obtained from Ison (1984) under paragraph (e)(7) of this section.
(v) Active avoidance test. Active avoidance testing shall be
conducted beginning at 60 to 61 days of age. Details on the apparatus
may be obtained in Brush and Knaff (1959) and on the conduct of testing
from Brush (1962), under paragraphs (e)(2) and (e)(4) of this section,
respectively; reviews on active avoidance conditioning by Brush (1971)
and McAllister and McAllister (1971) can be found under paragraphs
(e)(3) and (e)(9) of this section, respectively. In performing the
active avoidance task, the following measures should be made:
(A) Mean number of shuttles during the adaptation period preceding
each daily session.
(B) Mean number and latency of avoidances per session, presented in
blocks of 10 trials (2 blocks of 10 trials per session across 5
sessions).
(C) Mean number and latency of escapes per session, presented in
blocks of 10 trials as above.
(D) Mean duration of shocks per session, presented in blocks of 10
trials as above.
(E) Mean number of shuttles during the inter-trial intervals.
(8) Post-mortem evaluation--(i) Age of animals. One male and one
female per litter shall be sacrificed at weaning and the remainder
following the last behavioral measures. Neuropathology and brain weight
determinations shall be made on animals sacrificed at weaning and after
the last behavioral measures.
(ii) Neuropathology. Details for the conduct of neuropathology
evaluation may be obtained in the TSCA neuropathology guideline, in
Sec. 798.6400 of this chapter. At least 6 offspring per dose group shall
be randomly selected from each sacrificed group (weaning and adulthood)
for neuropathologic evaluation. These animals shall be balanced across
litters, and equal numbers of males and females shall be used. The
remaining sacrificed animals shall be used to determine brain weight.
Animals shall be perfused in situ by a generally recognized technique.
After perfusion, the brain and spinal cord shall be removed and gross
abnormalities noted. Cross-sections of the following areas shall be
examined: The forebrain, the center of the cerebrum and midbrain, the
cerebellum and pons, and the medulla oblongata; the spinal cord at
cervical and lumbar swelling; Gasserian ganglia, dorsal root ganglia,
dorsal and ventral root fibers, proximal sciatic nerve (mid-thigh and
sciatic notch), sural nerve (at knee), and tibial nerve (at knee).
Tissue samples from
[[Page 78]]
both the central and peripheral nervous system shall be further
immersion-fixed and stored in appropriate fixative for further
examination. After dehydration, tissue specimens shall be cleared with
xylene and embedded in paraffin or paraplast except for the sural nerve
which should be embedded in plastic. A method for plastic embedding is
described by Spencer et al. under paragraph (e)(12) of this section.
Tissue sections shall be prepared from the tissue blocks. The following
general testing sequence is recommended for gathering histopathological
data:
(A) General staining. A general staining procedure shall be
performed on all tissue specimens in the highest treatment group.
Hematoxylin and eosin (H&E) shall be used for this purpose. The staining
shall be differentiated properly to achieve bluish nuclei with pinkish
background.
(B) Special stains. Based on the results of the general staining,
selected sites and cellular components shall be further evaluated by use
of specific techniques. If H&E screening does not provide such
information, a battery of stains shall be used to assess the following
components in all appropriate required samples: Neuronal body (e.g.,
Einarson's gallocyanin), axon (e.g., Kluver's Luxol Fast Blue), and
neurofibrils (e.g., Bielchosky). In addition, nerve fiber teasing shall
be used. A section of normal tissue shall be included in each staining
to assure that adequate staining has occurred. Any changes shall be
noted and representative photographs shall be taken. If lesions are
observed, the special techniques shall be repeated in the next lower
treatment group until no further lesions are detectable.
(C) Alternative technique. If the anatomical locus of expected
neuropathology is well-defined, epoxy-embedded sections stained with
toluidine blue may be used for small sized tissue samples. This
technique obviates the need for special stains.
(iii) Brain weight. At least 10 animals that are not sacrificed for
histopathology shall be used to determine brain weight. The animals
shall be decapitated and the brains carefully removed, blotted, chilled,
and weighed. The following dissection shall be performed on an ice-
cooled glass plate: First, the rhombencephalon is separated by a
transverse section from the rest of the brain and dissected into the
cerebellum and the medulla oblongata/pons. A transverse section is made
at the level of the ``optic chiasma'' which delimits the anterior part
of the hypothalamus and passes through the anterior commissure. The
cortex is peeled from the posterior section and added to the anterior
section. This divides the brain into four sections, the telencephalon,
the diencephalon/mid-brain, the medulla oblongata/pons, and the
cerebellum. Sections shall be weighed as soon as possible after
dissection to avoid drying. Detailed methodology is available in
Glowinski and Iversen (1966) under paragraph (e)(6) of this section.
(d) Data reporting and evaluation. In addition to the reporting
requirements specified in part 792, subpart J of this chapter, the final
test report shall include the following information.
(1) Description of system and test methods. (i) A detailed
description of the procedures used to standardize observation and
operational definitions for scoring observations.
(ii) Positive control data from the laboratory performing the test
that demonstrate the sensitivity of the procedures being used. These
data do not have to be from studies using prenatal exposures. However,
the laboratory must demonstrate competence in testing neonatal animals
perinatally exposed to chemicals and establish test norms for the
appropriate age group.
(iii) Procedures for calibrating and assuring the equivalence of
devices and balancing treatment groups.
(iv) A short justification explaining any decisions where
professional judgement is involved such as fixation technique and choice
of stains.
(2) Results. The following information shall be arranged by test
group dose level.
(i) In tabular form, data for each animal shall be provided showing:
(A) Its identification number and litter from which it came.
(B) Its body weight and score on each developmental landmark at each
observation time; total session activity counts and intrasession
subtotals on
[[Page 79]]
each day measured; auditory startle response magnitude session counts
and intrasession subtotals on each day measured; avoidance session
counts and intrasession counts on each day measured; time and cause of
death (if appropriate); locations, nature or frequency, and severity of
the lesions; total brain weight; absolute weight of each of the four
sections; and weight of each section as a percentage of total brain
weight. A commonly used scale such as 1+, 2+, 3+, and 4+ for degree of
severity of lesions ranging from very slight to extensive may be used
for morphologic evaluation. Any diagnoses derived from neurologic signs
and lesions, including naturally occurring diseases or conditions, shall
also be recorded.
(ii) Summary data for each group shall include:
(A) The number of animals at the start of the test.
(B) Body weights of the dams during gestation and lactation.
(C) Litter size and mean weight at birth.
(D) The number of animals showing each observation score at each
observation time.
(E) The percentage of animals showing each abnormal sign at each
observation time.
(F) The mean and standard deviation for each continuous end point at
each observation time. These will include body weight, motor activity
counts, acoustic startle responses, performance in active avoidance
tests, and brain weights (both absolute and relative).
(G) The number of animals in which any lesion was found.
(H) The number of animals affected by each different type of lesion,
the average grade of each type of lesion, and the frequency of each
different type and/or location of lesions.
(3) Evaluation of data. An evaluation of the test results shall be
made. The evaluation shall include the relationship between the doses of
the test substance and the presence or absence, incidence, and severity
of any neurotoxic effect. The evaluation shall include appropriate
statistical analyses. The choice of analyses shall consider tests
appropriate to the experimental design and needed adjustments for
multiple comparisons.
(e) References. For additional background information on this test
guideline, the following references should be consulted:
(1) Adams, J., Buelke-Sam, J., Kimmel, C.A., Nelson, C.J., Reiter,
L.W., Sobotka, T.J., Tilson, H.A., and Nelson, B.K. ``Collaborative
behavioral teratology study: Protocol design and testing procedure.''
Neurobehavioral Toxicology and Teratology. 7: 579-586. (1985).
(2) Brush, F.R. ``The effects of inter-trial interval on avoidance
learning in the rat.'' Journal of Comparative Physiology and Psychology.
55: 888-892. (1962).
(3) Brush, F.R. ``Retention of aversively motivated behavior.'' In:
``Adverse Conditioning and Learning.'' Brush, F.R., ed., New York:
Academic Press. (1971).
(4) Brush, F.R. and Knaff, P.R. ``A device for detecting and
controlling automatic programming of avoidance-conditioning in a
shuttle-box.'' American Journal of Psychology. 72: 275-278 (1959).
(5) Dixon, W.J. and Massey, E.J. ``Introduction to Statistical
Analysis.'' 2nd ed. New York: McGraw-Hill. (1957).
(6) Glowinski, J. and Iversen, L.L. ``Regional studies of
catecholamines in the rat brain-I.'' Journal of Neurochemistry. 13: 655-
669. (1966).
(7) Ison, J.R. ``Reflex modification as an objective test for
sensory processing following toxicant exposure.'' Neurobehavioral
Toxicology and Teratology. 6: 437-445. (1984).
(8) Jensen, D.R. ``Some simultaneous multivariate procedures using
Hotelling's T2 Statistics.'' Biometrics. 28: 39-53. (1972).
(9) McAllister, W.R. and McAllister, D.E. ``Behavioral measurement
of conditioned fear.'' In: ``Adverse Conditioning and Learning.'' Brush,
F.R., ed., New York: Academic Press (1971).
(10) Neter, J. and Wasserman, W. ``Applied Linear Statistical
Models.'' Homewood: Richard D. Irwin, Inc. (1974).
(11) Sokal, R.P. and Rohlf, E.J. ``Biometry.'' San Francisco: W.H.
Freeman and Co. (1969).
(12) Spencer, P.S., Bischoff, M.C., and Schaumburg, H.H.,
``Neuropathological methods for the detection of neurotoxic disease.''
In: ``Experimental and
[[Page 80]]
Clinical Neurotoxicology.'' Spencer, P.S. and Schaumburg, H.H., eds.,
Baltimore, MD: Williams & Wilkins, pp. 743-757. (1980).
[53 FR 5957, Feb. 26, 1988]
PART 796--CHEMICAL FATE TESTING GUIDELINES--Table of Contents
Subpart A [Reserved]
Subpart B--Physical and Chemical Properties
Sec.
796.1050 Absorption in aqueous solution: Ultraviolet/visible spectra.
796.1950 Vapor pressure.
Subpart C--Transport Processes
796.2750 Sediment and soil adsorption isotherm.
Subpart D--Transformation Processes
796.3100 Aerobic aquatic biodegradation.
796.3500 Hydrolysis as a function of pH at 25 deg.C.
Authority: 15 U.S.C. 2603.
Subpart A [Reserved]
Subpart B--Physical and Chemical Properties
Sec. 796.1050 Absorption in aqueous solution: Ultraviolet/visible spectra.
(a) Introductory information--(1) Guidance information. (i)
Molecular formula.
(ii) Structural formula.
(2) Standard documents. The spectrophotometric method is based on
national standards and consensus methods which are applied to measure
the absorption spectra.
(b) Method--(1)(i) Introduction, purpose, scope, relevance,
application and limits of test. (A) The primary environmental purpose in
determining the ultraviolet-visible (UV-VIS) absorption spectrum of a
chemical compound is to have some indication of the wavelengths at which
the compounds may be susceptible to photochemical degradation. Since
photochemical degradation is likely to occur in both the atmosphere and
the aquatic environment, spectra appropriate to these media will be
informative concerning the need for further persistence testing.
(B) Degradation will depend upon the total energy absorbed in
specific wavelength regions. Such energy absorption is characterized by
both molar absorption coefficient (molar extinction coefficient) and
band width. However, the absence of measurable absorption does not
preclude the possibility of photodegradation.
(ii) Definitions and units. The UV-VIS absorption spectrum of a
solution is a function of the concentration, c1, expressed in
mol/L, of all absorbing species present; the path length, d, of the
spectrophotometer cell, expressed in cm; and the molar absorption
(extinction) coefficient, i, of each species. The
absorbance (optical density) A of the solution is then given by:
[GRAPHIC] [TIFF OMITTED] TC15NO91.045
For a resolvable absorbance peak, the band width is the
wavelength range, expressed in nm=10-9 m, of the peak at half
the absorbance maximum.
(iii) Reference substances. (A) The reference substances need not be
employed in all cases when investigating a new substance. They are
provided primarily so that calibration of the method may be performed
from time to time and to offer the chance to compare the results when
another method is applied.
(B) Reference compounds appropriate for the calibration of the
system are:
(1) Potassium dichromate (in 0.005 mol/L,
H2SO4 solution) from J.A.A. Ketelaar, paragraph
(d)(2) of this section:
log ............................... 3.56 3.63 3.16 3.50
in nm............................. 235 257 313 350
(2) Fluoranthene (in methanol) from C.R.C. Atlas of Spectral Data,
paragraph (d)(3) of this section:
log ........................ 4.75 4.18 4.73 3.91 3.92
in nm...................... 237 236 288 339 357
(3) 4-nitrophenol (in methanol) from C.R.C. Atlas of Spectral Data,
paragraph (d)(3) of this section:
log ........................................... 3.88 4.04
in nm......................................... 288 311
[[Page 81]]
See also paragraph (d)(1) of this section.
(iv) Principle of the test method. This method utilizes a double-
beam spectrophotometer which records only the absorption differences
between the blank and test solutions to give the spectrum of the
chemical being tested.
(v) Quality criteria--Reproducibility and sensitivity. (A)
Reproducibility and sensitivity, need not be measured directly. Instead,
the accuracy of the system in measuring the spectra of reference
compounds will be defined so as to assure appropriate reproducibility
and sensitivity. It is preferable to use a recording double-beam
spectrophotometer to obtain the UV-VIS spectrum of the test compound.
Such an instrument should have a photometric accuracy of
0.02 units over the absorbance range of 0 to 2 units. It
should be capable of recording absorbances at wavelengths of 200 to 750
nanometers nm with a wavelength accuracy of 0.5 nm. The
cells employed with the instrument must necessarily be transparent over
this wavelength range and must have a path length determined to within 1
percent. To ensure that the instrument is performing satisfactorily,
spectra for test solutions of K2Cr2O7
(for absorbance accuracy) and holmium glass (for wavelength accuracy)
should be run periodically.
(B) In the event that a recording double-beam instrument is not
available, it will be necessary to determine the absorbance of the test
solution in a single-beam instrument at 5-nm intervals over the entire
wavelength range and at 1-nm intervals where there are indicated
absorbance maxima. Wavelength and absorbance tests should be done as
with the double-beam instrument.
(2) Description of the test procedure--(i) Preparation--(A)
Preparation of test solutions. (1) Solutions should be prepared by
accurately weighing an appropriate amount of the purest form of the test
substance available. This should be made up in a concentration which
will result in at least one absorbance maximum in the range 0.5 to 1.5
units.
