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  INTRODUCTION TO ENVIRONMENTAL EFFECTS TESTING GUIDELINES

In order to assess the environmental  risk associated with a
chemical substance, a determination of ecotoxicity
potential, along with information on  the transport  and  fate
of the substance in the environment,  is needed.  The extent
and degree to which a chemical may pose a potential hazard
can be characterized in part by its ecotoxicity to  plant,
animal and microbial species which are valued  for economic
or ecological importance.  Ecotoxicity can be  evaluated on
the-basis of those acute, subacute or chronic  effects which
result in death, inhibition of reproduction, or an
impairment of growth and development  of an organism.
Ecotoxicity also can occur as a result of the.presence or
accumulation of a chemical in or on one organism which  is
not affected by the chemical but serves as basic food source
for another organism which is affected.

Whether a chemical substance will cause ecotoxic effects is
greatly dependent upon the organism and the stage in the
life cycle in which exposure occurs and the conditions  under
which exposure occurs.  The toxicity  of a chemical  may  not
be the same to all organisms or to all levels  of biological
organization.  Ideally, such testing  should employ  test
organisms or systems which provide for the broadest range of
taxonomic representation and biological processes within the
constraints of the costs and resources available.   The  Test
Guidelines for Environmental Effects  Testing have been
selected with these constraints in mind.
The Guidelines use single species of plants and animals and

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incorporate the best state-of-the-art methodologies for
testing purposes.  It is anticipated that the species and
methodologies used in the Guidelines will be reviewed and
revised as the state-of-the-art changes.  Such changes may
include other single species tests, multi-species tests or
microcosm tests.

The Test Guidelines and Support Documents ace identified by
the prefixes EG and ES, respectively, and are numbered
sequentially.  Where applicable, each Test Guideline is
supported by a document which provides the scientific
background and rationale used in the development, of the Test
Guideline.  In some cases, a Support Document provides
support for two Test Guidelines.

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INDEX TO  ENVIRONMENTAL EFFECTS  TEST GUIDELINES AND  SUPPORT DOCUMENTS
Document . Guideline
Daphnid Acute Toxic ity Test
Daphnid Chronic Toxicity Test
Mys id Shrimp Acute Toxicity Test
Mys id Shrimp Chronic Toxicity Test
Oyster Acute Toxicity Test
Oyster Bioconcentration Test
Penaeid Shrimp Acute Toxicity Test
Algal Acute Toxicity Test
Fish Acute Toxicity Test
Fish Bioconcentration Test
Fish Early Life Stage Toxicity Test
Seed Germination/Root Elongation
Toxicity Test
Early Seedling Growth Toxicity Test
Plant Uptake and Translocation Test
Avian Dietary Test
Bobwhite Reproduction Test
Mallard Reproduction Test
Daphnid Chronic Toxicity Test (OECD)
Algal Acute Toxicity Test (OECD)
Fish Acute Toxicity Test (OECD)
Fish Bioconcentration Test (OECD)
Support
EG-1
EG-2
EG-3
EG-4
E3G-5
EG-6
EG-7
EG-8
BG-9
EG-10
2G-11
EG-1 2
EG-13
EG-14
EG-15
EG-16
EG-17
BG-18
EG-19
EG-20
EG-21
Document
ES-1
ES-1
ES-2
ES-2
ES-3
ES-3
ES-4
ES-5
ES-6
ES-7
ES-8
ES-9
ES-10
ES-11
ES-12
ES-1 3
ES-14





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                                  BG-1
                                  August, 1982
       DAPHNID ACUTE  TOXICITY TEST
        OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES  AND  TOXIC SUBSTANCES
   U.S.  ENVIRONMENTAL PROTECTION  AGENCY
          WASHINGTON, D.C. 20460

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Office of Toxic Substances                                  EG-1
Guideline for Testing of Chemicals                  August,  1982


                   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 seg.).  This  guideline

prescribes an acute toxicity  test in  which daphnids  (Daphnia

magna or JD. 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 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

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                                                            EG-1
                                                    August,  1982
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  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 wate,r  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

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                                                            EG-1
                                                   August,  1982
concentration in the test chamber.  The test is started within  30

minutes afterthe 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 duringthe 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

def initive test.

     (ii)  The daphnids should be exposed to a series of

widelyspaced 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

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                                                            EG-1
                                                    August,  1982
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  should 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 rag/1').  An equal number  of daphnids

should be placed in two or more replicates.   If  solvents,

solubilizing agents or emuls if iers have to be used,  they should

be commonly used carriers and should not possess a  synergistic or

antagonistic effect on the toxicity of the test  chemical.  The

concentration of solvent should not exceed 0.1 ml/1.     The

concentration ranges should 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 should include controls consisting  of the

same dilution water, conditions,  procedures and  daphnids from the


                                4

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                                                            BG-1
                                                    August,  1982
same population (culture container), except that none o£  the

chemical is added.

     (iv)  The dissolved oxygen concentration,  temperature  and  pH

should be measured at the beginning of the test and at  24 and 48

hours in each chamber.

     (v)  The test duration is 48 hours.  The  test  is

unacceptable if more than 10 percent of the control organisms

appear to be immobilized, stressed or diseased  during the 48 hour

test period.  Each test chamber should be checked for immobilized'

daphnids at 3, 6, 12, 24 and 48 hours after the beginning of the

test.  Concentration-response curves and 24-hour and 48-hour EC50

values for immobilization should be determined  along with their

95 percent confidence limits.

     (vi)  In addition to immobility, any abnormal  behavior or

appearance should also be reported.

     (vii)  Distribution of daphnids among test chambers  should

be randomized.  In addition, test chambers within the testing

area should 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 dissolved test chemical (that

which passes through a 0.45 micron filter) in  the chambers  should

be measured as often as is feasible during the  test.  In  the

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                                                            EG-1
                                                  . August,  L932
static test the concentration of test chemical should 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 should be measured at  a

minimum; (A) in each chamber at the beginning of the test and at

24 and 43 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 should not vary more than 20 percent

(+ or -).

     (5)   [Reserved]

     (3)  Analytical measurements--( i)  Tes t chemical.  De ionized

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 should be

validated before beginning the test by appropriate.laboratory

practices.  An 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 should

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                                                            BG-1
                                                    August,  1982
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 should  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.

     (3)  Daphnids to be used  in acute  toxic'ity  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 should  have  originated from

the same source and be from the same population  (culture

container).

     (C)  Daphnids should not  be used for a  test (_!_) 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 two days  preceeding  the test;  (_4_) if  adults

in the culture do not produce  an average  of  at least three  young

per adult per day over the seven day period  prior  to the test


                                7

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                                                            EG-1
                                                    August,  1982
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)  Daphnids should 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.  Daphnids should be fed prior to the test.

     (3)  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 other signs of stress, physical damage and mortality.  Dead

and abnormal individuals should be discarded.  Organisms that

touch dry surfaces or are dropped or injured in handling should

be discarded.

     (3)  Smooth glass tubes (I.D. greater than 5 mm) equipped

with rubber bulb should be used for transferring daphnids

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                                                            EG-1
                                                   August,  1982
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 should 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 - 30 minute transition

period.  In addition, the flow-through system should contain

appropriate test chambers in which to expose daphnids to the test

chemical and an appropriate test substance delivery system.

     (3)  Facilities should be well ventilated and .free of fumes

and disturbances that may affect the test organisms.

     (C)  Test chambers should 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 particulatas into the

solutions.

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                                                            EG-1

                                                   August,  1982
     (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.



     (3)  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, daphn'ids can be exposed  in



glass or stainless steel containers  with stainless |steel or nylon



screen bottoms.  The containers should be suspended in the  test
  o


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 five 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:





                                10

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                                                      EG-1
                                              August, 1982
SUBSTANCE
                                        MAXIMUM CONCENTRATION
                                        20 mg/liter

                                         2 mg/liter

                                         5 mg/liter

                                         1 ug/liter

                                        20 ug/liter

                                        50 ng/liter



                                        50 ng/liter

                                        25 ng/liter
Particulate matter

Total organic carbon or               . .

chemical oxygen demand

Un-ionized ammonia

Res idual chlorine

Total organophosphorus pesticides

Total organochlorine pesticides plus

polychlorinated biphenyls (PCBs) or

organic chlorine

     (3)  The above water quality parameters under paragraph

(d)(2)(iii)(A) of this section should 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 should 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) should 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 u ohm/cm  is

acceptable as the diluent for making reconstituted.water.
                           11

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                                                            EG-1
                                                  : August,  1982

     (iv)  Cleaning.  All test equipment and test chambers  should

be cleaned before each test 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 should 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 daily during a test.  The 24-hour

flow through a test chamber should 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 any one test

chamber to another or from one time to any other.

    (3)  Test parameters.  Environmental parameters of the  water

contained in test chambers should be maintained as specified

below:

    (i)  Temperature of 20 ± i°c.

    (ii)  Dissolved oxygen concentration between 60 and 105

percent saturation.  Aeration, if needed to achieve this level,

should  be done before the addition of the test chemical. All

treatment and control chambers should be given the same aeration

trea tment.


                                12

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                                                           EG-1
                                                   August, .1982
    (iii)  The number of daphnids placed in a test chamber should

not affect test results.  Loading should not exceed forty

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 should not cause the dissolved

oxygen concentration to fall below the recommended levels.

    ( iv)  Photoperiod of 16 hours light and 8 hours darkness,

with a 15-30 minute transition period.

    (e)  Reporting.  The sponsor should submit to the US EPA all

data developed by the test that are suggestive or predictive of

acute  toxicity and all concomitant gross toxicological manifes-

tations.  In addition to the reporting requirements prescribed in

Part 792--Good Laboratory Practice Standards, the reporting of

test data should 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 con-centration

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
                                13

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                                                            EG-1
                                                   August,  1982
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 (in hours) of the daphnids used in

the test is also 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.


                                14

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                                                           EG-1
                                                   August, 1982
    (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.

    (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

f 1 u c tua t io ns , e tc . .
                                15

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                                  EG-2
                                  August, 1982
      DAPHNID CHRONIC  TOXICITY TEST
        OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES  AND  TOXIC SUBSTANCES
   U.S.  ENVIRONMENTAL PROTECTION AGENCY
          WASHINGTON, D.C. 20460

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Office of Toxic Substances                        ,          EG-2
Guideline for Testing of Chemicals                  August,  1982

                  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)  (P.L.

94-469, 90 Stat. 2003, 15 U.S.C. 2601 et seg.).  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 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 organisms over an extended period of

time.   In this test guideline, mortality and reproduction (and

optionally, growth) are the criteria of toxicity.

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                                                           EG-2
                                                   August, 1982
    (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 procedure—(1)  Summary of the test.  (i)  Test

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                                                            EG-2
                                                    August,  1982
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 (P < 0.05) in immobilization and

reproduction as compared to the control.

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                                                            EG-2
                                                   August,  1982
    (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/1), 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)  Def initive test.  (i)  The purpose of the  definitive

test is to determine concentration-response curves, EC50 values

and effects of a chemical on immobilization and reproduction

during chronic exposure.

    (ii)  A minumum of 20 daphnids per concentration should 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, 64 mg/1).  An equal number  of daphnids

should be placed in two or more replicates.  The concentration

ranges should be selected to determine the concentration-response

curves, EC50 values and MATC.  Solutions  should be  analyzed for

chemical concentration

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                                                            EG-2
                                                   August,  1982
prior to use and at designated times during the  test.

    (iii)  Every test should  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 test duration is 21 days.  The  test is unacceptable

if:

    (A)  more than 20 percent of the control  organisms  appear  to

be immobilized, stressed or diseased during the  test;

    (B)  each control daphnid living the full  21 days  produces an

average of less than 60 young;

    (C) any ephippia are produced by control  animals.

    (v)  The number of immobilized daphnids in each chamber

should be recorded on days 7, 14, 21 of the test.  After

offspring are produced, they should be removed from the test

chambers every two or three days.  Counts of  the cumulative

number of offspring per adult (number of young divided  by the

number of adults in each chamber) and the cumulative number  of

immobilized offspring per adult should be recorded on  days 14,

and 21 of the test.  Concentration-response curves, EC50  values

and associated 95 percent confidence limits for  adult

immobilization should be determined for days  7,  14 and  21.   A

MATC should be determined for the most sensitive  test  criteria

-------
                                                            EG-2
                                                   1 August,  1982
measured (number of adult animals  immobilized,  number  of  young

per adult and number of immobilized young per  adult).

    (vi)  In addition to immobility, any abnormal behavior or

appearance should also be reported.

    (vii)  Distribution of daphnids among the  test  chambers

should be randomized.  In addition, test chambers within  the

testing area should 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.

    (5)  [Reserved]

    (6)  Analytical measurements — (i)  Test chemical.  Deionized

water should be used in making stock solutions  of the  test

substance.   Standard analytical methods should  be used whenever

available in performing the analyses.  The analytical  method used

to measure the amount of test substance in a sample should be

validated before beginning the test by appropriate  laboratory

practices.   An analytical method is not acceptable  if  likely

degradation products of the test substance, 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 adults, total

offsring per adult and immobilized offspring per adult should be

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                                                           EG-2
                                                   August, 1982
counted during each definitive test.  Appropriate statistical


analyses should provide a goodness-of-fit determination for  the


adult immobilization concentration-response curves, calculated on


days 7, 14 and 21.  A 7-, 14- and 21-day EC50, based on adult


immobilization and corresponding 95 percent confidence intervals,


should be calculated.  Appropriate statistical tests (e.g.,


analysis of variance, mean separation test) should be used to


test for significant chemical effects on chronic test criteria


(cumulative number of immobilized adults, cumulative number  of


offspring per adult and cumulative number of  immobilized


offspring per adult) on days 7, 14 and 21.  An MATC should be
                                         o

calculated using these chronic test criteria.


    (d)  Test conditions — (1) Test species .   (i)  Selection.


(A)  The cladocerans, Daphnia magna or D. pulex, are the species


to be used in this test.  Either species can be utilized for


testing of a particular chemical.  The species identity of the


test organisms should be verified using appropriate systematic


keys .


    (3)  First ins tar daphnids, _<_ 24 hours old, are to be used to


start the test.


    (ii)  Acguis ition.  (A)   Daphnids to be used in chronic


toxicity tests should be cultured at the test facility.  Records


should be kept regarding the source of the initial stock and

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                                                            EG-2
                                                    August,  1982
culturing techniques.  All organisms used  for  a  particular test

should have originated from  the same population  (culture

container).

    (B)  Daphnids should not be used for  a test  if:

    (I) cultures contain ephippia;

    (_2_) adults  in the cultures do  not produce  young  before day

12;

    (_3_) more than 20 percent of the culture stock  die  in the two

days preceding the test;

    (_4_) adults  in the culture do not produce an  average  of at

least three young per adult per day over  the seven day period

prior to the tost;

    (_5_) daphnids have been used in any portion of  a  previous test

either in a treatment or in a control.

    (iii)  Feeding.  (A)  During the test  the  daphnids should be

fed the same diet and with the same frequency  as that used for

culturing and acclimation.  All treatments  and control(s)  should

receive, as near as reasonably possible,  the same  ration of  food

on a per-animal basis.

    (B)  The food concentration depends on the type  used.   Food

concentrations should be sufficient to support normal growth and

development and to allow for asexual (parthenogenic)

reproduction.  For automatic feeding devices,  a  suggested  rate is

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                                                            EG-2
                                                    August,  1982
5-7 mg food (either solids or algal cells, dry  weight) per  liter

dilution water or test solution.   For manual once-a-day  feeding,

a suggested rate is 15 mg food  (dry weight) per  liter dilution

water or test solution.

    (iv)  Loading.  The number  of  test organisms placed  in  a  test

chamber should not affect test  results.  Loading should  not

exceed forty daphnids per liter in the renewal  system.   In  the

flow-through test, loading limits  will vary depending on the  flow

rate of the dilution water.  Loading should not  cause the

dissolved oxygen concentration  to  fall below the recommended

level.

    (v)  Care and handling of test organisms.   (A)  Daphnids

should be cultured in dilution  water under similar environmental

conditions to those used in  the  test.  A variety of foods have

been demonstrated to be adequate for daphnid culture.  They

include algae, yeasts and a  variety of mixtures.

    (B)  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 other signs of

stress, physical damage and  mortality.  Dead and abnormal

individuals should be discarded.   Organisms that touch dry

surfaces or are dropped or injured during handling should be

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                                                            EG-2
                                                    August,  1982
discarded.

    (C)  Smooth glass tubes  (I.D. greater  than  5mm)  equipped  with

a rubber bulb can be used for transferring  daphnids  with  minimal

culture media carry-over.

    (D)  Care should be exercised to  introduce   the  daphnids

below the surface of any solution so  as not  to  trap  air under the

carapace.

    (vi)  Acclimation.  (A)  Daphnids should  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.  Daphnids should be fed  the same food during  the

test as is used for culturing and acclimation.

    (3)  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.

    (2)  Facilities — (i)  General.  (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


                                10

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                                                            EG-2
                                                    August,  1982
bubbles, or aerating the water when  water supplies contain

particulate matter, gas bubbles, or  insufficient dissolved

oxygen, respectively;

    (_4) an apparatus for providing a  16-hour  light and  8-hour

dark photoperiod with a 15- to 30-minute transition period;

    (_5_) an apparatus to introduce food  if continuous  or

intermittent feeding is used;

    (_6_) in addition, the flow-through test should contain

appropriate test chambers in which to expose  daphnids to the test-

substance and an appropriate test substance delivery  system.

    (B)  Facilities should be well ventilated and free  of fumes

and other disturbances that may affect  the test organisms.

    (ii)  Test chambers .  (A)  Materials and  equipment  that

contact test solutioas 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 test results.

    (3)  For renewal tests, daphnids  can be conveniently exposed

to the test solution 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.  Such containers should be suspended  in the test


                                11

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                                                            EG-2
                                                    August,  1982
chamber in such a manner to ensure 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.

    (D)  Test chambers should 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.

    (iii)  Test substance delivery system.   (A)   In the flow-

through test, proportional diluters, metering pump systems  or

other suitable systems should be used to  deliver  the  test

substance to the test chambers.

    (3)  The test substance delivery system  used  should be

calibrated before and after each test.   Calibration includes

determining the flow rate through each chamber  and the

concentration of the test substance in each  chamber.   The general

operation of the test substance delivery  system should be checked

twice daily during a test.  The 24-hour  flow rate through a test

chamber should 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 any one test chamber to another or from one


                                12

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                                                           SG-2
                                                   August, 1982
time to any other.  For the renewal test, test substance dilution

water should be completely replaced at least once every three

days.

    (iv)  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/1

Total  organic carbon or                           2 mg/1

  chemical oxygen demand                          5 mg/1

Un-ionized ammonia                               20 ug/1

Residual chlorine                                10 ug/1

Total  organophosphorus pesticides                50 ng/1

Total  organochlorine pesticides                   :

  plus polychlorinated biphenyls (PCBs)          50 ng/1

  or organic chlorine                            25 ng/1
                                13

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                                                    EG-2
                                            August, 1982
    (B)  The water quality characteristics listed above should be

measured at least twice a year or when it is suspected that these

characteristics may have changed significantly.  If dechlorinated

tap water is used, daily chlorine analysis should 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) should 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 of less than 1 microohm/cm is

acceptable as the diluent for making reconstituted water.

    (D)  If the test substance is not soluble  in water an

appropriate carrier should be used.

    (v)  Cleaning of test system.  All test equipment and test

chambers should be cleaned before each test following standard

laboratory procedures.  Cleaning of test chambers may be

necessary during the testing period.

    (3)  Test parameters.  (i)  Environmental  conditions of the

water contained in test chambers should be maintained as

specified below:

    (A)  Temperature of 20 ± i°c.

    (B) Dissoved oxygen concentration between  60 and 105 percent

saturation.  Aeration, if needed to achieve this level, should be


                                14

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                                                     EG-2
                                            August,  1932
done before the addition of the test substance.  All  treatment

and control chambers should be given the same  aeration  treatment.

    (C)  Photoperiod of 16-hours light and 8-hours darkness,, with

a 15-30 minute transition period.

    (ii)  Additional measurements include:

    (A)  The concentration of dissolved test substance  (that

which passes through a 0.45 micron filter) in  the chambers should

be measured during the test.

    (B)  At a minimum, the concentration of test substance should

be measured as follows:

    (_1_)  in each chamber before the test;

    J_2)  in each chamber on days 7, 14 and 21  of the  test;

    (3_)  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 substance

should not vary more than 20 percent.

    (C)  The dissolved oxygen concentration, temperature and  pH

should be measured at the beginning of the test and on days  7, 14

and 21 in each chamber.

    (e)  Reporting.  The sponsor should submit to the USEPA  all

data developed by the test that are suggestive or predictive  of

chronic toxicity and all associated toxicologic manifestations.


                                15

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                                                     EG-2
                                            August,  1982
In addition to the reporting requirements prescribed in  the   Part

792--Good Laboratory Practice Standards  the  reporting of  test

data should 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  substance including

its source, lot number, composition  (identity and concentration

of major ingredients and major impurities),  known physical and

chemical properties, and any carriers or other additives  used and

their concentrations.

    ^  '  The source of the dilution  water, its chemical

characteristics (e.g., conductivity, hardness, pH), 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 methods.  The age (in hours) of  the daphnids  used in

the test should 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 substance additions), the number of test

organisms per test chamber, the number of replicates per
                                16

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                                                     EG-2
                                            August,  1982
treatment, the lighting, the renewal process and schedule  for  the

renewal chronic test, the test substance delivery system and flow

rate expressed as volume additions per 24 hours for  the flow-

through chronic test, and the method of feeding (manual or

continuous) and type of food.

    (6)  The concentration of the  test substance in  test chambers

at times designated for renewal and flow-through tests.

    (7)  The number and percentage of organisms that show  any

adverse effect in each test chamber at each observation period.

    (8)  The cumulative adult and  offspring immobilization values

and the progeny produced at designated observation times,  the

time (days) to first brood and the number of offspring per adult

in the control replicates and in each treatment replicate.

    (9)  All chemical analyses of  water quality and  test

substance concentrations, including methods, method  validations

and reagent blanks.

    (10)  The data records of the  culture, acclimation, and  test

temperatures.

    (11)  Any deviation from this  test guideline, and anything

unusual about the test, (e.g., dilution failure, temperature

fluctuations ) .

    (12)  The MATC to be reported  is calculated as the geometric

mean between the lowest measured test substance concentration


                                17

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                                                    EG-2
                                            August, 1982
that had a significant (P<0.05) effect and the highest measured

test substance concentration that had no significant (P>0.05)

effect on day 7, 14 or 21 of the test.  The most sensitive of the

test criteria (number of adult animals immobilized, the number of

young per female and the number of immobilized young per female)

is used .to calculate the MATC.  The criterion selected for MATC

computation is the one which exhibits an effect (a, statistically

significant difference between treatment and control groups;

P<0.05) at the lowest test substance concentration for the

shortest period of exposure.  Appropriate statistical tests

(analysis of variance, mean separation test) should be used to

test for significant test substance effects.  The statistical

tests employed and the results of these tests should be reported.

    (13)  Concentration-response curves utilizing the average

measured test substance concentration should be fitted to

cumulative adult immobilization data at 7, 14, and 21 days.  A

statistical test of goodness-of-fit should be performed and the

results reported.                                  ,

    (14)  An EC50 value based on adult immobilization with

corresponding 95 percent confidence limits.when sufficient data

are present for days 7, 14, and 21.  These calculations should be

made using the average measured concentration of the test

substance.


                                18

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                                  ES-1
                                  Augus t,
1982
        TECHNICAL SUPPORT DOCUMENT

                    FOR

 DAPHNID ACUTE  AND  CHRONIC TOXICITY TEST
        OFFICE OF TOXIC  SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
   U.S.  ENVIRONMENTAL PROTECTION  AGENCY
          WASHINGTON, D.C.  20460

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                        TABLE OF CONTENTS

        Subject                                          Page
I.      Purpose                                           1
II.     Scientific Aspects                                1
        Test Procedures                                   1
        General                                           1
        Range-Finding Test                                7
        Definitive Test                                   7
        Test Condi tions                                   3
        Test Species                                      8
        Selection                                         8
        Sources                                           11
        Maintenance of Test Species                       11
        Handling and Acclimation                          11
        Feeding                                           15
        Facilities                                        18
        Construction Materials                            13
        Test Substance Delivery Syste-.n                    l.y
        Cleaning of Test System                           20
        Dilution Water                  .                  21
        Loading                                           22
        Controls                                          23
        Carriers                                          25
        Randomization                                     26
        Environmental Conditions                          26
        Dissolved Oxygen                                  26
        Light                                             31
        Temperature                                       31
        Reporting                                         34
III.     Economic Aspects                                  35
IV.     References                                        37

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Office of Toxic Substances                                ES-1
                                                  August,  1982

  Technical  Support Document foe Daphnid Acute and Chronic Tests
I.  Purpose
     The purpose of  this  document  is  to  provide  the
scientific background and rationale used  in  the  -levelopment
of Test Guideline  EG-1 which uses  Daphnia species  to
evaluate the acute and chronic  toxicity  of  chemical
substances.. The Document provides an account of  the
scientific evidence  and an  explanation of  the logic used  in
the selection of the test methodology, procedures  and
conditions prescribed in the Test  Guideline.   Technical
issues and practical considerations relevant to  the Test
Guideline are discussed.  In addition, estimates  of the cost
of conducting the  test are  provided.
II.  Scientific Aspects
     A.  Test Procedures
          1.  General.  Relatively i:ew industrial  chemicals,
compared to the vast number produced, have been  previously
tested by standard aquatic  bioassay methods.   As  a result,
many cannot be classified as to their toxicological
properties.   The acute and  chronic toxicity  tests  will
provide some of the  information needed to  evaluate the
hazard posed to aquatic organises  from a  chemical
substance.  Although assessment of effects on higher levels
of biological organization  is desirable,  it  is necessary  to
begin with effects on individuals  and small  test
populations.  Acute  effects can be considered as  those which
cause rapid damage to the organism by i:he  fastest  acting
mechanism of poisoning which can prove detrimental unless
the animal escapes the toxic environment  at  an early time.
     The static acute toxicity  test provides  the  most easily

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                                                         ES-1
                                                 August,  1982
obtainable measure of  toxicity.  This test provides
information on the immediate effects of short  term exposure
to potential toxicants under carefully controlled
conditions.  A single  exposure  to a potential  toxicant  in  a
confined system may represent the worst possible test case
for weak or non-motile organisms v/hich may be
incapable of avoiding  the chemical under natural conditions
(Curtis et al. 1979).
     The acute flow-through toxicity test  is especially
applicable for those test substances which display high
oxygen demand, are highly volatile, are unstable in  aqueous
solution, are biodegradable or  are readily assimilated by
the test organism.  By constantly maintaining  test solution
concentrations and environmental factors such  as dissolved
oxygen and pH within narrow limits, a more representative
indication of the potential toxic effects of the test
substance is obtained  as opposed to static test conditions
where degradation products and  metabolic wastes may  affect
the test organism as well as the test substance.
     The proposed Daphnia chronic toxicity test guidelines
are designed to assess the effects of tes i: substances on the
survival and reproduction of Daphnia as a representative
macroinvertabcate.  The duration of the test permits  the
organism to be exposed to a chemical from shortly after
birth until well into  adulthood.  The organisms are  exposed
long enough to allow the adults to produce several broods  oE
second generation progeny.  Initiating exposure shortly
after birth allows an assessment of the possible effects of
the test substance on such metabolic processes as
reproductive system, maturation/ fecundity, and growth.

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                                                         ES-1
                                                August,  1982
Exposure of the test organise for more than a complete
generation cycle (approximately ten days at the  test
temperature, Beislayer et al. 1974) allows the testing
facility to assess and predict the potential effect of  the
test compound on a representative Daphnia population.  The
number of first generation  test organisms unaffected at  the
termination of the test provide an indication of
survivorship.  Mathematical  treatment of the fecundity and
survival data provides an index of the inherent  ability  of a
population of test organisms to increase under similar
environmental conditions (Boughey, 1973).  Although, the
chronic assays require more  time and expense, they, pro-
vide much more accurate predictive information concerning
the effects of the test: substance (Liptak 1974).  The
potential disadvantages of the static assays, which exclude
feeding of test organisms,  include physico-chemical
modifications of the test substance and stress on the
organisms from their own metabolic wastes, dissolved oxygen
depletion, and starvation.   These disadvantages  are greatly
reduced by renewing the test solution-dilution water medium
at various rates while providing sufficient food.
     The replacement of the  medium can be continuous or  at
fairly frequent intervals.   Flow-through tests are more
expensive because they require more dilution water, more
test substance, and more expensive apparatus.  The setup,
breakdown, and maintenance  time for flow-through systems  is
greater and requires more experienced personnel.
     Intermediate results can be obtained between flow-
through and static bioassays by renewing the medium at
periodic intervals.  This system may be satisfactory if

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                                                         ES-1
                                                August,  1932
water is limited, if labor costs are not too high and if
bioassays are conducted relatively infrequently.
Laboratories that perform bioassays on a regular basis may
find flow-through tests to be cheaper and more effective
than renewal tests  (Liptak 1974).
     The difference in results between renewal and flow-
through chronic assays may be significant, depending in part
on the renewal frequency and the physical characteristics of
the test substance.  Nebeker and Puglisi (1974) investigated
the effects of several PCB's on Daphnia magna using weekly
renewal and flow-through techniques.  The LC50 values for
Arochlor 1254 for three-week assays were 31 ppb in the
renewal test and .1.3 ppb in the flow-through test.  The
authors attribute the thirty-fold difference in toxicity to
cumulative toxicity, the volatility of the PCB's and
sorption to bacteria, algal waste materials, test container
and food surfaces.
     The frequency of test chamber renewal may also affect
test results.  Bunner and Halcrow (1977) observed a
significant difference in ephippia production and mortality
with different dilution water replacement frequencies.
Daphnia magna maintained under the same photoperiod, feeding
rate, temperature, and density, but with renewal on an
alternate day basis, exhibited 1.2 percent ephippial
production and 3.8 percent mortality.  Daphnids maintained
under a weekly replacement scheme exhibited 8.7 percent
ephippial production and 35 percent mortality at the end of
the test period.  The renewal system test guideline
recommends alternate day renewal In order to maintain the
best practicable conditions for the test organisms.

