PB86-129335
Hazard Evaluation  Division,  Standard
Evaluation Procedure:  Acute  Toxicity
Test for Estuarine and Marine Organisms
(Mollusc 48-Hour Embryo Larvae Study)
(U.S.) Environmental  Protection Agency
Washington, DC
Jun 85
                   J.S. DEPARTMENT OF COMMERCE
                  ional Technical Information Service


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                                     Pabb-129335


                                   EPA-540/9-85-012
                                   June  1985
              HAZARD EVALUATION DIVISION

            STANDARD EVALUATION PROCEDURE

ACUTE TOXICITY TEST FOR ESTUARINE AND MARINE  ORGANISMS

        (Mollusc 48-Hour Embryo Larvae  Study)
                     Prepared by

                 Daniel Rieder, M.S.
    Standard Evaluation Procedures  Project  Manager
                  Stephen L. Johnson
              Hazard Evaluation  Division
             Office of Pesticide  Programs
    United States Environmental  Protection  Agency
             Office of Pesticide Programs
               Washington,  D.C.   20460

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 50273-101
 REPORT DOCUMENTATION
         PAGE
                        1. REPORT NO.
               3. Recipient** Accession No.
                 PBSfc   129335/AS
 4. Title and Subtitle
   HAZARD EVALUATION  DIVISION,  STANDARD EVALUATION PROCEDURE
   Acute Toxicity  Test for Estuarine and Marine Organisms  (Mollusc
   48-Hour Embryo  Larvae Study)
               5. Report Oat*
                  June 1985
                  EPA-5W9-85-012"
 7. Authord)
   Daniel  Reider
                8. Performing Organization Rapt. No.
 9. Performing Organization Nam* and Addrata
  U.S.  Environmental  Protection Agency/OPP/HED/(TS-769C)
  401  M Street SW
  Washington, D.C.   20460
                10. Pro|«et/T»«k/Work Unit No.
                H. Contracted or Grant(O) No.

                (O

                (G)
 12. Sponsoring Organization Nam* and Address
  Same  as #9.
                13. Type of Report & Period Covered
                                                                         14.
 IS. Supplementary Notes
  Supplement to  Pesticide Assessment Guidelines Subdivision E - Hazard Evaluation:
  Wildlife and Aquatic Organisms  (EPA-540/9-82-024 and  NTIS Order  Number PB83-153908),
  October, 1982.
 16. Abstract (Limit: 200 words)
       The Standard  Evaluation  Procedure  (SEP)  for the  Mollusc 48-Hour Embryo-Larvae Toxicity
 Test  is  a guidance  document primarily intended for Agency reviewers  and the  regulated in-i
 dustry who evaluate ecological  effects  data  specified ;in 4*0 CFR  Part: 1158.145.  ilhe^SEPits
 also  intended to  provide information to  the  general public indicating how the  Agency evalu-
 ates  these types  of studies.   As such,  it  is  designed to supplement  Subdivision E of the
 Pesticide Assessment Guidelines:  Hazard Evaluation - Wildlife and Aquatic Organisms.
 This  SEP provides  an Introduction, Materials  and Methods, Reporting  Requirements, Reviewer
 Evaluation, and Appendix of appropriate  methods to guide the review  and scientific evalu-
 ation of pesticide  effects on estuarine/marine fish.

       The SEP  for  the Estuarine Fish 9&--ffiour  Acute Toxicity Test  is only one  of a number
 of  SEP's published  by the National Technical  Information Service  as  a supplement to Sub-
 division E of the  Pestici'de Assessment  Guidelines.
 17. Document Analysis  a. Descriptors
                                                                         Reproduced from
                                                                         best available copy.
   b. Identlfiers/OpeivEnded Terms
   c. COSAT1 Field/Group
 18. Availability Statement
  Unclassified and  freely available.
19. Security Cless (This Report)
   Unclassified.
                                                         20. Security Class (This Page)
                                                            Unclassi fied.
21. No. of Pages
         17
                                                                                   22. Prlcfl
(See ANSI-239.18)
                                         See Instructions on Reverse
                         OPTIONAL FORy 272 (4-77)
                         (Formerly NTIS-3S)
                         Department of Commerce