(2) The absorption of a compound is due to its particular chemical
form. It is often the case that different forms are present, depending
on whether the medium is acidic, basic, or neutral. Consequently,
spectra under all three conditions are required where solubility and
concentration allow. Where it is not possible to obtain sufficient
concentrations in any of the aqueous media, a suitable organic solvent
should be used (methanol preferred).
(3) The acid medium should have a pH of less than 2, and the basic
medium should be at least pH 10. The solvent for the neutral solution,
and for preparing the acidic and basic ones, should be distilled water,
transparent to ultraviolet radiation down to 200 nm. If methanol must be
used, acidic and basic solutions can be prepared by adding 10 percent by
volume of HCl or NaOH in aqueous solution ([HCl], [NaOH]=1 mol/L).
(4) In theory, all chemical species other than that being tested are
present in both beams and would therefore not appear in the recorded
spectrum of a double-beam instrument. In practice, because the solvent
is usually present in great excess, there is a threshold value of
wavelength below which it is not possible to record the spectrum of the
test chemical. Such a wavelength will be a property of the solvent or of
the test medium. In general, distilled water is useful from 200 nm
(dissolved ions will often increase this), methanol from 210 nm, hexane
from 210 nm, acetonitrile from 215 nm and dichloromethane from 235 nm.
(B) Blank solutions. A blank must be prepared which contains the
solvent and all chemical species other than the test chemical. The
absorption spectrum of this solution should be recorded in a manner
identical to that of the test solution and preferably on the same chart.
This ``baseline'' spectrum should never record an absorbance reading
varying more than 0.05 from the nominal zero value.
(C) Cells. Cell pathlengths are usually between 0.1 cm and 10 cm.
Cell lengths should be selected to permit recording of at least one
maximum in the absorbance range of 0.5 to 1.5 units. Which set of cells
should be used will be governed by the concentration and the absorbance
of the test solution as indicated by the Beer-Lambert Law. The cells
should be transparent over the range of the spectrum being recorded, and
the
[[Page 82]]
path-lengths should be known to an accuracy of at least 1 per cent.
Cells should be thoroughly cleaned in an appropriate manner (chromic
acid is useful for quartz cells) and rinsed several times with the test
or blank solutions.
(ii) Performance of the test. Both cells to be employed should be
rinsed with the blank solution and then filled with same. The instrument
should be set to scan at a rate appropriate for the required wavelength
resolution and the spectrum of the blank recorded. The sample cell
should then be rinsed and filled with the test solution and the scanning
repeated, preferably on the same spectrum chart, to display the
baseline. The test should be carried out at 25 deg.C.
(c) Data and reporting--(1) Treatment of results. (i) The molar
absorption coefficient should be calculated for all absorbance
maxima of the test substance. The formula for this calculation is:
[GRAPHIC] [TIFF OMITTED] TC15NO91.046
where the quantities are as defined above (see Definitions and units).
(ii) For each peak which is capable of being resolved, either as
recorded or by extrapolated symmetrical peaks, the bandwidth should be
recorded.
(2) Test report. (i) The report should contain a copy of each of the
three spectra (3 pH conditions). If neither water nor methanol solutions
are feasible, there will be only one spectrum. Spectra should include a
readable wave-length scale. Each spectrum should be clearly marked with
the test conditions.
(ii) For each maximum in each spectrum, the value and
bandwidth (when applicable) should be calculated and reported, along
with the wavelength of the maximum. This should be presented in tabular
form.
(iii) The various test conditions should be included, such as scan
speed, the name and model of the spectrophotom-eter, the slit width
(where available), cell type and path length, the concentrations of the
test substance, and the nature and acidity of the solvent medium. A
recent test spectrum on appropriate reference materials for photometric
and wavelength accuracy should also be submitted (see Reproducibility
and sensitivity).
(d) Literature references. For additional background information on
this test guideline, the following references should be consulted:
(1) Milazzo, G., Caroli, S., Palumbo-Doretti, M., Violante, N.,
Analytical Chemistry, 49: 711 (1977).
(2) Katelaar, J.A.A., Photoelectric Spectrometry Group Bulletin, 8,
(Cambridge, 1955).
(3) Chemical Rubber Company, Atlas of Spectral Data, (Cliffland,
Ohio).
[50 FR 39472, Sept. 27, 1985]
Sec. 796.1950 Vapor pressure.
(a) Introduction--(1) Background and purpose. (i) Volatilization,
the evaporative loss of a chemical, depends upon the vapor pressure of
chemical and on environmental conditions which influence diffusion from
a surface. Volatilization is an important source of material for
airborne transport and may lead to the distribution of a chemical over
wide areas and into bodies of water far from the site of release. Vapor
pressure values provide indications of the tendency of pure substances
to vaporize in an unperturbed situation, and thus provide a method for
ranking the relative volatilities of chemicals. Vapor pressure data
combined with water solubility data permit the calculation of Henry's
law constant, a parameter essential to the calculation of volatility
from water.
(ii) Chemicals with relatively low vapor pressures, high
adsorptivity onto solids, or high solubility in water are less likely to
vaporize and become airborne than chemicals with high vapor pressures or
with low water solubility or low adsorptivity to solids and sediments.
In addition, chemicals that are likely to be gases at ambient
temperatures and which have low water solubility and low adsorptive
tendencies are less likely to transport and persist in soils and water.
Such chemicals are less likely to biodegrade or hydrolyze and are prime
candidates for atmospheric oxidation and photolysis (e.g., smog
formation or stratospheric alterations). On the other hand, nonvolatile
chemicals are less frequently involved
[[Page 83]]
in atmosphere transport, so that concerns regarding them should focus on
soils and water.
(iii) Vapor pressure data are an important consideration in the
design of other chemical fate and effects tests; for example, in
preventing or accounting for the loss of violatile chemicals during the
course of the test.
(2) Definitions and units. (i) ``Desorption efficiency'' of a
particular compound applied to a sorbent and subsequently extracted with
a solvent is the weight of the compound which can be recovered from the
sorbent divided by the weight of the compound originally sorbed.
(ii) ``Pascal'' (Pa) is the standard international unit of vapor
pressure and is defined as newtons per square meter (N/m2). A
newton is the force necessary to give acceleration of one meter per
second squared to one kilogram of mass.
(iii) The ``torr'' is a unit of pressure which equals 133.3 pascals
or 1 mm Hg at 0 deg.C.
(iv) ``Vapor pressure'' is the pressure at which a liquid or solid
is in equilibrium with its vapor at a given temperature.
(v) ``Volatilization'' is the loss of a substance to the air from a
surface or from solution by evaporation.
(3) Principle of the test methods. (i) The isoteniscope procedure
uses a standardized technique [ASTM 1978] that was developed to measure
the vapor pressure of certain liquid hydrocarbons. The sample is
purified within the equipment by removing dissolved and entrained gases
until the measured vapor pressure is constant, a process called
``degassing.'' Impurities more volatile than the sample will tend to
increase the observed vapor pressure and thus must be minimized or
removed. Results are subject to only slight error for samples containing
nonvolatile impurities.
(ii) Gas saturation (or transpiration) procedures use a current of
inert gas passed through or over the test material slowly enough to
ensure saturation and subsequent analysis of either the loss of material
or the amount (and sometimes kind) of vapor generated. Gas saturation
procedures have been described by Spencer and Cliath (1969) under
paragraph (d)(2) of this section. Results are easy to obtain and can be
quite precise. The same procedures also can be used to study
volatilization from laboratory scale environmental simulations. Vapor
pressure is computed on the assumption that the total pressure of a
mixture of gases is equal to the sum of the pressures of the separate or
component gases and that the ideal gas law is obeyed. The partial
pressure of the vapor under study can be calculated from the total gas
volume and the weight of the material vaporized. If v is the volume
which contains w grams of the vaporized material having a molecular
weight M, and if p is the pressure of the vapor in equilibrium at
temperature T (K), then the vapor pressure, p, of the sample is
calculated by
p=(w/M)(RT/v),
where R is the gas constant (8.31 Pa m2 mol-1
K-1) when the pressure is in pascals (Pa) and the volume is
in cubic meters. As noted by Spencer and Cliath (1970) under paragraph
(d)(3) of this section, direct vapor pressure measurements by gas
saturation techniques are more directly related to the volatilization of
chemicals than are other techniques.
(iii) In an effort to improve upon the procedure described by
Spencer and Cliath (1969) under paragraph (d)(2) of this section, and to
determine the applicability of the gas saturation method to a wide
variety of chemical types and structures, EPA has sponsored research and
development work at SRI International (EPA 1982) under paragraph (d)(1)
of this section. The procedures described in this Test Guideline are
those developed under that contract and have been evaluated with a wide
variety of chemicals of differing structure and vapor pressures.
(4) Applicability and specificity. (i) A procedure for measuring the
vapor pressure of materials released to the environment ideally would
cover a wide range of vapor pressure values, at ambient temperatures. No
single procedure can cover this range, so two different procedures are
described in this section, each suited for a different part
[[Page 84]]
of the range. The isoteniscope procedure is for pure liquids with vapor
pressures from 0.1 to 100 kPa. For vapor pressures of 10-5 to
10 3 Pa, a gas saturation procedure is to be used.
(ii) With respect to the isoteniscope method, if compounds that boil
close to or form azeotropes with the test material are present, it is
necessary to remove the interfering compounds and use pure test
material. Impurities more volatile than the sample will tend to increase
the observed vapor pressure above its true value but the purification
steps will tend to remove these impurities. Soluble, nonvolatile
impurities will decrease the apparent vapor pressure. However, because
the isoteniscope procedure is a static, fixed-volume method in which an
insignificant fraction of the liquid sample is vaporized, it is subject
to only slight error for samples containing nonvolatile impurities. That
is, the nonvolatile impurities will not be concentrated due to
vaporization of the sample.
(iii) The gas saturation method is applicable to solid or liquid
chemicals. Since the vapor pressure measurements are made at ambient
temperatures, the need to extrapolate data from high temperatures is not
necessary and high temperature extrapolation, which can often cause
serious errors, is avoided. The method is most reliable for vapor
pressures below 10 3 Pa. Above this limit, the vapor
pressures are generally overestimated, probably due to aerosol
formation. Finally, the gas saturation method is applicable to the
determination of the vapor pressure of impure materials.
(b) Test procedures--(1) Test conditions. (i) The apparatus in the
isoteniscope method is described in paragraph (b)(2)(i) of this section.
(ii) The apparatus used in the gas saturation method is described in
paragraph (b)(2)(ii) of this section.
(2) Performance of the tests--(i) Isoteniscope Procedure. The
isoteniscope procedure described as ANSI/ASTM Method D 2879-86 is
applicable for the measurement of vapor pressures of liquids with vapor
pressures of 0.1 to 100 kilopascals (kPa) (0.75 to 750 torr). ASTM D
2879-86 is available for inspection at the Office of the Federal
Register, 800 North Capitol Street, NW., suite 700, Washington, DC. This
incorporation by reference was approved by the Director of the Office of
the Federal Register. This material is incorporated as it exists on the
date of approval and a notice of any change in this material will be
published in the Federal Register. Copies of the incorporated material
may be obtained from the Non-Confidential Information Center (NCIC)
(7407), Office of Pollution Prevention and Toxics, U.S. Environmental
Protection Agency, Room B-607 NEM, 401 M St., SW., Washington, DC 20460,
between the hours of 12 p.m. and 4 p.m. weekdays excluding legal
holidays, or from the American Society for Testing and Materials (ASTM),
1916 Race Street, Philadelphia, PA 19103. The isoteniscope method
involves placing liquid sample in a thermostated bulb (the isoteniscope)
connected to a manometer and a vacuum pump. Dissolved and entrained
gases are removed from the sample in the isoteniscope by degassing the
sample at reduced presssure. The vapor pressure of the sample at
selected temperatures is determined by balancing the pressure due to the
vapor of the sample against a known pressure of an inert gas. The vapor
pressure of the test compound is determined in triplicate at
250.5 deg.C and at any other suitable temperatures
(0.5 deg.). It is important that additional vapor pressure
measurements be made at other temperatures, as necessary, to assure that
there is no need for further degassing, as described in the ASTM method.
(ii) Gas saturation procedure. (A) The test procedures require the
use of a constant-temperature box as depicted in the following Figure 1.
[[Page 85]]
[GRAPHIC] [TIFF OMITTED] TC01AP92.036
Figure 1--Schematic Diagram of Vapor Saturation Apparatus
The insulated box, containing sample holders, may be of any suitable
size and shape. The sketch in Figure 1 shows a box containing three
solid sample holders and three liquid sample holders, which allows for
the triplicate analysis of either a solid or liquid sample. The
temperature within the box is controlled to 0.5 deg. or
better. Nitrogen gas, split into six streams and controlled by fine
needle valves (approximately 0.79 mm orifice), flows into the box via
3.8 mm (0.125 in.) i.d. copper tubing. After temperature equilibration,
the gas flows through the sample and the sorbent trap and exits from the
box. The flow rate of the effluent carrier gas is measured at room
temperature with a bubble flow meter or other suitable device. The flow
rate is checked frequently during the experiment to assure that there is
an accurate value for the total volume of carrier gas. The flow rate is
used to calculate the total volume (at room temperature) of gas that has
passed through the sample and sorbent [(vol/time) x time = volume].
The vapor pressure of the test substance can be calculated from the
total gas volume and the mass of sample vaporized. If v is the volume of
gas that transported mass w of the vaporized test material having a
molecular weight M, and if p is the equilibrium vapor pressure of the
sample at temperature T, then p is calculated by the equation
p=(w/M)(RT/v).
In this equation, R is the gas constant (8.31 Pa m3mol-1
K-1). The pressure is expressed in pascals (Pa), the volume
in cubic meters (m3), mass in grams and T in kelvins (K).
T=273.15+t, if t is measured in degrees Celsius ( deg.C).
(B) Solid samples are loaded into 5 mm i.d. glass tubing between
glass wool plugs. The following Figure 2 depicts a drawing of a sample
holder and absorber system.
[[Page 86]]
[GRAPHIC] [TIFF OMITTED] TC01AP92.037
Figure 2--Solid Compound Sampling System
(C) Liquid samples are contained in a holder as shown in the
following Figure 3.
[GRAPHIC] [TIFF OMITTED] TC01AP92.038
Figure 3--Liquid Compound Sampling System
The most reproducible method for measuring the vapor pressure of liquids
is to coat the liquid on glass beads and to pack the holder in the
designated place with these beads.