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                                                         ES-1
                                                 Au,gus t,  1982
     The choice of' an acute test duration of  48-hours  was
chosen for the acuta tests because of  certain biological
characteristics of daphnids.  Adema  (1978) compared  the
mortality of one-day old daphnids which were  fed  an  algal
diet to those which were not fed, under identical
conditions.  Adema demonstrated that  43 hours  is  the maximum
time which ins tars can be deprived of  food without suffering
increased mortality.  The 48 hour test duration also allows
sufficient time for the ins tars to molt at least once.   Lee
and Buikema (1979) report there is a  molt-enhanced
sensitivity to certain compounds.
     The events in the daphnid life  cycle are dependent  on  a
number of factors including temperature, food  concentration
and type.  Pour distinct periods may  be recognized  in  the
daphnid life cycle (Pennak 1973): egg, juvenile,  adolescent,
and adult.  There are few juvenile ins tars.   Generally,  D.
pulex has 3-4 ins tars, and _D. magna  3-5.  The  adolescent
period is a single ins tar prior to the first  adult  ins tar
during which the eggs reach full development  in the  ovary.
The number of adult instars varies.   D. pulex  generally  has
18 - 25 instars, while D. magna from  6 - 22.   Molting  occurs
at an approximate rate of once a day  for the  juveniles and
at a rate of every two days for the  adults under favorable
condi tions .
     Richman (1958) observed that D.  pulex released  the
first brood at age eight days at 20°C.  Anderson and Jenkins
(1942) noted that D. magna released  its first brood  at the
age of seven days at 25°C.  Anderson  (1932) recorded sexual
maturity in _D. magna from six to ten  days when raised  in a
temperature range of 18 to 23°C.

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                                                         ES-1
                                                August,  1982
     The test duration of 21 days, starting with first
ins tar daphnids, will expose the daphnids to  the test
substance for approximately 11 total ins tars, including
approximately seven adult ins tars (Anderson and Je>nkins
1942, Richraan 1958).  The total number of ins tars In the
treatments will be a function of the effects  of the test
substance on juvenile growth and survival, reproductive
maturation, and adult growth and survival.-
     Andecson et al. (1937), using D. pulex,  and Anderson
and Jenkins (1942) using _D. magna, observed a peak in the
number of living young produced in both species at' the 10th
to llth ins tar which corresponds to days 20 to 23 after
experimental initiation.  Instars subsequent  to the 10th and
llth iastar generally produced less living young per molt.
Richman (1958) using D. pulex did not observe a decrease in
the number of young produced until approximately days 24 and
28.  The three studies indicate that the test period will
provide time for several adult ins tars and should encompass
or closely approach the life stages of maximal
reproduction.  Winner and Farrell (1976) assessed the
reproductive sensitivity of D. pulex to copper and concluded
that the experiment could have terminated anytime after the
third brood (approximately day 13) without altering
conclusions as to the effect of cooper on reproduction.
     It is important that treatment and controls be
monitored closely, especially after approximately day 5 to
determine the release time of the first brood, frequency of
subsequent broods and the number of living young produced
per brood.

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                                                         ES-1
                                                 August,  1982
          1.  Range-Finding Test
     The concentration range  for  the definitive  tests  is
normally chosen based on the  results of a range-finding
test.  Range-finding tests with daphnids are  usually short-
term bioassays which use fewer organisms per  test substance
concentration than required for the definitive test.   In  all
cases, the range-finding test is conducted  to reduce the
expense involved without having to repeat a definitive test
due to inappropriate test substance concentrations.
          2.  Definitive test
     The results of the definitive test will  be  used to
establish any statistically significant (PL 0.05)
differences between the treatments and the  control(s)
pertaining to survivorship and reproductive capabilities.
These parameters should provide an indication of the
potential effects of the test substance on  representative
populations of the test organism.
     Certain test parameters  as promulgated in the Test
Guidelines are more or less inflexible or have a narrow
range of acceptable values.   Slight variations of these
parameters have been demonstrated to have a significant
effect on the test organisms.  Small variations  of
temperature, for example, can produce changes in the
metabolic rate of daphnids (Kaestner 1970,  Bunting 1974), as
well as on their response to  toxicants (Bunting  and
Robertson, 1975).  Other test parameters establish maximum-
minimum criteria or a broad range of acceptable  values.
     ASTM (1930) concluded that, at present,  all conditions
do not warrant very precise control without invalidating
test results.  However, some  experimental conditions should

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be more narrowly controlled.  Therefore,  recommendations  for
test procedures are made which,  if  followed,  will .yield
results that are scientifically  sound  without causing
unnecessary testing costs.
     B.  Test conditions
          1.  Test species
              a.  Selection.  Daphnids  are  of ubiquitous
occurrence and are an important  link in many  aquatic.food
chains (Xring and O'Brien 1976,  Gulati  1978,  Makar'ewicz  and
Likens 1979).  Species of the genus Daphnia are  major
components of the freshwater zooplankton  throughout  the
world  (Hebert 1978).  Because of their predominantly
herbivorous nature, the daphnids represent  an intermediate
trophic linkage between the primary producers and the
carnivores and predators of higher  trophic  levels (Gulati
1978).
     Daphnia are found in a variety of aquatic environments,
except for rapid streams, brooks and grossly  polluted  water
(Pennak 1978).  Two of the most  common species found in
ponds, lakes and permanent pools are D. magna and D.
pulex_.  _D. magna, the largest of the daphnids,  is generally
found in the northern and western parts of  North America
while D. pulex is distributed over  the entire North  American
continent (Pennak 1978, Buikema  et  al.  1976).   Both  D. magna
and D. pulex have been used in toxicity tests.   D. magna  has
been used more extensively due to its  large size, ease of
culture, short generation time,  and sensitivity  to'toxic
compounds (Dewey and Parker 1964, Frear and Boyd 1967,
Builkema et al. 1976).  Buikema, _et_ _al_. (1976)  maintain  that
the cosmopolitan distribution of D. pulex,  as  well as  its

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                                                 August,  1982
adaptability to a wider range of habitats,  warrants  its  use
as a test organism.
     Daphnia have been used  to assess  the  toxicological
properties of a number of different  types  of compounds  for
                                                  i
both survival and the possible effects on  reproduction:
insecticides (Adema 1973); herbicides  (Schober and  Lampert
1977); organic compounds  (Adema 1978;  Canton et  al;  1975);
metals (Winner and Farrell 1976; Bertram and Hart 1979,
Biesinger and Chris tens en 1972); PCB's (Nebeker  and  Puglisi
1974); nitrilotriacetate  (NTA) (Biesinger  et al. 1974);
polyethyleneimine (PEI) (Stroganov et  al.  1977).
     Dap'nnids have been shown to be  very sensitive organisms
for assessing the possible deleterious effects of chemicals
on other aquatic forms.  D.  magna was  more  susceptible  to
several xenobiotics tested than other  invertebrates  and  fish
tested (Canton eet al. 1975, Leeuwangh 1973).  Kenaga  (1978)
used 75 insecticides and herbicides  to assess the
comparative toxicology of several different species
including birds, rats, fish, shrimp, honeybees,  and  daphnids
as indicators in toxicity screening  tests.  Comparisons
between the test organisms and test  substances indicated
that the daphnids were extremely sensitive  to a  number of
compounds .  Comparisons of the test  species, within  a
chemical class, indicated that the invertebrates (daphnids
and shrimp) were the most sensitive  test form for the  entire
spectrum of chemicals tested (Kenaga 1978).
     In a subsequent review  of the toxicity of more  than
30,000 test compounds, Kenaga and Moolenar  (1979)
demonstrated the enhanced sensitivity  of D. magna compared
to four species of fish, five species  of aquatic vascular

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                                                 August,  1982
plants, and the alga Chlorella.
     The sensitivity of D. magna was  compared  with  that of
D. pulex in two sets of experiments  (Winner and  Farrell
1976, Canton and Adema 1978).  The conclusions  indicated
that the two species do not significantly  differ  in  their
sensitivities to the compounds tested.
     Winner and Farrell (1976) studied the acute  a:nd  chronic
toxicity of copper  (as copper sulfate, pentahydrate)  to four
species of daphnids.  The daphnids used were divided  into
two groups, the larger species, D. magna and D.  pulex,  and
the smaller species, D. parvula and D. ambigua.   It  was
observed that the acute toxicity of D. magna and  D.  pulex
for copper did not  differ significantly (72-hour  LC50:  D.
mag n a, 86.5 ug/1; D. pulex, 86.0 ug/1; P > 0.05).   However,
two smaller species did display a slightly enhanced
sensitivity (72-hour LC5g: p. parvula, 72.0 ug/1; _D^_
ambigua, 67.7 ug/1.  Comparison of chronic exposure  data
demonstrated consistent similarities  in sensitivity  with
regard to survival.  Survivorship curves for the  two  lowest
test conditions (20 ug/1 and 40 ug/1) were never
significantly different (P > 0.05) from those  of  the
controls for the four species of Daphnia.  Enhanced
mortality was observed at the next highest concentration (60
ug/1) for all test  species.  The authors attribute  this
increase in toxicity between 40 ug/1 and 60 ug/1  to  a
saturation of the complexing capacity of the natural  pond
water used as dilution water.
     Canton and Adema (1978) investigated  the  sensitivity of
D. magna, D. pulex  and D.  cucullata to 15  different
compounds (13 organic, 2 inorganic).  Comparison  of  acute
                                10

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                                                         ES-1
                                                 August,  1982
toxicity test data pooled from  two different  laboratories
using the three test species  indicated  that with  only  two
exceptions (aniline and pentachlorophenol), there  were  no
significant differences (P >  0.05) between the  species.
Because of its size (manageability), as well  as its
comparable sensitivity, _p_. magna was designated the  daphnid
of choice for Dutch laboratory studies.
     As data are generated using both species under
carefully defined conditins,  the intercomparability  of  the
two test species for chronic  testing can  be ass as sod  in
future work.
              b.  Sources.  Daphnids as a group display
taxonomically troublesome variations in details of setation
and in carapace, head and postabdomen morpUoj.O'jy.
     Test species may be obtained from field  collections,
supply houses or established  cultures and should  be
identified and documented.  Verification  of either species
used should be performed using  the systematic keys of  Brooks
(1959) or Pennak (1978) for D.  magna, and Brandlova et  al.
(1974) for D. pulex.
          2.  Maintenance of  Test Species
              a.  Handling and Acclimation.   All  organisms
used in a test should be of the same species  and  from  the
same source to reduce variability of the  test results.
Laboratory culture of daphnids  from a single  innoculum
provides test organisms of similar history.   Reproduction
can be restricted to the parthenogenetic  production  of  only
females when suitable culture conditions  are  maintained.
This insures a supply of experimental animals with genetic
variability limited to the heterozygos ity of  the  parent
                                11

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                                                         ES-1
                                                August,  1982
     (APHA 1975, Kaestnar 1970, Pennak 1978).  Under proper
conditions, such as sufficient food, favorable temperature
and uncrowded conditions", D. magna cultures have been
maintained for many years (Kaestner 1970).  Daphnia from
cultures in which ephippia  (resting eggs) are being produced
should not be used for testing, as the production of
ephippia indicates unfavorable culture conditions and
production of males.
     Several culture methods have been described, with no
one method universally accepted.  Needham et al. (1959)
describes several historical methods of culture, some of
which are still utilized. .  Hutchinson (1967) reviews several
cultural schemes using primarily algae as food.
     The following references are not meant to be
exhaustive, but to provide  the testing facilities with some
recent information of various culture methods: D'Agastino
and Provasoli (1970), algae; Murphy (1970), algae; Burns
(1969), mixed algae; Kring  and O'Brien (1976), algae; Berge
(1978), algae; Canton et al. (1975), algae; Lee and Buikema
(1979), mixed algae; Winner and Farrell (1976), algae and
vitamins; Schultz and Kennedy (1976), algae and yeast; Dewey
and Parker (1954), algae and yeast; Buikema et al. (1976),
trout chow pellets; Beisinger and Christensen (1972), trout
fry food granules and grass; Fear and Boyd (1967), manure-
soil; and Whitten et al. (1976), hard-boiled egg yo'lk.
     It is left to the experience and discretion oE; the
testing facilities to decide which method(s) prove to be the
.most reliable.
     Acclimation to new environmental condition(s) is
accomplished by various biochemical and biophysical
processes.  Capacitive adaptations are those which permit
                                12

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                                                         ES-1
                                                 August,  1982
relative constancy of biological activity over a normal
range of environmental parameters.  To accommodate  the
necessary biochemical/physiological changes required  for the
test organism to adapt to new environmental conditions, the
rate of change of the factors should be such as to  avoid
additional stress.
     Once the desired conditions are established, the
animals should be held at these conditions for a period of
time to determine that no delayed symptoms of stress  appear
which could bias test results.
     The recommended rates of temperature acclimation
(l°C/day), plus culturing in dilution water for
approximately 21 days, are designed to allow the animals to
make the necessary physiological adjustments prior  to
exposure to the treatments.
     Food type, concentration,- and feeding rate should
approximate test conditions as closely as possible.   This
allows- the animals to adjust to test conditions and yields
to the investigator a preliminary assessment of the
effectiveness of the feeding regime to be used during the
extended test period.
     A culture should not be used as a source of test
organism if (a) the individuals appear stressed or  diseased;
(b) it possesses adults that do not produce young by  day 12,
which would indicate delayed maturation or infertility; (c)
it has adults that do not produce an average of at  least
three young per adult per day over a seven-day period, which
would indicate reproductive impairment due to genetic or
culture conditions such as crowding, inadequate diet  or some
pathogen; (d) ephippia are being produced, which would
indicate stressful conditions; or (e)  mortality exceeds 3
                                13

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                                                         ES-1
                                                 August,  1982
percent during the 48 hours  immediately  preceding  the  test.
     These criteria are designed  to prevent  bias  in the test
results caused by the use of reproductively  inferior
daphnids, which could result from  heredity or  a stressed
parent.  Stressed culture conditions causing metabolic
dysfunctions in the parent may be  reflected  in the tey t
organisms.  Such dysfunctions as reduced  yolk  synthesis
could result in an inferior  test daphnid.
     First ins tar D. magna or D. pulex are the initial test
stage.  Animals 0-24 hours old are to  be  used. This age
class can be collected by an overnight separation  of gravid
females, and insures that all the  test organisms will  be
f irs t- ins tar, pre-mo 11.
      o
     Dewy and Parker (1964) described  a  separation chamber
consisting of funnels with screen openings.  The instars
passed through the screen a:\-l were collected in receiving
jars while the adults remained in the funnel.  This  method
resulted in the production of animals of  known age with a
minimum of labor and time.
     Static acute assays indicate  an enhanced  sensitivity  of
first instar daphaids as compared to the  later juvenile or
early adult stages (Sanders and Cope 1956).
     Schultz and Kennedy (1976) and Lee and  Buikema (1979),
again using static acute assays, demonstrated  enhanced
sensitivity of Daphnia spp. at molting,   Such  mechanisms
as changes in permeability of the body surface and the
incorporation of large volumes of water  were postulated to
explain the enhanced toxic effect during  the molt  period.
     Exposure of the daphnids shortly after brood  release
insures exposure to the test substance prior to the first
molt.  Exposure of the test organism at an early age is
                                14

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                                                         ES-1
                                                 August,  1982
designed to assess any possible effects on  the organism
which may not manifest themselves until later  in  the  life
cycle.
     Subjecting the first  ins tar to  the test substance
allows  an evaluation of effects on such physiological
processes as ovary maturation  (reflected  in fertility),
fecundity, and the time from brood release  to  the first
post-release molt.  The effects on the parent  generation  can
also be reflected in growth rate and survivorship.
              b .  Feeding .  Daphnids should not be  fed
during  the acute tests.  The presence of  food  in  the  test
medium may have several effects: (1) The  test  substance may
be absorbed onto the food particles and either increase or
decrease its toxic effects (.Adema, 1978).   (2) It may alter
the dissolved oxygen content by increasing  BOD (biological
oxygen demand),' increase dissolved 'oxygen by pho tosyn the i:ic
activity, or reduce dissolved  oxygen by respiratory
demands.  (3) Feeding may alter the physiology of the
instars and change the uptake  and metabolism of the test
substance:  (4) It may introduce more variability into the
test.
     Feeding is required during culture of  daphnids and  in
renewal and flow-through chronic tests.   Food concentration
and type is extremely important because it
can affect: (1) the concentration of test substance needed
to elicit a. response, (2) diurnal dissolved oxygen  levels,
and (3) the physiological state of the test organism.
     A large number of variables concerning feeding are
evident from the literature.   These include food  type(s),
feeding rates, use of supplements, frequency of renewal of
                                15

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                                                         ES-1
                                                 August,  1982
test medium and dilution water and any  food  included  in
natural dilution waters.  Recommended feeding  regimes  for
daphnid testing may be based on the  literature  compilation
in Table 1.  Final feeding conditions are  left  to  the
discretion o.ir the individual laboratory based on experience
and the satisfaction of the  test guidelines  pertaining  to
control mortality and minimum number of control progeny.
These two criteria are designed to insure, in part, proper
feeding techniques during test conditions.
     The food used should be sufficient to maintain the  test
organism in a nutritional state which will support normal
metabolic activity and reproductive  capabilities.  This  is
advisable in order to avoid  introducing starvation as  a
variable into test results.
     Adema (1978) states that the feeding  of 4.0 x 107  to
6.0 x 107 Chlorella pyrenoidosa cells per  liter per adult D.
magna per day is the optimum amount  of  food  for reproduction
and was the same concentration used  in  cultures.
     Overfeeding may compromise test results through  (1)
excessive oxygen demands of  the food used; (2) preferential
sorption of the test compound as some critical  food
concentration is reached; (3) filtering rate of the daphnids
is reduced or goes to zero.  Kring and  O'Brien  (1976)
observed a reduction in the  filtering ratejipr D_. pulex at
concentrations in excess of  2.5 x 107 Ankistrodesmus
fallatus cells per liter.  McMahon (1965) observed a
leveling of the filtering rate for D. magna  at  1.5.x  10^
yeast cells per liter and 1.2 x 108  Chlorella vulgaris cells
per litec.  Less than optimum feeding can  be reflected  in
reduced production of progeny..  Richman (1958), using  D.
                                16

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                                                         ES-1
                                                 August,  1982
Table 1:  Several feeding regimes used  in  chronic  toxicity
           using Daphnia.
     Calculated Feeding Rate
Food Type (amount/I/daphnid/day)
                        Reference
Chlorella pyrenoidosa


Chiorel la pyrenoidosa
Yeast

Chlorella pyrenoidosa

Yeast Extract

Grass and Troui:
    Pellets


Yeast
Yeast
Scenedesmus obliquus
Yeast and
Scenedesraus acutus
Chlaymdomonas
reinhardti
   2.5 - 6.0 x 107
   cells

   1.3 x 107 cells
   6.5 x 109 cells

     4 x 107 cells
        solids
2 mg Yeast
1.2 x 105 cells
4 x 105 cells
4 mg
   7.5 rag
Adema (1978)
Bertram and
Hart (1979)

Serge (1978)
Biesinger and
Chris tens en
(1972)

Bunner and
Hal crow
(1977)

Dew:ey and
Pa rke r
(1964)

Schober and
Lampe r t
(1977)

Winner and
Parrel1
 ( 1976)
pulex and Chlamydomonas spp., observed  a  three-fold  change
in the cumulative number of  young produced  with
Chlamydomonas concentrations ranging  from 25  x  1Q3 cells  per

ml (30 young) to 100 x 103 cells per  ml  (92 young).
     Bunner and Halcrow (1977), using D.  magna  fed on  yeast
                                17

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                                                         E3-1
                                                 August,  1982
and maintained at the same photoperiod and  density,
observed an increase  in ephippia production of  18.6  percent
on a starvation diet  (0.025 mg yeast per animal per  day)  as
opposed to 9.6 percent ephippia on  a diet of  0.05  mg yeast
per animal per day.
        3.  Facilities
              a.  Construction Materials.   Construction
materials and any equipment that may contact  stock
solutions, test solutions or any water into which  the test
organisms will be placed should not contain any substances
that can be leached into the aqueous medii.ua.   Such
substances could introduce an error into the  test  results  or
stress the test organisms by direct or indirect toxic
effects.
     Materials and equipment should be chosen to minimize  or
eliminate the occurrance of sorption and leaching, which  may
reduce the effective  concentration  of the test substances
and introduce a potential error in  test results, or  which
may introduce contaminants into the system.
              b.  Test Substance Delivery System.  In flow-
through tests, the delivery of constant concentrations of
test substances is required to reduce variability  in test
results.  Large fluctuations in test substance concentration
will give abnormally high or low responses,  depending upon
the mechanism of toxic actions.  Proportional  diluters with
metering pumps or continuous-flow infusion  pumps have been
used extensively to maintain constant test  substance
concentration.  For the flow-through acute  and life-cycle
test guideline, all tests should be conducted  in
intermittent flows from a diluter or in continuous flow with
                                13

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                                                         E3-1
                                                 August,  1982
the test substance added by an  infusion pump.   The
procedures of Mount and Srungs  (1967) and  Hansen  et  al.
(1974) are recommended if the test substance can  be  added
without a carrier; the device described by Hansen et al.
(1974), if a carrier is necessary; or procedure of Bahner  et
al. (1975), if pumps are required for continuous  flow.
     Proportional diluters operate on a sequential filling
and emptying of water chambers.  The water chambers  are
calibrated to contain a measured amount of  water.  Separate
water chambers can be provided  for test substance ^nd
diluent water.  Diluent and test substance  waters are mixed
and delivered to  the test aquaria.  The cyclic  action of  the
diluent is regulated by a solenoid valve connected to the
inflow dilution water.  The system is subject  to  electrical
power failure, so an alternate  emergency power  source is
recommended .
     The proportional diluter is probably  the  best, system
!!or. routine use;  it is accurate over extended periods of
time, is nearly trouble free, and has fail-safe provisions
(Lemke et al. 1978).  A small chamber to promote  mixing of
test substance-containing water and dilution water may be
usorl between the  diluter and the tes t aquaria  for each
concentration.  If replicate chambers are  used  in this test,
separate delivery tubes should be run from this mixing
chamber to the appropriate replicate chambers.  If an
infusion pump is  used, a glass baffle should be employed  to
insure mixing of  the test substance and dilution  water.
Calibration of the test substance delivery  system should  be
checked carefully before and during each test.  This should
include determining the flow rate through  each  test  aquarium
and measuring the concentration of test substance in each

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                                                 August,  1982
test aquarium.  The general operation  of  the system should
be checked  twice daily.
     The use of municipal water supplies  is  not
recommended.  Municipal waters often contain high
concentrations of potentially  harmful  components such  as
chlorine, chloramines, copper, fluoride, . lead,  zinc
and iron.   A carbon filtered dechlorinated water may be
acceptable  if Daphnia can be cultured  in  it.   Caution  should
be exercised since municipal water may  vary  considerably  in
quality and chemical characteristics associated with
seasonal changes (e.g., extensive chlorination following
heavy storm activity), different sources  and modifications
or repairs  to the distribution system.
              c.  Cleaning of Test System.   Standard
laboratory  practices (e.g., USEPA 1974) are  recommended  to
                                                    :>
remove dust, dirt, other debris, and residues  from  test
facilities.  At the end of a test, test systems should be
washed in preparation for the next test.  This will prevent
chemical residues and organic matter from becoming  embedded
or absorbed into the equipment.  It is  also  recommended tht
any silicon cement which has been exposed to a test
substance is replaced prior to future  tests  to avoid
contamination due to sorption properties.
     Rinsing and priming the system with  dilution water
before use  (conditioning) allows equilibrium to be  reached
between the chemicals in the water and  the materials of the
test system.  The test system may sorb  or react with
substances  in the dilution water.  Allowing  this equilibrium
to take place before use lessens the chances of water
chemistry changes during a test.
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                                                         ES-1
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              d.  Dilution Water.  An adequate supply of
dilution water  is required in which the daphnids will
survive, grow, and reproduce.  This is necessary to insure
that the test organises will not be stressed or adversely
affected by the dilution water.
     Dilution water quality should be maintained within a
certain range to allow for standardization and comparability
of test data.  Changes outside the acceptable range
recommended may cause undue stress to the test organisms,
thus biasing test results.  Variations in water chemistry
from the recommended range may abso interfere chemically
with the test compound, either enhancing or diminishing the
toxicological properties.  Criteria have been established
for several heavy metals and pesticides which have been
known to produce adverse effects on aquatic organisms (ASTM
1930) .
     The dilution water should be vigorously aerated prior
to use Eor culturing and testing.  The recommended
saturation value of 90 to 100 percent should provide
sufficient oxygen under most conditions for daphnid
metabolic demands, as well as any chemical oxygen demand of
the test substance.
     Test chambers should not be aerated after the, test
organisms are introduced to prevent entrapment of air
bubbles under the daphnids1 carapace.
     Natural dilution water should be obtained from an
uncontaminated well, spring, or surface water source.  Wells
and springs generally provide water of fairly constant
quality.  Surface water sources are more likely to:be
subjected to point or non-point source loadings. Any
peculiarities in local ground and surface water due to
                                21

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                                                         ES-1
                                                 August,  L932
geologic conditions that are not listed in the
specifications of  the test guideline should be  investigated
in terms of their  effects on the test organisms.
     Natural dilution water is usually the most cost-
efficient type of  water, especially for extensive .testing
and flow-through systems.  Reconstituted water may be
prepared using ground or surface water which, in itself,
will not maintain  daphnids.  Reconstituted water has  the
advantage of having well defined chemical characteristics,
due to the specific chemical components defined in its
manufacture.  Reconstituted water is however, less; cost-
efficient than natural dilution water for large scale
renewal or flow-through testing due to the requirement oC a
distilling apparatus and labor required to measure and
mix the necessary  chemical components.
              e.   Loading .  The use of 10 ins tars per 200 ml
test solution is recommended for static acute tests.  This
loading should insure adequate dissolved oxygen for the
duration of the test period.  Adema (1973) recommended a
loading of 10 ml test solution per instar based on oxygen
consumption data for the instar at 20°C.  Adema suggested
this loading would result in a final dissolved oxygen
concentration of 80 percent saturation dilution water.  The
recommended loading provides twice the amount of test
solution suggested by Adema and should provide a margin of
safety for the dissolved oxygen of both the organism and
chemical.
     A recommended loading of one daphnid pec test chamber
for chronic, reproductive studies is designed to meet the
dissolved oxygen requirements of the organism and to allow
                                22

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                                                  :       ES-1
                                                 August,  1932
the observation of critical stages in the  life  cycle such  as
initial release of ins tars and  the subsequent  brood
frequency and size.
     Survivorship studies using five parent  daphn ids per  200
ml test solution may present some problems with  maintaining
dissolved oxygen levels above 60 percent saturation,
depending on the food  type and  feeding rate.   Should
dissolved oxygen values be observed below  60 percent
saturation, the use of a larger volume of  test  solution oc <*
different food, type and feeding rate is recommended.
     For acute and chronic flow-through assays  the near
constant changing of the test solution should  prove
sufficient to maintain all environmental conditions within
the criteria for a definitive test.
              f.  Controls.  Controls are  required for every
test to insure that the observed effects are due  to the  test
substance and not to other factors.
     In acute toxicity tests, a maximum of 10  percent
immobility is permissible in dilution water  control daphn ids
due to inherent biological factors and possible  handling-
induced stress.  Higher immobilization negates  the test
results and indicates  the need  to determine  the  cause of  the
increased immobilization in the control.   Possible sources
of increased control immobility include culturing
techniques, acclimation procedures, handling techniques, or
testing facilities or  procedures.
     In chronic tests, to insure that the  reproductive
capabilities of the test population are not  impaired, a.
lower limit of at least 60 young produced  per  control animal
(cumulative) has been  established for the  test duration.
                                23

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                                                         ES-1
                                                 August,  1982
Criteria proposed in the test guideline  for  culture

conditions are designed to reduce possible stress  conditions

which could be reflected in reproductive  impairment  of  the

organioi'.vs.  Similar criteria for the  experimental  procedures

are designed to insure that the observed  effects are  due  to

the test compound and not to crowding or  feeding. : Table  2

presents a summary of observed 21 day cumulative production

of young daphnids.  The range in values  can  be  attributed  to

such factors as loading, temperature, food type and

concentration, dilution water characteristics,  and in some

cases, the need to extract 21 day data from  the results of

experiments of longer duration.