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                  STANDARD EVALUATION PROCEDURE

                             PREAMBLE


     This Standard Evaluation Procedure (SEP) is one of a set of

guidance documents which explain the procedures used to evaluate

environmental and human health effects data submitted to the

Office of Pesticide Programs.  The SEPs are designed to ensure

comprehensive and consistent treatment of major scientific topics

in these reviews and to provide interpretive policy guidance

where appropriate.  The standard Evaluation Procedures will be

used in conjunction with the appropriate pesticide Assessment

Guidelines and other Agency Guidelines.  While the documents were

developed to explain specifically the principles of scientific

evaluation within the Office of Pesticide programs, they may also

be used by other offices in the Agency in the evaluation of

studies and scientific data.  The standard Evaluation procedures

will also serve as valuable internal reference documents and will

inform the public and regulated community of important consider-

ations in the evaluation of test data for determining chemical

hazards.  I believe the SEPs will improve both the quality of

science within EPA and, in conjunction with the pesticide Assess-

ment Guidelines, will lead to more effective use of both public

and private resources.
                                     [. Melone, Director
                               Hazard Evaluation Division

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                          TABLE OF CONTENTS
  I.  INTRODUCTION
        A. When Required 	   1
        B. Purpose 	   1
        C. Test Material 	   1
              1. Technical Grade 	   1
              2. End-Use Product 	   1
 II.  MATERIALS AND METHODS:  TESTING STANDARDS/
     RECOMMENDATIONS

        A. Acceptable Protocols	   2
        B. Test Organisms	   3
              1. Acceptable Species 	   3
              2. Size/Age/Physical Condition  	   3
              3. Source/Acclimation 	   3
        C. Test Solution 	   3
              1. Source of Dilution Water  	   3
              2. Temperature 	   4
        D. Testing System  	   4
              1. Test Vessels 	   4
              2. Photoperiod	   4
              3. Load i ng 	   4
              4. Solvents  	   5
              5. Aeration  	   5
        E. Test Design 	   5
              1. Test Levels 	   5
              2. Number of Test Animals  	   5
              3. Controls  	   6
              4. Beg inning the Test 	   6
              5. Measurement of Diluent  Characteristics  .   6
              6. Chemical Analysis	   6
III.  REPORTING REQUIREMENTS

        A. Test Material	   7
        B. Dilution Water 	   7
        C. Holding of Test Organisms  	   7
        D. Observable Effects Criteria  	   8
        E. Calculated EC50 	~.	   8
        F. Temperature/DO/pH 	   8
        G. Chemical Analysis 	   8
        H. Testing Protocols 	   8
 IV.  REVIEWER'S EVALUATION

        A. Review of Test Conditions  	   9
        B. Verification of Statistical Analysis  	   9

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            TABLE OF CONTENTS (Continued)


                                                  Page

C. Conclusions 	  9
      1. Categorization of Results 	  9
      2. Rationale 	  10
      3. Repairability 	  10
D. Descriptive Classification 	  10
E. References	  11

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       ACUTE TOXICITY TEST FOR ESTUARINE AND MARINE ORGANISMS;
                   MOLLUSC 48-HOUR EMBRYO LARVAE STUDY
I.  INTRODUCTION

    A.  When Required

    Acute toxicity studies with mollusc are required to support
registration of an end-use product intended for direct application
to the estuarine or marine environment.  This study is also
necessary when it is expected that the pesticide would enter this
environment in significant concentrations because of its expected
use or mobility.

     B.  Purpose

     0  To establish acute toxicity levels of the active
        ingredient to nontarget marine and estuarine organisms;

     0  To compare toxicity information with measured or
        estimated pesticide residues in the estuarine or marine
        environment to assess potential impact to invertebrates;

     0  To provide support for precautionary label statements
        to minimize adverse effects to estuarine or marine non-
        target organisms; and

     0  To indicate the need for further testing and/or field
        studies .

     C.  Test Material

          1.  Technical Grade

     Tests must be conducted with the technical grade of the
active ingredient.  If more than one active ingredient constitutes
a technical product then the technical grade of each active
ingredient must be tested separately.