(D) At very low vapor pressures and sorbent loadings, adsorption of
the chemical on the glass wool separating the sample and the sorbent and
on the glass surfaces may be a serious problem. Therefore, very low
loadings should be avoided whenever possible. Incoming nitrogen gas
(containing no interfering impurities) passes through a coarse frit and
bubbles through a 38 cm column of liquid sample. The stream passes
through a glass wool column to trap aerosols and then through a sorbent
tube, as described above. The pressure drop across the glass wool column
and the sorbent tube are negligible.
(E) With both solid and liquid samples, at the end of the sampling
time, the front and backup sorbent sections are analyzed separately. The
compound on each section is desorbed by adding the sorbent from that
section to 1.0 ml of desorption solvent in a small vial and allowing the
mixture to stand at a suitable temperature until no more test compound
desorbs. It is extremely important that the desorption solvent contain
no impurities which would interfere with the analytical method of
choice. The resulting solutions are analyzed quantitatively by a
suitable analytical method to determine the weight of sample desorbed
from each section. The choice of the analytical method, sorbent, and
desorption solvent is dictated by the nature of the test material.
Commonly used sorbents include charcoal, Tenax GC, and XAD-2. Describe
in detail the sorbent, desorption solvent, and analytical methods
employed.
(F) Measure the desorption efficiency for every combination of
sample, sorbent, and solvent used. The desorption efficiency is
determined by injecting a known mass of sample onto a sorbent and later
desorbing it and analyzing for the mass recovered. For each combination
of sample, sorbent, and solvent used, carry out the determination in
triplicate at each of three concentrations. Desorption efficiency may
vary with the concentration of the actual sample and it is important to
measure the efficiency at or near the concentration of sample under gas
saturation test procedure conditions.
[[Page 87]]
(G) To assure that the gas is indeed saturated with test compound
vapor, sample each compound at three differing gas flow rates.
Appropriate flow rates will depend on the test compound and test
temperature. If the calculated vapor pressure shows no dependence on
flow rate, then the gas is assumed to be saturated.
(c) Data and reporting. (1) Report the triplicate calculated vapor
pressures for the test material at each temperature, the average
calculated vapor pressure at each temperature, and the standard
deviation.
(2) Provide a description of analytical methods used to analyze for
the test material and all analytical results.
(3) For the isoteniscope procedure, include the plot of p vs. the
reciprocal of the temperature in K, developed during the degasing step
and showing linearity in the region of 298.15 K (25+C) and
any other required test temperatures.
(4) For the gas saturation procedure, include the data on the
calculation of vapor pressure at three or more gas flow rates at each
test temperature, showing no dependence on flow rate. Include a
description of sorbents and solvents employed and the desorption
efficiency calculations.
(5) Provide a description of any difficulties experienced or any
other pertinent information.
(d) References. For additional background information on this test
guideline the following references should be consulted:
(1) U.S. Environmental Protection Agency. Evaluation of Gas
Saturation Methods to Measure Vapor Pressures: Final Report, EPA
Contract No. 68-01-5117 with SRI International, Menlo Park, California
(1982).
(2) Spencer, W.F. and Cliath, M.M. ``Vapor Density of Dieldrin,''
Journal of Agricultural and Food Chemistry, 3:664-670 (1969).
(3) Spencer, W.F. and Cliath, M.M. ``Vapor Density and Apparent
Vapor Pressure of Lindane,'' Journal of Agricultural and Food Chemistry,
18:529-530 (1970).
[50 FR 39252, Sept. 27, 1985, as amended at 53 FR 12525, Apr. 15, 1988;
53 FR 21641, June 9, 1988; 60 FR 34466, July 3, 1995]
Subpart C--Transport Processes
Sec. 796.2750 Sediment and soil adsorption isotherm.
(a) Introduction--(1) Background and purpose. The adsorption of
chemicals to sediments and soils is an important process that affects a
chemical's distribution in the environment. If a chemical is adsorbed to
soil particles, it will remain on the soil surface and will not reach
ground water. If a chemical is not adsorbed, it will leach through the
soil profile and may reach ground waters and then surface waters.
Similarly, if a chemical adsorbed to sediment, it will accumulate in the
bed and suspended load of aquatic systems. If a chemical is not adsorbed
to sediment, it will accumulate in the water column of aquatic systems.
Information on the adsorption potential is needed under certain
circumstances to assess the transport of chemicals in the environment.
This section describes procedures that will enable sponsors to determine
the adsorption isotherm of a chemical on sediments and soils.
(2) Definitions and units. (i) The ``cation exchange capacity''
(CEC) is the sum total of exchangeable cations that a sediment or soil
can adsorb. The CEC is expressed in milliequivalents of negative charge
per 100 grams (meq/100g) or milliequivalents of negative charge per gram
(meq/g) of soil or sediment.
(ii) ``Clay mineral analysis'' is the estimation or determination of
the kinds of clay-size minerals and the amount present in a sediment or
soil.
(iii) ``Organic matter'' is the organic fraction of the sediment or
soil; it includes plant and animal residues at various stages of
decomposition, cells and tissues of soil organisms, and substances
synthesized by the microbial population.
(iv) ``Particle size analysis'' is the determination of the various
amounts of the different particle sizes in a sample (i.e., sand, silt,
clay), usually by sedimentation, sieving, micrometry, or combinations of
these methods. The names and diameter range commonly used in the United
States are:
------------------------------------------------------------------------
Name Diameter range
------------------------------------------------------------------------
Very coarse sand....................... 2.0 to 1.0 mm
[[Page 88]]
Coarse sand............................ 1.0 to 0.5 mm
Medium sand............................ 0.5 to 0.25 mm
Fine sand.............................. 0.25 to 0.125 mm
Very fine sand......................... 0.125 to 0.062 mm
Silt................................... 0.062 to 0.002 mm
Clay................................... <0.002 mm
------------------------------------------------------------------------
(v) The ``pH'' of a sediment or soil is the negative logarithm to
the base ten of the hydrogen ion activity of the sediment or soil
suspension. It is usually measured by a suitable sensing electrode
coupled with a suitable reference electrode at a 1/1 solid/solution
ratio by weight.
(vi) The adsorption ratio, ``Kd,'' is the amount of test
chemical adsorbed by a sediment or soil (i.e., the solid phase) divided
by the amount of test chemical in the solution phase, which is in
equilibrium with the solid phase, at a fixed solid/solution ratio.
(vii) ``Sediment'' is the unconsolidated inorganic and organic
material that is suspended in and being transported by surface water, or
has settled out and has deposited into beds.
(viii) ``Soil'' is the unconsolidated mineral material on the
immediate surface of the earth that serves as a natural medium for the
growth of land plants. Its formation and properties are determined by
various factors such as parent material, climate, macro- and
microorganisms, topography, and time.
(ix) ``Soil aggregate'' is the combination or arrangement of soil
separates (sand, silt, clay) into secondary units. These units may be
arranged in the soil profile in a distinctive characteristic pattern
that can be classified according to size, shape, and degree of
distinctness into classes, types, and grades.
(x) ``Soil classification'' is the systematic arrangement of soils
into groups or categories. Broad groupings are based on general soil
characteristics while subdivisions are based on more detailed
differences in specific properties. The soil classification system used
in this standard and the one used today in the United States is the 7th
Approximation-Comprehensive System. The ranking of subdivisions under
this system is: Order, Suborder, Great group, family, and series.
(xi) A ``soil horizon'' is a layer of soil approximately parallel to
the land surface. Adjacent layers differ in physical, chemical, and
biological properties such as color, structure, texture, consistency,
kinds and numbers of organisms present, and degree of acidity or
alkalinity.
(xii) ``Soil Order'' is the broadest category of soil classification
and is based on the general similarities of soil physical/chemical
properties. The formation of soil by similar general genetic processes
causes these similarities. The Soil Orders found in the United States
are: Alfisol, Aridisol, Entisol, Histosol, Inceptisol, Mollisol, Oxisol,
Spodosol, Ultisol, and Vertisol.
(xiii) ``Soil series'' is the basic unit of soil classification and
is a subdivision of a family. A series consists of soils that were
developed under comparable climatic and vegetational conditions. The
soils comprising a series are essentially alike in all major profile
characteristics except for the texture of the ``A'' horizon (i.e., the
surface layer of soil).
(xiv) ``Soil texture'' is a classification of soils that is based on
the relative proportions of the various soil separates present. The soil
textural classes are: clay, sandy clay, silty clay, clay loam, silty
clay loam, sandy clay loam, loam, silt loam, silt, sandy loam, loamy
sand, and sand.
(3) Principle of the test method. (i) The extent of adsorption of a
chemical onto sediment or soil is measured, using this test guideline,
by equilibrating aqueous solutions containing different, but
environmentally realistic, concentrations of the test chemical with a
known quantity of sediment or soil. After equilibrium is reached, the
distribution of the chemical between the water phase and the solid phase
is quantitatively measured by a suitable analytical method. Then,
sorption constants are calculated by using the Freundlich equation:
Equation 1
x/m=Cs=KCe l/n
where:
Ce=Equilibrium concentration of the chemical in the solution
phase
Cs=Equilibrium concentration of the chemical in the solid
phase
K=Freundlich adsorption coefficient
m=The mass of the solid in grams
l/n=Exponent where n is a constant
[[Page 89]]
x=The mass in micrograms of the chemical adsorbed by m grams of solid.
Logarithmetic transformation of the Freundlich equation yields the
following linear relationship:
Equation 2
log Cs=log K+(l/n) log Ce
(ii) In order to estimate the environmental movement of the test
chemical, the values K and l/n are compared with the values of other
chemicals whose behavior in soil and sediment systems is well-documented
in scientific literature.
(iii) The adsorption isotherm (AI) test has many desirable features.
First, adsorption results are highly reproducible. The test provides
excellent quantitative data readily amenable to statistical analyses.
Also, it has relatively modest requirements for chemicals, soils,
laboratory space, and equipment. It allows solution phase organic
chemical determinations that are relatively uncomplicated. A chemical
extraction-mass balance procedure to elicit information on chemical
transformations occurring at colloid interfaces can be incorporated into
this test. The ease of performing the isotherm test and mass balance
will depend upon the physical/chemical properties of the test chemical
and the availability of suitable analytical techniques to measure the
chemical.
(iv) The papers by Aharonson and Kafkafi (1975) under paragraph
(d)(1) of this section, Harvey (1974) under paragraph (d)(3) of this
section, Murray (1975) under paragraph (d)(4) of this section, Saltzman
(1972) under paragraph (d)(5) of this section, Weber (1971) under
paragraph (d)(6) of this section, and Wu (1975) under paragraph (d)(7)
of this section served as the basis for this section. The soil and
colloid chemistry literature and the analytical chemistry literature
substantiate the experimental conditions and procedures specified in
this guideline as accepted, standard procedures.
(4) Applicability and specificity. The AI Test Guideline can be used
to determine the soil and sediment adsorption potential of sparingly
water soluble to infinitely soluble chemicals. In general, a chemical
having a water solubility of less than 0.5 ppm need not be tested with
soil as the solid phase, since the literature indicates that these
chemicals are, in general, immobile in soils, see Goring and Hamaker
(1972) under paragraph (d)(2) of this section. However, this does not
preclude future soil adsorption/transformation testing of these
chemicals if more refined data are needed for the assessment process.
(b) Test procedures--(1) Test conditions--(i) Special laboratory
equipment. (A) Equilibrating solutions that contain, besides the test
chemical, 0.01M calcium nitrate dissolved in sterilized, distilled-
deionized H2O adjusted to neutral pH 7 by boiling to remove
CO2.
(B) Containers shall be composed of material that (1) adsorb
negligible amounts of test chemical, and (2) withstand high speed
centrifugation. The volume of the container is not a major
consideration; however, it is extremely important that the amount of
soil or sediment and the solid/solution ratio used in the study result
in minimal container headspace. It is also extremely important that the
containers be sterilized before use.
(C) A 150 micron (100 mesh) stainless-steel or brass sieve.
(D) Drying oven, with circulating air, that can attain 100 deg.C.
(E) Vortex mixer or a comparable device.
(F) Rotary shaker or a comparable device.
(G) High speed temperature-controlled centrifuge capable of
sedimenting particles greater than 0.5 micron from aqueous solution.
(ii) Temperature. The test procedure shall be performed at
235 deg.C.
(iii) Replications. Three replications of the experimental
treatments shall be used.
(iv) Soil pretreatment. The following soil pretreatment steps shall
be performed under the following conditions:
(A) Decrease the water content, air or oven-dry soils at or below 50
deg.C.
(B) Reduce aggregate size before and during sieving, crush and grind
dried soil very gently.
(C) Eliminate microbial growth during the test period using a
chemical or physical treatment that does not alter
[[Page 90]]
or minimally alters the soil surface properties.
(D) Sieve soils with a 100 mesh stainless-steel or brass sieve.
(E) Store all solutions and soils at temperatures between 0 and 5
deg.C.
(v) Sediment pretreatment. The following sediment pretreatment steps
shall be performed under the following conditions:
(A) Decrease the H2O content by air or oven-drying
sediments at or below 50 deg.C. Sediments should not be dried
completely and should remain moist at all times prior to testing and
analysis.
(B) Eliminate microbial growth during the test period by using a
chemical and/or physical treatment that does not alter or minimally
alters the colloid surface's properties.
(C) Store at temperatures between 0 and 5 deg.C.
(vi) Solid/solution ratio. The solid/solution ratio shall be equal
to or greater than 1/10. If possible, the ratios should be equal to or
greater than 1/5. The sediment or soil dry weight after drying for a 24-
hour minimum at 90 deg.C is recommended for use as the weight of the
solid for ratio and data calculations. If an insufficient amount of
chemical remains in the water phase for quantification, the solid/
solution ratio should be adjusted so that measurable amounts of the test
chemical remain in solution.
(vii) Equilibration time. The equilibration time will depend upon
the length of time needed for the parent chemical to attain an
equilibrium distribution between the solid phase and the aqueous
solution phase. The equilibration time shall be determined by the
following procedure:
(A) Equilibrate one solution containing a known concentration of the
test chemical with the sediment or soil in a solid/solution ratio equal
to or greater than \1/10\ and preferably equal to or greater than \1/5\.
It is important that the concentration of the test chemical in the
equilibrating solution (1) does not exceed one-half of its solubility
and (2) should be 10 ppm or less at the end of the equilibration period.
(B) Measure the concentration of the chemical in the solution phase
at frequent intervals during the equilibration period.