     Table 2: Cumulative production of young _D. magna during
              a period of 21 days after  birth.

     Average Cumulative     T^s r. Temperature
     Production of Young          °C                    Reference
     	Pe r Dap hn id	

            67-122              20                 ,     Berge
                                                        (1978)

             73                 25                 '     Anderson
                                                        and Jenkins
                                                        (1942)

           38-43                18                      Nebecker
                                                        and Puglisi
                                                        (1974)

            63                  20                      Schober and
                                                        Lampert
                                                        (1977)

           30-92                20                      Richman
                                                        (1953)

            83                  -                       Canton
                                                        et  al.
                                                        (1975)


                                24

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                                                         ES-1
                                                 August,  1982
          g.  Carriers.  Carriers should only be used  when
they are necessary to  solubilize hydrophobia test  compounds.
Dimethylforraamide and  triethylene glycol are the carriers of
choice due to their low volatility and  toxicity, as  well  as
their ability to dissolve many organic  compounds (ASTM
1930).  Sax  (1979) suggests  that dimethylformamide not come
in contact with halogenated  hydrocarbons and inorganic
nitrates due to the reactivity of the compounds.   Schobet:
and Lampert  (1977) observed  a significant effect of  the use
of ethanol as a carrier for  Atrazin (chlorinated triazine
herbicide) using D. pulex for a 28-day  exposure.  lAlthough
no effect was observed with  0.1 percent ethanol carrier,  the
use of 0.5 percent carrier with the herbicide produced
effects greater than the sum of the individual effects.
Significant differences (P < 0.05) were observed on  such
parameters as number of young per animal and mean  length.
     Comparison of dilution  water controls and 0.5 percent
ethanol controls indicated that use of  the ethanol control
resulted in approximately a  40 percent  reduction in  the
number of young per animal over the experimental period.
                                                  i
Mean length data also  indicated a carrier effect.
     The investigation of Schober and Lampert (1977)
demonstrates possible errors associated with the use of a
carrier and reinforces the recommendation that a carrier  be
used only when necessary.  The investigation also  emphasizes
the need for a carrier control and the  investigation of the
effects of two different concentrations of the same  carrier
on the test organism.
     Krugel et al. (1978) describe an apparatus for  the
continuous dissolution of poorly soluble compounds for
b io as 3 ays .
                                25

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                                                         ES-1
                                                 August,  1982
          h.  Randomization.  Randomization  is  required  to
prevent conscious or unconscious  biases  from being
introduced.  These biases can be  in environmental conditions
such as temperature and lighting, daphnid  selection  and
distribution.
        4.   Environmental Conditions
              a.  Dissolved Oxygen.   Daphnija respond  to
partial anoxia by synthesizing hemoglobin  (Hoar 1966,
Lockwood  1967, Kaestner 1970).  This  adaptation has
significant survival value  (increased  life span and
increased egg production) when compared  to those organisms
that lack or possess reduced concentrations  of  hemoglobin.
In mature females, considerable hemoglobin enters the  eggs,
accelerating embryonic development.   After egg  laying, the
level of  hemoglobin in the  adult's blood is  approximately
two-thirds of its normal value.   The  concentration in  the
hloovl iacreases during the  time the young  are developing  in
the brood pouch.  The cycle is then repeated with the
release of the young and production of a new batch of
parthenogenic eggs.
     Considering its molecular size,  incorporation into  the
eggs and possible replacement rates,  hemoglobin synthesis
may represent a considerable energy demand on the
organism.  Such demands can be inferred  from the increase
rate of feeding at partial  anoxic conditions.   No chronic
toxicity  test data could be located comparing Daphnia
response  to varying concentrations of  dissolved oxygen.
     Adema (1970) determined the  oxygen consumption  of 25
egg-bearing adult .daphnids  at 20°C to  be about  850
ug/02/day.  The ins tars released  from  adults (25) in 24
                                26

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                                                         ES-1
                                                August,  1982
hours consume an additional 600 ug/02/day.  Adema suggests  a
minimum of 2.5 liters of oxygen-saturated medium for  25
daphnid on an alternate day renewal scheme will satisfy
daphnid oxygen demands, as well as any other oxygen sinks
such as food remnants, excreta and test compound oxygen
demands .
     The solubility of oxygen in freshwater at  20°C is
approximately 9 rag/1.  The test guidelines recommend  that
dissolved oxygen not fall below 60 percent saturation or
approximately 5.5 mg/1 at any time during the test.   Kring
and O'Brien (1976), using _D. pulex at 22°C, observed  that
when the oxygen concentration dropped below 3 mg/1 the
filtering rate of D. pulex decreased drastically.  The same
authors cite unpublished data of a critical oxygen
concentration of about 3 mg/1 for D. magna.  The recommended
minimum concentration of 60 percent .saturation  is well above
the critical oxygen concentrations observed.
     Kring and O'Brien (197-5) observed that exposure  for
less than one hour to oxygen concentrations of  1 mg/1 caused
a negligible depression in the filtering rate of JD. pulex.
Longer exposures to dissolved oxygen values less than the 3
mg/1 critical concentration resulted in depressed filtering
rates (60 percent reduction) for an eight-hour  period.
After 24 hours of exposure, the filtration rate increased to
near normal values.  Continued exposure to dissolved  oxygen
values less than the critical concentration resulted  in the
ability of the animals to resume and surpass the initial
high filtering rates, presumably because the low oxygen
concentration stimulated the production of hemoglobin.  The
daphnids synthesized increa>3ing amounts of hemoglobin
                                27

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                                                         ES-1
                                                 August,  1982
associated with the time they were exposed  to  low  dissolved
oxygen values  increased.  Consideration of  the increase  in
hemoglobin synthesis and the high filtering rates  associated
with long-term exposure to  low dissolved oxygen suggests
that hemoglobin synthesis is energetically  inefficient.
Maintenance of test dissolved oxygen values above  60  percent
saturation should prevent any biochemical stress on the  test
organism associated with hemoglobin synthesis  which could
decrease the energy available for other metabolic  processes.
     The presence or absence of dissolved molecular oxygen
in the test solution may also affect the form  of the  metals
and ions in the medium.  Several general types of  redox
reactions of ionic species  of mat a Is have been demonstrated,
depending in part on pH, the presence of organic complexing
agents, the presence of other ionic species such as the
carbonate ion and the presence of molecular oxygen (Faust
an;] Hunter 1967).  Several  of the reactions are  reversible;
thus various equilibria can be established, depending  on  the
chemical composition of the test medium.  Maintenance  of
dissolved oxygen levels in  excess of 60 percent saturation
should reduce the variability of the ionic shifting for
metallic test substances.   No data could be located to allow
comparison of the toxicity  of the different ionic  forms  of a
given metal.  Due to the chemical complexity of  the test
medium, such as food type and concentration, metabolic
products and test system-test substance interaction,
maintaining dissolved oxygen values at a given ninimum
should help to reduce variability in test results.
     The concentration of dissolved oxygen should  be
monitored closely in -static acute test chambers  to insure
                                28

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                                                           ES-1
                                                   August, 1982
that the  oxygen level  is  above the required  minimum.  During
a test,  the chambers are  not  to be aerated under any
circuinstances.  This is specified to prevent  air  entrapment
under  the ins tar carapace as  well as not  to  enhance
volatilization  of the  test substance.  If levels cannot be
maintained  above 60 percent,  the static flow-through method
should be used.
     The  potential for oxygen depletion in the  renewal test
does exist,  and depends on loading, food  type and
concentration,  feeding frequency, and renewal frequency.
Several renewal studies listed in Table 3 show a range of
values for these parameters.   Only one study, Winner and
Farrell  (1976), presents  any  dissolved oxygen data.  These
authors state that observe^ dissolved oxygen values were
always in excess of 95 percent saturation.

     Table  3.  Summary of  Renewal Chronic Assay  Techniques.
    Initial loading   Replacement           Feeding
    (daphnids/volume) Frequency     Food    Frequency
                               Reference
       1/40 ml

       1/40 ml


       1/100 ml


       1/50 ml


       1/40 ml
4 Days   C. pyrenoidosa  1 day    Bertram and
        ~    and Yeast         Hart (1979)
1 Week   Trout Pellets   1 v-feek   Biesinger and
                              Chris tens en
                              (1972)
2 Days   Scenedesmus^
        and Yeas't
2-3  Days Chlorella
2 Days   Schober and
        Lampert
        (1977)
2-3  Days Stroganov
        et al.
        (1977)
3 Days   hlamydonpnas    1 Day    Winner and
                              Farrell
                              (1976)
                                 29

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                                                       ES-1
                                               August, 1932
        The use of photosynthetically active algae can
provide significant oxygen to the test medium during  the
light photoperiod.  During the dark photoperiod, algal
respiration competes with the other oxygen sinks for
dissolved oxygen.  Again, depending on a number of
variables, significant oxygen depletion is a possibility.
Animals maintained on non-photosynthetic food with extended
replacement schemes, such as in Biesinger and Christensen
(1972), may be exposed to extended periods of- low dissolved
oxygen.  No data on the effects of low dissolved oxygen on
reproduction and survivorship of the test organisms could be
located .
        The suggested scheme of alternate day renewal in
chronic testing should aid in maintaining dissolved oxygen
levels above the recommended minimum.  Dissolved oxygen
readings should be taken several tmes during the first two
days of testing to determine that the proper levels are
maintained.  It is especially important to determine.
dissolved oxygen values at the end of the dadc photoperiod
when using algal food supplies.
        The inability to maintain sufficient dissolved
oxygen values in a renewal system indicates the need  for a
flow-through test or larger volume test chambers.
        The flow-through techniques should not present any
significant dissolved oxygen problems due to (1) the
constant replacement of the test medium and (2) the volume
of the test chamber and loading ratio, which should provide
sufficient oxygen according to the data of Adema (1978).
                                                  |
This is not to imply that dissolved oxygen need not be
monitored.
                                30

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                                                        ES-1
                                               Augur, t.  1982
              b_.  Light.  A 16-hour light, 8-hour dark
photoperiod with a 15 to 30-minute transition has been
suggested to meet biological requirements of daphnids and to
increase test standardization (ASTM, 1930).  The recommended
photoperiod approximates the temperate summer light regime
v;hich would support parthenogenic reproduction.  The
transition period is recommended i:o simulate natural
conditions.  A device for maintaining the photoperiod has
been described by Drummond and Dawson (1970).
     Stross and Hill (1968), using D. pulex at five
individuals per 50 ml density at 19°C for 30 days duration,
observed decreasing sexual reproduction with changing
photoperiod.  At 12L:12D, approximately 90 percent sexual
reproduction was recorded, while increasing the light period
to 14L:10D resulted in 0 percent sexual reproduction.
Extrapolation to the re com tended photoperiod at 16L:8D
should insure parthenogenic reproduction.
     Wide spectrum fluorescent bulbs (Color Rendering Index
greater than 90) and a light intensity at the surface of the
test chambers not exceeding 800 lux (74 ft. candles) is
essentially equivalent to the average tab let op conditions.
These facilities would allow the use of room lighting for
experimental conditions, provided it was controlled to the
recommended photoperiod and transition period.
              Temperature.  The selected test temperature
(20 ± 1°C)  approximates room temperature, thus minimizing
the requirement for extensive temperature controlling
devices in culturing, acclimating and testing facilities.
                                31

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                                                        ES-1
                                               August,  1982
An accurate device controlling room temperature should
maintain the daphnids within the proper range of
temperatures.
     The selected temperature also approximates summer  water
temperatures for temperate lakes.  The test temperature  is
within the range in which _D. magna reproduces
parthenogenically.  Kaestner (1970) reports asexual
reproduction for D. magna in the temperature range 11°C  to
27°C.  It was also reported that temperatures below 15°C,
together with stress conditions, resulted  in sexual
reproduction.  Sexual reproduction was also reported as
temperatures approached 30°C.
     Variations in test temperatures beyond those suggested
could bias test results.  Bunting (1974) observed a 50
percent reduction in the growth rate of juvenile D. magna at
15°C as compared to 20°C.  This rate reduction manifested
itself in increased time periods between molts.  At 25°C an
increased growth rate was observed as compared to 20°C,  but
beyond 25°C a reduction in the growth rate was observed,
indicating a potential thermal stress. Bunting and Robertson
(1975) observed a significant difference in the acute
effects of two herbicides, Aiainotrizole and Amitrole, on
juvenile D. pulex at two temperatures.  It required
approximately twice the herbicide concentration at the. lower
temperature, 15°C, to produce the same effect as at 20°C.
The narrow range of specified temperatures for these test
guidelines should reduce significant differences in reported
test results.
     The information presented in Table 4 was derived from
                                32

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                                                       B5-1
                                               August, 1982
a search of articles concerning control mortality during
chronic toxicity tests.  In some cases, the 21-day control
mortality was interpolated from long-term survivorship
curves.  Based on the studies the recommendation of a 20
percent maximum control mortality criterion for 21 day
chronic tests should .provide a reasonable testing
requirement.

     Table 4: Percent Mortality in Controls for 21-
                day experiments using daphnids.
Percent
Mortality
12
8-20
0
15
0
11
Test
Temperature
20
18
19
20
20
20
Species
D . mag n a
D. magna
D. pulex
D. magna
D. pulex
D. pulex

trance
Berge
(1978)
Nebecker
Puglis i
(1974)
Bertram
and Hart
(1976)
Winner
and
Farreil
(1976)
Schober
and
Lamoert
(1977)
Vfinner
and
Fa.c<:e 1. 1
(1976)
                                33

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                                                        ES-1
                                                August,  1982
     C.  Reporting
          1.  Acute Tests.  For each set  of data,  with  a
minimum of the 24- and 48- hour observations, the  EC50  and
95 percent confidence limits should be calculated  based on
the mean measured concentration of the toxicant.   A
concentration-response curve for each observation  period
shall also be constructed.  It is strongly suggested  that a
statistician be consulted before the test is  initiated  to
insure that the specific test procedures  used will satisfy
the statistical requirements of the methods of data
analys is.
     Acute toxicity tests usually produce quantal  data, that
is,- counts of the number of organisms in  two  mutually-
exclusive .categories - alive/dead; affected/not affected.  A
variety of methods can be used to calculate an EC50 and 95
percent confidence limits from quantal data containing  two
or more concentrations at which the percent affected  is
between zero and one hundred.  The most widely used are the
probit moving average, and Litchfield-Wilcoxon methods
(Finney, 1964 and 1971, Stephan 1977, Litchfield and
Wilcoxon 1949).  The method of Litchfield  and Wilcoxon
(1949) produces a slope function */hich together with  EC50
value allows reconstruction of the probit  line,  The  slope
of the straight line can be useful for interpolation  of the
potential effects of concentrations other  than those  near
the EC50.  The slope may also provide indications  as  to the
mode of toxicity or any change in the toxic effects over the
experimental period.  Sprague (1969) discusses the
construction and interpretation of concentration-response
curves using several methods of data analysis.  Thus, both
the EC50 values and the dose-response curves are necessary

                                34

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                                                        ES-1
                                                August,  1982
for evaluating the hazard potential of a  given  test
substance.
          2.  Chronic Tests.  The statistical methods  used
to evaluate the effects of a test compound on survival  and
reproduction should be described in full.  The  choice  oC
methods is left to the testing facilities, but  it  is
suggested that a statistician be consulted prior  to  the
initiation of the test program.
     Suggested methods include analysis of variance  (ANOVA)
and appropriate mean separation tests  (Sokal and  Rahlf  1969,
Steele and Torrie 1960).
III.  Economic Aspects.
     The Agency awarded a contract to  Enviro Control,  Inc.
to provide us with an estimate of the  cost for  performing
static and flow-through acute toxicity tests and  renewal  and
flow-through chronic toxicity tests.   Enviro Control
supplied us with two estimates; a protocol estimate  and a
laboratory survey estimate.

                     Protocol Estimates
                                     range            mean
Acute (static and flow-through)     $322-$965         $643
Chronic (renewal and flow-through) $2021-$6064       $4043

     These estimates were prepared by separating  the
guidelines into  individual tasks  and estimating  the  hours
used to accomplish each task.  Hourly rates  were  then
applied to yield a total direct labor charge.  An overhead
rate of 115 percent, other direct costs  ($50 - acute,  $450  -
chronic), a general and administrative rate  of 10 percent
and a fee of 20 percent were then added  to the direct labor
charge to yield  the final estimate.
                                35

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                                                       ES-1
                                               August,- 1982
                 Laboratory  Survey  Estimates
                                      range          mean
Acute (static and flow-through)     $340-$1250       $743
Chronic (renewal and flow-through) $750-$10,000     $4178

     The. laboratory survey estimates were compiled from
three laboratories for the acute guideline and five
laboratories for the chronic guideline.
                                36

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                                                        ES-1
                                                August, 1982
IV.   INFERENCES
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     acute and chronic  toxicity tests.   Hydrobiologia
     59:125-134.

     Anderson BG .   1932.   The number of pre-adult'ins tars,
     growth,  relative growth and variation in Daphnia
     magna.  Biol. Bull.   63:81-98.

     Anderson BG  and Jenkins JC.  1942.  A time study of
     events in the life span of D.  magna.   Biol. Bull.
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     Anderson BG,  Lumer H and Zupancic  L J.  1937,  Growth
     and variability in Daphnia pulex.   Biol.. Bull. 73:444-
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     APHA.  1975.   American Public  Health  Association.
     Standard Methods for the Examination  of  Water and
     Wastewater.   14th  ed.   New York, N.Y.  1193 p.

     ASTM.  .1980.   American Society for Testing and Material
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     with  Fishes,  Macroinvertebrates and Amphibians.
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     Gerge WF.  1978.  Breeding Daphnia magna.
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     Bertram  PE and Hart  BA.  1979.  Longevity and
     reproduction  of Daphnia pal ex  exposed to cadmium
     contaminated  food  or water.  Environ. Pollut. 19:295-
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     Biesinger KE  and Christensen GM.  1972.   Effects of
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     Brandlova J,  Bramdl  A and Fernando CH.  1974.  The
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     Brooks JL.  1959.   Cladocera.   In  Freshwater Biology.
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                                            Augus t,
                                                   SS-1
                                                   1982
 Buikema AL,  Lee DR and Cairns  J.   1976.   Screening.
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 Bunting DL.   1974.   Zooplankton:  Thermal regulation and
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 Bunting DL and  Robertson  EB.   1975.   Lethal  and
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•Burns  CW.  1969.  Relation between filtering rate,
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 Daphnia] .  Limnol.  Oceanogr.  14:693-700.
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 Daphnia magna and  comparison of  the sensitivity of
 Daphnia magna with Daphnia pulex and Daphnia cucullata
 in  short-term experiments.   Hydrobiologia  59:135-140.

 Curtis  MW, Copland TL and  Ward CH.   1979.  Acute
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 D'Agostino AS and  Provasoli L.  1970.   Dixenic culture
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 Dewey TE and Parker BL.   1964.  Mass rearing of Daphnia
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Ooudoroff P.  1942.  The resistance and ci
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                                          .83 : 219-244.
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                                           August,  1992
Doudoroff P.  1945.  The resistance and acclimation of
marine flushes to temperature changes.   II,  Experiments
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Drummond RA and Dawson WF.  1970.  An  inexpensive
method for simulating diel patterns of  lighting  in the
laboratory.  Trans. Amer. Fish. Soc. 99;434-435.
Faust SD and Hunter JV.  1967.  Principles  and
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Finney DJ.  1964.  Statistical Methods  in Biological
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Finney DJ.  1971.  Probit Analysis.  3rd ed. Cambridge
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Frear DE and Boyd JE.  1967.  Use oC Daphnia magna for
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Gulati RD.  1973.  The ecology of the  common planktonic
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Hebert PDN.  1978.  The population biology  of Daphnia.
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Hoar WS.  1966.  General and Comparative Physiology.
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Hutch inson GE.  1967.  A Treatise on Limnology.  Volume
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Kaestner A.  1970.  Invertebrate Zoology. Vol. III.
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Kenaga EE.  1978.  Test organisms and  methods useful
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                           39

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                                                    ES-1
                                           August,  1982
Kenaga EE and Moolenar RJ.  1979.  Fish and Daphnia
toxicity as surrogates for aquatic plants and algae.
Env. Sci. Tech. 13:1479-1430.

Kring RL and O'Brien WJ.  1976.  Effects of varying
oxygen concentrations on the filtering rate of Daphnid
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Krugel S, Jenkins D and Klein S.  1978.  Apparatus for
the continuous dissolution of poorly water-soluble
compounds for bioassays.  Water Research  12:269-272.

Lee DR and Buikema AL.  .1979.  Molt-related sensitivity
of Daphnia oulex in toxicity testing.  J. Fish. Res.
Bd. Canada.  36:1129-1133.

Leeuwangh P.  1973.  Toxicity tests with Daphnids:   Its
application in the management of water quality.
Hydrobiologia 59:145-148.

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Lockwood APM.   1967.   Physiology of Crustacea.
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Makarewicz JD and Likens GE.  1979.  Structure and
function of the zooplankton community of Mirror Lake,
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McMahon JW.  1965.  Some physical factors influencing
the feeding behavior of Daphnia magna.  Can. J. Zool.
43:603-611.

Murphy JS.  1970.  A general method for the monaxenic
cultivation of the Daphindae.  Biol. Bull. 139:312-332.

Nebeker AV and Puglisi FA.  1974.  Effects of
polychlorinated biphenyls (PCB's) on survival and
reproduction of Daphnia, Gammarus and Tanytarsus.
Trans.  Amer. Fish. Soc. 103:722-728.
                           40

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                                                    ES-1
                                            August,- 1932
Needham JG, Galtsoff  PS,  Lutz  FE and  Welch PS.   1959.
Culture Methods  Hoc  Invertebrate Animals.   Dover Press,
New York, N.Y. 590 p.

Pennak RW.  1973,  Freshwater  Invertebrates  of  the
United States.   John  Wiley.  New York,  N.Y.  303 p.

Richman S.  1958.  The  trans forma tic a of  energy by
Daphnia pulex.   Eco.  Monog.  28:273-291.

Sanders HO.   1970.   Toxicities  of some  herbicides to
six species of freshwater crustaceans.   J.  Wat. PO.! kit.
Contr. Fed.   42:1544-1550.

Sax NI.   1979.   Dangerous Properties  of  Industrial
Material.   Reinhold,  New  York,  N.Y.  1343  p.
Schober U and Lampert W.  1977.   SlZuects  of  sublethal
concentrations of the herbicide Atrazin on growth and
reproduction  of  Daphnia pulex.   Bull.  Environ.  Contam.
Tox. 17:269-277.

Schultz TW  and Kennedy JR.   1976.  Cytotoxic effects of
the herbicide 3-amino-l,  2,  4-triazole  on  Daphnia
pulex .  Biol. Bull.   151:370-385.

Sokal RR and  Rohlf FJ.  1969.   Biometry.   W.H.  Freeman
Co., San Francisco,  CA.   776 p.

Sprague JB.   1969.   Measurement of  pollution toxicity
to fish.  I.  Bioassay methods  for acute  toxicity.
Water Research 3:793-821.

Steele RGD  and Torcie JH.   1960.   Principles and
Procedures  of Statistics.   McGraw Hill,  Inc. New York,
N.Y.

Stephan CE.   1977.   Methods  f.or calculating  and LC^.
In Aquatic  Toxicology and Hazard Evaluation. A.STM STP
~6~T4.  F.L.  Mayer and  J.L. Hamelink, eds  Philadelphia,
PA.  pp. 65-84.

Stroganov NS, Maks imova NN  and  Isakova  YI.   1977.
Long-term residual effects  of  polyethyleneamine on
Daphnia.  Hydrobiological J.   13:74-83.
                           41

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                                                   ES-1
                                           August,- 1982
Stross RG and Hill JC.  1968.  Photoperiod control of
winter diapause in the freshwater crustacean,
Daphnia.  Biol. Bull.  134:176-198.

Winner RW and Farrell MP. • 1976.  Acute and chronic
toxicity of copper to four species of Daphnia.  J.
Fish. Res. 3d. Canada.  33:1635-1691.

Whitten RH, Penergrass WR and Best RL.  1976.  Care of
Living Invertebrates.  Carolina Biological Supply Co.
Burlington, NC. 25 p.
                           42

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                                  BG-3
                                  August,  1982
     MYSID SHRIMP ACUTE TOXICITY TEST
        OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES  AND  TOXIC SUBSTANCES
  U.S.  ENVIRONMENTAL PROTECTION AGENCY
          WASHINGTON, D.C. 20460

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Office of Toxic Substances                                  EG-3
Guideline for Testing of Chemicals                 August,  1982


                 MYSID SHRIMP 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 ^t_. seg.) .  This guideline

prescribes a test using mys id shrimp as test organisms to develop

data on the acute toxicity of chemicals.  The United States

Environmental Protection Agency  (EPA) will  use data from these

tests in- assessing the hazard of a chemical to the aquatic

environment.

    (b)  Definitions.  The definitions in Section 3 of the  Toxic

Substances Control Act (TSCA) and  in Part 792—Good Laboratory

Practice Standards apply to this test guideline.  The  following

definitions also apply to this guideline.

    (1)  "Death" means the lack of reaction of a test  organism to

gentle prodding.

    (2)  "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.

    (3)  "LC50"  means that experimentally  derived concentration

of test substance that is calculated to kill 50 percent of  a test

population during continuous  exposure over  a specified period of

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                                                            EG-3
                                                    August,  1982
t ime.

    (4)  "Loading"  means  the  ratio  of  test  organisms  biomass

(grams, wet weight) to the volume  (liters) of  test  solution  in a

test chamber.

    (5)  "Retention chamber"   means  a structure  within a flow-

through test chamber which confines  the  test organisms,

facilitating observation of  test organisms and eliminating  loss

of organisms in outflow water.

    (6)  "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.  The test  substance is  introduced

into each test chamber.  In  a  flow-through test, the rate at

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  my s ids

acclimated in accordance with  the  test  design  into  the  test

chambers.  Mys ids in the test  chambers  are observed periodically

during the test, the dead mys ids removed and the findings

recorded.  Dissolved oxygen  concentration, pH, temperature,

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                                                            EG-3
                                                   August,  1982
salinity, the concentration of test substance, and other  water

quality characteristics are measured at specified  intervals  in

test chambers.  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 determine:

    (A)  which life stage (juvenile or young adult)  is to be

utilized in the definitive test.

    (B)  the test solution concentrations for the  definitive

test.

    (ii)  The mys ids should be exposed to a  series of  widely

spaced concentrations of test substance (e.g., 1,  10,  100 mg/1,

etc.), usually under static conditions.

    (iii)  This test should be conducted with both newly-hatched

juvenile (< 24 hours old) and young adult (5-6 days  old)

mysids.  For each age class (juvenile or young adult), a  minimum

of ten mys ids should be exposed  to each concentration  of  test

substance for up to 96 hours.  The exposure  period may be

shortened if data suitable for the purpose of the  range-finding

test can be obtained in less time.  The age  class  which is most

sensitive to the test substance  in the range-finding test should

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                                                            EG-3
                                                    August,  1982
be utilized in the definitive  test.  When  no  apparent  difference

in sensitivity of the  two  life stages  is found,  juveniles  should

be utilized in the definitive  test.  No replicates  are  required

and nominal concentrations of  the  chemical  are  acceptable.

    (4)  Definitive test.  (i)  The purpose of  the  definitive7

test is to determine the concentration-response  curves  and  the

48- and 96- hour LC50  values'with  the  minimum amount of  testing

beyond the range-finding test.

    (ii)  The definitive test  should be conducted on the mys id

life stage (juveniles  or young adults) which  is  most sensitive to

the test substance being evaluated.

    (iii)  A minimum of 20 mysids  per  concentration should  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/1).   An equal  number  of mys ids

should be placed in two or more replicates.   If  solvents,

solubilizing agents or emulsifiers have to  be used, they should

be commonly used carriers and should not possess a synergis tic or

antagonistic effect on the toxicity of the  test  substance.   The

concentration of solvent should not exceed  0.1 ml/1.     The

concentration ranges should  be selected to  determine the

concentration-response curves  and  LC50 values at 48 and  96

hours.  The concentration of test  substance in  test solutions

should be analyzed prior to  use.

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                                                            EG-3
                                                   August,  1982
    (iv)  Every test should include controls consisting of the

same dilution water, conditions, procedures, and mys ids from  the

same population or culture container, except that none of the

chemical is added.