          2.  End-Use Product

     The applicant may be required to test the end-use product as
well if:

     0  The end-use product will be introduced directly into the
        marine or estuarine environment when used^as directed;
        The mollusc ECso of the technical grade of the active
        ingredient is equal to or less than the expected environ-
        mental concentration in the marine or estuarine environment
        when the end-use product is used as directed;

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                                  -2-
      0  An ingredient of the end-use product is expected to
         enhance the toxicity of the end-use product beyond that
         expected from the active ingredient(s); or

      0  The technical product is insoluble in water but the
         formulated product is soluble in water.  In that case the
         carriers or inerts of the formulated product must be tested
         in a solvent control.


II.   MATERIALS AND METHODS:  TESTING STANDARDS/RECOMMENDATIONS

      A.  Acceptable Protocols

      EEB does not endorse any one protocol.  It is sometimes
 necessary and desirable to alter the procedures presented in
 published protocols to meet the needs of the chemical or test
 organisms used.  However, EEB does recommend some protocols as
 guidance for developing mollusc acute toxicity tests.  These
 protocols include:

      American Public Health Association, American Water Works
      Association and Water Pollution Control Federation. 1981.
      Standard Methods for the Examination of Water and Wastewater.
      Fifteenth edition.  Publication office:  American Public
      Health Association, 1015 18th Street-NW, Washington, DC
      20036.

      American Society Testing Materials. 1980.  Standard Prac-
      tice for Conducting Static Acute Toxicity Tests with Larvae
      of Four Species of Bivalve Molluscs.  E 724-80, Published by
      the ASTM Committee on Standards, 1916 Race Street, Philadelphia,
      PA, 19103.

      Committee on Methods for Toxicity Tests with Aquatic
      Organisms.  1975.  Methods for Acute Toxicity Tests with
      Fish, Macroinvertebrates and Amphibians.  U.S. Environmental
      Protection Agency, Ecol. Res. Series, EPA 660/375-009. 61 pp.

      Woelke, C.E. 1967. "Measurement of Water Quality with the
      Pacific Oyster Embryo Bioassay."  Water Quality Criteria, ASTM
      STP 416, p. 112.

      These referenced protocols are presented as flexible guidance
 to help researchers design scientific protocol and to help the
 reviewer validate studies.  It is important to reeognize that
 mollusc tests are validated based on whether they provide scienti-
 fically sound information on the acute toxicity of the test
 material to mollusc that is useful in risk assessments and whether
 they fulfill guideline requirements.  This is more important than
 whether they follow a referenced protocol step-by-step.

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                                 -3-
     B.  Test Organisms

          1.  Acceptable Species

     The selected species should have a demonstrated sensitivity to
known toxicants.  If possible they should be species that occur
in the area of exposure or that represent exposed species.

     Numerous species are specifically referenced in "Methods of
Acute Toxicity Tests with Fish, Macroinvertebrates, and
Amphibians" (Committee on Methods for Toxicity Tests with Aquatic
Organisms, 1975, and ASTM E 729-80).  The following species are
preferred because there is a substantial data base available on
them:

         Crassostrea giga                    Pacific oyster
         Crassostrea virginica               Eastern oyster
         Mytilus edulis                      Mussel
         Mercenaria mercenaria               Quahog

Some other species may be acceptable if they have been shown to be
sensitive in acute toxicity tests.

          2.  Size/Age/Physical Condition

     Embryos should be tested within one hour of spawning and after
fertilization.  The eggs are fertilized as they are discharged from
the female; they are then added to treated test solution.

     The adults must be from clean water that is not subject to point
or non-point pollution so they will not have developed genetic
resistance to toxicants that would make their offspring less
sensitive to toxicants.

          3.  Source/Acclimation

     Mollusc larvae are obtained from adult brood stock in the labora-
tory.  This brood stock is obtained either from the wild or from
commercial sources and caused to spawn.  The referenced protocols
provide procedures for causing molluscs to spawn.

     All organisms must be from the same source.  This may include
laboratory or commercial stocks.  Organisms captured in the wild
are acceptable provided they meet the requirements pertaining to
physical condition and age/size criteria mentioned above.