(C) Determine the equilibration time by plotting the measured
concentration versus time of sampling; the equilibration time is the
minimum period of time needed to establish a rate of change of solution
concentration of 5 percent or less per 24 hours.
(viii) Centrifugation time. Calculate the centrifugation time,
tc, necessary to remove particles from solution greater than
approximately 0.5 m (5 x 10-5 m) equivalent
diameter (which represents all particles except the fine clay fraction)
using the following equation:
Equation 3
tc(min)=1.41 x 109 [log(R2/
R1)]/N2
where:
tc=centrifuge time in minutes
R2=distance from centrifuge spindle to deposition surface of
centrifuge
R1=distance from spindle to surface of the sample
N=number of revolutions of the centrifuge per minute.
(ix) Storage of solutions. If the chemical analysis is delayed
during the course of the experiment, store all solutions between 0 and 5
deg.C.
(x) Solvents for extraction. It is important that the solvent used
to extract the chemical from the sediment or soil is reagent grade or
better. Solvents shall contain no impurities which could interfere with
the determination of the test compound.
(2) Test procedure--(i) Equilibration. Add six solutions containing
different concentrations of the test chemical to at least one gram of
each solid. The initial concentration of the test chemical in these
solutions will depend on the affinity the chemical has for the sediment
or soil. Therefore, after equilibrium is attained, it is extremely
important that the highest concentration of the test chemical in the
equilibrating solution does not exceed 10 ppm, is at least one order of
magnitude greater than the lowest concentration reported, and does not
exceed one half of its solubility.
(A) Immediately after the solutions are added to the solids, tightly
cap the containers and vigorously agitate them for several minutes with
a vortex mixture or similar device.
[[Page 91]]
(B) Shake the containers throughout the equilibration period at a
rate that suspends all solids in the solution phase.
(ii) Centrifugation. When the equilibration time has expired,
centrifuge the containers for tc minutes.
(iii) Chemical extraction. (A) After centrifugation, remove the
supernatant aqueous phase from the solid-solution mixture.
(B) Extract the chemical adsorbed on the sediment or soil colloid
surfaces with solvent.
(iv) Chemical analysis. Determine the amount of parent test chemical
in the aqueous equilibrating solution and organic solvent extractions.
Use any method or combination of methods suitable for the identification
and quantitative detection of the parent test chemical.
(c) Reporting. Report the following information:
(1) Temperature at which the test was conducted.
(2) Detailed description of the analytical technique(s) used in the
chemical extraction, recovery, and quantitative analysis of the parent
chemical.
(3) Amount of parent test chemical applied, the amount recovered,
and the percent recovered.
(4) Extent of adsorption by containers and the approach used to
correct the data for adsorption by containers.
(5) The individual observations, the mean values, and graphical
plots of x/m as a function of Ce for each sediment or soil
for (i) the equilibration time determination and (ii) the isotherm
determination.
(6) The quantities K, n, and l/n.
(7) Soil information: Soil Order, series, texture, sampling
location, horizon, general clay fraction mineralogy.
(8) Sediment information: sampling location, general clay fraction
mineralogy.
(9) Sediment and soil physical-chemical properties: percent sand,
silt, and clay (particle size analysis); percent organic matter; pH (1/1
solids/H2O); and cation exchange capacity.
(10) The procedures used to determine the physical/chemical
properties listed under paragraphs (c) (7) through (9) of this section.
(d) References. For additional background information on this test
guideline the following references should be consulted:
(1) Aharonson, N., Kafkafi, U. ``Adsorption, mobility and
persistence of thiabendazole and methyl 2-benzimidasole carbamate in
soils,'' Journal of Agricultural and Food Chemistry, 23:720-724 (1975).
(2) Goring, C.A.I., Hamaker, J.W., (eds). Organic Chemicals in the
Soil Environment. Vol. I & II (New York: Marcel Dekker, Inc., 1972).
(3) Harvey, R.G. et al. ``Soil adsorption and volatility of
dinitroaniline herbicides,'' Weed Science, 22:120-124 (1974).
(4) Murray, D.S. et al. ``Comparative adsorption, desorption, and
mobility of dipropetryn and prometryn in soil,'' Journal of Agricultural
and Food Chemistry, 23:578-581 (1973).
(5) Saltzman, S.L. et al. ``Adsorption, desorption of parathion as
affected by soil organic matter,'' Journal of Agricultural and Food
Chemistry, 20:1224-1226 (1972).
(6) Weber, J.B. ``Model soil system, herbicide leaching, and
sorption,'' Weed Science, 19:145-160 (1971).
(7) Wu, C.H., et al. ``Napropamide adsorption, desorption, and
movement in soils,'' Weed Science, 23:454-457 (1975).
[50 FR 39252, Sept. 27, 1985, as amended at 52 FR 19058, May 20, 1987;
54 FR 29715, July 14, 1989]
Subpart D--Transformation Processes
Sec. 796.3100 Aerobic aquatic biodegradation.
(a) Introduction--(1) Purpose. (i) This Guideline is designed to
develop data on the rate and extent of aerobic biodegradation that might
occur when chemical substances are released to aquatic environments. A
high biodegradability result in this test provides evidence that the
test substance will be biodegradable in natural aerobic freshwater
environments.
(ii) On the contrary, a low biodegradation result may have other
causes than poor biodegradability of the test substance. Inhibition of
the microbial inoculum by the test substance at the test concentration
may
[[Page 92]]
be observed. In such cases, further work is needed to assess the aerobic
aquatic biodegradability and to determine the concentrations at which
toxic effects are evident. An estimate of the expected environmental
concentration will help to put toxic effects into perspective.
(2) Definitions. (i) ``Adaptation'' is the process by which a
substance induces the synthesis of any degradative enzymes necessary to
catalyze the transformation of that substance.
(ii) ``Ultimate Biodegradability'' is the breakdown of an organic
compound to CO2, water, the oxides or mineral salts of other
elements and/or to products associated with normal metabolic processes
of microorganisms.
(iii) ``Ready Biodegradability'' is an expression used to describe
those substances which, in certain biodegradation test procedures,
produce positive results that are unequivocal and which lead to the
reasonable assumption that the substance will undergo rapid and ultimate
biodegradation in aerobic aquatic environments.
(3) Principle of the test method. This Guideline method is based on
the method described by William Gledhill (1975) under paragraph (d)(1)
of this section. The method consists of a 2-week inoculum buildup period
during which soil and sewage microorganisms are provided the opportunity
to adapt to the test compound. This inoculum is added to a specially
equipped Erlenmeyer flask containing a defined medium with test
substance. A reservoir holding barium hydroxide solution is suspended in
the test flask. After inoculation, the test flasks are sparged with
CO2-free air, sealed, and incubated, with shaking in the
dark. Periodically, samples of the test mixture containing water-soluble
test substances are analyzed for dissolved organic carbon (DOC) and the
Ba(OH)2 from the reservoirs is titrated to measure the amount
of CO2 evolved. Differences in the extent of DOC
disappearance and CO2 evolution between control flasks
containing no test substance, and flasks containing test substance are
used to estimate the degree of ultimate biodegradation.
(4) Prerequisites. The total organic carbon (TOC) content of the
test substance shall be calculated or, if this is not possible,
analyzed, to enable the percent of theoretical yield of carbon dioxide
and percent of DOC loss to be calculated.
(5) Guideline information. (i) Information on the relative
proportions of the major components of the test substance will be useful
in interpreting the results obtained, particularly in those cases where
the result lies close to a ``pass level.''
(ii) Information on the toxicity of the chemical may be useful in
the interpretation of low results and in the selection of appropriate
test concentrations.
(6) Reference substances. Where investigating a chemical substance,
reference compounds may be useful and an inventory of suitable reference
compounds needs to be identified. In order to check the activity of the
inoculum the use of a reference compound is desirable. Aniline, sodium
citrate, dextrose, phthalic acid and trimellitic acid will exhibit
ultimate biodegradation under the conditions of this Test Guideline
method. These reference substances must yield 60 percent of theoretical
maximum CO2 and show a removal of 70 percent DOC within 28
days. Otherwise the test is regarded as invalid and shall be repeated
using an inoculum from a different source.
(7) Reproducibility. The reproducibility of the method has not yet
been determined; however it is believed to be appropriate for a
screening test which has solely an acceptance but no rejective function.
(8) Sensitivity. The sensitivity of the method is determined by the
ability to measure the endogenous CO2 production of the
inoculum in the blank flask and by the sensitivity limit of the
dissolved organic carbon analysis. If the test is adapted to handle
\14\C-labeled test substances, test substance concentrations can be much
lower.
(9) Possibility of standardization. This possibility exists. The
major difficulty is to standardize the inoculum in such a way that
interlaboratory reproducibility is ensured.
(10) Possibility of automation. None at present, although parts of
the analyses may be automated.
[[Page 93]]
(b) Test procedures--(1) Preparations--(i) Apparatus. The shake
flask apparatus under the following Figure 1 contains 10 mL of 0.2N
Ba(OH)2 in an open container suspended over 1 liter of
culture medium in a 2-liter Erlenmeyer flask.
[GRAPHIC] [TIFF OMITTED] TC01AP92.039
Figure 1--Shake-Flask System for Carbon Dioxide Evolution
The Ba(OH)2 container is made by placing a constriction just
above the 10 mL mark of a 50 mL heavy-duty centrifuge tube and attaching
the centrifuge tube to a 2 mm I.D. x 9 mm O.D. glass tube by means of 3
glass support rods. The centrifuge tube opening is large enough to
permit CO2 to diffuse into the Ba(OH)2, while the
constriction permits transferal of the flask to and from the shaker
without Ba(OH)2 spillage into the medium. For periodic
removal and addition of base from the center well, a polypropylene
capillary tube, attached at one end to a 10 ml disposable syringe, is
inserted through the 9 mm O.D. glass tube into the Ba(OH)2
reservoir. The reservoir access port is easily sealed during incubation
with a serum bottle stopper. Two glass tubes are added for sparging,
venting, and medium sampling. The tops of these tubes are connected with
a short section of flexible tubing during incubation.
(ii) Reagents and stock solutions. (A) Stock solutions, I, II, and
III under the following Table 1.
(B) Yeast extract.
(C) Vitamin-free casamino acids.
(D) 70 percent O2 in nitrogen or CO2-free air.
(E) 0.2N Ba(OH)2.
(F) 0.1 N HCl.
(G) 20 percent H2SO4.
(H) Phenolphthalein.
(I) Dilution water--distilled, deionized water (DIW).
(iii) Soil inoculum. A fresh sample of an organically rich soil is
used as the inoculum in the ultimate biodegradation test. Soil is
collected, prepared, and stored according to the recommendations of
Pramer and Bartha (1972) under paragraph (d)(2) of this section. The
soil surface is cleared of litter and a soil sample is obtained 10 to 20
cm below the surface. The sample is screened through a sieve with 2 to 5
mm openings and stored in a polyethylene bag at 2 to 4 deg.C for not
more
[[Page 94]]
than 30 days prior to use. The soil is never allowed to air-dry, and
shall not be frozen during storage.
Table 1--Medium Employed for Assay of CO2 Evolution
------------------------------------------------------------------------
Stock
Solution
Solution \1\ Compound Conc. (g/
L)
------------------------------------------------------------------------
I NH4Cl.......................... 35
KNO3........................... 15
K2HPO43H2O........... 750
NaH2PO4H2O........... 25
II \2\ KCl............................ 10
MgSO4.......................... 20
FeSO47H2O............ 1
III CaCl2.......................... 5
ZnCl2.......................... 0.05
MnCl24H2O............ 0.5
CuCl2.......................... 0.05
CoCl2.......................... 0.001
H3 BO3......................... 0.001
MoO3........................... 0.0004
------------------------------------------------------------------------
\1\= Each liter of test medium contains 1 mL of each solution.
\2\= Final pH is adjusted to 3.0 with 0.10 N HCl.
(iv) Acclimation Medium. Acclimation medium is prepared by adding,
for each liter of distilled, deionized water (DIW): 1 mL each of
solutions I, II, and III in Table 1 in paragraph (b)(1)(iii) of this
section, 1.0 gm of soil inoculum (prepared according to paragraph
(b)(1)(iii) of this section), 2.0 mL of aerated mixed liquor (obtained
from an activated sludge treatment plant not more than 2 days prior to
commencing the acclimation phase, and stored in the interim at 4 deg.C)
and 50 mL raw domestic influent sewage. This medium is mixed for 15
minutes and filtered through a glass wool plug in a glass funnel. The
filtrate is permitted to stand for 1 hour, refiltered through glass
wool, and supplemented with 25 mg/L each of Difco vitamin-free casamino
acids and yeast extract. Appropriate volumes are added to 2-liter
Erlenmeyer flasks. Test compounds are added incrementally during the
acclimation period at concentrations equivalent to 4, 8, and 8 mg/L
carbon on days 0, 7, and 11, respectively. On day 14, the medium is
refiltered through glass wool prior to use in the test. For evaluating
the biodegradability of a series of functionally or structurally related
chemicals, media from all inoculum flasks may be combined before final
filtration.
(2) Procedures. (i) Inoculum (100 mL of acclimation medium) is added
to 900 mL DIW containing 1 mL each of solutions I, II, and III in Table
1 under paragraph (b)(1)(iii) of this section in a 2-liter Erlenmeyer
flask. Test compound equivalent to 10 mg/liter carbon is added to each
of the replicate flasks containing the test medium. Ten mL of 0.2 N Ba
(OH)2 are added to the suspended reservoir in each flask and
duplicate 10 mL samples of Ba(OH)2 are also saved as
titration blanks for analysis with test samples. Flasks are sparged with
CO2-free air (for volatile test materials, sparging is done
prior to addition of the chemical), sealed, and placed on a gyrotary
shaker (approximately 125 rpm) at 20 to 25 deg.C in the dark. For each
set of experiments, each test, reference, inhibited, and control system
should be analyzed at time zero and at a minimum of four other times
from time zero through day 28. Sampling must be made with sufficient
frequency to allow for a smooth plot of biodegradation with time.
Sampling times should be varied by the investigator as deemed
appropriate to match the rate of degradation of the test substance.
Tests may be terminated when biodegradation reaches a plateau and is
consistent (10 percent) over 3 consecutive days or on day
28, whichever occurs first. For chemicals which are water soluble at the
test concentration, an adequate volume (5 to 10 mL) of medium is removed
for DOC analysis. Each sample for DOC analysis should be filtered
through a membrane filter of 0.45 micrometer pore diameter before DOC
analysis. For all test and reference compounds, Ba(OH)2 from
the center well is removed for analysis. The center well is rinsed with
10 mL CO2-free DIW and is refilled with fresh base. Rinse
water is combined with the Ba(OH)2 sample to be analyzed.