    (v)  The dissolved oxygen concentration, temperature,

salinity, and pH should be measured at the beginning of the test

and at 24, 48, 72 and 96 hours  in each chamber.

    (vi)  The test duration is  96 hours.  The test is

unacceptable if more than 10 percent of the control  organisms die

or exhibit abnormal behavior during the 96 hour test period.

Each test chamber should be checked for dead mys ids  at 3, 6,  12,

24, 43, 72 and 96. hours after the beginning of the test.

Concentration-response curves and 43- and 96- hour LC50 values

should be determined along with their 95 percent confidence

limits.

    (vii)  In addition to death, any abnormal behavior or

appearance should also be reported.

    (viii)  Distribution of mys ids among test chambers should be

randomized.   In addition, test  chambers within the testing area

should 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.

    (ix)  The concentration of  dissolved test substance (that

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                                                            EG-3
                                                    August,  1982
which passes through a 0.45 micron filter)  in the  chambers should

be measured as often as  is feasible during  the  test.   At  a

minimum, during static tests, the concentration of test substance

should be measured in each chamber at  the beginning  and at the

end of the test.  During the flow-through test, the  concentration

of test substance should be measured (A) in each chamber  at  the

beginning of the test and at 48 and 96 hours after the start of

the test; (B) in at least one chamber  containing the next to the

lowest test substance concentration at least once  every 24 hours .

during the test; and (C) 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

substance should not vary more than 20 percent.

    (5)  [Reserved]

    (6)  Analytical measurements — (i)  Test chemical.  Deionized
                                                   i
water should be used in making stock solutions  of  the  test

substance.  Standard analytical methods should  be  used whenever

available in performing the analyses.  The analytical  method used

to measure the amount of test substance in  a sample  should be

validated before beginning the test by appropriate laboratory

practices.  An analytical method is not acceptable if  likely

degradation products of the test substance, such as  hydrolysis

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                                                            EG-3

                                                   August,  1982
and oxidation products, give positive or negative interferences


which cannot be systematically  identified and corrected


mathematically.


    (ii)  Numerical.  The number of dead mys ids should be  counted


during each definitive test.  Appropriate statistical analyses


should provide a goodness-of-fit determination for  the


concentration-response curves.  A 48- and 96- hour  LC50 and


corresponding 95 percent interval should be  calculated.


    (d)  Test conditions — (1)   Test species — (i)  Selection.


(A)  The mys id shrimp, My s id ops is bahia, is  the organism


specified for these tests.  Either juvenile  (< 24 hours old) or


young adult (5-6 days old) mys ids are to be  used to start  the


test.


    (3)  Mys ids to  be used in acute toxicity  tests  should


originate from laboratory cultures in order  to assure that the


individuals are of  similar age  and experiential history.   Mys ids


used for establishing laboratory cultures may be purchased
     /

commercially or collected from  appropriate natural  areas.


Because oE similarities with other mysid species, taxonomic


verification should be obtained from the commercial supplier or


through an appropriate systematic key.


    (C)  Mysids used in a particular test should be of similar


age and be of normal size and appearance for  their  age.  Mys ids

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                                                            EG-3
                                                    August,  1982
should not be used for a test if they  exhibit  abnormal  behavior

or if they have been used  in a previous  test,  either in a

treatment or in a control  group.

    (ii)  Acclimation.  (A)  Any change  in  the temperature and

chemistry of the dilution  water used for  holding or  culturing  the

test organisms to those of the test should  be  gradual.   Within  a

24-hour period, changes in water temperature should  not exceed

1°C,  while salinity changes should not exceed  5 Percent.

    (B)   During acclimation mysids should be maintained in

facilities with background colors and  light intensities similar

to those of the testing areas.

    (iii)  Care and handling.  Methods for  the care  and handling

of mys ids such as those described in USEPA  (1978)  can be  used

during holding, culturing  and testing periods.

    ( iv)  Feeding.  Mys ids should be fed  during testing.   Any

food utilized should support survival, growth  and  reproduction  of

the mysids.  A recommended food is live Artemia spp.  (48-hour-old

nauplii).

    (2)   Facilities — (i)  Apparatus.   (A)   Facilities which  may

be needed to perform this  test include:  (I)  flow-through  or

recirculating tanks for holding and acclimating mys ids ;  (J_)  a

mechanism for controlling and maintaining the  water  temperature

during the holding, acclimation and test  periods;  (_3_) apparatus


                                8

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                                                            EG-3
                                                    August,  1982
for straining particulate matter, removing gas  bubbles, or

aerating the water, as necessary; and  (_4_) an  apparatus  for

providing a 14-hour light and 10-hour  dark photope.riod  with  a  15

to 30 minute transition period. In addition,  for  flow-through

tests, flow-through chambers and a test substance delivery system

are required.  Furthermore, it  is recommended  that mys ids be held

in retention chambers within test chambers to  facilitate

observations and eliminate  loss of test organisms through outflow

water.  For static tests, suitable chambers for exposing test

mys ids to the test substance are required.  Facilities  should  be

well ventilated and free of fumes and  disturbances that may
                                         o
affect the test organisms.

    (3)  Test chambers should 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)  Cleaning .  Test substance delivery systems and test

chambers should be cleaned  before each test following standard

laboratory practices.

    (iii)  Construction materials.  (A)  Materials and  equipment

that contact test solutions should be  chosen  to minimize sorption

of test chemicals from dilution water  and should  not contain

substances that can be leached into aqueous solution in

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                                                            EG-3
                                                   August,  1982
quantities that can affect test results.

    (3)  For use in the flow-through test, retention chambers

utilized for confinement of test organisms can be constructed

with netting material of appropriate mesh size.

    (iv)  Dilution water.  (A)  Natural or artificial seawater is

acceptable as dilution water if mys ids will survive and

successfully reproduce in it for the duration of the holding,

acclimating and testing periods without showing signs of stress,

such as reduced growth and fecundity.  Mys ids should be cultured •

and tested in dilution water from the same origin.

    (B)  Natural seawater should be filtered through a filter

with a pore size of < 20 microns prior to use in a:test.

    (C)  Artificial seawater can be prepared by adding

commercially available formulations or by adding specific amounts

of reagent-grade chemicals to deionized water.  Deionized water

with a conductivity less than 1 u ohm/cm at 12°C is acceptable

for making artificial seawater.  When deionized water is prepared

from a ground or surface water source, conductivity and total

organic carbon (or chemical oxygen demand) should be measured on

each batch.

    (v)  Test substance delivery system'.  In flow-through tests,

proportional diluters, metering pumps or other suitable systems

should be used to deliver test substance to the test chambers.


                                10                  :

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                                                            BG-3
                                                   August,  1982
The system used should be calibrated before each test.

Calibration includes determining the flow rate  through  each

chamber and the concentration of the test substance in  each

chamber.  The general operation of the test substance delivery

system should be checked twice daily during a test.  The 24-hour

flow through a test chamber should 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 among test chambers or

across time.

    (3)  Test parameters.  Environmental parameters of  the water

contained in test chambers should be maintained as specified

below:

    (i)  Temperature of 25 ± 2°C.

    (ii)  Dissolved oxygen concentration between 60 and 105

percent saturation.  Aeration, if needed to achieve this level,

should be done before the addition of the test substance.  All

treatment and control chambers should be given  the same aeration

treatment.

    (iii)  The number of mys ids placed in a test solution should

not be so great as to affect results of the test.  Thirty mys ids

per liter is the recommended level of loading for  a static

test.  Loading requirements for the flow-through test will vary

depending on the flow rate of dilution water.   The loading should


                                11

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                                                           EG-3
                                                   August, 1982
not cause the dissolved oxygen concentration to fall below the

recommended levels.

    (iv)  Photoperiod of 14 hours light and 10 hours darkness,

with a 15-30 minute transition period.

    (v)  Salinity of 20 ± 3 o/oo.
    (e)  Reporting.  The sponsor should submit to the EPA all

data developed during the test that are suggestive or predictive

of acute toxicity and all concomitant toxicologic

manifestations.  In addition, to the general reporting

requirements prescribed in Part 792--Good Laboratory Practice

Standards, the reporting of test data should include the

following:

    (1)  The source of the dilution water, its chemical

characteristics (e.g., salinity, pH, etc.) and a description of

any pretreatment.

    (2)  Detailed information about the test organisms, including

the scientific name and method of verification, age, source,

history, abnormal behavior, acclimation procedures; and food used.

    (3)  A description of the test chambers, the depth and volume

of solution in the chamber, the way the test was begun (e.g.,

conditioning, test substance additions, etc.), the number of

organisms per treatment, the number of replicates, the loading,

the lighting, the test substance delivery system and the flow


                                12

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                                                            EG-3
                                                   August,  1982
rate expressed as volume additions per 24 hours.

    (4)  The measured concentration of test substance  in  test

chambers at the times designated.

    (5)  The number and percentage of organisms that died or

showed any other adverse effects in the control and in each

treatment at each observation period.

    (6)  Concentration-response curves should be fitted to

mortality data collected at 24, 48, 72 and 96 hours.   A

statistical test of goodness-of-fit should be performed and the  .

results reported.

    (7)  The 48-hour and 96-hour LC50, and when sufficient data
o
have been generated, the 24-hour and 72-hour LC50's and the

corresponding 95 percent confidence limits and the methods used

to calculate the values.  These calculations should be made using

the average measured concentration of the test substance.

    (8)  Methods and data records of all chemical analyses of

water quality and test substance concentrations, including method

validations and reagent blanks.

    (9)  The data records of the holding, acclimation  and test

temperature and salinity.

    (f)  References.  U.S. Environmental Protection Agency,

1978.   Bioassay Procedures for the Ocean Disposal Permit

Program.  Environmental Research Laboratory, Office of Research

and Development.  Gulf Breeze, Fl. EPA-600-9-78-010.


                                13

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                                 EG-4
                                 August,  1982
    MYSID SHRIMP CHRONIC TOXICITY TEST
        OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC  SUBSTANCES
   U.S.  ENVIRONMENTAL PROTECTION AGENCY
          WASHINGTON, D.C.  20460

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Office of Toxic Substances                            '      SG-4
Guideline for Testing Chemicals                     August,  1982


                Mys id  shrimp 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 seg.).  This  guideline

prescribes tests using mys ids as test organisms  to develop data

on the chronic toxicity of chemicals.  The United States

Environmental Protection Agency  (EPA) will use data from these

tests in assess ing the hazard of a chemical to the aquatic

environment.

    (b)  Def initions .  The definitions in section 3 of  the Toxic

Substances Control Act (TSCA) and  in Part 792—Good Laboratory

Practice Standards apply to this test guideline.  The  following

definitions also apply to this guideline:

    (1)  "Chronic toxicity test" means a method  used to determine

the concentration of a substance that produces an adverse effect

from prolonged exposure of an organism to that substance.  In

this test, mortality, number of  young per female and growth  are

used as measures of chronic toxicity.

    (2)  "Death" means the lack  of reaction of a test  organism to

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                                                            SG-4
                                                   August,  1982
gentle prodding.

    (3)  "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.

    (4)  "Gl (Generation 1)" means those mysids which are used to

begin the test, also referred to as adults; G2  (Generation  2) are

the young produced by Gl.

    (5)  "LC50" means that experimentally derived concentration

of test substance that is calculated to kill 50 percent of  a test

population during continuous exposure over a specified period of

t ime.

    (6)  "Loading" means the ratio of test organism biomass

(gram, wet weight) to the volume (liters) of test solution  in a

tes t chamber.

    (7)  "MATC" (Maximum Acceptable Toxicant Concentration) means

the maximum concentration at which a chemical can be present and

not be toxic to the test organism.

    (8)  "Retention chamber" means a structure  within a flow-

through test chamber which confines the test organisms,

facilitating observation of test organisms and  eliminating

washout from test chambers.

    (c)  Test procedures — (1)  Summary of the test..  (i)  In

preparation for the test, the flow of test solution through each

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                                                            EG-4
                                                    August,  1982
chamber is adjusted to the rate desired.  The test substance  is

introduced into each test chamber.  The  rate at 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 mysids acclimated in

accordance with the test design into retention chambers within

the test and the control chambers.  Mysids in the test and

control chambers are observed periodically during the  test, the

dead mysids removed and the findings reported.

    (ii)  Dissolved oxygen concentration, pH, temperature,

salinity, the concentration of test substance and other water

quality characteristics are measured at  specified intervals in

selected test chambers.

    (iii)  Data collected during the test are used to  develop a

MATC (Maximum Acceptable Toxicant Concentration) and quantify

effects on specific chronic parameters.

    (2)  [Reserved]

    (3)  Range-finding test.  (i)  A range-find ing test should be

conducted to establish test solution concentrations for the

definitive test.

    (ii)  The rays ids should be exposed to a series of  widely

spaced concentrations of the test substance (e.g., 1,  10, 100

mg/1), usually under static conditions.

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                                                            EG-4
                                                    August,  1982
    (iii)  A minimum of 10 mys ids should be exposed  to each  con-

centration 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,  LC50 values,

and effects of a chemical on growth and reproduction during

chronic exposure.

    (ii)  A minimum of 40 mys ids per concentration should be

exposed to four 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/1). .An equal  number of mys ids

should be placed in two or more replicates.   If solvents,

solubilizing agents or emulsifiers have to be used,  they should

be commonly used carriers and should not possess  a synergistic or

antagonistic effect on the toxicity of the test substance.   The

concentration of solvent should not exceed 0.1  ml/1.     The

concentration ranges should be selected to determine the

concentration-response curves, LC50 values and  MATG.

Concentration of test substance in test solutions should be

analyzed prior to use.

    (iii)  Every test should include controls consisting of  the

same dilution water, conditions, procedures and mysids from  the

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                                                            EG-4
                                                    August,  1982
same population or culture container, except  that none  of  the

chemical is added.

    (iv)  The dissolved oxygen concentration,  temperature,

salinity and pH should be measured at the beginning  of  the  test

and on days 7, 14, 21 and 28 in each chamber.

    (v)  The test duration is 28 days.  The test is  unacceptable

if more than 20 percent of the control organisms die, appear

stressed or are diseased during the test.  The  number of dead

mys ids in each chamber should be recorded on  days 7, 14, 21 and

28 of the test.  At the time when sexual characteristics are

discernable in the mysids (approximately 10^12  days  in  controls;

possible delays may occur in mys ids exposed to  test  substances),

the number of males and females (identified by  ventral  brood

pouch) in each chamber should be recorded.  Body length (as

measured by total mid line body length, from the anterior tip of

the carapace to the posterior margin of the uropod)  should  be

recorded for males and females at the time when sex  can be

determined simultaneously for all mysids in control  and  treatment

groups.  This time cannot be specified because  of possible  delays

in sexual maturation of mys ids exposed to test  substances.  A

second observation of male and female body lengths should  be

conducted, on day 28-of the test.  To reduce stress.on the mys ids ,

body lengths can be recorded by photography through  a stereo-

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                                                            EG-4
                                                    August,  1982
microscope with appropriate scaling information.   As  offspring

are produced by the Gl rays ids  (approximately  13-16  days  in

controls), the young should be counted and separated  into

retention chambers at the  same test substance concentration as

the chambers where they originated.   If  available  prior  to

termination of the test, observations on the  mortality,  number of

males and females and male and female body length should be

recorded for the G2 mysids.  Concentration-response curves,  LC50

values and associated 95 percent confidence limits  for the number

of dead mys ids (Gl) should be  determined  for  days  7,  14, 21 and

28.  An MATC should be determined for the most sensitive test

criteria measured (cumulative  mortality  of adult mys ids, number

of young per female, and body  lengths of  adult males  and

f emales).

    (vi)  In addition to death, any abnormal  behavior or

appearance should also be  reported.

    (vii)  Distribution of mys ids among  test  chambers should be

randomized.  In addition,  test chambers  within the, testing area

should be positioned in a  random manner  or in a way in which

appropriate statistical analyses, can be  used  to determined the

variation due to placement.

    (viii) - The concentration  of dissolved tes t subs tance-  ( that

which passes through a 0.45 micron filter) in the  chambers  should

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                                                            EG-4
                                                   August,  1982
be measured as often as is feasible during the test.  The

concentration of test substance should be measured:  (a)  in  each

chamber at the beginning of the test and on days 7,  14,  21  and

28; and (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 substance

should 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

substance.   Standard analytical methods should be employed

whenever available in performing the analyses.  The  analytical

method used to measure the amount of test substance  in a sample

should be validated before beginning the test by appropriate

laboratory practies.  An analytical method is not acceptable  if

likely degradation products of the test substance, such  as

hydrolysis and oxidation products, give positive or  negative

interferences which cannot be systematically identified  and

corrected mathematically.

    (ii)  Numerical.  (A)  The number of dead mys ids, cumulative

young per female and-body .lengths of male and female mysids

should be recorded during each definitive test.  Appropriate

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                                                            EG-4
                                                    August,  1982
statistical analyses should provide a goodness^-of-fit

determination for the day 7, 14,  21 and  28  adult  (Gl) death

concentration-response curves.

    (B)  A 7-, 14-, 21- and 28- day LC50, based on  adult  (Gl)

death, and corresponding 95 percent confidence intervals  should

be calculated.  Appropriate statistical  tests  (e.g., analysis  of

variance, mean separation test) should be used to test  for

significant chemical effects on chronic  test criteria (cumulative

mortality of adults, cumulative number of young per female and

body lengths of adult male and females)  on  designated days.  An

MATC should be calculated using these chronic  test  criteria.

    (d)  Test conditions — (1)  Test species — (i)  Selection.

(A)  The mys id shrimp, Mysidopsis bahia, is the organism

specified for these tests.  Juvenile mys ids, _£ 24 hours old, are

to be used to start the test.

    (3)  Mys ids to be used in chronic toxicity tests should

originate from laboratory cultures in order to ensure the

individuals are of similar age and experiential history.  Mys ids

used for establishing laboratory  cultures may  be purchased

commercially or collected from appropriate  natural  areas.

Because of similarities with other mysid species, taxonomic

determinations..should be verified by the commercial- supplier or

by an appropriate individual.

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                                                            EG-4
                                                    August, 1982
    (C)  Mys ids used in a particular  test  should  be of  similar

age and be of normal size and  appearance  for their age.

    (D)  Mys ids should not be  used  for  a  test if  they exhibit

abnormal behavior, or  if they  have  been used in a previous test,

either in a treatment or in a  control group.

    (ii)  Acclimation.  (A)  Any  change in the temperature and

chemistry of the water used for holding or culturing the  test

organisms to those of  the test should be  gradual.  Within a 24-

hour period, changes in water  temperature  should  not exceed 1°C,

while salinity changes should  not exceed  5 Percent.

    (B)  During acclimation mysids  should  be maintained in

facilities with background colors and light intensities similar

to those of the testing areas.

    (iii)  Care and handling.  Methods  for the care and handling

of mys ids such as those described in  US EPA (1978) can be  used

during holding, culturing and  testing periods.

    (iv)  Feeding.  Mysids should be  fed  during testing.   Any

food utilized should support survival,  growth and reproduction of

the mysids.  A recommended food is  live Artemia spp. nauplii

(approximately 48 hours old).

    (2)  Facilities—(i)  Apparatus.  (A)   Facilities which may

be needed to perform this test include:  (_L_) flow-through or

recirculat.ing tanks for holding and acclimating mys ids;  (_2_) a

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                                                            EG-4

                                                    August,  1982
mechanism for controlling and maintaining the  water  temperature



during the holding, 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 14-hour light and 10-hour  dark photoperiod  with  a  15-



to 30-minute transition period.   In  addition,  flow-through



chambers and a test substance delivery system  are  required.   It



is recommended that mys ids be held in  retention chambers  within



test chambers to facilitate observations and eliminate  loss



through outflow water.



    (B)  Facilities should be well ventilated  and  free  of fumes
                                                 o


and disturbances that may affect  the test organisms.



    (C)  Test chambers should 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)  Cleaning.  Test substance  delivery systems and  test



chambers should be cleaned before each test following standard



laboratory practices.



    (iii)  Construction materials.   (A)  Materials and  equipment



that contact test solutions should be  chosen to minimize sorption



of tes t chemicals --from-the dilution  water and  should not contain



substances that can be leached into  aqueous solution in





                                10

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                                                            EG-4
                                                   August,  1982
quantities that can affect the test results.

    (B)  Retention chambers utilized for confinement of  test

organisms can be constructed with netting material of appropriate

mesh size.

    (iv)  Dilution water.  (A)  Natural or artificial seawater  is

acceptable as dilution water if mysids will survive and

successfully reproduce in it for the duration of the holding,

acclimating and testing periods without showing signs of stress,

such as reduced growth and fecundity.  Mys ids should be  cultured

and tested in dilution water from the same origin.

    (3)  Natural seawater should be filtered through a filter

with a pore size of < 20 microns prior to use in a test.

    (C)  Artificial seawater can be prepared by adding

commercially available formulations or by adding specific amounts

of reagent-grade chemicals to deionized or glass-distilled

water.  Deionized water with a conductivity less than 1  u ohm/cm

at 12°C is acceptable as the diluent for making artificial

seawater.  When deionized water is prepared from a ground or

surface water source, conductivity and total organic carbon (or

chemical oxygen demand) should be measured on each batch.

    (v)  Test substance delivery system.  Proportional diluters,

metering pumps or other suitable systems should be used  to

deliver test substance to the test chambers.  The system used


                                11

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                                                            EG-4
                                                   August,  1982
should be calibrated before each test.  Calibration includes

determining the flow rate and the concentration of the  test

substance in each chamber.  The general operation of the test

substance delivery system should be checked twice daily during  a

test.  The 24-hour flow rate through a chamber should be equal  to

at least five times the volume of the chamber.  The flow rates

should not vary more than 10 percent among chambers or  across

time.

    (3)  Test parameters.  Environmental parameters of  the water'

contained in test chambers should be maintained as specified

below:

    (i)  Temperature of 25 _+ 2°C.

    (ii)  Dissolved oxygen concentration between 60 and 105

percent saturation.  Aeration, if needed to achieve this level,

should be done before the addition of the test substance.  All

treatment and control chambers should be given the same aeration

treatment.                •,

    (iii)  The number of mysids placed in a test solution should

not be so great as to affect results of the test.  Loading

requirements for the 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 14 hours light and 10 hours darkness,


                                12

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                                                            EG-4
                                                   August,  1982
with a 15-30 minute transition period.

    (v)  Salinity of 20 _+• 3 °/oo.

    (e)  Reporting. 'The sponsor should submit to the  EPA all

data developed by the test that are suggestive or predictive of

chronic toxicity and all concomitant toxicologic

manifestations.  In addition to the general reporting

requirements prescribed in Part 792--Good Laboratory Practice

Standards, the reporting of test data should include the

following:

    (1)  The source of the dilution water, its chemical

characteristics (e.g., salinity, pH, etc.) and a description of

any pretreatment.

    (2)  Detailed information about the test organisms, including

the scientific name and.method of verification, average length,

age, source, history, observed diseases, treatments acclimation

procedures and food used.

    (3)  A description of the test chambers, the depth and volume

of solution in the chamber, the way the test was begun (e.g.,

conditioning, test substance additions, etc.), the number of

organisms per treatment, the number of replicates, the loading,

the lighting, the test substance delivery system, and  the flow

rate expressed as volume additions, per 24-hours.  ._. ..
                                13

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                                                            EG-4
                                                    August,  1982
    (4)  The measured concentration of test substance  in  test

chambers at the  times designated.

    (5)  The first time  (day) that sexual characteristics  can  be

observed in controls and  in each test substance  concentration.

    (6)  The length of time for the appearance of the  first brood

for each concentration.

    (7)  The means (average of replicates) and respective  95

percent confidence intervals for:
                                                    /
    (A)  Body length of males and females at  the  first

observation day  (depending on time of sexual  maturation)  and on

day 28.

    (3)  Cumulative number of young produced  per  female on day

28.

    (C)  Cumulative number of dead adults on  day  7, 14, 21 and

28.

    (D)  If available prior to test termination  (day 28),  effects

on G2 mysids (number of males and females, body  length of  males

and females and  cumulative mortality).

    (8)  The MATC is calculated as the geometric  mean  between  the

lowest measured  test substance concentration  that had  a

significant (P<0.05) effect and the highest measured test

substance concentration that had .no significant  (P>0.05)  effect

in the chronic test.  The most sensitive of the  test criteria  for



                                14

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                                                            EG-4

                                                   August,  1982



adult  (Gl) mysids  (cumulative number of dead mysids, body  lengths



of males and females or the number of young per  female)  is  used



to calculate the MATC.  The criterion selected for MATC



computation is the one which exhibits an effect  (a statistically



significant difference between treatment and control groups;



P<0.05) at the lowest test substance concentration for the



shortest period of exposure.  Appropriate statistical tests



(analysis of variance, mean separation test) should be used  to



test for significant chemical effects.  The statistical  tests



employed and the results of these tests should be reported.



    (9)  Concentration-response curves should be fitted  to  the



cumulative number of adult dead for days 7, 14,  21 and 28.   A
                        o


statistical test of goodness-of-fit should be performed  and  the



results reported.



    (10)  An LC50 value based on the number of dead adults  with



corresponding 95 percent confidence intervals for days 7,. 14, 21



and 28.  These calculations should be made using the average



measured concentration of the test substance.



    (11)  Methods and data records of all chemical analyses  of



water quality and test substance concentrations, including  method



validations and reagent blanks.



    (12)  The data records of the holding, acclimation and  test



temperature and .salinity.