     C.  Test Solution

          1.   Source of Dilution Water

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                                -4-
     An adequate supply of dilution water that meets the minimum
requirements must be available.  The water may be natural or
reconstituted but must be able to support the test animals without
stress.  Specifically, control larvae spawned by adults conditioned
in the dilution water must not incur more than 10% abnormal
development or 30% mortality in 48 hours.

     Natural or reconstituted seawater of 30 to 34% salinity and
pH of 8 to 8.3 should be used when testing marine (stenohaline)
mollusc, and 10 to 17% salinity and pH 7.7 to 8.0 with estuarine
(euryhaline) mollusc species.  Natural seawater is considered to
be of constant quality if the weekly range of salinity is less
than 6% and if the monthly pH range is less than 0.8 of a pH
unit.  See the protocols by the American Society for Testing
Materials (1980) or the Committee on Methods for Toxicity Tests
with Aquatic Organisms (1975) for guidance on specific amounts of
minerals in reconstituted seawater.  Commercial sources of seawater
mixture are acceptable provided they do not adversely affect the
test organism or alter the toxicity of the test material.

         2.  Temperature

     Most protocols recommend 20°C as the temperature.  However
it is possible that a higher temperature (e.g., 25°C) may be accept-
able when the adult molluscs were collected from warmer waters as
in southern areas.  Temperature should not vary more than 2°C
during the test.  Laboratories using molluscs collected in the
south where ambient temperatures may already be 27-28°C may have
difficulty raising the temperature the 5-10°C necessary to induce
spawning without exceeding the lethal limit (33-34°C) for adult
molluscs.

     D.  Testing System

          1.  Test Vessels

     Glass 1 Liter beakers are preferred.  It is strongly recommended
that each treatment be tested in duplicate or even triplicate.
There should be 20,000 to 30,000 fertilized eggs per liter of
test solution.

          2.  Photoperiod

     A 16-hour light and an 8-hour dark photoperiod should be
provided, with a 15 to 30 minute transition period between light
and dark.

          3.   Loading

     There should be 20,000 to 30,000 fertilized eggs per liter of
test solution.

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                              -5-
          4.  Solvents

     If solvents other than water are necessary, they should be
used sparingly, not to exceed 0.5 ml/L in any test solution.
The following solvents are preferred:

                   dimethyl formamide
                   triethylene glycol
                   methanol
                   acetone
                   ethanol

          5.  Aeration

     Aeration is not recommended because it could cause.volatili-
zation of test material.  However if aeration is necessary, the
residues in the test solution must be measured at the beginning
and near the end of the study.

     E.  Test Design

          1.  Test Levels (Nominal/Measured)

     Initially, range finding tests may be necessary to define
concentrations of the toxicant needed for definitive studies.
Test reports should provide information describing range finding
study procedures and results.  The information should include
sample sizes, dosage levels, and mortality data.  If there is a
problem with solubility or volatibility, if the test containers
are aerated, or if it is a flow-through test, the residue levels
must be measured at the beginning and at the end of the test
period.

     If it can be shown that a chemical will have an £€50 greater
than 100 ppm, a definitive study need not be performed.

     Definitive acute toxicity tests normally are designed to
include one or more control groups and a geometric series of at
least five toxicant concentrations to be tested.  Each designated
treatment group should be exposed to a concentration of toxicant
that is at least 60% of the next highest concentration.  If a
formulated product is tested, it should be clearly stated in the
test report whether results are expressed in terms of active
ingredient alone or as total formulated product.

          2.  Number of Test Animals

     Numbers of mollusc larvae per level are determined as a
certain density per volume of test solution.  If oyster larvae
are used, normally, 20,000 to 30,000 are tested per liter.
Replicate test chambers may be used, but the number of organisms
would still be measured as number of larvae per volume of test
solution.

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                                 -6-
          3.  Controls

     Each test requires a concurrent control using the same dilution
water and same concentrations of larvae at each test level.  The
number of control replicates should be four or the equivalent of
10% of the total number of treatment replicates, whichever is
greater.  If any solvent other than water is used a solvent
control should be conducted.  This solvent control should contain
the highest concentration of the solvent that was added to any of
the test solutions.  A test is not acceptable if there is more
than 30% mortality in the control.  If mean mortality exceeds 2%
in controls, each treatment response should be corrected for the
mean level of control using the following equation:

    Net Percent   (  percent of surviving larvae for    )
     Mortality = (	each treatment replicate	) x iQO
     For Each    (   mean percent of surviving control   )
      Level       (    larvae per control replicate     )

          4.  Beginning the Test

     Fill each test container with the same volume of test solution
and toxicant.  The larvae are then mixed with the test solution at
approximately 20,000 to 30,000 per liter.