Flasks are resealed and placed on the shaker. On the day prior to
terminating the test, 3 mL of 20 percent H2SO4 are
added to the medium to release carbonate bound CO2.
(ii) For each set of experiments, each test substance shall be
tested in triplicate.
(iii) For each set of experiments, one or two reference compounds
are included to assess the microbial activity of the test medium.
Duplicate reference flasks are prepared by adding
[[Page 95]]
reference compound equivalent to 10 mg/liter carbon to each of two
flasks containing the test medium. Reference compounds which are
positive for ultimate biodegradability include: sodium citrate,
dextrose, phthalic acid, trimellitic acid, and aniline.
(iv) For each test set, triplicate controls receiving inoculated
medium and no test compound, plus all test and reference flasks, are
analyzed for CO2 evolution and DOC removal. Results from
analysis of the control flasks (DOC, CO2 evolution, etc.) are
subtracted from corresponding experimental flasks containing test
compound in order to arrive at the net effect due to the test compound.
(v) A test system containing a growth inhibitor should be
established as a control for each substance tested for biodegradation by
this method. That inhibited system must contain the same amount of
water, mineral nutrients, inoculum, and test substance used in the
uninhibited test systems, plus 50 mg/L mercuric chloride
(HgCl2) to inhibit microbial activity.
(vi) Flasks shall be incubated in the dark to minimize both
photochemical reactions and algal growth. Appropriate sterile controls
or controls containing a metabolic inhibitor, such as 50 mg/1
HgCl2, are needed to correct for interferences due to
nonbiological degradation. With volatile organic materials, sparging
with CO2-free air is performed only once, just prior to
addition of the test chemical. Analyses for CO2 evolution and
DOC removal are conducted within 2 to 3 hours of sampling to minimize
interferences which may occur in storage. All glassware should be free
of organic carbon contaminants.
(3) Analytical measurements. The quantity of CO2 evolved
is measured by titration of the entire Ba(OH)2 sample (10 mL
Ba(OH)2+10 mL rinse water) with 0.1 N HCl to the
phenolphthalein end point. Ba(OH)2 blanks are also
supplemented with 10 mL CO2-free DIW and titrated in a
similar manner. Samples (5 mL) for DOC are centrifuged and/or filtered
and supernatant or filtrate analyzed by a suitable total organic carbon
method.
(c) Data and reporting--(1) Treatment of results. (i) Test compound
(10 mg carbon) is theoretically converted to 0.833 mmol CO2.
Absorbed CO2 precipitates as BaCO3 from
Ba(OH)2, causing a reduction in alkalinity by the equivalent
of 16.67 mL of 0.1 N HCl for complete conversion of the test compound
carbon to CO2. Therefore, the percent theoretical
CO2 evolved from the test compound is calculated at any
sampling time from the formula:
Percent CO2 evolution=[(TF-CF)/16.67] 100 (for 10 mg/L test
compound carbon)
where:
TF= mL 0.1 N HCl required to titrate Ba(OH)2 samples from the
test flask
CF= mL 0.1 N HCl required to titrate Ba(OH)2 samples from the
control flask.
(ii) The cumulative percent CO2 evolution at any sample
time is calculated as the summation of the percent CO2
evolved at all sample points of the test.
(iii) The percent DOC disappearance from the test compound is
calculated from the following equation:
Percent DOC Removal=[1-(DTFx- DCFx)/
(DTFo- DCFo)] 100
where:
DTF= Dissolved organic carbon from test flask
DCF= Dissolved organic carbon from control flask
o= Day zero measurements
x= Day of measurements during test.
(iv) The difference between the amount of 0.1 N HCl used for the
Ba(OH)2 titration blank samples and the Ba(OH)2
samples from the control units (no test compound) is an indication of
the activity of the microorganisms in the test system. In general, this
difference is approximately 1 to 3 mL of 0.1 N HCl at each sampling
time. A finding of no difference in the titration volumes between these
two samples indicates a poor inoculum. In this case, the validity of the
test results is questionable and the test set shall be rerun beginning
with the acclimation phase.
(v) CO2 evolution in the reference flasks is also
indicative of the activity of the microbial test system. The suggested
reference compounds should all yield final CO2 evolution
values of at least 60 percent of theoretical CO2. If, for any
test set, the percent theoretical CO2 evolution value for the
reference
[[Page 96]]
flasks is outside this range, the test results are considered invalid
and the test is rerun.
(vi) Inhibition by the test compound is indicated by lower
CO2 evolution in the test flasks than in the control flasks.
If inhibition is noted, the study for this compound is rerun beginning
with the acclimation phase. During the test phase for inhibitory
compounds, the test chemical is added incrementally according to the
schedule: Day 0--0.5 mg/liter as organic carbon, Day 2--1 mg/liter C,
Day 4--1.5 mg/liter C, Day 7--2 mg/liter C, Day 10--5 mg/liter C. For
this case, the Ba(OH)2 is sampled on Day 10, and weekly
thereafter. The total test duration remains 28 days.
(vii) The use of 14C-labeled chemicals is not required.
If appropriately labeled test substance is readily available and if the
investigator chooses to use this procedure with labeled test substance,
this is an acceptable alternative. If this option is chosen, the
investigator may use lower test substance concentrations if those
concentrations are more representative of environmental levels.
(2) Test report. (i) For each test and reference compound, the
following data shall be reported.
(ii) Information on the inoculum, including source, collection date,
handling, storage and adaptation possibilities (i.e., that the inoculum
might have been exposed to the test substance either before or after
collection and prior to use in the test).
(iii) Results from each test, reference, inhibited (with
HgCl2) and control system at each sampling time, including an
average result for the triplicate test substance systems and the
standard deviation for that average.
(iv) Average cumulative percent theoretical CO2 evolution
over the test duration.
(v) Dissolved organic carbon due to test compound at each sampling
time (DTF-DCF).
(vi) Average percent DOC removal at each sampling time.
(vii) Twenty-eight day standard deviation for percent CO2
evolution and DOC removal.
(d) References. For additional background information on this test
guideline the following references should be consulted:
(1) Gledhill, W.E. ``Screening Test for Assessment of Ultimate
Biodegradability: Linear Alkyl Benzene Sulfonate,'' Applied
Microbiology, 30:922-929 (1975).
(2) Pramer, D., Bartha, R. `'Preparation and Processing of Soil
Samples for Biodegradation Testing,'' Environmental Letters, 2:217-224
(1972).
[50 FR 39252, Sept. 27, 1985, as amended at 52 FR 19058, May 20, 1987]
Sec. 796.3500 Hydrolysis as a function of pH at 25 deg.C.
(a) Introduction--(1) Background and purpose. (i) Water is one of
the most widely distributed substances in the environment. It covers a
large portion of the earth's surface as oceans, rivers, and lakes. The
soil also contains water, as does the atmosphere in the form of water
vapor. As a result of this ubiquitousness, chemicals introduced into the
environment almost always come into contact with aqueous media. Certain
classes of these chemicals, upon such contact, can undergo hydrolysis,
which is one of the most common reactions controlling chemical stability
and is, therefore, one of the main chemical degradation paths of these
substances in the environment.
(ii) Since hydrolysis can be such an important degradation path for
certain classes of chemicals, it is necessary, in assessing the fate of
these chemicals in the environment, to know whether, at what rate, and
under what conditions a substance will hydrolyze. Some of these
reactions can occur so rapidly that there may be greater concern about
the products of the transformation than about the parent compounds. In
other cases, a substance will be resistant to hydrolysis under typical
environmental conditions, while, in still other instances, the substance
may have an intermediate stability that can result in the necessity for
an assessment of both the original compound and its transformation
products. The importance of transformation of chemicals via hydrolysis
in aqueous media in the environment can be determined quantitatively
from data on hydrolysis rate constants. This hydrolysis Test Guideline
represents a test to
[[Page 97]]
allow one to determine rates of hydrolysis at any pH of environmental
concern at 25 deg.C.
(2) Definitions and units. (i) ``Hydrolysis'' is defined as the
reaction of an organic chemical with water, such that one or more bonds
are broken and the reaction products of the transformation incorporate
the elements of water (H2O).
(ii) ``Elimination'' is defined in this Test Guideline to be a
reaction of an organic chemical (RX) in water in which the X group is
lost. These reactions generally follow the same type of rate laws that
hydrolysis reactions follow and, thus, are also covered in this Test
Guideline.
(iii) A ``first-order reaction'' is defined as a reaction in which
the rate of disappearance of the chemical substance being tested is
directly proportional to the concentration of the chemical substance and
is not a function of the concentrations of any other substances present
in the reaction mixture.
(iv) The ``half-life'' of a chemical is defined as the time required
for the concentration of the chemical substance being tested to be
reduced to one-half its initial value.
(v) ``Hydrolysis'' refers to a reaction of an organic chemical with
water such that one or more bonds are broken and the reaction products
incorporate the elements of water (H2O). This type of
transformation often results in the net exchange of a group X, on an
organic chemical RX, for the OH group from water. This can be written
as:
RX+HOH ROH+HX.
(A) Another result of hydrolysis can be the incorporation of both H
and OH in a single product. An example of this is the hydrolysis of
epoxides, which can be represented by
(B) The hydrolysis reaction can be catalyzed by acidic or basic
species, including OH- and H3O+
(H+). The promotion of the reaction by
H3O- or OH- is called specific acid or
specific base catalysis, respectively, as contrasted with general acid
or base catalysis encountered with other cationic or anionic species.
Usually, the rate law for chemical RX can be written as:
Equation 1
-d[RX]/d+ = kh[RX]=kA[H+]
[RX]
+kB[OH-] [RX]+k,N
[H2O] [RX],
where KA, kB and k'N are the second-
order rate constants for acid and base catalyzed and neutral water
processes, respectively. In dilute solutions, such as are encountered in
following this Test Guideline, water is present in great excess and its
concentration is, thus, essentially constant during the course of the
hydrolysis reaction. At fixed pH, the reaction, therefore, becomes
pseudo first-order, and the rate constant (kh) can be written
as:
Equation 2
kh=kA [H+]+kB
[OH-]+kN,
where kN is the first-order neutral water rate constant.
Since this is a pseudo first-order process, the half-life is independent
of the concentration and can be written as:
Equation 3
t1/2=0.693/kh.
At constant pH, Equation 1 can be integrated to yield the first order
rate expression
Equation 4
log10C=- (kh t/
2.303)+log10Co,
where C is the concentration of the test chemical at time t and
Co is the initial chemical concentration (t=0).
(C) At a given pH, Equation 2 under paragraph (a)(2)(v)(B) of this
section contains three unknowns, kA, kB, and
kN. Therefore, three equations (i.e., measurements at three
different pH's at a fixed temperature) are required if one wishes to
solve for these quantities. Making suitable approximations for
quantities that are negligible, the expressions for kA,
kB, and kN using values of kh measured
at pH 3, 7, and 11 are:
Equation 5
kA=103 [kh (3)-kh
(7)+10-4 kh (11)]
[[Page 98]]
kB=103 [kh (11)-kh
(7)+10-4 kh (3)]
kN=kh (7)-10-4 [kh
(3)+kh (11)]
The calculated rate constants from equation 5 under this paragraph can
be employed in equation 2 under paragraph (a)(2)(v)(B) of this section
to calculate the hydrolysis rate of a chemical at any pH of
environmental concern.
(D) The equations under paragraph (a)(2) of this section apply
whether the test chemical has one or more hydrolyzable groups. In the
latter case, the rate may be written as:
Equation 6
-d[RX]/dt= [RX]+k2 [RX]+ . . . . +kn
[RX]=(k1+k2+ . . . . . kn)
[RX]=kh [RX].
Equation 6 applies to the hydrolysis rate of a molecule having n
hydrolyzable groups, each of which follows first-order reaction
kinetics. The measured kh is now the sum of the individual
reaction rates and is the only rate constant required in this section.
(3) Principle of the test method. Procedures described in this
section enable sponsors to obtain quantitative information on hydrolysis
rates through a determination of hydrolysis rate constants and half-
lives of chemicals at pH 3.00, 7.00, and 11.00 at 25 deg.C. The three
measured rate constants are used to determine the acidic, basic, and
neutral rate constants associated with a hydrolytic reaction. The latter
constants can then be employed in determining the hydrolysis rates of
chemicals at any pH of environmental concern at 25 deg.C.
(4) Applicability and specificity. There are several different
common classes of organic chemicals that are subject to hydrolysis
transformation, including esters, amides, lactones, carbamates,
organophosphates, and alkyl halides. Processes other than nucleophilic
displacement by water can also take place. Among these are elimination
reactions that exhibit behavior similar to hydrolysis and, therefore,
are also covered in this section.
(b) Test procedures--(1) Test conditions--(i) Special laboratory
equipment. (A) A thermostatic bath that can be maintained at a
temperature of 251 deg.C.
(B) A pH meter that can resolve differences of 0.05 pH units or
less.
(C) Stoppered volumetric flasks (no grease) or glass ampoules that
can be sealed.
(ii) Purity of water. Reagent-grade water (e.g., water meeting ASTM
Type IIA standards or an equivalent grade) shall be used to minimize
biodegradation. ASTM Type IIA water is described in ASTM D 1193-77
(Reapproved 1983), ``Standard Specification for Reagent Water.'' ASTM D
1193-77 (Reapproved 1983) is available for inspection at the Office of
the Federal Register, 800 North Capitol Street, NW., suite 700,
Washington, DC. This incorporation by reference was approved by the
Director of the Office of the Federal Register. This material is
incorporated as it exists on the date of approval and a notice of any
change in this material will be published in the Federal Register.
Copies of the incorporated material may be obtained from the Non-
Confidential Information Center (NCIC) (7407), Office of Pollution
Prevention and Toxics, U.S. Environmental Protection Agency, Room B-607
NEM, 401 M St., SW., Washington, DC 20460, between the hours of 12 p.m.
and 4 p.m. weekdays excluding legal holidays, or from the American
Society for Testing and Materials (ASTM), 1916 Race Street,
Philadelphia, PA 19103.
(iii) Sterilization. All glassware shall be sterilized. Aseptic
conditions shall be used in the preparation of all solutions and in
carrying out all hydrolysis experiments to eliminate or minimize
biodegradation. Glassware can be sterilized in an autoclave or by any
other suitable method.