                                15

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                                                           EG-4
                                                   August, .1982
    (f)  References.  U.S. Environmental Protection Agency,

1978.   Bioassay Procedures for the Ocean Disposal Permit

Program.   Environmental Research Laboratory, Office of Research

and Development Gulf Breeze, FL: EPA-600/9-78-010.
                                16

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                                   ES-2
                                   August,  1982
         TECHNICAL  SUPPORT DOCUMENT

                     FOR

MYSID SHRIMP ACUTE  AND CHRONIC TOXICITY TEST
         OFFICE OF  TOXIC  SUBSTANCES
  OFFICE OF PESTICIDES AND TOXIC  SUBSTANCES
    U.S. ENVIRONMENTAL  PROTECTION AGENCY
           WASHINGTON,  D.C.  20460

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                        Table of Contents

        Subject                                         Page
I.      Purpose                                          1
II.     Scientific Aspects                               1
        Test Procedures                                  1
        General                                          1
        Range-Finding Test                               3
        Definitive Test                                  4
        Test Conditions                                  6
        Test Species                                     6
        Selection                                        6
        Sources                                          7
        Maintenace of Test Species                       8
        Handling and Acclimation                         8
        Fe ed i ng                                          8
        Facilities                                       10
        General                                          10
        Construction Materials                           11
        Test Substance Delivery System                   13
        Test Chambers                                    1.4
        Cleaning of Test System                          15
        Dilution Water                                   16
        Controls                                         18
        Carriers                                         18
        Randomization                                    19
        Environmental Conditions                         19
        Dissolved Oxygen                                 19
        Light                                            20
        Temperature and Salinity                         20
        Reporting                                        21
III.     Economic Aspects                                 24
IV.     References                                       26

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Office of Toxic Substances                               ES-2
                                                 August,  1932
I.  Purpose
    The purpose of this document  is  to provide  the
scientific background and rationale used  in  the development
of Test Guideline EG-2 which uses Mysid shrimp  to evaluate
the toxicity of chemical substances.  The Document provides
an account of the scientific evidence and an explanation  of
the logic used in the selection of the test  methodology,
procedures and conditions prescribed  in the  Test
Guideline.  Technical issues and practical considerations
relevant  to the Test Guideline are discussed.   In;addition,
estimates of the cost of conducting the test  are provided.
II.  Scientific Aspects
    A.  Test Procedures
          1. .General
    The choice of rays id toxicity  tests (96-hour static, 96-
hour flow-through or 28-day chronic is based  on several
considerations.  A static test requires less  equipment,
fewer chemical analyses and disposal of smaller quantities
of contaminated wastewater than flow-through  systems.
Static tests are a relatively easy means  to  evaluate and
compare the acute effects of a test substance.
    The flow-through system more closely simulates the
natural exposure process, eliminating problems  associated
with accumulation of organic material and toxic metabolic
products.  Test substances are more thoroughly mixed in a
flow-through system and problems of sorption  to suspended
sediments, feces and uneaten food are reduced.  In order  to
produce valid toxicity test results, the  flow-through test
should be used with test substances which have  a high oxygen
demand, are highly volatile, are unstable, biodegradable or

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                                                         ES-2
                                                 August,  1982
are removed in significant amounts  by the  test organisms.
    A more comprehensive evaluation of  the potential
environmental hazard of a test substance is  available  from
chronic toxicity testing.  The chronic  test using  mys ids
provides two important advantages over  other  toxicity
testing regimes.  First, it permits an  evaluation  of
response to chronic exposure to a test  substance.   Second,
it allows a determination of effects of the test substance
throughout sequential life stages of the organism  (juvenile,
adult, egg).  These data can be used to estimate potential
adverse population and community changes associated with
shifts in growth and reproductive potential.
    For the acute tests, 96 hours is a  convenient  interval
of time for starting and completing a test in a normal  five-
                                           0
day work week, and is better than shorter  periods  for
estimating accumulative and other chronic  effects.  Because
set-up is the most expensive portion of a  test, a  96-hour
test is only slightly more expensive than  24  or 48  hour
tests.  Yet additional data on the  LCSO's  over time and the
observations of other abnormal effects  that do not  appear  in
shorter tests are gained for this slight increase  in cost.
Although the 43 hour test can reduce costs, eliminate  the
necessity of feeding of the mys ids  during  the test, and make
the test more comparable to the 48  hour Daphnia acute
toxicity tests, the 96-hour toxicity test  was selected  for
the mys id test guidelines because of greater  potential  for
determining the incipient LC50 (threshold  limit for acute
toxicity) through extension of the  toxicity curve.  In
situations where the 96 hour LC50 does  not permit  estimation
of an incipient LC50 (lethal threshold  concentration),

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                                                         ES-2
                                                August,  1982
continuation of the test may allow better estimation  of  the
toxic effects of the test substance.   In a  review of  375
toxicity tests, Sprague (1969) found that a lethal threshold
clearly had not been reached in 42 tests, while  in 122 other
tests the threshold was reached in four days or  longer.
Because of the importance of the  incipient  LC50  in hazard
assessment, continuation of the acute  toxicity tests  past
the 96 hours is recommended for those  test  substances which
do not elicit a threshold concentration within the four  day
test period.
    For the life cycle test, a 28-day  experimental period  is
used to permit testing through at least one complete  life
cycle in Mysidopsis bahia at 25°C.  Juveniles utilized for
tests reach sexual maturity within 12-14 days under normal
conditions at this temperature (USEPA  1978).  However, test
substances may delay sexual maturity several days (Nimmo et
al. in press c).  Tests longer than 28 days are  not
recommended because of possible fouling of  retention
chambers with subsequent decreases in  the efficiency  of  the
flow-through system.
         2.  Range-Finding Test
    The concentration range for the definitive test is
normally chosen based on the results of a range-finding
test.  In the acute static and flow-through test, the range-
finding test also serves as a means of determining: which
life stage (juvenile or young adult) is most sensitive to
the test substance and should be used  in the definitive
test.  For the acute tests, range-finding tests  are normally
short-term (24-96 hour-)-, static or flow-through-  bioass ays ,"
which utilize fewer organisms per test substance

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                                                         ES-2
                                                 August,  1982
concentration than the definitive test.   For the  life  cycle
test, range-finding tests may  take  the  form of  acute,  flow-
through tests using different  life stages or determination
of incipient LCSO's to allow selection  of appropriate
definitive test concentrations.  In all cases,  the range-
finding test is conducted to reduce the expense  involved
with having to repeat a definitive test due to  inappropriate
test substance concentrations.
         3.  Definitive Test
    The concentration range for the definitive  test  is
chosen based on the results of preliminary range-finding
tests.  By testing a minimum of five concentrations  in  the
acute bioassays, partial kills both above and below  the
median 50 percent mortality level are probable  and will help
define the concentration-response relationship.   The more
partial kills, the better the  definition  of the
concentration-response curve.  The slope  and shape of the
concentration-response curve may give insight into possible
mechanisms of action of a chemical and will allow estimation
of the effects of lower concentrations upon test  :
organisms.  In addition, by having partial kill data, a
greater array of statistical methods can  be used  to
determine the LC50.
    The utilization of the most sensitive of the  two life
stages (juvenile or young adult), as required for the
definitive acute mys id tests,  is based on evidence that
these two life stages exhibited differential mortality to
eleven pesticides (Nimmo et al. in press  b).  The procedure
of testing both stages in the  range-finding test  and using
the most sensitive life stage  in the definitive test permits

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                                                         ES-2
                                                August,  1982
determination of toxicity data at less expense than
conducting two complete definitive tests.
    Because of the increased expense of the 28-day mysid
life cycle test, the nature of the recorded data and
equipment limitations, testing of a minimum of only four
concentrations is required.  These observations will allow
determination of the MATC (maximum acceptable toxicant
concentration) limits for the most sensitive life cycle
criterion recorded (mortality, body lengths of males and
females and numbers of young per female).
    The number of mys ids exposed to each test substance
concentration (e.g. 20 in acute tests and 40 in chronic
test) is designed to allow adequate numbers for statistical
evaluation even with the presence of partial mortality
(Nimmo et al. 1977, 1978, USEPA 1978).
    Measurement of test substance concentrations at
designated periods during static and flow-through tests
allows documentation of real test concentrations at
appropriate periods under acute and chronic conditions.
    Chemical and physical parameters (temperature, pH,
dissolved oxygen and salinity) are recorded at specified
times to permit evaluation of the biological conditions
present for mysid survival in test solutions.
    Specified observations on mortality and life cycle
characteristics are designed to allow an adequate evaluation
of concentration-response effects in both acute and chronic
mysid tests.  In addition, these defined observation times
allow greater comparability of dose-response data between
different chemicals and laboratories.

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                                                         ES-2
                                                 August,  1982
    B.  Test Conditions
         1.  Test Species
              a.  Selection
    The primary considerations  in  the selection  of  this
organism for toxicity testing were:  (a) sensitivity  to a
variety of chemical substances;  (b)  geographical
distribution and abundance, (c)  ecological  importance and
(d) existence of established culture methods  for laboratory
rearing.
    The test species, Mysidopsis bahia, is  a  member  of the
family Mysidae, which is found  in  most of the neritic zones
of the world's oceans.  Mysidopsis bahia  inhabits shallow
water grass flats along the eastern  and western  Gulf of
Mexico.  They are particularly  abundant from  the Galveston
  o
Bay system to southern Florida.
    Mysids occupy an  important  position in  near  shore food
webs.  They constitute a major  source of  food for many fish
species, including catfish, flounder, anchovy, silverside,
sunfish and seatrout  (Darnell 1958,  Schuster  1959, Odum and
Herald 1972, Powell and Schwartz 1979).   In addition to
their role in food chains of fish, mysids are important in
the conversion of organic detritus to living  tissue  in
estuarine environments (Hopkins, 1965).
    To date, Mysidopsis bahia has  been the  most  extensively
tested mys id shrimp (Nimmo et al.  1977, 1978, In press a, in
press b, in press c, USEPA 1978, Gripe et al. in press).
During its development as a test species  (since  1977)
methods of culturing, holding and  testing have been
established, and the methodologies developed  at  the  EPA Gulf
Breeze Environmental Research Laboratory  were considered

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                                                         ES-2
                                                 August,  1982
heavily in the design of this guideline.   This  laboratory,
and others, have established Mysidopsis  bahia  as  a  test
organism and have stressed the  importance  of the  following
qualities: (1) small size; (2)  short  (18 day)  life  cycle;
(3) small brood size; (4) readily reared through  all  life
stages in culture;  (5) commercially available  (6)
representative of an ecologically important family  and  (7)
extremely sensitive to a variety of test substances.   In
addition, this species has been selected as a  test  organism
for a variety of assessment programs  in  EPA and other
government agencies, as well as private  laboratory
testing.  The sensitivity characteristic of mys ids  was
dramatically reported by Bionomics (EPA  Contract  No.  68-01-
4646).  In testing  of the acute toxicity (no effects
concentrations) of  35 priority  pollutants, mysids were found
to be on the average more sensitive than any of the other
species tested (i.e., Selenestrum capricornutum,  Skeletonema
cos tatum, Daphnia magna, Cypr inodon variegatus  and  Lepomis
inachrochirus) .
              b.  Sources
    It is recommended that the  mys ids used for  laboratory
testing as specified in this test guideline be obtained
commercially from a supplier willing  to  certify proper
taxonomic identification.  Although field  collection  is
acceptable, it is highly recommended  that  test organisms
originate from culture stock.   Definitive  identification of
Mysidopsis bahia (Molenock, 1969), is difficult without
expertise, and it occurs sympatrieally with two other
species of Mys id op sis .  Reliable use  of  Mys id ops is -bahia is
required for this testing procedure,  and it is therefore

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                                                         ES-2
                                                 August,  1982
suggested that the mysids be  cultured  in  the  laboratory  to
meet all testing needs.  The  ease with which  this  species
can be cultured in the laboratory has  been demonstrated
(Nimmo et al. 1977, 1978).
    Furthermore, laboratory culturing  of  mys ids  permits  the
isolation of newly-hatched juveniles.   This  allows  control
of the size, age and experiential history of  mys ids  used  for
acute and chronic testing.
         2.  Maintenance of Test Species
              a.  Handling and Acclimation
    The bay mys id, _M. bahia,  may be  cultured  in  aquaria
supplied with either filtered flowing  or  recirculating
seawater.  Details of _M. bahia culture can be obtained from
Nimmo et al. (1977) and USEPA (1978).   A  salinity  of  20  °/oo
for mys id culture allows optimal reproductive conditions
(Nimmo et al. 1977, USEPA 1978) and  reduces  acclimation
problems related to transfer  of animals from  culture to  test
water.
              b.  Feeding
    Artemia spp. nauplii suggested for mys id  feeding, can  be
reared in the laboratory from commercially available  eggs.
These eggs or any other appropriate  food  used for  mys ids,
should not be used if the total organochlorine pesticide
plus polychlorinated biphenyls exceeds  0.3 ug/g  (wet
weight), or if organic chlorine exceeds 0.15  ug/g  (wet
weight).  A recent study by Johns and  Waiten  (1979)  reported
that adult Mys id ops is bahia fed Arteinia spp.  from  San Pablo
Bay, California exhibited increased  mortality, did  not
reproduce and'showed reduced  growth  rates. ~ "In contras t,
both juvenile and adult mys ids fed Artemia spp..  strains

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                                                        ES-2
                                                August, 1982
collected from Brazil, Australia, Italy and Utah maintained
high survival and growth rates.  Additional studies have
indicated major differences in the nutritional value of
Artemia to brachyuran crustaceans (Johns et al. in press).
These results strongly imply that nutritional quality of
Artemia, possibly associated with pesticide or heavy metal
contamination, can significantly influence test results and,
therefore, should be considered.
    In order to separate Artemia spp. nauplii  (used for
mys id feeding) from their egg cases and other debris, a
light box may be employed.  This system makes use of the
positive phototropism of Artemia to separate nauplii from
unwanted materials.  It is important to isolate the nauplii
from the egg cases and to deliver only nauplii to the test
chambers in order to minimize build-up of organic debris
within the chambers.  The decomposition of the entrapped egg
cases may directly or indirectly enhance or reduce the
toxicity of the test substance.
    To isolate juvenile mys ids, ovigerous females may be
placed within a retention chamber, which is then submerged
into a five-liter glass battery jar or other suitable vessel
(USEPA 1978).  The retention chamber should be slightly
smaller than the battery jar and should extend above the
water level of the battery jar.  A slow flow of salt water
(approximately 4 drops/second) should be dripped into the
jar to sustain proper dissolved oxygen levels and prevent
stagnation.  Juveniles pass through the cylinder mesh (one
millimeter mesh opening) at birth and attach to the walls of
the battery-ja-r; thereby minimizing cannibalism by adult
females and facilitating capture.  During this isolation

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                                                 !        ES-2
                                                August,  1982
procedure, mysids should be given a supply of 24-hr old
Artemia spp.  (nauplii).  Juvenile mys ids may be  removed from
the sides of the jar every 4-12 hours during this period
using a glass tube.
         3.  Facilities                          .
              a.  General
    In flow-through systems it may be - necessary  to have  the
capability to vary and maintain the water temperature.
Since the temperature of the dilution water can  be expected
to vary both daily and seasonally, facilities for adjusting
temperature may be needed to maintain the desired culturing,
holding, and testing temperatures.  Filters are  needed to
remove particulates and biological material from the
dilution water so the diluter system and retention chambers
will not become clogged, cause a change in test  substance
concentrations, or lead to stagnation and oxygen depletion
within the retention chambers.  The primary concern is to
minimize the confounding of results associated with the
differential sorption of the test substance on cell walls,
clay particles, etc. which in turn may enhance or reduce the
availability of the test substance to the mys ids.
    To minimize these problems, the dilution water should be
filtered through a 20 micrometer or smaller pore-size filter
to sufficiently reduce the amount of suspended sediments,
organic material and biological organisms (phytoplankton,
zooplankton, fungi, bacteria, etc.).
    Requiring filtration through a 20 micrometer or smaller
filter is based on recent modifications to the testing
procedure developed at the EPA Gulf Breeze Environmental
Research Laboratory (USEPA 1978).  In addition, filtration
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                                                         ES-2
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to five micrometers more effectively controls fouling of the
retention chamber mesh walls.  Minimizing this problem also
prevents dissolved oxygen depletion and stagnation within
the retention chambers.  Filtration through five micrometer
filters is attainable for the recommended flow rate and
quantity of water needed to conduct the test.
    Gas accumulation may also cause adverse effects and
therefore, a device which removes air bubbles may be
necessary.  A suitable device is described by Penrose and
Squires (1976).  When the dissolved oxygen in the1 dilution
water is less that 60 percent, a device is needed to aerate
the water.  Culture techniques recommend 70-100 percent
saturation for other marine crustaceans (Forster and Beard
of the test substance during aeration through
volatilization, aeration should be conducted prior to
introduction of the test substance.
    In order to attain optimal test results, it is necessary
to culture arid test the organisms in an environment
considerate of both their behavioral and physiological
needs.  Mys id shrimp are extremely sensitive to fluctuations
in these parameters which may be reflected in a number of
ways, all of which can affect test validity (Bahner et al.
1975).
              b.  Construction Materials
    All pipes, tanks, holding chambers, mixing chambers,
metering devices, and test chambers should be made of
materials that minimize the release of chemical contaminants
into the dilution water or the adsorption of the test
substances.  Chemicals.-that may leach from construction
materials can stress test organisms, or possibly act
                                11

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                                                August, 1982
synergistically or antagonistically with test substances to
give inaccurate results.  Generally, undesirable substances
are not leached from borosilicate glass, titanium, and
perfluorocarbon plastic.  In addition, the tendency of these
materials to absorb substances is minimal.  Rubber,
polyvinyl chloride, copper, brass, galvanized metal, lead
and epoxy resins should not come in contact with dilution
water,, stock solution, or test solutions because of the
toxic substances they contain.  Cast iron should not be used
in water systems since rust may develop and result in
fouling.  Teflon (Algoflon), Perspex, Polyethylene, Tygon,
Polypropylene, Polycarbonates (Makrolor) and Polyester
(Gabraster) have been shown to be non-toxic and suitable for
experiments with marine organisms (APHA 1975, USEPA 1978).
    Retention chambers, aquaria delivery systems, pipes and
any tank exposed to solutions that may come in contact with
the organism should not be soldered or brazed, since lead,
tin, copper or zinc may be leached.  Silicone adhesive is
the preferred bonding agent for all construction
materials.  It is inert, and the solvent it generally
contains (acetic acid) is easily washed away or volatilized
from the system.  A minimum amount of the adhesive should
contact the test solution because it may absorb test
materials.  If large amounts of the adhesive are needed for
strength, it should be applied to the outs ides of chambers
and apparatus to minimize contact.                 ;
    In static testing, borosilicate glass, crystallizing
dishes, or similar containers may be used as test chambers.
Use of these dishes will minimize sorption of the test
substance into the chamber walls and minimize residues of
                                12

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                                                         ES-2
                                                 August,  1982
test substances or metabolites remaining after cleaning.
Glass plates can be placed over  the  test chambers  to  allow
for stacking and to minimize space requirements and
evaporative loss of test solution.   Chamber size should  be
adequate to insure proper loading and to minimize
cannibalism.
              c.  Test Substance Delivery System
    In flow-through tests, the delivery of constant
concentrations of test substances is required to reduce
variability in test results.  Large  fluctuations in test
substance concentration will give abnormally high  or  low
responses, depending upon the mechanism of toxic actions.
Proportional diluters with metering pumps or continuous  flow
infusion pumps have been used extensively to maintain
constant test substance concentration.  For the flow-through
acute and life cycle test guideline, all tests should be
conducted in intermittent flows  from a diluter or  in
continuous flow with the- test substance added by an infusion
pump.  The procedures of Mount and Brungs (1967) and  Hansen
et al. (1974) are recommended if the test substance can  be
added without a carrier; the device described by Hansen et
al. (1974) if a carrier is necessary; or procedure of  Banner
et al. (1975) if pumps are required  for continuous, flow.
    Proportional diluters operate on a sequential  filling
and emptying of water chambers.  The water chambers are
calibrated to contain a measured amount of water.  Separate
water chambers can be provided for test substance  and
diluent water.  Diluent and test substance waters  are mixed
and delivered to the -test-aquaria.  The cyclic action of- the
diluent is regulated by a solenoid valve connected to the
                                13

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                                                         ES-2
                                                August,  1982
inflow dilution water.  The system is subject to electrical
power failure, so an alternate emergency power source  is
recommended.  In addition,.the mysids should to be shielded
from the clicking sound of solenoid valves.  If unshielded
from this disturbance, mys ids may jump out of the test
solution and stick to the  sides of the retention chambers
(US EPA 1978).
    The proportional diluter is probably the best system  for
routine use; it is accurate over extended periods of time,
is nearly trouble free, and has fail-safe provisions (Lemke
et al. 1978).  A small chamber to promote mixing of test
substance-containing water and dilution water may be used
between the diluter and the test aquaria for each
concentration.  If replicate chambers are used in this  test,
separate delivery tubes should be run from this mixing
chamber to the appropriate replicate chambers.  If an
infusion pump is used, a glass baffle should be employed  to
insure mixing of the test  substance and dilution water.
Calibration of the test substance delivery system should  be
checked carefully before and during each test.  This should
include determining, the flow rate through each test aquarium
and measuring the concentration of test subs tance in each
test aquarium.  The general operation of the system should
be checked twice daily.
              d.  Test Chambers
    Retention chambers are suggested to prevent escape  of
mysids from the test system, reduce cannibalism and
facilitate counting and observation.  Overcrowding enhances
cannibalism and • ass ignment of five mys ids per retention
chamber is recommended to  minimize this problem.  The mesh
                                14

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                                                         ES-2
                                                 August,  1982
size (315 micrometer mesh opening)  of  the  screen  used  in
construction of  the retention  chambers  at  the  EPA laboratory
in Gulf Breeze minimizes the problems  associated  with
fouling by fungal, bacterial and  algal  growth,  yet is  still
small enough to  retain the mysids and  food  organisms.   This
mesh size is slightly more porous than the  200  micrometer
opening mesh recommended by Nimmo et al.  (1977).   Use  of the
larger mesh size combined with 20 micrometer filtration
obviates the need for continuous  lighting,  which  was
employed in early testing protocols  (USEPA  1978)  to minimize
fouling of the retention chambers.
              e.  Cleaning of  Test  System
    Standard laboratory Practices (e.g, USEPA  1974) are
recommended to remove dust, dirt, other debris, and residues
from test facilities.  At the  end of a  test,  test systems
should be washed in preparation for  the next  test.   This
will prevent chemical residues  and  organic  matter from
becoming embedded or absorbed  into  the  equipment.   It  is
also recommended that any silicon cement which  has  been
exposed to a test substance is  replaced prior  to  future
tests to avoid contamination due  to  sorption properties.
    Rinsing and  priming the system  with dilution  water
before use (conditioning) allows  equilibrium to be  reached
between the chemicals in the water  and  the  materials of the
test system.  The test system  may sorb  or  react with
substances in the dilution water.   Allowing  this  equilibrium
to take place before use lessens  the chances  of water
chemistry changes during a test.
    Even after extens ive" washing y new -facilities"  may~s*t ill'
contain toxic residues.  The best way  to determine: whether
                                15

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                                                         ES-2
                                                August,  1982
toxic residues remain is to rear mys ids through at least one
complete life cycle. If the rays ids survive and successfully
reproduce the test facililties can be considered to be free
of toxic residues.
              f.  Dilution Water
    A constant supply of dilution water is required to
maintain constant experimental conditions.  An interruption
in flow or change in water quality can change the chemistry
of the test system and possibly the response of the test
population.  Therefore, the results of a  test with variable
dilution water quality are not comparable to tests run under
constant conditions and they are more difficult to
interpret.
    For acute and chronic toxicity tests, a minimum
criterion for acceptable dilution water is that healthy
mys ids will survive and reproduce in it without showing
signs of stress such as abnormal behavior (erratic swimming,
loss of equilibrium or lack of feeding activity). :
    Natural seawater, obtained from a source with similar
characteristics as those designated for the test species or
water from an area where the test organisms were obtained,
is preferable to artificial seawater. Dilution water should
be of constant quality and should be uncontaminated.
Contaminated water can affect results both directly and
indirectly.  if natural seawater is used, it should meet the
following specifications for contaminant  levels (APHA 1975).
                                16

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                                                        ES-2
                                                 August,  1982
         Suspended solids                       <  20 mg/1
         Total organic carbon                   <  10 mg/1
         Un-ionized ammonia                     <  20 mg/1
         Residual chlorine                      <   3 ug/1
         Total Organ ©phosphorous pesticides     <  50 ug/1
         Pesticides plus PCB's                  <  50 ug/1

    In addition, water used to make reconstituted  seawater
should meet or exceed the same water quality  criteria.
    Maintenance of desired salinities in the  test  aquaria
throughout the duration of the test may pose  a  problem.
When possible, water from an area of high salinity shoulds
be used; low salinities can then be obtained  by adding
distilled or deionized water as needed.  To increase
salinity, a strong, natural brine, which can  be obtained by
freezing and then partially thawing seawater, can  be used.
This procedure is suitable if limited amounts of  seawater
are needed; however, it is recommended that artificial
seawater salts be used when large increases in  salinities
are required (APHA 1975).
    Due to the volume of water necessary to conduct a
chronic test and technical problems associated  with
conditioning of the dilution water, the use of  reconstituted
seawater for these tests may not be currently feasible
because of high costs and lack of information on ;proper
aging processes.  Research is needed to determine  methods in
which reconstituted water can be conditioned  and  aged with
the use of appropriate storage and selective filtration
before use of reconstituted" s"e'awate'r~c"an become "a "viable
alternative.                                   ' :
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                                                        ES-2
                                                August,  1982
              g.  Controls
    Controls are required for every  test  to assure  that  any
effects which are observed are due to the test substance and
not to other factors.  These may include  effects  from
construction materials, environmental factors, nutritional
quality of food, vapors, stressed test organisms, etc.
Within the acute, 96-hour tests ten percent control
mortality may be present due to inherent  biological
factors.  Any increase above this is considered to be due to
conditiions of the test or the test organisms.  The  ten
percent mortality figure is representative for a  wide
variety of organisms,  including both fish and invertebrates
(ASTM 1979) and is generally utilized for 96-hour testing.   .
Some of this mortality in invertebrates may be associated
with injury during handling.
    In an analysis of  thirteen lifa cycle tests which
utilized M_. bahia (Nimmo, unpublished laboratory  data; Gulf
Breeze EPA Laboratory), control mortality over the  testing
period (20-28 days) ranged from 0-31 percent.  The mean
control mortality of these studies was 11 percent, with  34
percent of the test results greater than  the 11 percent.
Control mortality of 20 percent in the chronic test  will be
considered to be due to conditions of the test or'the test
organisms .
              h.  Carriers
    Carriers can effect test organisms and can possibly
alter the form of the  test substance in water.  For  these
reasons it is preferable to avoid the use of carriers in
toxicity tests unless  absolutely necessary to dissolve the
test substance.  Since carriers can stress or adversely
                                18

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                                                        E3-2
                                                 August,  1982
effect test organisms, the amount of carrier should be  kept
to a minimum.  Recommended maxima are  0.5 ml/1  in static
tests and 0.1 ml/1 in flow-through tests  (APHA  1975).
    Triethlyene glycol has been found  to  exert  the least
influence on mys id response to test substances  of several
carriers that have been used.  Acetone and ethanol have a
stronger tendency to reduce the surface tension of the
water, and therefore, decrease oxygen saturation (Veith and
Comstock 1975, Krugel et al. 1973, APHA 1975).
              i.  Randomization
    The test chamber position in the testing area and
assignment of mys ids to test chambers are randomized to
prevent conscious or unconscious biases from being
introduced.  These biases can be in environmental conditions
and distribution', dilutor system function, etc.
         4.  Environmental Conditions
              a.  Dissolved Oxygen
    In flow-through testing, large variations in flow rates
to aquaria will result in undesirable differences in
exposure and test conditions between aquaria.   Parameters
such as dissolved oxygen and test substance concentration
can decrease more rapidly in aquaria with low flow rates;
similarly metabolic products can build-up under these
conditions.  Proper dilution water filtration and mesh size
of retention chambers are of utmost importance  in
maintaining flow rate of test solutions and exchange within
retention chambers.  Previous studies  (Nimrao et al. 1977,
USEPA 1973) have found that a flow rate which allows five
test chamber volume changes per 24 hours is adequate to
obtain necessary conditions for mys id testing.
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                                                        ES-2
                                                 August,  1982
              b.  Light
    A 14-hour light and 10-hour  dark  photoperiod  is
recommended in an effort  to approximate  representative
natural conditions under  which M_. bahia  is  found  and  thus
reduce test-related stress.   If  used,  this  photoperiod
should remain constant throughout culturing,  holding  and
testing, as any deviations could effect  test  results.
Mys ids appear very sensitive  to  light changes and
intensity.  At very high  intensities  swimming is  inhibited,
with mys ids sinking to the tank  bottom.   In very  dim  light,
shoaling behavior is disrupted (Steven 1961).   With use of  a
light-dark regime a transition period is  recommended.
Mys ids are known to react to sudden light changes  by  jumping
out of the water.  Gradual transition will  avoid  mortality
caused by mys ids "sticking" to test chamber walls.
              c .  Temperature and Salinity
    Test temperature and  salinity choices (25°± 2°C and 20
± 3 °/oo, respectively) were made after  review  of  reports on
the habitat characteristics of _M. bahia  (Nimmo  et al. 1977,
Price 1978).  These temperature  and salinity  ranges were
also found to produce the greatest reproductive success in
laboratory cultures of _M. bahia  (Nimmo et al.  1977, US EPA
1978).  Furthermore, minimizing  variability in  testing
conditions through specific temperature  and salinity  values
allows greater comparability of  interlaboratory test  results
and the development of a  comparative  toxicology data  base.
An acceptable method for  maintaining  desired  temperature and
salinity ranges in flow-through  bioassays with  marine
organisms is described in Bahner and  Nimmo  (1975).
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                                                         ES-2
                                                August,  1982
    C.  Reporting
    A coherent theory of the dose-response  relationship,  on
which the acute toxicity tests are based, was  introduced  by
Bliss (1935), and is widely accepted  today.  This  theory  is
based on four assumptions:
    (1)  Response is a positive function of dosage,  i.e.  it
    is expected that increasing treatment rates should
    produce  increasing responses.
    (2)  Randomly selected animals are normally distributed
    with respect to their response to a toxicant.
    (3)  Due to homeostasis, response magnitudes are
    proportional to the logarithm of  the dosage, i.e.  it
    takes geometrically increasing dosages  (stresses)  to
    produce  arithmetically increasing responses (strains)  in
    test animal populations.
    (4)  In  the case of a direct dosage of  animals,  their
    resistance to effects' is proportional to body  mass.
    Stated another way, the treatment needed to produce a
    given response is proportional to the size of  the
    animals  treated.
    The concentration-response curve, where percent
mortality is plotted as a function of the logarithm  of test
solution concentration, can be interpreted  as  a cumulative
distribution of tolerance within the population (Hewlett  and
Plackett 1979).  Experiments designed to measure tolerance
directly (Bliss 1944) have shown that tolerance is
lognormally distributed within an experimental population in
most cases.  Departures from the lognormal  pattern of
distribution are generally associated with  mixtures  of ve~ry~"
susceptible and very resistant individuals  within  a
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                                                         ES-2
                                                August,  1932
population (Hewlett and Plackett 1979).  In addition,
mixtures of toxicants can produce tolerance curves which
deviate significantly from the lognormal pattern  (Finney
1971).
    If tolerances are lognormally distributed within  the
experimental population, the resulting concentration-
response curve will be sigmoidal in shape, resembling a
logistic population curve (Hewlett and Plackett 1979).
While estimates for the mean lethal dose can be made
directly from the dose response curve, a linear
transformation often is possible, using probit (Bliss 1934,
Finney 1971) or logit (Hewlett and Plackett 1979)
trans formations .
    Once the mortality data have been  transformed', a
straight line can be fitted to the points by-a least
squares
linear regression equation and confidence limits  can  be
determined for predicted mortality values.  An additional
advantage is that the significance of  the slope of the
regression line can be determined (Draper and Smith 1976).
    While, the mean lethal dose. (LC50)  can be estimated
graphically from the linearized dose-response curve (APHA
1975), other techniques are preferable since the  graphical
method does not permit the calculation of confidence  limits.
    The probit method (Finney  1971), which is recommended in
the acute toxicity test guideline, uses the probit
transformation and the maximum likelihood curve-fitting
technique.  Other appropriate  tests used in data  reduction
include the modified probit method of  Litchfield  and
Wilcoxon (1949), the logit method (Ashton 1972) and the
                                22

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                                                August,  1982
moving average method (Thompson 1947).
    If there are no partial kills in an experiment,
determination of the concentration-response curve is not
possible.  In situations where there are no partial kills,
the binominal test (Siegel 1956) can be used to estimate the
LC50 and confidence limits around the LC50 value  (Stephan
1977).
    If concentration-response data are plotted for each 24-
hour interval throughout the test, the LC50 determined from
each curve can be plotted as a function of time,  yielding  a
time-acute toxicity curve (APHA 1975).  This curve
approaches a line parallel to the time axis asymptotically,
indicating a constant or threshold value for LC50.  The
absence of a threshold LC50 may indicate the need for an
acute test of longer duration.
    The statistical tests recommended for analyses of mys id
life-cycle data (mortality, body lengths of males and
females and young per female) were chosen to permit as
complete an interpretation of the quantifiable data as
possible.  Under most conditions, the analysis of variance
(ANOVA) is a powerful statistical test allowing the
determination of significant differences between  treatment
means through incorporation of data variability.  This
statistical examination is especially important in
biological experimentation due to the presence of many
sources of inherent variability.  In previous chronic mys id
testing, Nimrao et al.  (In press b) employed the  analysis of
variance with subsequent comparisons between means utilizing
Student-New-Kuels7 Duncan's, Dunnett's, or Bonferron's
tests.  Futhermore, the use of analysis of variance (ANOVA)
                                23

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                                                         ES-2
                                                August,  1982
and mean separation tests have been employed in mysid
testing at the EPA Environmental Research Laboratory at Gulf
Breeze, Florida, and have given consistent results under  the
experimental conditions stated in the  test guideline
document.
III.   Economic Aspects
    The Agency awarded a contract to Enviro Control, Inc.  to
provide us with an estimate of the cost for performing
static and flow-through acute toxicity tests and flow-
through chronic toxicity tests.  Enviro Control supplied  us
with two estimates; a protocol estimate and a  laboratory
survey estimate.