          5.  Measurement of Diluent Characteristics

     Temperature should be recorded hourly throughout the acclimation
and test periods in at least one test chamber if the test containers
are not in a temperature controlled water bath because air temperature
may change more frequently and to a greater extent than water, thus
affecting the test container temperature.  If the temperature is
controlled by a water bath, the temperature of the bath may be
recorded every six hours.  The range and average temperature must
be recorded.

     The dissolved oxygen (DO) concentration must be measured at the
beginning of the test and at the end of the test in the control
and the high, medium, and low concentration.  The DO level must
be between 60% and 100% of saturation.

     The pH should be measured at the beginning and end of the test in
the control and the high, medium, and low toxicant concentrations.

          6.  Chemical Analysis

     It may be necessary to chemically analyze test solutions to
determine exact concentrations of pesticides.  It is particularly
important that residues are measured if:

     0  The test solutions were aerated;

     0  The test material was volatile, insoluble, or precipitated
        out of solution;

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                                   -7-
       0  The test containers were not made of glass or stainless
          steel;

       0  The test chemical is known to adsorb to the test container's
          structural material; or

       0  A flow-through system is used (measurement verifies accuracy
          of metering system).


III.   REPORTING REQUIREMENTS

         The test report submitted to the Agency must fully describe
  the materials and methods used to perform the study.   The reviewer
  must be able to determine from the report that the study was performed
  under conditions that render the results acceptable for use in a
  risk assessment and/or for fulfilling a guideline requirement.
  The following information is particularly important for a complete
  evaluation.

       A. . Test Material

       If the study is to be performed with the technical grade product,
  the test material should be clearly identified as to  source,
  batch, and exact purity.  Simply identifying the material as
  technical may not be sufficient because the percent active
  ingredient of some newer products may increase with time as the
  manufacturing process is improved to produce greater  purity.

       For studies involving the end-use product, the exact percent
  of  the active ingredient and the type of formulation  (e.g.,
  granular, wettable powder) of the test material should be described.
  It  should clearly state in the test report whether the results
  are expressed in terms of active ingredient or as formulation.

       B.  Dilution Water

       Test reports submitted to the Agency should include a complete
  description of  dilution waters used in the test.  Descriptions
  should include  identification of the source, the chemical
  characteristics of the water, and information on any  pretreatment.

       C.  Holding of Test Organisms

       Test reports should include complete information on holding and
  acclimation conditions including feeding schedules and treatment
  for diseases.

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                                 -8-
     D.  Observable Effects Criteria

     According to Woelke, fertilized eggs develop into free-swimming,
fully shelled veliger larvae in the 48 hours covered by this
protocol.  He assumes that failure to .do so will break the life
cycle of, in this case, the Pacific oyster.

     The measured response in this test should be the percentage
of larvae dying or failing to develop complete shells.  Larvae
possessing misshapen shells or otherwise malformed shells are
considered normal (survivable) as long as the shell is complete.
Incidences of misshapen shells or otherwise malformed shells
should be reported.

     To determine percent response per level, samples containing
about 150 to 250 larvae taken from each culture are examined.  The
number of fully developed larvae compared to not fully developed
larvae are counted and used in the following equation to obtain
percent response per level.

   Percent     ( # fully dev. in control -    )
  reduction  = ( ft fully dev. in test culture ) x IQO
  per test     ( # fully dev. in control      )
    level      (         culture              )

     E.  Calculated EC50

     The statistically calculated ECsg with 95% confidence limits
and the method of calculation must be presented.  In lieu of a
calculated EC$Q the study may show that the ECso is greater than
100 ppm.  The mean percent abnormal larvae from each test level
should be plotted on probability paper, with the concentrations
converted to logs.  The EC^Q may then be calculated by linear
regression.