(iv) Precautions for volatility. If the chemical is volatile the
reaction vessels shall be almost completely filled and sealed.
(v) Temperature controls. All hydrolysis reactions shall be carried
out at 25 deg.C (1 deg.C) and with the temperature
controlled to 0.1 deg.C.
(vi) pH conditions. It is recommended that all hydrolysis
experiments be performed at pH 3.00, 7.00, and 11.00 0.05
using the appropriate buffers described in paragraph (b)(2)(i)(A) of
this section.
(vii) Concentration of solutions of chemical substances. The
concentration of the test chemical shall be less than one-half the
chemical's solubility in water but not greater than 10-3 M.
[[Page 99]]
(viii) Effect of acidic and basic groups. Complications can arise
upon measuring the rate of hydrolysis of chemicals that reversibly
ionize or are protonated in the pH range 3.00 to 11.00. Therefore, for
these chemicals, it is recommended that these hydrolysis tests be
performed at pH 5.00, 7.00, and 9000.05 using the
appropriate buffers described in paragraphs (b)(2)(i) (A) and (B) of
this section. If a test chemical reversibly ionizes or protonates in the
pH range 5.00 to 9.00, then it is recommended that additional hydrolysis
tests should be carried out at pH 6.00 and 8.000.05 using
the buffers described in paragraph (b)(2)(i)(B) of this section.
(ix) Buffer catalysis. For certain chemicals, buffers may catalyze
the hydrolysis reaction. If this is suspected, hydrolysis rate
determination shall be carried out with the appropriate buffers and the
same experiments repeated at buffer concentrations lowered by at least a
factor of five. If the hydrolysis reaction produces a change of greater
than 0.05 pH units in the lower concentration buffers at the end of the
measurement time, the test chemical concentrations also shall be lowered
by at least a factor of five. Alternatively, test chemical
concentrations and buffer concentrations may both be lowered
simultaneously by a factor of five. A sufficient criterion for
minimization of buffer catalysis is an observed equality in the
hydrolysis rate constant for two different solutions differing in buffer
or test chemical concentration by a factor of five.
(x) Photosensitive chemicals. The solution absorption spectrum can
be employed to determine whether a particular chemical is potentially
subject to photolytic transformation upon exposure to light. For
chemicals that absorb light of wavelengths greater than 290 nm, the
hydrolysis experiment shall be carried out in the dark, under amber or
red safelights, in amber or red glassware, or employing other suitable
methods for preventing photolysis. The absorption spectrum of the
chemical in aqueous solution can be measured under Sec. 796.1050.
(xi) Chemical analysis of solutions. In determining the
concentrations of the test chemicals in solution, any suitable
analytical method may be employed, although methods which are specific
for the compound to be tested are preferred. Chromatographic methods are
recommended because of their compound specificity in analyzing the
parent chemical without interferences from impurities. Whenever
practicable, the chosen analytical method should have a precision within
5 percent.
(2) Preparation--(i) Reagents and solutions--(A) Buffer solutions.
Prepare buffer solutions using reagent-grade chemicals and reagent-grade
water as follows:
(1) pH 3.00: use 250 mL of 0.100M potassium hydrogen phthalate; 111
mL of 0.100M hydrochloric acid; and adjust volume to 500 mL with
reagent-grade water.
(2) pH 7.00: use 250 mL of 0.100M potassium dihydrogen phosphate;
145 mL of 0.100M sodium hydroxide; and adjust volume to 500 mL with
reagent-grade water.
(3) pH 11.00: use 250 mL of 0.0500M sodium bicarbonate; 113 mL of
0.100M sodium hydroxide; and adjust volume to 500 mL with reagent-grade
water.
(B) Additional buffer solutions. For chemicals that ionize or are
protonated as discussed in paragraph (b)(1)(viii) of this section,
prepare buffers using reagent-grade water and reagent-grade chemicals as
follows:
(1) pH 5.00: use 250 mL of 0.100M potassium hydrogen phthalate; 113
mL of 0.100M sodium hydroxide; and adjust volume to 500 mL with reagent-
grade water.
(2) pH 6.00: use 250 mL of 0.100M potassium dihydrogen phosphate; 28
mL of 0.100M sodium hydroxide; and adjust volume to 500 mL with reagent-
grade water.
(3) pH 8.00: use 250 mL of 0.100M potassium dihydrogen phosphate;
234 mL of 0.100M sodium hydroxide; and adjust volume to 500 mL with
reagent-grade water.
(4) pH 9.00: use 250 mL of 0.0250M borax (Na2
B4O7); 23 mL of 0.100M hydrochloric aid; and
adjust volume to 500 mL with reagent-grade water.
(C) Adjustment of buffer concentrations. (1) The concentrations of
all the above buffer solutions are the maximum concentration to be
employed in carrying out hydrolysis measurements. If the
[[Page 100]]
initial concentration of the test chemical is less than 10-3
M, the buffer concentration shall be lowered by a corresponding amount;
e.g., if the initial test chemical concentration is 10-4 M,
the concentration of the above buffers shall be reduced by a factor of
10. In addition, for those reactions in which an acid or base is not a
reaction product, the minimum buffer concentration necessary for
maintaining the pH within +0.05 units shall be employed.
(2) Check the pH of all buffer solutions with a pH meter at 25
deg.C and adjust the pH to the proper value, if necessary.
(D) Preparation of test solution. (1) If the test chemical is
readily soluble in water, prepare an aqueous solution of the chemical in
the appropriate buffer and determine the concentration of the chemical.
Alternatively, a solution of the chemical in water may be prepared and
added to an appropriate buffer solution and the concentration of the
chemical then determined. In the latter case, the aliquot shall be small
enough so that the concentration of the buffer in the final solution and
the pH of the solution remain essentially unchanged. Do not employ heat
in dissolving the chemical. The final concentration shall not be greater
than one-half the chemical's solubility in water and not greater than
10-3 M.
(2) If the test chemical is too insoluble in pure water to permit
reasonable handling and analytical procedures, it is recommended that
the chemical be dissolved in reagent-grade acetonitrile and buffer
solution and then added to an aliquot of the acetonitrile solution. Do
not employ heat to dissolve the chemical in acetonitrile. The final
concentration of the test chemical shall not be greater than one-half
the chemical's solubility in water and not greater than 10-3
M. In addition, the final concentration of the acetonitrile shall be one
volume percent or less.
(3) Performance of the test. Carry out all hydrolysis experiments by
employing one of the procedures described in this paragraph. Prepare the
test solutions as described in paragraph (b)(2)(i) of this section at pH
3.00, 7.00, and 11.000.05, and determine the initial test
chemical concentration (Co) in triplicate. Analyze each
reaction mixture in triplicate at regular intervals, employing one of
the following procedures:
(i) Procedure 1. Analyze each test solution at regular intervals to
provide a minimum of six measurements with the extent of hydrolysis
between 20 to 70 percent. Rates should be rapid enough so that 60 to 70
percent of the chemical is hydrolyzed in 672 hours.
(ii) Procedure 2. If the reaction is too slow to conveniently follow
hydrolysis to high conversion in 672 hours but still rapid enough to
attain at least 20 percent conversion, take 15 to 20 time points at
regular intervals after 10 percent conversion is attained.
(iii) Procedure 3. (A) If chemical hydrolysis is less than 20
percent after 672 hours, determine the concentration (C) after this time
period.
(B) If the pH at the end of concentration measurements employing any
of the above three procedures has changed by more than 0.05 units from
the initial pH, repeat the experiment using a solution having a test
chemical concentration lowered sufficiently to keep the pH variation
within 0.05 pH units.
(iv) Analytical methodology. Select an analytical method that is
most applicable to the analysis of the specific chemical being tested
under paragraph (b)(1)(xi) of this section.
(c) Data and reporting--(1) Treatment of results. (i) If Procedure 1
or 2 were employed in making concentration measurements, use a linear
regression analysis with Equation 4 under paragraph (a)(2)(v)(B) of this
section to calculate kh at 25 deg.C for each pH employed in
the hydrolysis experiments. Calculate the coefficient of determination
(R2) for each rate constant. Use Equation 3 under paragraph
(a)(2)(v)(B) of this section to calculate the hydrolysis half-life using
kh.
(ii) If Procedure 3 was employed in making rate measurements, use
the mean initial concentration (Co) and the mean
concentration of chemical (C) in Equation 4 under paragraph (a)(2)(v)(B)
of this section to calculate kh for each pH used in the
experiments. Calculate the hydrolysis half-life using kh in
Equation 3 under paragraph (a)(2)(v)(B) of this section.
[[Page 101]]
(iii) For each set of three concentration replicates, calculate the
mean value of C and the standard deviation.
(iv) For test chemicals that are not ionized or protonated between
pH 3 and 11, calculate kA, kB, and kN
using Equation 5.
(2) Specific analytical and recovery procedures. (i) Provide a
detailed description or reference for the analytical procedure used,
including the calibration data and precision.
(ii) If extraction methods were used to separate the solute from the
aqueous solution, provide a description of the extraction method as well
as the recovery data.
(3) Test data report. (i) For Procedures 1 and 2, report
kh, the hydrolysis half-life (t1/2), and the
coefficient of determination (R2) for each pH employed in the
rate measurements. In addition, report the individual values, the mean
value, and the standard deviation for each set of replicate
concentration measurements. Finally, report kA,
kB, and kN.
(ii) For Procedure 3, report kh and the half-life for
each pH employed in the rate measurements. In addition, report the
individual values, the mean value, and the standard deviation for each
set of replicate concentration measurements. Finally, report
kA, kB, and kN.
(iii) If, after 672 hours, the concentration (C) is the same as the
initial concentration (Co) within experimental error, then
kh cannot be calculated and the chemical can be reported as
being persistent with respect to hydrolysis.
[50 FR 39252, Sept. 27, 1985, as amended at 53 FR 10391, Mar. 31, 1988;
53 FR 12526, Apr. 15, 1988; 53 FR 22323, June 15, 1988; 60 FR 34467,
July 3, 1995]
PART 797--ENVIRONMENTAL EFFECTS TESTING GUIDELINES--Table of Contents
Subpart A [Reserved]
Subpart B--Aquatic Guidelines
Sec.
797.1050 Algal acute toxicity test.
797.1300 Daphnid acute toxicity test.
797.1330 Daphnid chronic toxicity test.
797.1400 Fish acute toxicity test.
797.1600 Fish early life stage toxicity test.
797.1930 Mysid shrimp acute toxicity test.
797.1950 Mysid shrimp chronic toxicity test.
Authority: 15 U.S.C. 2603.
Source: 50 FR 39321, Sept. 27, 1985, unless otherwise noted.
Subpart A [Reserved]
Subpart B--Aquatic Guidelines
Sec. 797.1050 Algal acute toxicity test.
(a) Purpose. The guideline in this section is intended for use in
developing data on the acute toxicity of chemical substances and
mixtures (``chemicals'') subject to environmental effects test
regulations under the Toxic Substances Control Act (TSCA) (Pub. L. 94-
469, 90 Stat. 2003, 15 U.S.C. 2601 et seq.). This guideline prescribes
test procedures and conditions using freshwater and marine algae to
develop data on the phytotoxicity of chemicals. The United States
Environmental Protection Agency (U.S. EPA) will use data from these
tests in assessing the hazard of a chemical to the environment.
(b) Definitions. The definitions in section 3 of the Toxic
Substances Control Act (TSCA) and the definitions in part 792--Good
Laboratory Practice Standards of this chapter apply to this test
guideline. The following definitions also apply to this guideline:
(1) Algicidal means having the property of killing algae.
(2) Algistatic means having the property of inhibiting algal growth.
(3) ECx means the experimentally derived chemical concentration that
is calculated to effect X percent of the test criterion.
(4) Growth means a relative measure of the viability of an algal
population based on the number and/or weight of algal cells per volume
of nutrient medium or test solution in a specified period of time.
(5) Static system means a test container in which the test solution
is not renewed during the period of the test.
(c) Test procedures--(1) Summary of the test. (i) In preparation for
the test, fill test containers with appropriate volumes of nutrient
medium and/or test solution. Start the test by introducing algae into
the test and control containers in the growth chambers. Environmental
conditions within the growth
[[Page 102]]
chambers are established at predetermined limits.
(ii) At the end of 96 hours enumerate the algal cells in all
containers to determine inhibition or stimulation of growth in test
containers compared to controls. Use data to define the concentration-
response curve, and calculate the EC10, EC50, and
EC90 values.
(2) [Reserved]
(3) Range-finding test. (i) A range-finding test should be conducted
to determine:
(A) If definitive testing is necessary.
(B) Test chemical concentrations for the definitive test.
(ii) Algae are exposed to a widely spaced (e.g., log interval)
chemical concentration series. The lowest value in the series, exclusive
of controls, should be at the chemical's detection limit. The upper
value, for water soluble compounds, should be the saturation
concentration. No replicates are required; and nominal concentrations of
the chemical are acceptable unless definitive testing is not required.
(iii) The test is performed once for each of the recommended algal
species or selected alternates. Test chambers should contain equal
volumes of test solution and approximately 1 x 104
Selenastrum cells/ml or 7.7 x 10\4\ Skeletonema cells/ml of test
solution. The algae should be exposed to each concentration of test
chemical for up to 96 hours. The exposure period may be shortened if
data suitable for the purposes of the range-finding test can be obtained
in less time.
(iv) Definitive testing is not necessary if the highest chemical
concentration tested (water saturation concentration or 1000 mg/l)
results in less than a 50 percent reduction in growth or if the lowest
concentration tested (analytical detection limit) results in greater
than a 50 percent reduction in growth.
(4) Definitive test. (i) The purpose of the definitive test is to
determine the concentration response curves, the EC10's,
EC50's, and EC90's for algal growth for each
species tested, with a minimum amount of testing beyond the range-
finding test.
(ii) Algae should be exposed to five or more concentrations of the
test chemical in a geometric series in which the ratio is between 1.5
and 2.0 (e.g., 2, 4, 8, 16, 32, and 64 mg/l). Algae shall be placed in a
minimum of three replicate test containers for each concentration of
test chemical and control. More than three replicates may be required to
provide sufficient quantities of test solution for determination of test
substance concentration at the end of the test. Each test chamber should
contain equal volumes of test solution and approximately
1 x 104 Selenastrum cells/ml or 7.7 x 104
Skeletonema cells/ml of test solution. The chemical concentrations
should result in greater than 90 percent of algal growth being inhibited
or stimulated at the highest concentrations of test substance compared
to controls.