                     Protocol Estimates
                                         range         mean
    Acute (static and flow through)  $ 322-$ 965  .    $ 643
    Chronic                          ?1653-$4959      $3306

    These estimates were prepared by separating the
guidelines into individual tasks and estimating the  hours
used to accomplish each task.  Hourly rates were  then
applied to yield a total direct labor charge.  An overhead
rate of 115 percent, other direct costs  ($50-acute,  $415-
chronic), a general and administrative rate of 10 percent
and a fee of 20 percent were then added  to the direct labor
charge to yield the final estimate.
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                                                        ES-2
                                                August, 1982
                 Laboratory  Survey  Estimates

                                        range
    Acute (static and flow through)  $ 340-$ 1250
    Chronic
    The laboratory survey estimates were compiled from three
laboratories for the acute guideline and one laboratory for
the chronic guideline.
                                25

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                                                August,
                                                    ES-2
                                                    1982
IV.   References
    APHA.  1975.  American Public Health Association.
    American Water Association and Water Pollution Control
    Federation.  Standard methods for examination of water
    and waste water,  14th ed.  New York: American Public
    Health Association.
    Ashton WD.  1972.
    Publishing Co.  New
                   The logit transformation.
                   York:
Hafner
    ASTM.   1979.  American Society for Testing and
    Materials.   New standard practice for conducting basic
    acute  toxicity tests with fishes, macroinverte'orates and
    amphibians.  Philadelphia, PA: American Society for
    Testing and Materials.

    Banner LH and Nimmo DW. 1975.  Methods to assess effects
    of combinations of toxicant, salinity and temperature on
    estuarine animals.  Proc. Univ. Mo. Ann. Conf. Trace
    Subst. Environ. Health .pp :. -16-7-177.

    Bahner LH,  Wilson AJ, Shepppard JM, Patrick JM, Goodman
    LR, and Walsh GE.  1977.  Kepone bioconcentration,
    accumulation, loss and transfer through estuarine food
    chains.  Chesapeake Sci. 13:299-308.

    Bahner LH,  Craft CD, Nimmo DR.  1975.  A saltweater
    flow-through bioassay method with controlled temperature
    and salinity.  Prog. Fish. Cult.  37 ( 3 ) : 126-1 29 .

    Berkson J.   1949.  The minimum chi-square and maximum
    likelihood  solution in terms of a linear transform, with
    particular  reference to bioassay.  J. Amer.  Stat.
    Assoc. 44:273-278.

    Bionomics Inc.  1978.  In-depth studies on health and
    environmental impacts of selected water pollutants.  EPA
    Control No.  68-01-4646, 1977-1978.

    Bliss  CI.  1934.  The method of probits.  Science 79:38-
    39.
Bliss CI.  1934.
mortality curve.
                      The calculatlon-of -the dosage-
                      Ann.  Appl.  Biol.  22:134-307.
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                                            August,  1982


Bliss CI.  1944.  The U.S.P.  collaborative rat assay
for digitalis.  J.  Amer.  Pharm.  Ass. 33:225-245.

Cripe GM, Nimmo DR, Hamaker TL.  In press.  Effects  of
two organophosphate pesticides on swimming stamina of
the mysid, Mys id ops is bahia.  In: Vernberg FJ, Calabrese
A, eds .  Pollution and physiology of marine organisms.
New York: Academic Press.

Darnell RM.  1958.  Food habits of fishes and large
invertebrates of Lake Ponchatrain, Louisiana, an
estuarine community.  Publ. Inst.  Marine Sci.  Univ.
Texas, 5:353-416.

Draper NR. and Smith H.  1966.  Applied regression
analysis.  New York: John Wiley and Sons.

Drummond RA and Dawson WF.  1970.  An  inexpensive method
for simulating diel pattern of lighting in the
laboratory.  Trans.  Amer.  Fish.  Soc.  99:434-435.

Finney AJ.  1971.  Probit analysis.  London, England:
Cambridge University Press.

Foster JRM and Beard TW.  1974.  Experiments to assess
the suitability of nine species of prawns for intensive
cultivation. Aquaculture.  3:355-368.

Hans en D J,  Schimmel SE, Matthews E.   1974.  Avoidance
of Aroclor 1254 by shrimp and fishes.  Bull. Environ.
Contain. Tox .  12( 2) : 243-256.

Hewlett PS. and Plackett RL.  1979.  The interpretation
of quantal responses in biology.  University Park Press,
Baltimore, MD: 82 pp.

Hopkins TL.  1965.  Mysid shrimp abundance in.surf ace
waters of Indian River Inlet, Delaware.  Chesapeake  Sci.
6:86-91.

Johns DM and Walton W.  1979.  International Study on
Artemia; X. Effects of food source on survival, growth,
and reproduction in the mysid, Mys id ops is bahia.
Abstract cited in Amer. Zoolog. - 19(3):906.    ,
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                                                     ES-2
                                            August,  1982
John DM, Peters ME, Beck AD.  In press.  International
Study on Artemia  (1):  Nutritional value of geographical
and temporal strains of Artemia.  Effects on survival
and growth of two species of brachyuran larvae.   In:
Personne, Sorgeloos, Roels, Jaspers, eds.  The brine
shrimp Artemia Vol. 3. Wetterner, Belgium: Universa
Press.
Klein-Mac Phee C, Howell WH, Beck AD.  In press.
International study on Artemia.  Nutritional value of
five geographical strains of Artemia to winter  flounder
(Psuedopleuronectes Americanus"!  larvae.  In: Personne,
Sorgeloos, Roels, Jaspers, eds.  The Brine shrimp
Artemia  Vol. 3.  Wetterner, Belgium: Universa  Press.

Krugel Sf Jenkins D, Kleen SA.  1978.  Apparatus for  the
continuous dissolution of poorly water-soluble  compounds
for bioassays.  Water Res.  12:269-272.

Lemke SE, Brungs WA, Hilligan BJ.  1978.  Manual for
construction and operation of toxicity-testing
proportional diluters.  EPA Report No.  600/3-78-072.

Litchfield JT, Jr. and Wilcoxon F.  1949.  A simplified
method of evaluating dose-effect experiments. J. Pharm.
Exp. Ther.  96:99-113.

Molenock J.  1969.  My s id ops is bahia, a new species of
mys id (Crustacea: Mysidacea) from Galveston Bay,
Texas.  Tulane Studies in Zool.  15: 113-116.

Mount DI. and Brungs WA.   1967.  A s implif ied dos ing
apparatus for fish toxicology studies.  Water Res. 1:21-
29.

Nimmo DR, Bahner LH, Rigby RA, Sheppard JM, Wilson A J,
Jr.  1977. Mysidopsis bahia;  an estuarine species
suitable for life-cycle toxicity tests to determine the
effects of a pollutant.  In: Aquatic toxicology and
hazard evaluation, ASTM STP 634. FL Mayer and Hamelink
JL, eds. Philadelphia, PA: American Society for Testing
and Materials, pps. 109-116.

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                                                     ES-2
                                            August,  1982
Nimmo DR, Rigby RA, Bahner LH, Sheppard JM.   1978.  The
acute and chronic effects of cadmium on the estuarine
mysid, Mys id ops is bah i a. Bull. Environ. Contam. Tox . 19:
80-85.

Nimmo DR, Hamaker TL, Matthews E, Young WT.   In press
a.  The long-term effects of suspended sediment on
survival and reproduction of the mysid shrimp,
Mys id ops is bahia in the laboratory.  Marine Ecosystems
Analys is .  National Oceanograpnic and Atmospheric
Administration, Department of Commerce.  Boulder, CO.

Nimmo DR, Hamaker TL, Matthews E, Moore JC.   In press
b.  Effects of eleven pesticides on Mys id ops is bahia
throughout its life cycle.   In: Vernberg FJ and
Calabrese A, eds.  Pollution and physiology of marine
organisms.  New York: Academic Press.

Nimmo DR, Hamaker TL, Moor JC, Wood RA.  In press c.
Acute and chronic effects of Dimilin on survival  and
reproduction of Mys id ops is bah i a.  Philadelphia,  PA:
American Society for Testing and Materials.

Odum WE and Heald EJ.  1972.  Trophic analysis of an
estuarine mangrove community.  Bull. Marine Sci.  22:
671-738.

Olney CE, Schauer PS, Simpson KL.  In press.
International study on Artemia.  Comparison of the
chlorinated hydrocarbons and heavy metals  in  five
different strains of newly hatched Artemia  In:
Personne, Sorgeloss, Roels,  Jaspers, eds.  The
brineshrimp Artemia.  Wetterner, Belgium:  Uaiversa
Press.

Penrose WR and Spuires WR.   1976.  Two devices for
removing supersaturated gases in aquarium  systems.
Trans. Am. Fish Soc. 105: 116-118.

Powell AB and Schwartz FJ.   1979.  Food of Paralichthys
dentatus and _P. 1 ethos tig ma  (Pices: Bothidae)  in  North
Carolina estuaries.  Estuaries 2:276-279.

Price WW.  1978.  Occurence  of Mys id ops is "almyra  Bowman
(Crustacea, Mysidacea) from  the eastern coast of
Mexico.  Gulf Research Reports 6:173-175.
                            29

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                                                    ES-2
                                            August, 1982
Schuster CN.  1959.  A biological evaluation of the
Delaware River Estuary.  Info. Series Public No. 3.
Univ.  Delaware Marine Laboratories.

Siegel,  1956.  Nonparametrie statistics for the
behavioral sciences.  McGraw - Hill. Publ. Co. New York:

Sokal RR and Rohlf FJ.  1969.  Biometry. San Francisco,
CA: W.H. Freeman and Co.

Sprague JB.  1969.  Measurement of pollutant toxicity to
£ish.  I. Bioassay methods for acute toxicity.  Water
Res. 3:793-821.

Stephan CF.  1977.  Methods for calculating an LC50.
In: Mayer FL and Hamelink, eds. Aquatic toxicology and
hazard evaluation.  ASTM STP 634.  Philadelphia, PA:
American Society for Testing and Materials,  pp. 65-84.

Supplee VC and Lightner DV.  1976.  Gas-bubble disease
due to oxygen supersaturation in raceway-reared
California brown shrimp. Prog. Fish Cult.  38:159.

Thompson WR.  1947.  Use of moving averages and
interpolation to estimate median effective dose.  I.
Fundamental formulae, estimation and error, and relation
to other methods. Bacterial. Rev.  11:115-145.

US EPA.  1974.  U.S. Environmental Protection Agency.
Manual of analytical methods for the analysis of
pesticide residues in human and environmental samples.
Research Triangle Park, NC: U.S. Environmental
Protection Agency.

USEPA.  1978.  U.S. Environmental Protection Agency.
Bioassay Procedures for the Ocean Disposal Permit
Program.  Environmental Research Laboratory.  Gulf
Breeze, Fl:  U.S. Environmental Protection Agency.  EPA-
600/9-78-010.

Veith GD and Cornstock VM.  1975.  Apparatus for
continuously saturating water with hydrophobic organic
chemicals.   J. Res. Fish. Bd. Canada  32: 1349-1851.
                            30

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                                  EG-5
                                  August, 1982
        OYSTER ACUTE TOXICITY TEST
        OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES  AND  TOXIC SUBSTANCES
   U.S.  ENVIRONMENTAL PROTECTION  AGENCY '""
          WASHINGTON, D.C. 20460

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Office of Toxic Substances                                  03-5
Guideline for Testing Chemicals                     August,  1982
                    OYSTER ACUTE TOXICITY TEST


    (a)  Purpose.  This guideline will  be used  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

tests to be used to develop data on  the  acute toxicity of

chemicals to Eastern oysters, Crassostrea virginica  (Gmelin).

The United States Environmental Protection Agency  (USEPA)  will

use data from these tests in assessing  the hazard  of  a chemical

to the environment.

    (b)  Def in it ions .  The definitions  in section  3  of the Toxic

Substances Control Act (TSCA) and in Part 792—Good  Laboratory

Practice Standards are applicable to this test  guideline.   The

following definitions also apply:

    (1)  "Acute toxicity" is the discernible  adverse  effects

induced in an organism within a short period  of time  (days) of

exposure to a chemical.  For aquatic animals  this  usually  refers

to continuous exposure to the chemical  in water for  a period  of

up to four days.  The effects (lethal or sublethal)  occurring may

usually be observed within the period of exposure  with aquatic

organisms.  _In..this-..tes t.. .guideline, -shel-1 deposition  is used  as •

the measure of toxicity.

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                                                            EG-5
                                                   August,  1982
    (2)  "EC 50" is that experimentally derived concentration of

a chemical in water that is calculated to  induce shell  deposition

50 percent less than that of the controls  in a test batch  of

organisms during continuous exposure within a particular exposure

period which should be stated.

    (3)  "Shell deposition" is the measured length of shell

growth that occurs between the time the shell is ground at test

initiation and test termination 96 hours later.

    (4)  "Umbo" means the narrow end (apex) of the oyster  shell.

    (5)  "Valve height" means the greatest linear dimension of

the oyster as measured from the umbo to the ventral edge of the

valves (the farthest distance from the umbo).

    (c)  Test procedures — (1)  Summary of  the test.   (i)   The

water solubility and the vapor pressure of the test chemical

should be known.  Prior to testing, the structural formula of the

test chemical, its purity, stability in water and light, n-

octanol/water partition coefficient, and pKa values should be

known prior to testing.  The results of a  biodegradability test

and the method of analysis for the quantification of  the chemical

in water should also be known.

    (ii)  For chemicals with limited solubility under the  test

conditions, it may not be possible to determine an EC SO.  If it

is observed that the stability or homogeneity of the  test

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                                                            EG-5

                                                   August,  1982
chemical cannot be maintained, then care should be  taken  in  the



interpretation of the results and a note made  that  these  results



may not be reproducible.



    (iii)  Test chambers are filled with appropriate  volumes  of



dilution water.  The flow of dilution water through each  chamber



is adjusted to the rate desired.  The test chemical is  introduced



into each test chamber and the flow-rate adjusted to  establish



and maintain the desired concentration in each test chamber.



Test oysters which have been acclimated and prepared  by grinding



away a portion of the shell periphery are randomly  introduced



into the test and control chambers.  Oysters in the test  and
          o


control chambers are observed daily during the test for evidence



of feeding or unusual conditions, such as shell gaping, excessive



mucus production or formation of fungal growths in  the  test



chambers.  The observations are recorded and dead oysters



removed.  At the end of 96 hours the increments of  new shell



growth are measured in all oysters.  The concentration-response



curve and EC 50 value for the test chemical are developed from



these data.



    (2)  [Reserved]



    (3)  Range-finding test.  A range-finding  test should be



conducted to establish test chemical concentrations for the



definitive test.  The test is conducted in the same way as the

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                                                            EG-5
                                                   August,  1982
definitive test except a widely spaced chemical concentration

series (i.e. log-interval) is used.

    (4)  Definitive test.  (i)  Oysters which meet condition

criteria (age, size, reproductive status, health) and which have

been acclimated to test conditions should have approximately 3 to

5 mm of the shell periphery, at the rounded (ventral) end, ground

away with a small electric disc grinder or other appropriate

device, taking care to uniformly remove the shell rim to produce

a.smooth, rounded blunt profile.  The oyster's valves should be

held together tightly during grinding to avoid vibrating the

shell and injuring the adductor muscle.  Oysters of which so much

of the shell rim has been removed that an opening into the shell

cavity is visible should not be used.

    (ii)  It is desirable to have shell growth values for the low

and high concentrations relatively close to, but different from,

0 and 100 percent.  Therefore, the range of concentrations to

which the oysters are exposed should be such that in 96 hours

relative to the controls, very little shell growth occurs in

oysters exposed to the highest concentration and shell growth is

slightly less than controls at the lowest concentration.  Oysters

in the remaining concentrations should have increments of shell

growth, such that ideally, the concentration producing 50 percent

shell growth relative to the controls is bracketed with at least

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                                                            EG-5'
                                                    August,  1982
one concentration above and one below it.

    (iii)  The test should be carried out without  adjustment of

pH unless there is evidence of marked change  in the p.H  of  the

solution.  Then it is advised that the  test be repeated  with pH

adjustment to that of the dilution water and  the results

reported .

    (iv)  The test begins when at least 20 prepared oysters  are

placed in each of the test chambers containing the appropriate

concentrations of test substance and controls.  The steady-state

flows and test chemical concentrations should be documented.  At

least 5  test chemical concentrations should be used.  The

dilution factor between concentrations should not  exceed 1,8.

    (v)  The distribution of individual oysters among the  test

chambers should be randomized.  The oysters should be spread  out

equidistantly from one another so that  the entire:test  chamber  is

used.  The oysters should also be placed with the  left  (cupped)

valve down and the open, unhinged ends  all oriented in  the same

direction facing the incoming flow of test solution.

    (vi)  The oysters are inspected at  least  after 24,  48, 72 and

96 hours.  Oysters are considered dead  if touching of the  gaping

shell produces no reaction.  Dead oysters are removed when

observed and mortalities are recorded.  Observations at  three

hours and six hours are also desirable.

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                                                            EG-5
                                                   August,  1982 .
    (vii)  Shell growth is the primary criterion used in this

test guideline to evaluate the toxicity of the test chemical.

Shell growth increments in all oysters should be measured after

96 hours exposure.  Record the length of  the longest "finger" of

new shell growth to the nearest 0.5 mm.   Oysters should be

handled very gently at this stage to prevent damage to the new

shell growth.

    (viii)  Records should be kept of visible abnormalities such

as loss of feeding activity (failure to deposit f eces), excessive

mucus production (stringy material floating suspended from

oysters), spawning or appearance of shell (closure or gaping).

    (ix)  The criteria for a valid definitive test are:

    (A)  The mortality in the controls should not exceed 10

percent at the end of the test.

    (B)  The dissolved oxygen concentration should be at least 60

percent of air saturation throughout the  test.

    (C)  Oysters should not spawn during  test.  If they do the

test should be repeated with prespawn oysters.

    (D)  There should be evidence that the concentration of the

substance being tested has been satisfactorily maintained (e.g.,

within 80 percent of the nominal concentration) over the test

period.  The total concentration of test substance-( i-.e. both

dissolved and suspended undissolved particulates) should be

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                                                           EG-5
                                                   August, 1982
measured; (I)  in each chamber at 0-hour, (_2_)  in each chamber at

96-hours and (3_) in at least one appropriate chamber whenever a

malfunction is  detected in any part of the test chemical delivery

sys tern.

    (E)  Dissolved oxygen, temperature, salinity and pH

measurements should be made at the beginning of the test, at 48

hours, and at the end of the test in the control chambers and in

those test chambers containing the highest, lowest and a middle

concentration of the test substance.

    (5)  Test results.  (i)  At the end of the test, a one-way

analysis of variance followed with an appropriate ad hoc test

(the studentized Neuman-Keul's or Duncan's multiple range tests;

or Dunnetts1 or Williams'  pairwise comparison tests) should be

conducted on the oyster shell deposition test data.  The probit

transformation should then be applied to the response variable

and then regressed, using  least squares regression, on dose or

log-dose.  An F Test for linearity should be conducted to

determine whether the chosen regression technique adequately

describes the experimental data.

    (ii)  Calculate the ratio of the mean shell growth for each

group of test oysters (exposed to each of the test chemical

concentrations) to the mean shell growth of the group of control

oysters.  From  these data  the concentration-response curve is

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                                                             EG-5
                                                    August,  1982
 drawn  and  an EC 50  along with the 95 percent confidence limits on

 the  value  are determined from the curves.  The mean measured

 concentration of  test chemical should be used to .calculate the EC

'50 and to  plot the  concentration-response curve.

     (6)   [Reserved]

     (d)  Test conditions—(1)  Test species — (i)  Selection.

 (A)  The Eastern  oyster, Crassostrea virginica, should be used as

 the  test organism.

     (B)  Oysters  used in the same test should be 30 to 50

 millimeters  in valve  height and should be as similar in age

 and/or size  as possible to reduce variability.  The standard

 deviation  of  the  valve height should be less than' 20 percent of

 the  mean.

     (C)  Oysters  used in the same test should be from the same

 source and  from the same holding and acclimation tank(s).

     (D)  Oysters  should be in a prespawn condition of gonadal

 development  prior to  and during the test as determined by direct

 or histological observation of the gonadal tissue for the

 presence of  gametes.

  .   (ii)   Acquis ition.  Oysters may be cultured in the

 laboratory,  purchased from culture facilities or commercial

 harves ters ,.-O-r--colle.cted-.-f.r.om—a..na.tural-population. - in -an

 unpolluted  area free  from epizootic disease.


                                 8

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                                                            EG-5
                                                   August,  1982
    (iii)  Acclimation.   (A)  Oysters should be attended  to

immediately upon arrival.  Oyster shells should be  brushed clean

of fouling organisms and  the transfer of the oysters  to the

holding water should be gradual to reduce stress  caused by

differences in water quality characteristics and  temperature.

Oysters should be held for at least 12 to 15 days before

testing.  All oysters should be maintained in water of the

quality to be used in the test for at least seven days before

they are used.

    (B)  During holding,  the oysters should not be  crowded and

the dissolved oxygen concentration should be above  60 percent

saturation.  The temperature of the holding water should  be  the

same as that used for testing.  Holding tanks should  be kept

clean and free of debris.  Cultured algae may be  added to

dilution water sparingly, as necessary to support life and growth

and such that test results are not affected as. confirmed  by

previous testing. •

    (C)  Oysters should be handled as little as possible.  When

handling is necessary, it should be done as gently, carefully,

and quickly as possible.

    (D)  A batch of- oysters is acceptable for testing if  the

percentage mortality over the seven day period prior  to testing

is less than five percent.  If the mortality is between 5 and

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                                                           EG-5
                                                   August, 1982
10%, acclimation should continue for seven additional days.  If

the mortality is greater than 10%, the entire batch of oysters

should be rejected.  Oysters should not be used which appear

diseased or otherwise stressed.  Oysters infested with mudworms

(Polydora sp.) , boring sponges (Cliona cellata) or which have

cracked, chipped, bored, or gaping shells should not be used.

    (2)  Test facilities—(i)  Apparatus.  (A)  In addition to

normal laboratory equipment, an oxygen meter, equipment for

delivering the test chemical, adequate apparatus for temperature

control, and test tanks made of chemically inert material are

needed.

    (B)  Constant conditions in the test facilities should be

maintained as much as possible throughout the test.  The

preparation and storage of the test material, the holding of the

oysters and all operations and tests should be carried out in an

environment free from harmful concentrations of dust, vapors and

gases  and in such a way as to avoid cross-contamination.  Any

disturbances that may change the behavior of the oysters should

be avoided.

    (ii)  Dilution water.  A constant supply of good quality

unfiltered seawater should be available throughout the holding,

acclimation and testing periods.  Natural seawater is

recommended, although artificial seawater with food added may be


                                10

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                                                           EG-5
                                                   August, 1982
used.  In either case, to ensure each oyster is provided equal

amounts of food, the water should come from a  thoroughly mixed

common source and should.be delivered at a flowrate of at least

one and preferably five liters per hour per oyster.  The flowrate

should be +_ 10 percent of the nominal flow.  A dilution water is

acceptable if oysters will survive and grow normally for 14 days

without exhibiting signs of stress; i.e. excessive mucus

production (stringy material floating suspended from oysters),

lack of feeding, shell gaping, poor shell closing in response to

prodding, or excessive mortality.  The dilution water should have

a salinity in excess of 12 parts per thousand, and should be

similar to that in the environment from which  the test oysters

originated.  A natural seawater should have a  weekly range in

salinity of less than 10 parts per thousand and a monthly range

in pH of less than 0.8 units.  Artificial seawater salinity

should not vary more than 2 parts per thousand nor more than 0.5

pH units.  Oysters should be tested in dilution water from the

same origin.

    (3)  Test parameters  (i)  Carriers .  Stock solutions of

substances of low aqueous solubility may be prepared by

ultrasonic dispersion or, if necessary, by use of organic

solvents, emulsifiers or dispersants of low toxicity to

oysters.  When such carriers are used the control oysters should


                                11

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                                                            EG-5
                                                    August,  1982
be exposed to the same concentration  of  the  carrier  as 'that used

in the highest concentration of  the  test substance.  The

concentration of such carriers should not exceed  0.1 ml/1.

    (ii)  Dissolved oxygen.  The dissolved oxygen concentrations

should be at least 60 percent of the saturation value and should

be recorded daily.

    (iii)  Loading.  The loading rate should not  crowd oysters

and should permit adequate circulation of  water while avoiding

physical agitation of oysters by water current.

    ( iv)  Temperature.  The test temperature is 20°C _+_ 1°C.

Temporary fluctuations (less than  8 hours)  within 15°C  to  25°C

are permissible.  Temperature should be  recorded  continuously.

    (v)  pH.  The pH should be recorded  twice weekly in  each test

chamber.

    (e)  Reporting.  In addition to  the  reporting  requirements

prescribed in Part 792—Good Laboratory  Practice  Standards,  the

report should contain the following:

    (1)  The source of the dilution  water, the mean,  standard

deviation and range of the salinity, pH,  temperature, and

dissolved oxygen during the test period.

    (2)  A description of the test procedures  used  (e.g.  the

flow-through system, test chambers.,—chemical de.l.i.very .system,
                                12

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                                                           EG-5
                                                   August, 1982
aeration, etc.).

    (3)  Detailed information about the oysters used, including

the age and/or size (i.e. height), source, history, method of

confirmation of prespawn condition, acclimation procedures, and

food used.

    (4)  The number of organisms tested, the loading rate, and

the flowrate.

    (5)  The methods of preparation of stock and test solutions,

and the test chemical concentrations used.

    (6)  The number of dead and. live test organisms, the

percentage of organisms that died, and the number that showed any

abnormal effects in the control and in each test chamber at each

observation period.

    (7)  The 96-hour shell growth measurements of each oyster;

the mean, standard deviation and range of the measured shell

growth at 96 hours of oysters in each concentration of test

substance and control.

    (8)  The calculated 96 hour EC 50 and its 95 percent

confidence limits and the statistical methods used to calculate

these values.

    (9)  When observed, the 96 hour observed no-effect

concentration (the highest concentration tested at which there

were no mortalities, abnormal behavioral or physiological effects


                                13

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                                                           'EG-5
                                                   August, 1982
and at which shell growth did not di fer from controls).

    (10)  A graph of the concentratJ n-response curve based on

the 96 hour chemical concentration a d shell growth measurements

upon which the EC 50 was calculated.

    (11)  iMethods and data records c  all chemical analyses of

water quality parameters and test su stance concentrations,

including method validations and rea ent blanks.

    (12)  Any incidents in the cours  of the test which might

have influenced the results.

    (13)  A statement that the test  as carried out in agreement

with the prescriptions of the test c ideline given above

(otherwise a description of any devi tions occuring).
                                14

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                                  EG-6
                                  August, 1982
       OYSTER BIOCONCENTRATION TEST
        OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND  TOXIC  SUBSTANCES
  U.S.  ENVIRONMENTAL" PROTECTION AGENCY
          WASHINGTON,  D.C. 20460

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Office of Toxic Substances                                  EG-6
Guidelines for Testing Chemicals                   August,  1982
                   OYSTER BIOCONCENTRATION TEST


    (a)  Purpose.  This guideline is to be used for assessing  the

propensity of chemical substances to bioconcentrate in tissues of

estuarine and marine molluscs.  This guideline describes a

bioconcentration test procedure for the continuous exposure of

Eastern oysters (Grassestrea virginica) to a test substance in a

flow-through system.  The United States Environmental Protection

Agency (USEPA) will use data from this test in assessing the

hazard a chemical may present to the environment.