     F.  Temperature/DO/pH

     Dissolved oxygen, pH measurements and the range and average
temperature during the study should be reported.

     G.  Chemical Analysis

     The test report should provide information on the methods
(references) utilized and the results of analyses.  Residues
found at the beginning and end of the study should be reported.

     H.  Testing Protocols

     The test report should include reference to the testing
protocol(s) used during the study.

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                                   -9-
IV.   REVIEWER'S EVALUATION

      A.  Review of Test Conditions

      The reviewer should identify each aspect of the reported procedure
 that is inconsistent with recommended protocol.  The significance
 of these deviations must be determined.  The number of deviations
 and their severity will determine the validity of the study and
 the interpretation of the results.

      B.  Verification of Statistical Analysis
      The reviewer should ensure that the ECso has been properly derived
 by recalculating these values.   An acceptable acute toxicity test
 should provide not only an EC$Q but also a NOEL (no observed
 effect level) and a slope of the dose/effect response.  The
 NOEL is the highest test level  at which no effects were observed.

      If the recalculated results differ substantially from the submitted
 results, the reviewer should note this and attempt to explain the
 differences .

      C.  Conclusions

           1.  Categorization of Results

      The significance of inconsistencies in the test procedures must
 be determined by the reviewer so that the results of the test can
 be categorized as to whether they fulfill Part 158 regulations
 and are useful in performing a  risk assessment.  Categories are
 described as:

      0  Core;  All essential information was reported and the study
         was performed according to recommended protocols.  Minor
         inconsistencies with standard methodologies may be apparent/
         however the deviations  do not detract from the study's sound-
         ness or intent.  Studies within this category fulfill the
         basic requirements of current guidelines and are acceptable
         for use in a risk assessment.

      0  Supplemental;  Studies  in this category are scientifically
         sound, however, they were performed under conditions that
         deviated substantially  from recommended protocols.  Results
         do not meet guideline requirements, however, the information
         may be useful in a risk assessment.  Some of the conditions
         that may place a study  in a supplemental category i.nclude:

              Unacceptable test  species;
              Inappropriate test material;
              Concentrations tested were less than 100 ppm but
              not high enough to produce an effect on the organ-
              isms or a precise  EC^Q; and

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                                 -10-
             Deviations from recommended test solution character-
             istics (variations in DO, temperature, hardness,
             and pH can affect toxicological response).

     0  Invalid;  These studies provide no useful information.
        They may be scientifically unsound, or they were performed
        under conditions that deviated so significantly from
        recommended protocols that the results will not be useful
        in a risk assessment.

        Examples of studies placed in this category commonly include
        those where the test system was aerated, test vessels were
        constructed from materials other than glass, or there were
        problems of solubility or volatility of the test material.
        Unless acceptable chemical analyses of actual toxicant
        concentrations were performed in studies such as these, the
        reviewer cannot be sure that test organisms were actually
        exposed to nominally designated concentrations.

        A study where the test material was not properly identified
        can also be made invalid.

          2.  Rationale

     Identify what makes the study supplemental or invalid.  While
all deviations from recommended protocol should be noted, the
reviewer is expected to exercise judgment in the area of study
categorization.

          3.  Repairability

    Indicate whether the study may be upgraded or given a higher
validation category if certain conditions are met.  Usually this
would involve the registrant submitting more data on the study.

     D.  Descriptive Classification

     The reviewer should indicate what the results were and how
much information can be drawn from them.  At a minimum, an acute
toxicity test will provide an ECso with 95% confidence limits.
This should allow classifying the test material based on the
following scheme:
                                        Category
                    (ppm)               Description

                    < 0.1             very highly toxic
                 0.1-1             highly toxic
                    1-10            moderately toxic
                  10 - 100           slightly toxic
                    > 100            practically non-toxic

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                                 -11-
These descriptive categories are for inter-chemical comparison
only and do not reflect actual environmental hazard to the test
organism.  The results may provide other useful information such
as slope or a no observed effect level (NOEL).  These additional
data are useful in a risk assessment.

     E.  References

     The reviewer should reference any information used in the
validation procedure.  This should include protocol documents,
statistical methods, or information taken from files of other
branches.

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