(iii) Every test shall include a control consisting of the same
nutrient medium, conditions, procedures, and algae from the same
culture, except that none of the test substance is added. If a carrier
is present in any of the test chambers, a separate carrier control is
required.
(iv) The test begins when algae from 5- to 10-day-old stock cultures
are placed in the test chambers containing test solutions having the
appropriate concentrations of the test substance. Algal growth in
controls should reach the logarithmic growth phase by 96 hours. If
logarithmic growth cannot be demonstrated, the test shall be repeated.
At the end of 24, 48, 72, and 96 hours the algal growth response (number
or weight of algal cells/ml) in all test containers and controls shall
be determined by an indirect (spectrophotometry, electronic cell
counters, dry weight, etc.) or a direct (actual microscopic cell count)
method. Indirect methods shall be calibrated by a direct microscopic
count. The percentage inhibition or stimulation of growth for each
concentration, EC10, EC50, EC90 and the
concentration-response curves are determined from these counts.
(v) At the end of the definitive test, the following additional
analyses of algal growth response shall be performed:
(A) Determine whether the altered growth response between controls
and test algae was due to a change in relative cell numbers, cell sizes
or both. Also note any unusual cell shapes,
[[Page 103]]
color differences, flocculations, adherence of algae to test containers,
or aggregation of algal cells.
(B) In test concentrations where growth is maximally inhibited,
algistatic effects may be differentiated from algicidal effects by the
following two methods for Skeletonema and by the second method for
Selenastrum.
(1) Add 0.5 ml of a 0.1 percent solution (weight/volume) of Evans
blue stain to a 1 milliliter aliquot of algae from a control container
and to a 1 milliliter aliquot of algae from the test container having
the lowest concentration of test chemical which completely inhibited
algal growth (if algal growth was not completely inhibited, select an
aliquot of algae for staining from the test container having the highest
concentration of test chemical which inhibited algal growth). Wait 10 to
30 minutes, examine microscopically, and determine the percent of the
cells which stain blue (indicating cell mortality). A staining control
shall be performed concurrently using heat-killed or formaldehyde-
preserved algal cells; 100 percent of these cells shall stain blue.
(2) Remove 0.5 ml aliquots of test solution containing growth-
inhibited algae from each replicate test container having the
concentration of test substance evaluated in paragraph (c)(4)(v)(B)(1)
of this section. Combine these aliquots into a new test container and
add a sufficient volume of fresh nutrient medium to dilute the test
chemical to a concentration which does not affect growth. Incubate this
subculture under the environmental conditions used in the definitive
test for a period of up to 9 days, and observe for algal growth to
determine if the algistatic effect noted after the 96-hour test is
reversible. This subculture test may be discontinued as soon as growth
occurs.
(5) [Reserved]
(6) Analytical measurements--(i) Chemical. (A) Glass distilled or
deionized water shall be used in the preparation of the nutrient medium.
The pH of the test solution shall be measured in the control and test
containers at the beginning and at the end of the definitive test. The
concentration of test chemical in the test containers shall be
determined at the beginning and end of the definitive test by standard
analytical methods which have been validated prior to the test. An
analytical method is unacceptable if likely degradation products of the
chemical, such as hydrolysis and oxidation products, give positive or
negative interference.
(B) At the end of the test and after aliquots have been removed for
algal growth-response determinations, microscopic examination, mortal
staining, or subculturing, the replicate test containers for each
chemical concentration may be pooled into one sample. An aliquot of the
pooled sample may then be taken and the concentration of test chemical
determined. In addition, the concentration of test chemical associated
with the algae alone should be determined. Separate and concentrate the
algal cells from the test solution by centrifuging or filtering the
remaining pooled sample and measure the test substance concentration in
the algal-cell concentrate.
(ii) Numerical. Algal growth response (as percent of inhibition or
stimulation in the test solutions compared to the controls) is
calculated at the end of the test. Mean and standard deviation should be
calculated and plotted for each treatment and control. Appropriate
statistical analyses should provide a goodness-of-fit determination for
the concentration response curves. The concentration response curves are
plotted using the mean measured test solution concentrations obtained at
the end of the test.
(d) Test conditions--(1) Test species. Species of algae recommended
as test organisms for this test are the freshwater green alga,
Selenastrum capricornutum, and the marine diatom, Skeletonema costatum.
Algae to be used in acute toxicity tests may be initially obtained from
commercial sources and subsequently cultured using sterile technique.
Toxicity testing shall not be performed until algal cultures are shown
to be actively growing (i.e., capable of logarithmic growth within the
test period) in at least 2 subcultures lasting 7 days each prior to the
start of the definitive test. All algae used for a particular test shall
be from the same source and the same stock culture.
[[Page 104]]
Test algae shall not have been used in a previous test, either in a
treatment or a control.
(2) Facilities--(i) General. (A) Facilities needed to perform this
test include: a growth chamber or a controlled environment room that can
hold the test containers and will maintain the air temperature, lighting
intensity and photoperiod specified in this test guideline; apparatus
for culturing and enumerating algae; a source of distilled and/or
deionized water; and apparatus for carrying out analyses of the test
chemical.
(B) Disposal facilities should be adequate to accommodate spent
glassware, algae and test solutions at the end of the test and any bench
covering, lab clothing, or other contaminated materials.
(ii) Test containers. Erlenmeyer flasks should be used for test
containers. The flasks may be of any volume between 125 and 500 ml as
long as the same size is used throughout a test and the test solution
volume does not exceed 50 percent of the flask volume.
(iii) Cleaning and sterilization. New test containers may contain
substances which inhibit growth of algae. They shall therefore be
cleaned thoroughly and used several times to culture algae before being
used in toxicity testing. All glassware used in algal culturing or
testing shall be cleaned and sterilized prior to use according to
standard good laboratory practices.
(iv) Conditioning. Test containers should be conditioned by a rinse
with the appropriate test solutions prior to the start of the test.
Decant and add fresh test solutions after an appropriate conditioning
period for the test chemical.
(v) Nutrient medium. (A) Formulation and sterilization of nutrient
medium used for algal culture and preparation of test solutions should
conform to those currently recommended by the U.S. EPA for freshwater
and marine algal bioassays. No chelating agents are to be included in
the nutrient medium used for test solution preparation. Nutrient medium
should be freshly prepared for algal testing and may be dispensed in
appropriate volumes in test containers and sterilized by autoclaving or
filtration. The pH of the nutrient medium shall be 7.5 (0.1)
for Selenastrum and 8.1 (0.1) for Skeletonema at the start
of the test and may be adjusted prior to test chemical addition with
0.1N NaOH or HC1.
(B) Dilution water used for preparation of nutrient medium and test
solutions should be filtered, deionized or glass distilled. Saltwater
for marine algal nutrient medium and test solutions should be prepared
by adding a commercial, synthetic, sea salt formulation or a modified
synthetic seawater formulation to distilled/deionized water to a
concentration of 30 parts per thousand.
(vi) Carriers. Nutrient medium shall be used in making stock
solutions of the test chemical. If a carrier other than nutrient medium
is absolutely necessary to dissolve the chemical, the volume used shall
not exceed the minimum volume necessary to dissolve or suspend the
chemical in the test solution.
(3) Test parameters. (i) The test temperature shall be 24 deg.C for
Selenastrum and 20 deg.C for Skeletonema. Excursions from the test
temperature shall be no greater than 2 deg.C. Temperature
should be recorded hourly during the test.
(ii) Test chambers containing Selenastrum shall be illuminated
continuously and those containing Skeletonema shall be provided a 14-
hour light and 10-hour dark photoperiod with a 30 minute transition
period under fluorescent lamps providing 300 25 uEin/m\2\
sec (approximately 400 ft-c) measured adjacent to the test chambers at
the level of test solution.
(iii) Stock algal cultures should be shaken twice daily by hand.
Test containers shall be placed on a rotary shaking apparatus and
oscillated at approximately 100 cycles/minute for Selenastrum and at
approximately 60 cycles/minute for Skeletonema during the test. The rate
of oscillation should be determined at least once daily during testing.
(iv) The pH of nutrient medium in which algae are subcultured shall
be 7.5 (0.1) for Selenastrum and 8.1 (0.1) for
Skeletonema, and is not adjusted after the addition of the algae. The pH
of all test solutions shall be measured at the beginning and end of the
test.
[[Page 105]]
(v) Light intensity shall be monitored at least daily during the
test at the level of the test solution.
(e) Reporting. The sponsor shall submit to the EPA all data
developed by the test that are suggestive or predictive of acute
phytotoxicity. In addition to the general reporting requirements
prescribed in part 792--Good Laboratory Practice Standards of this
Chapter, the following shall be reported:
(1) Detailed information about the test organisms, including the
scientific name, method of verification, and source.
(2) A description of the test chambers and containers, the volumes
of solution in the containers, the way the test was begun (e.g.,
conditioning, test substance additions, etc.), the number of replicates,
the temperature, the lighting, and method of incubation, oscillation
rates, and type of apparatus.
(3) The concentration of the test chemical in the control and in
each treatment at the end of the test and the pH of the solutions.
(4) The number of algal cells per milliliter in each treatment and
control and the method used to derive these values at the beginning, 24,
48, and 72 hours, and end of the test; the percentage of inhibition or
stimulation of growth relative to controls; and other adverse effect in
the control and in each treatment.
(5) The 96-hour EC10, EC50, and
EC90 values, and when sufficient data have been generated,
the 24, 48, and 72 hour LC50's and 95 percent confidence
limits, the methods used to derive these values, the data used to define
the shape of the concentration-response curve and the goodness-of-fit
determination.
(6) Methods and data records of all chemical analyses of water
quality and test substance concentrations, including method validations
and reagent blanks.
(7) The results of any optional analyses such as: Microscopic
appearance of algae, size or color changes, percent mortality of cells
and the fate of subcultured cells, the concentration of test substance
associated with algae and test solution supernate or filtrate.
(8) If the range-finding test showed that the highest concentration
of the chemical tested (not less than 1000 mg/l or saturation
concentration) had no effect on the algae, report the results and
concentration and a statement that the chemical is of minimum phytotoxic
concern.
(9) If the range-finding test showed greater than a 50 percent
inhibition of algal growth at a test concentration below the analytical
detection limit, report the results, concentration, and a statement that
the chemical is phytotoxic below the analytical detection limit.
[50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19058, May 20, 1987]
Sec. 797.1300 Daphnid acute toxicity test.
(a) Purpose. This guideline is intended for use in developing data
on the acute toxicity of chemical substances and mixtures
(``chemicals'') subject to environmental effects test regulations under
the Toxic Substances Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003,
15 U.S.C. 2601 et seq.). This guideline prescribes an acute toxicity
test in which daphnids (Daphnia magna or D. pulex) are exposed to a
chemical in static and flow-through systems. The United States
Environmental Protection Agency will use data from this test in
assessing the hazard a chemical may present in the aquatic environment.
(b) Definitions. The definitions in section 3 of the Toxic
Substances Control Act (TSCA) and part 792--Good Laboratory Practice
Standards of this chapter apply to this test guideline. In addition, the
following definitions apply to this guideline:
(1) Brood stock means the animals which are cultured to produce test
organisms through reproduction.
(2) EC50 means that experimentally derived concentration
of test substance in dilution water that is calculated to affect 50
percent of a test population during continuous exposure over a specified
period of time. In this guideline, the effect measured is
immobilization.
(3) Ephippium means a resting egg which develops under the carapace
in response to stress conditions in daphnids.
(4) Flow-through means a continuous or an intermittent passage of
test solution or dilution water through a test
[[Page 106]]
chamber or culture tank with no recycling.
(5) Immobilization means the lack of movement by the test organisms
except for minor activity of the appendages.
(6) Loading means the ratio of daphnid biomass (grams, wet weight)
to the volume (liters) of test solution in a test chamber at a point in
time, or passing through the test chamber during a specific interval.
(7) Static system means a test system in which the test solution and
test organisms are placed in the test chamber and kept there for the
duration of the test without renewal of the test solution.
(c) Test procedures--(1) Summary of the test. (i) Test chambers are
filled with appropriate volumes of dilution water. In the flow-through
test, the flow of dilution water through each chamber is adjusted to the
rate desired. The test chemical is introduced into each treatment
chamber. The addition of test chemical in the flow-through system is
conducted at a rate which is sufficient to establish and maintain the
desired concentration in the test chamber. The test is started within 30
minutes after the test chemical has been added and uniformly distributed
in static test chambers or after the concentration of test chemical in
each flow-through test chamber reaches the prescribed level and remains
stable. At the initiation of the test, daphnids which have been cultured
and acclimated in accordance with the test design are randomly placed
into the test chambers. Daphnids in the test chambers are observed
periodically during the test, the immobile daphnids removed, and the
findings recorded.
(ii) Dissolved oxygen concentration, pH, temperature, the
concentration of test chemical and other water quality parameters are
measured at specified intervals in selected test chambers. Data are
collected during the test to develop concentration-response curves and
determine EC50 values for the test chemical.
(2) [Reserved]
(3) Range-finding test. (i) A range-finding test should be conducted
to establish test solution concentrations for the definitive test.
(ii) The daphnids should be exposed to a series of widely spaced
concentrations of the test chemical (e.g., 1, 10, 100 mg/1, etc.),
usually under static conditions.
(iii) A minimum of five daphnids should be exposed to each
concentration of test chemical for a period of 48 hours. The exposure
period may be shortened if data suitable for the purpose of the range-
finding test can be obtained in less time. No replicates are required
and nominal concentrations of the chemical are acceptable.
(4) Definitive test. (i) The purpose of the definitive test is to
determine the concentration-response curves and the 24- and 48-hour
EC50 values with the minimum amount of testing beyond the
range-finding test.
(ii) A minimum of 20 daphnids per concentration shall be exposed to
five or more concentrations of the chemical chosen in a geometric series
in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, and 64
mg/l). An equal number of daphnids shall be placed in two or more
replicates. If solvents, solubilizing agents or emulsifiers have to be
used, they shall be commonly used carriers and shall not possess a
synergistic or antagonistic effect on the toxicity of the test chemical.
The concentration of solvent should not exceed 0.1 mg/l. The
concentration ranges shall be selected to determine the concentration-
response curves and EC50 values at 24 and 48 hours.
Concentration of test chemical in test solutions should be analyzed
prior to use.
(iii) Every test shall include controls consisting of the same
dilution water, conditions, procedures and daphnids from the same
population (culture container), except that none of the chemical is
added.