    (b)  Definitions .  The definitions in section 3 of the Toxic

Substances Control Act (TSCA) and in Part 792--Good Laboratory

Practice Standards are applicable to this test guideline.  The

following definitions also apply:

    (1)  "Acclimation" is the physiological compensation by test

organisms to new environmental conditions (e.g., temperature,

salinity, pH) .

    (2)  "Bioconcentration" is the net accumulation of a chemical

directly from water into and onto aquatic organisms.

    (3)  "Bioconcentration factor (BCF)" is the quotient of the

concentration of a test chemical in tissues of aquatic organisms

at or over a discrete time period of exposure divided by the

concentration of test chemical in the test- water at or duri-ng -the.

same time period.

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                                                            EG-6
                                                    August,  1982
    (4)  "Depuration" is the elimination  of  a  test  chemical  from

a test organism.

    (5)  "Depuration phase" is  the portion of  a  bioconcentration

test after the uptake phase during which  the organisms  are  in

flowing water to which no  test  chemical  is added.

    (6)  "EC 50" is that experimentally derived  concentration  of

a chemical in water that is calculated to induce shell  deposition

50 percent less than that  of the controls in a test batch of

organisms during continuous exposure within  a  particular period   '

of exposure (which should  be stated).

    (7)  "Loading" is the  ratio of the number  of oysters to  the

volume (liters) of test solution passing  through the  test chamber

per hour.

    (8)  "Steady-state" is the  time period during which the

amounts of test chemical being  taken up and  depurated by the test

oysters are equal, i.e., equilibrium.

    (9)  "Steady-state bioconcentration factor"  is  the  mean

concentration of the test  chemical in test organisms  during

steady-state divided by the mean concentration of;the test

chemical in the test solution during the  same  period.

    (10)   "Umbo" is the narrow  end (apex) of the oyster shell.

    (11)   "Uptake" is the .sorp tion. of a test chemical into.,and

onto aquatic organisms during exposure.

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                                                            EG-6
                                                    August,  1982
    (12)  "Uptake phase" is the  initial portion  of  a

bioconcentration test during which  the organisms  are  exposed  to

the test solution.

    (13)  "Valve height" is the  greatest  linear  dimension of  the

oyster as measured from the umbo  to  the ventral  edge  of  the

valves (the farthest distance from  the umbo).

    (c)  Test procedures — (1)  Summary of  the  test.   Oysters  are

continuously exposed to a minimum of  one  constant,  sublethal

concentration of a test chemical  under flow-through conditions

for a maximum of 28 days.  During this time,  test solution and

oysters are periodically sampled  and  analyzed  using appropriate

methods to quantify the test chemical concentration.   If,  prior

to day 28, the tissue concentrations  of the  chemical  sampled  over

three consecutive sampling periods  have been shown  to be

statistically similar (i.e., steady-state  has  been  reached),  the

uptake phase of the test is terminated, and  the  remaining oysters

are transferred to untreated flowing  water until  95 percent of

the accumulated chemical residues have been  eliminated,  or for  a

maximum depuration period of 14  days.  The mean  test  chemical

concentration in the oysters at  steady-state is  divided  by the

mean test solution concentration  at  the same time to  determine

the bioconcentration-factor-.(-B.CE)-. ..If steady-state-is not

reached during 28 days of uptake, the steady-state  BCF should be

-------
                                                            EG-6
                                                    August,  1982
calculated using non-linear parameter estimation methods.

    (2)   [Reserved]

    (3)  Range-finding test.  The oyster acute  toxicity  test  is

used to determine the concentration levels to be used  in the

oyster bioconcentration test.

    (4)  Definitive test.  (i)  The following data on  the  test

chemical should be known prior to testing:

    (A)  Solubility in water.

    (B)  Stability in water.

    (C)  Octanol-water partition coefficient.

    (D)  Acute toxicity (e.g. propensity to inhibit shell

deposition) to oysters.

    (E)  The validity, accuracy and minimum detection  limits  of

selected analytical methods.

    (ii)  At least one or more concentrations should be  tested to

assess the propensity of the compound to bioconcentrate.   The

concentrations selected should not stress or adversely affect the

oysters and should be less than one-tenth the EC 50 determined in

either the range-finding or 96-hour definitive  test in the Oyster

Acute Toxicity Test Guideline (USEPA 1981).  The test

concentration should be less than the solubility limit of  the

test substance in water, and should Joe close to.  th.e .potential-, or. - -

expected environmental concentration. The limiting factor  of  how

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                                                            BG-6
                                                   August,  1982
low one can test is based on the detection limits of the

analytical methods.  The concentration of the test material  in

the test solution should be at least ten times greater than  the

detection limit in water.

    (iii)  If it is desirable to document that the potential to

bioconcentate is independent of the test chemical concentration,

at least two concentrations should be tested which are at least a

factor of 10 apart.

    (iv)  To determine the duration of this test, an estimation

of the uptake phase should be made prior to testing based upon

the water solubility or octanol-water partition coefficient  of

the test chemical.   This estimate should also be used to

designate a sampling schedule.

    (v)  The following criteria should be met for a valid test:

    (A)  If it is observed that the stability or homogeneity of

the test chemical cannot be maintained, then care should be  taken

in the interpretation of the results and a note made that these

results may not be reproducible.

    (B)  The mortality in the controls should not exceed 10

percent'at the end of the test.

    (C)  The dissolved oxygen concentration should be > 60

percent of .saturation, throughout-the . tes t. ....

    (D)  There should be evidence that the concentration of  the

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                                                            EG-6
                                                   August,  1982
chemical being tested has been satisfactorily mainained  (e.g.

within 80 percent of the nominal concentration) over  the  test

period.

    (E)  Results are invalid and the test should be repeated if

the oysters spawn during the test.

    (F)  Temperature variations from 20°C should be held  to a

minimum.

    (vi)  The following methodology should be followed:

    (A)  The test should not be started until the test chemical

delivery system has been observed to be functioning properly and

the test chemical concentrations have equilibrated (i.e.  the

concentration does not vary more than 20 percent).  Analyses of

two sets of test solution samples taken prior to test initiation

should document this equilibrium.  At initiation (time 0), test

solution samples should be collected immediately prior to the

addition of oysters to the test chambers.

    (B)  The appropriate number of oysters should be brushed

clean and should be randomly distributed to each test chamber.

The oysters should be spread out equidistant from one another and

placed with the left (cupped) valve down and the unhinged ends

(opposite from umbo) all oriented in the same direction facing

the incoming flow.	                             -  -

    (C)  Oysters should ba exposed to the test chemical during

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                                                            EG-6
                                                    August,  1982
the uptake phase until steady state has been  reached  or  for a

maximum of 28 days.  The uptake phase should  continue for at

least 4 days.  Then the remaining oysters should  be  transferred

to untreated flowing water  and sampled periodically  to determine

if depuration of the test chemical occurs.  Every test should

include a control consisting of the same dilution water,

conditions, procedures, and oysters from the  same group  used in

the test, except that none  of the test chemical  is added.   If a

carrier is present in the test chamber, a separate carrier

control is required.

    (D)  Oysters should be  observed (and data recorded)  at least

daily for feeding activity  (deposition of feces)  or  any  unusual

conditions such as excessive mucus production (stringy material

floating suspended from oysters), spawning, or appearance  of

shell (closure or gaping).  If gaping is noted,  the  oyster(s)

should be prodded.  Oysters which fail to make any shell

movements when prodded are  to be considered dead,  and should be

removed promptly with as little disturbance as possible  to the

test chamber(s) and remaining live oysters.

    (E)  For oysters sampled, careful examination of  all  the

tissues should be made at the time of shucking for any unusual

conditions, such as a._wa.tery appearance,-or_ ,di.f feren.c.es._i.n-color -

from the controls.

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                                                            EG-6
                                                    August,  1982
    (F)  Observations on compound solubility should  also  be

recorded.  These include the appearance  of surface slicks,

precipitates, or material adsorbing  to the test  chamber.

    (vii)  Sampling.  (A)  At each of the designated  sampling

times, triplicate water samples and  enough oysters should be

collected from the  test chamber(s) to allow for  tissue  analyses

of at least four oysters.  The concentration of  test  chemical

should be determined  in a minimum of 4 oysters analyzed

individually at each sampling period.  If individual  analysis  is

not possible, due to  limitations of  the  sensitivity  of  the

analytical methods, then pairs, triplicates or more  oysters may

be pooled to constitute a sample for measurement.  A similar

number of control oysters should also be collected at each  sample

point, but only those collected at the first sampling period and

weekly thereafter, should be analyzed.   Triplicate control  water

samples should be collected at the time  of test  initiation  and

weekly thereafter.  Test solution samples should be  removed from

the approximate center of the water  column.

    (B)  At each sampling period the appropriate numbers  of

oysters are removed and treated as follows:

    (_1_)  The valve height of each oyster should  be measured.

    (_2_)  Oysters should be s.hucked as soon .as-practical-..after

removal and should never be refrigerated or frozen in the

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                                                            EG-6
                                                    August,  1982
shell.  The shell should be opened at  the  hinge,  the  adductor

muscle severed and the  top valve  removed.   The  remaining  adductor

muscle should be severed where  it attaches  to the lower valve and

the entire oyster removed.

    (_3_)  The shucked oysters should  then  be drained  three

minutes, blotted dry, weighed and analyzed  immediately  for the

test chemical.  If analyses are delayed,  the shucked  oysters

should be wrapped individually  in aluminum  foil  (for  organic

analysis) or placed  in  plastic  or glass containers  (for metal

analysis) and frozen.

    (C)  If a radiolabelled test  compound  is used,  a  sufficient

number of oysters should also be sampled at termination to permit

identification and quantitation of any major (greater than 10

percent of parent) metabolites  present.   It is crucial  to

determine how much of the activity present  in the oyster  is

directly attributable to the parent  compound.

    (5)  Test results   (i)  Steady-state has been reached when

the mean concentrations of test chemical  in whole oyster  tissue

for three consecutive sampling periods are  statistically  similar

(F test, P=0.05).  A BCF is then calculated by dividing the mean

tissue residue concentration during  steady-state  by  the mean  test

solution concentration  d.ur-i.ng -th.is,.s ame_ period... ...A -9-5 percent

confidence interval should also be derived  from  the BCF.   This

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                                                            EG-6
                                                    August,  1982
should be done by calculating the mean oyster tissue

concentration at steady-state (XQ) and its 97.5 percent

confidence interval Xo ± t  (S.E.) where t is the  t statistic  at

P=0.025 and S.E. is the one standard error of the mean.   This

calculation would yield lower and upper confidence limits  (Lo and

UQ).  The same procedure should be used to calculate  the  mean and

97.5 percent confidence interval for the test solution concen-

trations at steady-state, Xs ± t(S.E.), and the resulting  upper

and lower confidence limits (Ls and Us)•  The 95 percent

confidence interval of the 3CF would then be between  LO/US  and

UO/LS.   If steady-state was not reached during the maximum 28 day

uptake period, the maximum BCF should  be calculated using  the

mean tissue concentration from that and the previous  sampling

day.  An uptake rate constant should then be calculated using

appropriate techniques.  This rate constant is used to estimate

the steady-state BCF and the time to steady-state.

    (ii)  If 95 percent elimination has not been observed  after

14 days depuration then a depuration rate constant should  also be

calculated.  This rate constant is used to estimate the time  to

95 percent elimination.

    (iii)  Oysters used in the same test should be 30 to  50

millimeters in, .valve_height .and ..should .be ^as. similar,  in~..age-

and/or size as possible to reduce variability.  The standard


                               10

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                                                            SG-6

                                                    August,  1982
deviation of the height should be  less  than  20  percent  of  the



mean (N=30).



    (6)  Analytical measurements.   (i)  All  samples  should be



analyzed using USEPA methods and guidelines  whenever feasible.



The specific methodology used should  be validated  before the test



is initiated.  The accuracy of the  method should be  measured by



the method of known additions.  This  involves adding a  known



amount of the test chemical to three  water samples  taken from an



aquarium containing dilution water  and  a number of  oysters equal



to that to be used in the test.  The  nominal concentration of



these samples should be the same as  the concentration to be used
                   o


in the test.  Samples taken on two  separate  days should be



analyzed.  The accuracy and precision of the analytical method



should be checked using reference or  split samples or suitable



corroborative methods of analysis.   The accuracy of  standard



solutions should be checked against other standard solutions



whenever possible.



    (ii)  An analytical method should not be used  if likely



degradation products of the test chemical, such as hydrolysis and



oxidation products, give positive or  negative interferences,



unless it is shown that such degradation products  are not  present



in the test chambers during ->the--tes-t»—-Atomic^-absorp-tion	.,__....



spectrophotometric methods for metal  and gas chromatographic





                                11

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                                                            EG-6
                                                    August,  1982
methods for organic compounds are preferable  to colorimetrie

methods .

    (iii)  In addition to analyzing samples of test  solution  at

least one reagent blank should also be analyzed when a  reagent  is

used in the analysis.

    (iv)  When radiolabelled test compounds are used, total

radioactivity should be measured in all samples.  At the  end  of

the uptake phase, water and tissue samples should be analyzed

using appropriate methodology to identify and estimate  the  amount

of any major (at least 10 percent of the parent compound)

degradation products or metabolites that may  be present.

    (d)  Test conditions — (1)  Test species.  (i)  The  Eastern

oyster, Crassostrea virginica, should be used as the test

organism.

    (ii)  Oysters used in the same test should be 30 to 50

millimeters in valve height and should be as  similar in age

and/or size as possible to reduce variability.  The  standard

deviation of the valve height should be less  than 20 percent  of

the mean.

    (iii)  Oysters used in the same test should be from the same

source and from the same holding and acclimation tank(s).

    (iv)  Oysters .should be .-in a_prespawn cond:i tionvof.. go.nadal	

development prior to and during the test as determined  by direct


                                12

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                                                            EG-6
                                                    August, 1982
or histological observation  of  the  gonadal tissue for the

presence of gametes.

    (v)  Oysters may  be  cultured  in the laboratory, purchased

from culture facilities  or commercial  harvesters, or collected

from a natural population  in an unpolluted area free from

epizootic disease.

    (vi)  The holding  and  acclimation  of  the oysters should be as

follows:

    (A)  Oysters should  be attended to immediately upon

arrival.  Oyster shells  should  be brushed  clean of fouling

organisms and the  transfer of  the oysters  to the holding water

should be gradual  to  reduce  stress  caused  by differences in water

quality characteristics  and  temperature.   Oysters should be held

for at least 12 to 15  days before testing.   All oysters should be

maintained in water of the quality  to  be  used in the test for at

least seven days before  they are used.

    (3)  During holding, the oysters should not be crowded and

the dissolved oxygen  concentration  should  be above 60 percent

saturation.  The temperature of the holding waters should be the

same as that used  for  testing.  Holding tanks should be kept clean

and free of debris.   Cultured  algae may be added to dilution

water sp.aring.ly.,i as...necessary_.to_.sup.po.rt^ 1 i.f e;-.a.-nd-igrowth,- such

that test results  are  not  affected,  as confirmed by previous


                                13

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                                                            EG-b
                                                    August, 1982
testing.  Oysters should  be  handled  as  little as possible.  When

handling is necessary,  it should  be  done as gently, carefully,

and quickly as possible.

    (C)  A batch of  oysters  is  acceptable for testing if the

percentage mortality over the seven  day period prior to testing

is less than five percent.   If- the mortality is between 5 and 10

percent, acclimation should  continue for seven additional days.

If the mortality is  greater  than  10  percent, the entire batch of

oysters should be rejected.  Oysters should not be used which

appear diseased or otherwise stressed.   Oysters infested with

mudworms (Polydora sp.),  boring sponges (Cliona cellata) or which

have cracked, chipped,  bored, or  gaping shells should not be

used.

    (2)  Facilities — (i)   Apparatus.  (A)   An oxygen meter,

equipment for delivering  the test chemical, adequate apparatus

for temperature control,  test  tanks  made of. chemically inert

material and other normal laboratory equipment are needed.

    (3)  Constant conditions in the  test facilities should be

maintained as much as  possible  throughout the test.  The

preparation and storage of the  test  material, the holding of the

oysters and all operations and  tests should be carried out in an

environment. £r.,e.e.J:r.-Qm» harmf.ul:.co-n.cen,trat.ions: ~of—du-s t-,..-vapors and-

gases  and in such a  way as to avoid  cross-contamination.  Any


                                14

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                                                            EG-6
                                                    August,  1982
disturbances that may  change  the behavior  of  the  oysters  should

be avoided.

    (ii)  Dilution water  A constant  supply of  good  quality

unfiltered seawater should be  available  throughout  the  holding,

acclimation, and testing periods.   Natural seawater  is

recommended, although  artificial seawater  with  food  (algae) added

may be used.  In either case,  to ensure  each  oyster  is  provided

equal amounts of food, the water should  come  from a  thoroughly

mixed common source and should  be delivered at  a  flow  rate of  at "

least one, and preferably five  liters per  hour  per  oyster.  The

flowrate should be +_ 10 percent of  the nominal  flow.   A dilution

water is acceptable if oysters  will survive and grow normally

over the period in which the  test is  conducted  without  exhibiting

signs of stress, i.e.  excessive mucus production  (stringy

material floating suspended from oys ters ) , lack of  feeding, shell

gaping, poor shell closing in response to prodding,.or  excessive

mortality.  The dilution water  should have a  salinity  in  excess

of 12 parts per thousand, and should  be  similiar  to  that  in the

environment from which the test oysters  originated.  A  natural

seawater should have a weekly range in salinity of  less than 10

parts per thousand and a monthly range in  pH  of less than 0.8

units .  , -Artif icial\s-e:awa±er~\s hould ~.n.at-...varyr.mor_e ^than -.2 -pa.rts  per

thousand nor more than 0.5 pH units.  Oysters should be tested in


                                15

-------
                                                            EG-6
                                                    August, 1982
dilution water  from  the same  origin.

    (3)  Test parameters—(i)   Carriers .   Stock solutions of

substances of low  aqueous solubility  may  be prepared by

ultrasonic dispersion  or,  if  necessary, by use of organic

solvents, emulsifiers  or dispersants  of low toxicity to

oysters.  When  such  carriers  are used,  the control oysters should

be exposed to the  same concentration  of the carrier as that used

in the highest  concentration  of  the test  substance. The

concentration of such  carriers  should not exceed 0.1 ml/1.

    (ii)  Dissolved  oxygen.   The dissolved oxygen concentrations

should be at least 60  percent  of the  air  saturation value and

should be recorded daily.

    ( iii)  Loading.  The loading rate should not crowd oysters

and should permit  adequate circulation  of water while avoiding

physical agitation of  oysters  by water current.

    ( iv)  Temperature.  The test temperature should be 20°C +_

1°C.  Temporary excursions (less than eight hours) within 15°C to

25°C are permissible.  Temperature  should be recorded continu-

ously.

    (v)  pH.  The  pH should be  recorded  twice weekly in each test

chamber.

    (e)  Rep.ort-i.ng...._ ..In---.,additloji..-to. th,e--rep.ort.i.ng~. req.ul,reraen.ts. .— .

prescribed in Part 792—Good  Laboratory Practice Standards, the


                                 16

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                                                            EG-6
                                                    August,  1982
report should contain the following:

    (1)  The source of the dilution water,  the  mean,  standard

deviation and range of the salinity, pH,  temperature  and

dissolved oxygen during the test period.

    (2)  A description of the test procedures used  (e.g.  the

flow-through system, test chambers, chemical delivery system,

aeration, etc.).

    (3)  Detailed information about the oysters  used,  including

age, and/or size (i.e. height), weight  (blotted  dry),  source,

history, method of confirmation of prespawn condition,

acclimation procedures ,and food used.

    (4)  The number of organisms tested,  loading  rate and

flowrate.

    (5)  The methods of preparation of stock and  test solutions

and the test chemical concentrations used.

    (6)  The number of dead and live organisms,  the percentage of

oysters that died and the number that showed any  abnormal affects

in the control and in each test chamber at  each  observation

period.

    (7)  Methods and data records of all  chemical analyses  of

water quality parameters and test chemical  concentrations,

including .method .valid.ati.o.ns. .and. ..r.eag.e.nt  bla.nks .—. —	
                                17

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                                                            EG-6
                                                   August,  1982
    (8)  Description of sampling, sample storage  (if required)

and analytical methods of water and tissue analyses for  the  test

chemical.

    (9)  The mean, standard deviation and range of the concentra-

tion of test chemical in the test solution and oyster tissue at

each sampling period.

    (10)  The time to steady-state.

    (11)  The steady-state or maximum BCF and the 95 percent

confidence limits.

    (12)  The time to 95 percent elimination of accumulated

residues of the test chemical from test oysters.

    (13)  Any incidents in the course of the test which  might

have influenced the results.

    (14)  If the test was not done in accordance with the

prescribed conditions and procedures, all deviations should be

described in full.

    (f)  References.  U.S. Environmental Protection Agency.

1981.   Oyster Acute Toxicity Test Guideline, Toxic Substances

Control Act, section 4.  Office of Pesticides and Toxic

Substances, Washington, D.C.: U.S. Environmental Protection

Agency.
                                18

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                                       ES-3
                                       August, 1982
             TECHNICAL  SUPPORT DOCUMENT

                         FOR

OYSTER ACUTE TOXICITY TEST  AND BIOCONCENTRATION TEST
             OFFICE OF  TOXIC  SUBSTANCES
      OFFICE  OF PESTICIDES AND TOXIC SUBSTANCES
        U.S.  ENVIRONMENTAL  PROTECTION AGENCY
               WASHINGTON,  D.C.  20460

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                        TABLE OF CONTENTS

        Subject                                         Pagt

I.       Purpose                                          1
II.      Scientific Aspects                               1
        General                                          1
        Test Procedures                                  5
        Range Finding .                          .         6
        Acute Test                                       6
        Bioconcentration Test                            7
        Definitive Test                                  3
        Acute Test                                       8
        Biocentcation Test.                               10
        Analytical                                       15
        Water Quality                                    15
        Collection of Test Solution Samples              15
        Test Substance Measurement                       16
        Test Data                                        18
        Analysis                                         18
        Acute Toxicity Test                              13
        Bioconcentration Test                            23
        Temperature Measurements                         28
        Test Conditions                                  28
        Test Species                                     28
        Selection                                        28
        Sources                                          30
        Size                                             31
        Condition                            s            31
        Maintenance of Test species                      33
        Feeding                                          33
        Facilities                                       35
        General                                          35

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        Subject                                         Page
        Construction Materials                           36
        Test Substance Delivery System                 .  36
        Test Chambers and Loading                        38
        Flow-through System                              39
        Cleaning                                         40
        Dilution Water                                   40
        Carriers                                         44
        Environmental Conditions                         45
        Dissolved Oxygen (See Section 2.1.3,             45
        Dilution Water)
        Temperature                                      45
        Light              '                              43
        Salinity (See Section 2.1.3,                     48
        Dilution Water)
        Reporting                                        48
III.     Economic Aspects                                 48
IV.      References                                       51

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Office of Toxic Substances                               ES-3
                                                 August, 1982
I.  Purpose
    The purpose of  this  document  is  to provide the
scientific background and  rationale  used  in the development
of Test Guidelines  EG-5  and  EG-6  which uses the Eastern
oyster ,Crassostrea Virginia,  to  evaluate toxicity and bio-
concentration of chemical  substances.   The Document provides
an account of the logic  used  in the  selection of the test
methodology, procedures  and  conditions prescribed in the
Test Guidelines.  Technical  issues and practical
considerations relevant  to the Test  Guidelines are
discussed.  In addition, estimates of  the cost of conducting
i:he tests are provided.
II.  Scientific Aspects
    A.  General
    The test guidelines  represent  a synthesis of  testing
procedures and the current  laboratory  practices  of various
researchers.  Increased  interest and research in .aquatic
toxicology and bioconceatration and their use as  monitoring
tools has led to  the  need for  standardized procedures for
testing the sublethal responses of  marine and estuarine
bivalve molluscs.  Mortality testing is  not practical
because bivalves  are  able to close  their valves  and seal
themselves off from environmental  stress for long periods of
time.  The results of such  tests would be difficult, if not
impossible, to interpret.   A more  useful test is  the shell
deposition test,  which employs concentrations that will
produce an adverse effect,  but will not  cause the animal to
close up.   The shell  deposition test is  intended  to provide
a short-term. .as.s.essme-n.t_of-_the~ haz.a.r.d  .whaoiv-a-.-tes t ..ch-emical -

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                                                         ES-3
                                                 August,  1982
may present to oysters, and  to serve as a range-finding  test
for the bioconcentration  test.  Therefore,  the  test should
be of short duration, and be similar in conditions  to  the
bioconcentration test.
    Butler et al.  (1960)  demonstrated  that  shell  growth  in
juvenile oysters could be employed as  a sensitive method for
the continuous monitoring of physiological  stress occurriag
in bivalves exposed to various concentrations of
pesticides.  In Butler's  studies, shell growth  was  used  as  a
measure of reversible inhibitory effect.  The advantage  of
shell growth lies  in  the  ability to" use the shell as an
ongoing physiological stress monitor without the  need  for
periodic sacrificing  of organisms.  In addition,  the test  is
rapid, reliable, reproducible and requires  no specialized
equipment or personnel training.
    The method first  developed by Butler which  utilized
shell deposition as a bioassay technique has been
successfully employed by  numerous researchers (Schuster  and
Priagle 1969, Tinsman and Maurer 1974, Frazier  1976, Conger
et al. 1978, Cunningham 1976, Epifanio and  Mootz  1976, Lowe
et al. 1972).  Epifanio (1979) showed  that  growth of hard
and soft tissues in oysters  was closely coupled, as
determined by correlation analysis.  This indicates  that
shell deposition serves as a usaful indicator of  oyster
physiological response rather than just as  a singular
response to calcium carbonate deposition.   Studies  conducted
by Conger et al. (1978) indicated a statistically
significant difference (P<_ 0.001) in the level  of inhibition
of shell depos it ion_. in_ oys tersr- which were .subj.ec ted;.-, to. ..0-..25:
mg/1 cadmium.  Schimmel et al. (1976)  compared  a  50  percent

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                                                         ES-3
                                                 August,  1982
median shell deposition  reduction  (SC50)  in  Crassostrea
virginica and  the  LC50  in  five  fish  species.   In this study,
shell deposition was at  least as sensitive a  test  as  those
employing fish.  In  tests  conducted  by  Butler (1965)  the
shell deposition test was  more  sensitive  than other  acute
toxicity tests  employing other  estuarine  organisms.   The
oyster shell deposition  methodology  is  also  described in
Standard Methods (APHA  1975), Bioassay  Procedures  for the
Ocean Disposal  Permit Program (USSPA 1978) and American
Society for Testing  and  Materials  (ASTM 1980).
    Interest in bioconcentration began  with  the  discovery
that levels of  heavy metals  in  many  animals  v/ere much higher
than in the surrounding  water.   In oysters,  the  phenomenon
was observed early in this  century (Hiltner  and  Wickman
'1919), and elaborated upon 'by subsequent  workers (Galtsoff
1942, Chipman  et al. 1958).  The  increased impacts of
hydrocarbons and various organic compounds, such as
insecticides,  in recent  years,  led to  Eie.~i.ii  studies  of
bioconcentration by oysters  of  such  chemicals (Stegeman and
Tecil 1973, Butler  1967,  Hansen  et  al.  1976,  3rodtmann 1970).
    Laboratory  testing  of  oyster bioconcentration  is  a
relatively recent  development.   Schinvmel  et  al.  (1977),
Banner et al.  (1977), Frazier (1979a,b),  Hansen  et al.
(1976), Lee et  al. (1978),  Parrish et  al.  (1976),  and
Stegeman (1974) have all investigated  bioconcentration  using
oystecs in controlled laboratory settings.   However,  the
experimental methodology of  each investigator was  often
substantially different.
    Other flow-through "testing-, particul*arly*corrcerned-~v7ith ~
sublethal effects on fish  and bivalves  in the late 1960's