(iv) The dissolved oxygen concentration, temperature and pH shall be
measured at the beginning and end of the test in each chamber.
(v) The test duration is 48 hours. The test is unacceptable if more
than 10 percent of the control organisms are immobilized during the 48-
hour test period. Each test chamber shall be checked for immobilized
daphnids at 24 and 48 hours after the beginning of the test.
Concentration-response curves and 24-hour and 48-hour EC50
values for
[[Page 107]]
immobilization shall be determined along with their 95 percent
confidence limits.
(vi) In addition to immobility, any abnormal behavior or appearance
shall also be reported.
(vii) Test organisms shall be impartially distributed among test
chambers in such a manner that test results show no significant bias
from the distributions. In addition, test chambers within the testing
area shall be positioned in a random manner or in a way in which
appropriate statistical analyses can be used to determine the variation
due to placement.
(viii) The concentration of the test chemical in the chambers should
be measured as often as is feasible during the test. In the static test
the concentration of test chemical shall be measured, at a minimum, at
the beginning of the test and at the end of the test in each test
chamber. In the flow-through test the concentration of test chemical
shall be measured at a minimum:
(A) In each chamber at the beginning of the test and at 48 hours
after the start of the test;
(B) In at least one appropriate chamber whenever a malfunction is
detected in any part of the test substance delivery system.
Among replicate test chambers of a treatment concentration, the measured
concentration of the test chemical shall not vary more than
20 percent.
(5) [Reserved]
(6) Analytical measurements. (i) Test chemical. Deionized water
should be used in making stock solutions of the test chemical. Standard
analytical methods should be used whenever available in performing the
analyses. The analytical method used to measure the amount of test
chemical in a sample shall be validated before beginning the test by
appropriate laboratory practices. Any analytical method is not
acceptable if likely degradation products of the test chemical, such as
hydrolysis and oxidation products, give positive or negative
interferences which cannot be systematically identified and corrected
mathematically.
(ii) Numerical. The number of immobilized daphnids shall be counted
during each definitive test. Appropriate statistical analyses should
provide a goodness-of-fit determination for the concentration-response
curves. A 24- and 48-hour EC50 and corresponding 95 percent
interval shall be calculated.
(d) Test conditions--(1) Test species--(i) Selection. (A) The
cladocerans, Daphnia magna or D. pulex, are the test species to be used
in this test. Either species may be used for testing of a particular
chemical. The species identity of the test organisms should be verified
using appropriate systematic keys. First instar daphnids, 24
hours old, are to be used to start the test.
(B) Daphnids to be used in acute toxicity tests should be cultured
at the test facility. Records should be kept regarding the source of the
initial stock and culturing techniques. All organisms used for a
particular test shall have originated from the same culture population.
(C) Daphnids shall not be used for a test (1) if cultures contain
ephippia; (2) if adults in the cultures do not produce young before day
12; (3) if more than 20 percent of the culture stock die during the 2
days preceding the test; (4) if adults in the culture do not produce an
average of at least 3 young per adult per day over the 7-day period
prior to the test and (5) if daphnids have been used in any portion of a
previous test, either in a treatment or in a control.
(ii) Acclimation. (A) Brood daphnids shall be maintained in 100-
percent dilution water at the test temperature for at least 48 hours
prior to the start of the test. This is easily accomplished by culturing
them in the dilution water at the test temperature. During production of
neonates, daphnids should not be fed.
(B) During culturing and acclimation to the dilution water, daphnids
should be maintained in facilities with background colors and light
intensities similar to those of the testing area.
(iii) Care and handling. (A) Daphnids should be cultured in dilution
water under similar environmental conditions to those used in the test.
Organisms should be handled as little as possible. When handling is
necessary it should be done as gently, carefully, and quickly as
possible. During culturing and acclimation, daphnids should be observed
carefully for ephippia and
[[Page 108]]
other signs of stress, physical damage and mortality. Dead and abnormal
individuals shall be discarded. Organisms that touch dry surfaces or are
dropped or injured in handling shall be discarded.
(B) Smooth glass tubes (I.D. greater than 5 mm) equipped with rubber
bulb should be used for transferring daphnids with minimal culture media
carry-over. Care should be exercised to introduce the daphnids below the
surface of any solution to avoid trapping air under the carapace.
(iv) Feeding. A variety of foods (e.g., unicellular green algae)
have been demonstrated to be adequate for daphnid culture. Daphnids
shall not be fed during testing.
(2) Facilities--(i) Apparatus. (A) Facilities needed to perform this
test include: (1) Containers for culturing and acclimating daphnids; (2)
a mechanism for controlling and maintaining the water temperature during
the culturing, acclimation, and test periods; (3) apparatus for
straining particulate matter, removing gas bubbles, or aerating the
water as necessary; and (4) an apparatus for providing a 16-hour light
and 8-hour dark photoperiod with a 15 to 30 minute transition period. In
addition, the flow-through system shall contain appropriate test
chambers in which to expose daphnids to the test chemical and an
appropriate test substance delivery system.
(B) Facilities should be well ventilated and free of fumes and
disturbances that may affect the test organisms.
(C) Test chambers shall be loosely covered to reduce the loss of
test solution or dilution water due to evaporation and to minimize the
entry of dust or other particulates into the solutions.
(ii) Construction materials. (A) Materials and equipment that
contact test solutions should be chosen to minimize sorption of test
chemicals from the dilution water and should not contain substances that
can be leached into aqueous solution in quantities that can affect the
test results.
(B) For static tests, daphnids can be conveniently exposed to the
test chemical in 250 ml beakers or other suitable containers.
(C) For flow-through tests, daphnids can be exposed in glass or
stainless steel containers with stainless steel or nylon screen bottoms.
The containers should be suspended in the test chamber in such a manner
to insure that the test solution flows regularly into and out of the
container and that the daphnids are always submerged in at least 5
centimeters of test solution. Test chambers can be constructed using 250
ml beakers or other suitable containers equipped with screened overflow
holes, standpipes or V-shaped notches.
(iii) Dilution water. (A) Surface or ground water, reconstituted
water or dechlorinated tap water are acceptable as dilution water if
daphnids will survive in it for the duration of the culturing,
acclimation and testing periods without showing signs of stress. The
quality of the dilution water should be constant and should meet the
following specifications:
------------------------------------------------------------------------
Substance Maximum concentration
------------------------------------------------------------------------
Particulate matter........................ 20 mg/liter.
Total organic carbon or................... 2 mg/liter.
Chemical oxygen demand.............. 5 mg/liter.
Un-ionized ammonia........................ 1 g/liter.
Residual chlorine......................... <3 g/liter.
Total organophosphorus pesticides......... 50 ng/liter.
Total organochlorine pesticides plus 50 ng/liter.
polychlorinated biphenyls (PCBs) or.
Organic chlorine.......................... 25 ng/liter.
------------------------------------------------------------------------
(B) The above water quality parameters under paragraph
(d)(2)(iii)(A) of this section shall be measured at least twice a year
or whenever it is suspected that these characteristics may have changed
significantly. If dechlorinated tap water is used, daily chlorine
analysis shall be performed.
(C) If the diluent water is from a ground or surface water source,
conductivity and total organic carbon (TOC) or chemical oxygen demand
(COD) shall be measured. Reconstituted water can be made by adding
specific amounts of reagent-grade chemicals to deionized or distilled
water. Glass distilled or carbon-filtered deionized water with a
conductivity less than 1 ohm/cm is acceptable as the diluent
for making reconstituted water.
(iv) Cleaning. All test equipment and test chambers shall be cleaned
before
[[Page 109]]
each use using standard laboratory procedures.
(v) Test substance delivery system. In flow-through tests,
proportional diluters, metering pump systems, or other suitable devices
should be used to deliver test chemical to the test chambers. The system
shall be calibrated before each test. Calibration includes determining
the flow rate through each chamber and the concentration of the test
chemical in each chamber. The general operation of the test substance
delivery system should be checked twice during a test. The 24-hour flow
through a test chamber shall be equal to at least 5 times the volume of
the test chamber. During a test, the flow rates should not vary more
than 10 percent from any one test chamber to another.
(3) Test parameters. Environmental parameters of the water contained
in test chambers shall be maintained as specified below:
(i) The test temperature shall be 20 deg.C. Excursions from the
test temperature shall be no greater than 2 deg.C.
(ii) Dissolved oxygen concentration between 60 and 105 percent
saturation. Aeration, if needed to achieve this level, shall be done
before the addition of the test chemical. All treatment and control
chambers shall be given the same aeration treatment.
(iii) The number of daphnids placed in a test chamber shall not
affect test results. Loading shall not exceed 40 daphnids per liter test
solution in the static system. In the flow-through test, loading limits
will vary depending on the flow rate of dilution water. Loading shall
not cause the dissolved oxygen concentration to fall below the
recommended levels.
(iv) Photoperiod of 16 hours light and 8 hours darkness.
(e) Reporting. The sponsor shall submit to the U.S. EPA all data
developed by the test that are suggestive or predictive of acute
toxicity and all concomitant gross toxicological manifestations. In
addition to the reporting requirements prescribed in part 792--Good
Laboratory Practice Standards of this chapter, the reporting of test
data shall include the following:
(1) The name of the test, sponsor, testing laboratory, study
director, principal investigator, and dates of testing.
(2) A detailed description of the test chemical including its
source, lot number, composition (identity and concentration or major
ingredients and major impurities), known physical and chemical
properties and any carriers or other additives used and their
concentrations.
(3) The source of the dilution water, its chemical characteristics
(e.g., conductivity, hardness, pH, etc.) and a description of any
pretreatment.
(4) Detailed information about the daphnids used as brood stock,
including the scientific name and method of verification, age, source,
treatments, feeding history, acclimation procedures, and culture method.
The age of the daphnids used in the test shall be reported.
(5) A description of the test chambers, the volume of solution in
the chambers, the way the test was begun (e.g., conditioning, test
chemical additions), the number of test organisms per test chamber, the
number of replicates per treatment, the lighting, the method of test
chemical introduction or the test substance delivery system and the flow
rate (in flow-through test) expressed as volume additions per 24 hours.
(6) The concentration of the test chemical in each test chamber at
times designated for static and flow-through tests.
(7) The number and percentage of organisms that were immobilized or
showed any adverse effects in each test chamber at each observation
period.
(8) Utilizing the average measured test chemical concentration,
concentration-response curves should be fitted to immobilization data at
24 and 48 hours. A statistical test of goodness-of-fit should be
performed and the results reported.
(9) The 24- and 48-hour EC50 values and their respective
95 percent confidence limits using the mean measured test chemical
concentration and the methods used to calculate both the EC50
values and their confidence limits.
(10) All chemical analyses of water quality and test chemical
concentrations, including methods, method validations and reagent
blanks.
[[Page 110]]
(11) The data records of the culture, acclimation and test
temperatures.
(12) Any deviation from this test guideline and anything unusual
about the test, e.g., diluter failure, temperature fluctuations, etc.
[50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19059, May 20, 1987]
Sec. 797.1330 Daphnid chronic toxicity test.
(a) Purpose. This guideline is intended for use in developing data
on the chronic toxicity of chemical substances and mixtures
(``chemicals'') subject to environmental effects test regulations under
the Toxic Substances Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003,
15 U.S.C. 2601 et seq.). This guideline prescribes a chronic toxicity
test in which daphnids are exposed to a chemical in a renewal or a flow-
through system. The United States Environmental Protection Agency will
use data from this test in assessing the hazard a chemical may present
to the aquatic environment.
(b) Definitions. The definitions in section 3 of the Toxic
Substances Control Act (TSCA), and the definitions in part 792 Good
Laboratory Practice Standards of this chapter apply to this test
guideline. In addition, the following definitions apply to this
guideline:
(1) Brood stock means the animals which are cultured to produce test
organisms through reproduction.
(2) Chronic toxicity test means a method used to determine the
concentration of a substance in water that produces an adverse effect on
a test organism over an extended period of time. In this test guideline,
mortality and reproduction (and optionally, growth) are the criteria of
toxicity.
(3) EC50 means that experimentally derived concentration
of test substance in dilution water that is calculated to affect 50
percent of a test population during continuous exposure over a specified
period of time. In this guideline, the effect measured is
immobilization.
(4) Ephippium means a resting egg which develops under the carapace
in response to stress conditions in daphnids.
(5) Flow-through means a continuous or intermittent passage of test
solution or dilution water through a test chamber or culture tank with
no recycling.
(6) Immobilization means the lack of movement by daphnids except for
minor activity of the appendages.
(7) Loading means the ratio of daphnid biomass (grams, wet weight)
to the volume (liters) of test solution in a test chamber at a point in
time or passing through the test chamber during a specific interval.
(8) MATC (Maximum Acceptable Toxicant Concentration) means the
maximum concentration at which a chemical can be present and not be
toxic to the test organism.
(9) Renewal system means the technique in which test organisms are
periodically transferred to fresh test solution of the same composition.
(c) Test procedures--(1) Summary of the test. (i) Test chambers are
filled with appropriate volumes of dilution water. In the flow-through
test the flow of dilution water through each chamber is then adjusted to
the rate desired. The test substance is introduced into each test
chamber. The addition of test substance in the flow-through system is
done at a rate which is sufficient to establish and maintain the desired
concentration of test substance in the test chamber.
(ii) The test is started within 30 minutes after the test substance
has been added and uniformly distributed in the test chambers in the
renewal test or after the concentration of test substance in each test
chamber of the flow-through test system reaches the prescribed level and
remains stable. At the initiation of the test, daphnids which have been
cultured or acclimated in accordance with the test design, are randomly
placed into the test chambers. Daphnids in the test chambers are
observed periodically during the test, immobile adults and offspring
produced are counted and removed, and the findings are recorded.
Dissolved oxygen concentration, pH, temperature, the concentration of
test substance, and other water quality parameters are measured at
specified intervals in selected test chambers. Data are collected during
the test to determine any significant differences (p0.05) in
immobilization and reproduction as compared to the control.
[[Page 111]]
(2) [Reserved]
(3) Range-finding test. (i) A range-finding test should be conducted
to establish test solution concentrations for the definitive test.
(ii) The daphnids should be exposed to a series of widely spaced
concentrations of the test substance (e.g., 1, 10, 100 mg/l), usually
under static conditions.
(iii) A minimum of five daphnids should be exposed to each
concentration of test substance for a period of time which allows
estimation of appropriate chronic test concentrations. No replicates are
required and nominal concentrations of the chemical are acceptable.
(4) Definitive test. (i) The purpose of the definitive test is to
determine concentration-response curves, EC