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                                                         ES-3
                                                 August, 1982
and early 1970's  led  to  a  general  recognition of the need
for standardization  in  flow-through testing.  Several
authors proposed  methodologies  (Sprague 1969, Cairns and
Dickson 1973,  Esvelt  and Connors  1971,  Lichatowich et al.
1973, LaRoche et  al.  1970).   These were synthesized into the
bioassay section  of Standard  Methods  (APH/A 1975) and
sections of the Ocean Disposal  Bioassay Manual (Butler and
Lowe 1978).  However, neither publication considered
bioconcentration.  Bioconcentration methodologies have
emerged only in the last few  years, and have been drafted as
proposed standards by the  American Society for Testing and
Materials (ASTM 1980).
    The test guidelines  adapt,  to  the extent possible, the
procedures of Standard  Methods,  EPA and ASTM to the specific
requirements of the Eastern oyster, Crassostrea Virginia
Gmelin.
    Many industrial chemicals have not  been previously
tested by standard aquatic bioassay methods and, as a
result, cannot be classified  as  to their toxicolpgical
properties or propensity to bioconcentrate.
    The oyster shell  deposition  test provides informa±ion on
the effects of short-term  exposure of the test oysters to
the test chemical under  controlled conditions (ASTM
1980b).  Continuous administration of the test chemical in
this 96-hour flow-through  system  represents a practial
simulation of chemical sui.lls of  effluent discharges to non-
motile organisms  which  are incapable of avoiding the
perturbation ( APHA 1975).  As such, the oyster shell
depos ition tes-t -is. .part.icula-rly-^us4if-ul  .£o.r_,evaiu.a-tl-:iA!>-Uu->. -..-,
short-term toxicity of specific substances or was,tes oa

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                                                         ES-3
                                                 August, 1982
marine molluscs.  This  test  is  employed  primarily as an
appropriate range-f inding  test  for the more complex
bioconcentration test.   As a range-finding test,  it provides
information- on  the  upper limit  of  exposure that is not
anticipated to cause  adverse effects  during the
bioconcentration test.
    Oysters, as filter  feeders,  can be exposed to relatively
large amounts of a  potential toxicant.   This  is because the
oyster pumps large  volumes of water and  removes both living
and non-living particulate matter  from that water.  A
potential toxicant  can  be  accumulated in the oyster tissues
in concentrations much  greater  than occur in the  ambient
water or particulate  matter.  This accumulation,  known as
bioconcentration, has been demonstrated  LOC a number of
petrochemicals (Anderson and .Anderson.. 1976, Anderson 1978,
Banner et a1. 1977, Lee  et al.  1978,  Stegeman 1974),
pesticides (Brodtmann 1970,  Sutler 1967, Parrish et al.
1976, Schimmel et al. 1977),  and metals  (Fraziar   1975, 1976,
1979 a,b).  The contaminated organism can, in turn, pass its
body burden of toxicant  on to the  next trophic lavel in a
concentrated form.  Sinne  humans are major consumers of
oysters, the potential  for oysters to bioconcentrate a
potentially toxic substance  is  of  additional concern.  In
addition, bioconcentration of a substance by oysters may be
an indication that  the  substance is biologically active and
could affect other  elements  of  the aquatic system.
    The bioconcentration tes t provides  an estimate of that
potential.  The results  of the  test can  provide a basis' for
decis io.ns. .concerning, .what: con.c.ejitra-tio.Tis/,-"~i:L"any;,J.7_o£r,:the:;«_ '._
test chemical in vater  may be bioconcentrated to  potentially

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                                                         ES-3
                                                 August, 1982
hazardous concentrations  in  the  aquatic biota.
    3.  Test procedures
         1.  Range-Finding Test
              a.  Acute  test
    For the oyster  acute  toxicity  test, a range-f inding test
is recommended  to determine  the  appropriate concentrations
of test chemical to  be used  for  a  definitive test when the
acute toxicity  of the substance  is  unknown or cannot be
elucidated from existing  toxicity  data.  This approach
should minimize the  possibility  that .an inappropriate
concentration series will be utilized  in the definitive test
arid under certain circumstances  may even preclude the need
to conduct the  definitive test.   In order to minimize the
cost and time required to obtain the requisite  data nominal
concentrations.-are  permitted,-  test duration may be
shortened, replicates are not  required, and other test
procedures and  conditions are  relaxed.
    The range-finding test (or other available  information)
needs to be accurate enough  to ensure  that dose levels in
the definitive  test  are spaced to  result in concentrations
above and below the  EC50  values  for shell deposit ion-.  IF
the substance has no measurable  effect  at the saturation
concentration (at least  1000 mg/1),  it  is considered
relatively non-toxic to oysters  and definitive  testing is
deemed unnecessary.  In  all  cases,  the  range-finding test is
conducted to reduce  the expanse  involved in having to repeat
a definitive test because of  inappropriate ttfs i: chemical
concentrations.
    In the .range-f.indi.ng-  -tes t:;... g.r-oup,s -of. _f-ive :.or. .:no-r:&: fey t
oysters are exposed  to a  broad range of concentrations of

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                                                         ES-3
                                                 August,  L982
the substance.  Sufficient concentrations should be  tested
such that the concentration which  inhibits shell deposition
by 50 percent of the control organisms can be approximated.
The number of concentrations will  normally range from  3-6
depending upon the shape of the toxicity curve  for that
substance and prior knowledge of its approximate toxicity.
Only concentrations less than the  solubility limit in  water
are tested.
    •N         b.  BioconcentratJ.oa Test
    The oyster acute toxicity test is used as the  cange-
finding test for the oyster bioconcentration test.   The
concentration of test chemical in  the test solution  should
not stress, irritate, or otherwise adversely effect  the
organisms during the bioconcentration test.  To meet this
criteria, the ASTM (19-30) recommends that the highest  -	-
concentration be no more than one-tenth the 96  hour  SCcQ
based on reduced shell deposition.
    If stress, irritation, or other adverse effects  are
observed, the bioconcentration test should be repeated at a
lower concentration.
    In the bioconcentration test,  it would be most u.y.-Cul
for the hazard and risk assessment processes to use  an
exposure concentration that approximates the expected  or
estimated environmental concentration.  One should take
care, however, that the selected concentration  is  at least
three times above its Detection limit and will  allow
quantification of the residues in  tissue.  Test
concentrations of 1-10 ug/1 would  be appropxiate tfot: many
compounds.                   •

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                                                         ES-3
                                                 August, 1982
         2.  Definitive test
              a.  Acute Test
    The specific requirements of  the  definitive  oyster acute
toxicity test (USEPA  1980) are  the  analytical  determinations
of chemical concentrations,  the unbiased  selection of
oysters for each treatment, the use of  controls,  the
assessment of test validity, and  the  recording,  analysis,
and presenta-tion of  data.  These requirements assure  that
the chemical concentration - oyster response  relationship  is
accurately known, that chemical effects are not  confounded
by differential oyster sensitivity, and that  the
relationships are clearly presented.   Reporting  the
occurrence of such effects as abnormal  shell  movement  and
feeding behavior provide qualitative  data that further
assist the assessment-of. toxicity.
    The results of a  definitive test  are  used  to calculate
the 96 hour EC50 and  the concentration-response  relationship
of the test chemical  and the test oysters.  If the con-
centrations of  test chemical which  produce no effect,  a
partial inhibition of shell deposition, and 100  percent
inhibition have been  determined during  the range-finding
test, then five or six test chemical  concentrations should
be sufficient to estimate the appropriate EC50 value in a
definitive test.  In  some cases however,  to obtain two
partial inhibitions bracketing  the  50 percent level, it may
be necessary to test  8-10 concentrations.
    The slope of the  concentration-response curve provides
an indication of the  range of sensitivity of  the test
oys ters . to .the ".t.es-t.-.ch.emica-1-' andvimay-~al Low^es-t.-imat-io.ns.ucof.-. - --•
lower concentrations  that will  affect the test organism.

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                                                         ES-3
                                                 August, 1982
For example,- if the slope of the concentration-response
curve is very steep,  then a slight  increase  in concentration
of the test chemical  will affect a  much  greater portion or
the test oysters than would a similar  increase if  the slope
of the curve was very shallow.  The  slope  of  the
concentration-response curve reveals the extent of
sensitivity of the test oysters over a range  of
concentrations .
    The exposure of two or more replicate  groups having a
minimum of 20 oysters each, to each  test chemical
concentration is required in the guideline.   That  minimum is
based on an optimum number of test  oysters needed  for
statistical confidence, equipment requirements, and
practical considerations of handling the test organisms.
    At least two repl-icates should -be  included in  order to -
demonstrate the level of precision  in  the  data and  indicate
the significance of variations.  Test  chambers holding
replicate groups should have no water  connections  between
them.  The distribution of test oysters  to  the test  chambers
should be randomized  to prevent bias from  being introduced
into the test results.
    The exposure time of 96 hours in the oyster acute
toxicity test guideline is specified in  order to permit a
comparison of data developed through the use  of this test
guideline with the acute toxicity data in  the published
literature (see Soot ion 1.5) The use of  the  96-hour  exposure
period was proposed initially in 1951  by an  aquatic  bioassay
committee (Doudoroff  et al. 1951) and  was  selooted,  in large
part, as--.a matter -of ^convenience/ since,.. it.:is-.easily"-.^-'—", :.:..'
scheduled within the  five-day work  week.  The OS-hour

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                                                         ES-3
                                                 August,  1982
exposure period is also required  in  the  flowing  seawater
toxicity test using oysters  as  a  bioassay  procedure for the
US SPA Ocean Disposal Permit  Program  (US SPA 1978).
              b.  Bioconcentration Test
    The bioconcentratioa  test guideline  recommends  that the
uptake phase last no more than  28 days and the depuration
phase last no more than 14 days for  a  total maximum test
duration of: 42 days.  This is based  on the experience  of
researchers who have found that, generally speaking,
substances are either rapidly taken  up or  very slowly  taken
up.  Krieger et al. (1979) demonstrated  attainment  of
steady-state for antipyrine  uptake in  less than  90  minutes
using mussels.  Schimmel  et  al. (1978) showed  that  oysters
reached uptake equilibrium with respect  to sodium
pentachlorophenate in .4-days .   On .the  other hand,  Stegeman  •
(1974) postulated that while low  molecular weight
hydrocarbons are rapidly  taken  up and  released,  high
molecular weight compounds are  taken up  and released much
more slowly and, in fact, may never  be completely
eliminated.  Hydrocarbons apparently reach equilibrium with
the lipid fraction of the animal, so that  the physiological
state of the organism has a  great influence on
bioconcentration and depuration.
    Because of the role of the  lipid fraction  in modifying
bioconcentration, it is possible  to  generate estimates of
the bioconcentration factor  of organic chemicals from  a
knowledge of their lipophillc nature.  Veith et  al.  (1979)
analyzed the correlation  between the n-octanol/water
partition coefficient -.(Jl-)_.> :.a. commo.nl.y:,us,ed:-.me,as.ar-e.v..o£i;a .: ....
substance's lipophilic nature, and the experimentally
                                10

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                                                         ES-3
                                                 August,  1982
derived biooo-ioentration factor  (BCF).   They  show that the
log BCF and the log ? ace  linearly  related  by the equation:

              log BCF = 0.85 log  ?  -  0.70

    They suggest that the  high correlation  of the equation
(r2 = 0.897) means that the log BCF can  be  estimated  to
within an order of magnitude for  substances having  a  broad
range of partition coefficients.  Approximately  5 percent of
the substances tested had  low log BCFs despite high log P,
thus falling outside of the general relation.   However, as
Veith et al. (1979) point  out, none of the  substances  with
high BCF values had low log P values.  This means that use
of the relationship should not lead to an underestimation of
the bioconcentrati-on f-actor.
    Chiou et al. (1977) present support  for estimating the
partition coefficient from the aqueous solubility.   Their
relationship states that:

              log P = 5.00 - 0.670  log S

where S is  the aqueous solubility in  micromol/liter.   They
found that  the log P values for 34  organic  substances  ranged
from 1.26 (for phenoxyacetic acid)  to 6.72  (for  2,  3,  4, 2",
4', 5',-PCB).
    On the  basis of these  known relationships between
solubility, lipophilic nature and bioconcentration,  Neeley
(1978) developed the equations for  estimating the times to
steady-s-ta-te.. - The- es timates/are :based on--f-ish.,  but are": •  •••
applicable  to molluscs.  In  the bioconcentration test, the
                                11

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                                                        KS-3
                                                August, 1932
exposure period should be long enough to demonstrate that
steady-state has been reached.
    Before starting a bioconcentration test, an estimation
of the BCF or the time to steady-state should be made in
order to avoid running the test for the maximum time
period.  A summary by Kenaga and Goring (1980) presents data
and methods to estimate the BCF.  The two uost commonly used
factors for predicting bioconceatration potential are water
solubility and octanol-water partitioning.  Water solubility
can be determined empirically in the laboratory, or in some
cases, taken from the literature (Chiou et al. 1977, Kenaga
and Goring 1930).  Octanol-water partition coefficients can
be determined empirically, estimated by reverse-phase high
pressure liquid chroma tog raphy according to Veith et al.
(1979), calculated, according to Leo et al. (1971) or.-taken
from  the literature (Chiou et al.  1977, Hanch. et al. 1972,
Kenaga and Goring, 1980).  However, soiae of the reported
data  are highly variable and may not be appropriate for use.
    An estimate of the time to steady-state (5 in hours) can
be estimated from the water solubility or octanol-water
partition coefficient using the equations developed by ASTM
(1930): S=3.0/antilog (0.431 log W-2.11) or S=3.0/antilog (-
0.414 log P + 0.122) where W = water solubility (mg/1) and
P=octanol-water partition coefficient.
    Presented below is a summary of data correlating various
exposure times to the corresponding estimates of the
partition coefficient and BCF.
                                12

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                                                          ES-3
                                                 August,  1982
                    Log  P
                           BCF
                           S
1,585
8,710
33,113
120,226
316,228
524,807
933,254
3.2
3.94
4.52
5.08
5.5
5.72
5.97
105
446
1387
4150
10,000
14,521
23,636
2
4
7
12
18
22
28
Log BCF

 .02
2.65
3.14
4.62
4.0
4.16
4.37
    Log BCF was estimated  using the equation of Veith et  al.
(1970) where  log BCF=0.85  log  P-0.70.
    3ased on  the estimate  of  the time to steady state, one
of the following sampling  schemes may be used  to generate
appropriate data.
                         Sampling Days
   Test
   Period/  3a<4
34-14
S>15-21
              S>21
Exposure
lb 4b
^b i
1 3
2 7
3 10
4 12
14
Depuration
lb 1
£-Q n
12h 4
1 6

a. length estimated time
b. hours.

1
3
7
10
14
18
22
1
3
7
10

to s ceaiiy s tate in days .

1
3
7
10
14
21
28
1
3
7
10 •
14

                                 13

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                                                         ES-3
                                                 Augus t,•1982
    There are  two methodologies  in  use  today to estimate
bioconcentration potential;  the  kinetic approach and the
steady-state approach and both are  based  on  research
conducted with fish.  Bishop  and  Maki  (1980) and Harnelink
(1977) give a review of both,  Using the  kinetic approach,
Bishop and Maki (1930), Branson  et  al.  (1975),  Cember et al.
(1978) and Krseminsky (1977)  proposed  the use of first-order
kinetic expressions from relatively short (_<_ 5  days) fish
exposures, and a subsequent  depuration  period,  to calculate
uptake and depuration rate constants.   These rate constants
are then u.s^d  to estimate the BCF at the  time of apparent
steady-state, and the time to 50  percent  elimination.  The
steady-state method, in more  widespread use, exposes fish
for a longer period of  time  until steady-state  in the tissue
is experimentally observed (Barrows et  al. -1980, Bishop and
Maki 1980, Ve i th et al. 1979) and continues  with a
depuration phase until  SOpercent  or 95  percent  elimination
has been observed.  The estimation  of  bioconcentration using
the kinetic approach cannot  account and adjust  for changes
in the rates of uptake  and depuration  such as those observed
by Barrows et al. (1980) an-1  Melancon  and Lech  (1979).   The
use of the kinetic approach  also  requires access to a
sophisticated computer system, apparatus  not readily
available to many laboratories.
    Although Bishop and Maki  (1930) and Branson et al.
(1975) have shown excellent  agreement  between estimates of
bio-concentration factors for some  compounds using both
approaches, the agency has recommended  a  modified steady-
state method -for determrnat.ir>n;"of'_^b±oco,ncexi-trat.ion.^. -The--  ~-
empirical nature of the data, the relative ease with which
                                14

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                                                         ES-3
                                                Jarmay,- 1982
the test can be performed  and  the  number of  researchers and
laboratories that have performed such  tests  make this test
more appropriate at this time.  As  the data  base for
comparisons of BCFs' between  the  two methods  grows,  the
kinetic approach may become  ^aore useful and  valuable.  Under
TSCA the Agency is  required  to review  all test guidelines
annually, and in the future  the Agency will  consider
adopting the kinetic approach.
         3.  Analytical
              a.  Water Quality Analysis
    Measurement of  certain water quality parameters of the
dilution water such as dissolved oxygen, temperature,
salinity and pH is  important,  Quantification of these
parameters at the beginning, during, and at  the end of the
exposure period for flow-through  tests- is neces-sary -in- order
to determine if the water  quality  varied during the test.
If significant variation occurs, the resulting data should
be interpreted in light of the estimated toxicity values.  A
decrease in dissolved oxygen indicates that  the flow rate
should be increased.
              b.  Collection of Test Solution Samples. . - -   -
    The objective of the recommended sampling procedure is
to obtain a representative sample  of the test solution for
use in measuring the concentration of  the test chemical.
Although there is mixing in  the test chamber, material can
concentrate near the sides and bottom  of the chamber due  to
physical or chemical properties of  the substance, or to
interactions with organic  materials  associated with the test
animalsv. .For-_this..reason.rvvater. s.amplesvs-h-ou-rd /bB:;t.a-ken-	.-«•
near the center of  the test  chamber.   The hand liny  and
                                15

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                                                         ES-2
                                                 August, 1982
s to cage of the samples requires  care  to  prevent  the loss  of
the test chemical from the sample  before analysis.
    Standardized methods should  be used  in  collecting the
samples and performing the analyses  to develop  chemical and
physical data.  Appropriate sources  foe  such  methodology
include, but are not  limited  to  Hedgpeth 1966,  Strickland
and Parsons 1972, AOAC 1975,  APHA  1975,  USEPA .1974, and ASTM
1979.
              c.  Test Chemical  Measurement
    The actual substance concentration used in  the
definitive test should be determined  v/lch the best  available
analytical precision.  Analysis  of stock solutions  and test
solutions just prior  to use will minimise problems  with
storage (e.g., formation of degradation  products,
adsorption, transformation, etc.).   Nominal concentrations-
are not adequate for  the purposes  of  the definitive tests.
If definitive testing is not  required because the substance
elicits an insufficient response at  the  1000  mg/1  level in
the range-EiiuUivj test, the concentration of  substance in
the test solution should be determined to confirm  the actual
exposure lavel.  The pH of the test  solution  should be..
measured prior to testing to  determine if it  lies  outside of
the species1 optimal range.   This  test guideline does not
include pH adjustment for the following  reasons: the use of
acid or base may chemically alter  the test  chemical making
it more or less toxic, the amount  of  acid or  base  needed  to
adjust the pH may vary from one  test  solution concentration
to the next, and the  effect the  test  chemical has  on pH may
indirectly af f.ect-the ..physiology .of.-.the'-tes.t-oy.a'ters..•••r^:.:. .. -
                                16

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                                                         ES-3
                                                 August,-  1982
    To assess and quantify any possible  changes  in test
chemical concentration, whenever  a. malfunction of  the
toxicant delivery system is detected,  all  potentially
affected test: chambers should be  sampled  at that time.
    If the measured concentrations  of  dissolved  test
chemical are 50 percent more or less  than  the  nominal
concentration, steps should be taken  to  determine  the cause
for this deviation.  A sample of  the  stock solution as  well
as influent samples to various test chambers should be
analyzed to determine if the reduction in  test chemical
occurs prior to delivery of the test  solution' to the
aquaria.  If results of these analyses  indicate  that the
proper amounts of test sbstance are entering the test
chambers, then the total test chemical  concentration should
be measured in at least the chambers.-containing-..the highest
test chemical concentration.  These data will  give
indications if the difference between  nominal  and  measured
test concentrations is due to volatilization or  degradation
of the test chemical, or to insolubility o.C  the  test
chemical in the dilution water.
    If the tox.i.cant delivery, sys-tem has  been prope'r-ly.~
calibrated and the oysters randomly introduced into each
test chamber, the measured differences between replicates  at
each concentration should ba less  than 20  percent.   If  the
differences exceed this, the test should be repeated.
    The concentrations of test chemical  measured after
initiation should be within 30 percent of  the  concentrations
measured prior to introduction of  the  oysters.   If the
difference exceeds this-, -the-test-should-be repeated.-us-ing . a
higher flow rate.
                                17

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                                                         GS-3
                                                 August, 1982
    Use of reliable and  validated  analytical techniques and
methods is essential  to  the  usefulness of the test data in
assessing the environmental  hazard of  the substance.
Significant variation in the measured  concentrations lessens
the value of the toxicity data  generated.
         4.  Test Data
              a.  Analysis
    A coherent  theory of  the dose-response relationship, on
which acute toxicity  tests are  based,  was introduced by
Bliss (1935), and is  widely  accepted  today.   This theory is
based on four assumptions:
    (.1)  Response is  a positive function of  dosage, i.e. it
    is expected that  increasing treatment rates sho.uld
    produce increasing responses.
    (2).  Randomly, s-elected animals-are .normal ly • dis-t-r ibuted
    with respect to their response to  a toxicant.
    (3)  Due to homeostasis, response  magnitudes  are pro-
    port ioiial i:o the  logarithm  of  the  dosage, i.e. it  takes
    geometrically increasing dosages  (stresses) to produce
    arithmetically increasing responses (strains) in test
    animal populations.
    (4)  In the case  of  a direct dosage of animals, theic
    resistance  to effects  is proportional to body mass.
    Stated another way,  the  treatment  needed to produce a
    given response is proportional to  the size of the
    animals treated.
              b.  Acute  Toxicity Test
    Oyster shell deposition  data have  been analyzed by
Cunningham.-..(.-19Jj5.) a ad .S.chimmel.-.et. al..  -,(-197-6., 1.9,7-8.),. -•„    .  .
    Cunningham  (1976) evaluated all shell growth data  to the
                                18

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                                                         SS-3
                                                 August, 1982
P _<_0.05 level of confidence using  an  analysis  of  variance
coupled with a Duncan Multiple-Range  test.   A detailed
summary of the procedure  for ranking  the  means  and computing
the Duncan Multiple Range  (DMR)  values  is given in Steel and
Torrie (1960) .
    Schimmel et al. (1976) developed  an EC50,  which is that
concentration of toxicant  which  produces  a  50  percent median
shell deposition reduction in  test oysters  as  compared to
control oysters.   During  other studies,  Schimmel  et al.
(1978), analyzed oyster shell  deposition  data by  linear
regression with probit transformation to  determine the RC50
and 95 percent confidence  intervals.
    Two types of statistical techniques should  be employed
for.- analyzing oyster shell deposition data:   1) analysis of
variance and 2) linear or  non-linear-  *reyress ion.-- -The test -
design that  is assumed is  control,  carrier  control (if
solvent carrier is utilized),  and  five  test  concentrations
giving a total of  seven treatments.   For  each  treatment, 20
similar-sized oysters are  tested for  96  hours.  At the end
of the test, each  oyster's shell deposition  is  measured  and
recorded, giving 20 separate growth rsoonses  for  each .  ....
treatment.
    At this point, it is  appropriate  to  conduct a one-way
ANOVA on these data to determine if there is  a  significant
effect on shell deposition due to  the treatments  (test
concentrations).   A significant  F  value  (P  less than or
equal to 0.05) would indicate  such an effect and  should  be
following with an  appropriate  post hoc  test  (e.g., the
s.tude n tized;. -Neoman- Keuls Vi' or., Dun can ls_mul t:irpl:e;.~raageu tey.ts>. -
or Dunnetts' or Williams'  pairwise comparison  tests).  These
tests are designed to indicate
                                19

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                                                         SS-3
                                                 August, 1982
which test concentrations  caused  significant effects.  For
some situations  this  information  may  be all that is
necessary; i.e., proof  that  a statistically significant
effect has occured due  to  the  test substance.   On the other
hand, if the ANOVA shows no  significant effect due to
treatments, then the  criteria  requiring effects on both
sides of 50 percent will not have been  met.  If the control
and control with carrier are different, then there are
severe test problems  that  should  be rectified.  Either the
solvent is toxic at the concentrations  tested, or there is
large variability among oysters,  probably undiagnosed
disease or improper test apparatus.
    The second method of analysis to  be utilized is
regress ion—eithoc linear  with  possible data transformations
or non-linear leas-t s-quares.. -Growth  data collected -from an
oyster: shell deposition test is dose-response  in which the
responses are graded  (or continuous)  as opposed to quantal
(discrete or binomial).  Due to this  fact,  the distribution
of measurements  at each concentration level is generally
assumed to be normally  distributed and  the response curve is
sigiaoid (slant S shaped);  in the  center of  the response
curve, the curve is typically relatively straight, while at
each end of the  curve,  the curve  becomes asymptotic to the
100 percent (control  growth  or  no-effect) and  0 percent (no
growth or full-effect)  levels.  Use of  the guideline's
proposed test design  causes  the following to occur:  two
controls and one no-effect concentration groups 60 oyster
growth values at the  no-effe'ct  end of the rsponse curve.
The highes t. .or-J: ull-ef f e.ct concentra.tio.n;',;'gr.aiip:s-. '..-2;0.,:,oys tec.
growth values at the  ota-ec- end  of the response curve.  That
                                20

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                                                          ES-3
                                                 August,  1932
leaves 20 growth  values  :   each of three treatments  to
describe  the  linear cent;  .1 portion of .the curve.   Linear
regression, if used,  shoi  ' depend only on data from the
three central  treatments,  since 0 and 100 percent  responses
may be far out on the GUI  -ed ends of the response  curve.
Improper  use  of  linear r<  ression on data from all
treatments will  likely o^  restimate the EC$Q and widen  the
associated confidence ini  :rval, especially if the  highest
test concentration was c'c  'Sen to be very high as compared
with the  other test cone*  .trations.  On the other:  hand,  if
all 5'concentrations  pro''  de partial response, then  simple
linear regression on grov  h data is an appropriate model  if
the fit is reasonable.
    The alternate approac.  es to straight linear regression
are:. 1)  regular • pro-bit  e  alys-is- regress ton-(-us ing- nvaximum -
likelihood or  minimum     , 2) various transformations  prior
to least squares  linear  i  gression, and 3) non-linear
regress ion.
    Probit analysis a^:;ic  s relatively small weights to
response  values near 0 ar   100 percent.  This is one of  the
primary reasons why this._ ..nalys is .is_ acceptable - fo-r-use  on •
dose-response  data that  c  ntain no-effect and full-effect
concentrations.   Although  probits do not exist for 0 a;vl  100
percent effects,  they re;  aced with close estimates  and used
in the regression calculc  .ions.  Another reason ilor  using
the probit transformation  is that it linearizes the
integrated normal signmo:   curve.  If all five test
concentrations provide p.?  tial responses, then one  can
1 ikely .expe.ct -_pr.o.bi.t'.:anal-.TS is-to^gxveL^r.e"li'ab.l:e-.»cfisaai;t.si.iWh.eri--.
estimating the ECcg and  i  3 confidence interval provided  the
                                 21

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                                                         ES-3
                                                 August, 1982
fit is reasonable.   Generally,  orobits  are regressed on log-
dose as opposed  to  dose.
    The major drawback  to  using probit analysis in this
situation is that the method  does  not make full use of the
continuous aspect of  the  response  variable.  Probit analysis
only requires that  the  response variable be quantal.
Consequently, the approach literally wastes information by
not using it.
    The probit  transformation can  be applied to the response
variable and then regressed,  using least squares regress ion,
on dose or log-dose.  This approach has the same advantage
as probit analysis  in that It tends to  linearize sigmoid-
type curves.  Therefore,  it is  appropriate to utilize data
with response rates  a-t -or  near  0 and 100 percent.   In
addition, this..approach .makes, use...of. .the .f.ac-t.. tha.t. .-the .
response variable is a  continuous  measure.  This approach,
when the fit is  reasonable, should give the most reliable
EC5Q estimate and possibly .H  narrower confidence interval
than the other  approaches.
    Several other transformational approaches that might be
tried (when the  probit  transformation regressed on dose or
log-dose doesn't fit the data)  are regress response on log-
dose, response on the square  root  of dose or response on the
inverse of dose.
    If none of  the  above-mentioned linear regression
transformations  produce an acceptable linear function, then
a non-linear sigmoid  function such as:   GROWTH = a/(l +
bcDOSE) or GROWTH =  I/(a + b"0*0032) could be fit  to the
data.  Th.e .problem•.wi.tirr.us.ing, aoniin.ear, JLan.ctio.ns'-~..i.s-• .that. ..in
theso cases three parameters,  a, b, and o (rather  than two
                                22

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                                                         ES-3
                                                 August, 1982
as in simple linear models),  should  be  estimated.   This
generally requires more  data  for comparable fits.   However,
these functions will  fit  the  data.
    For each of the above  methods--s imple linear,  probit
analysis, linear regression via  the .various transformations,
and non-linear regression—generalized  lack-of-fit tests can
be conducted to determine  whether t'<\~-i  chosen regression
technique adequately  describes  the experimental data.  Since
there are twenty shell growth values  foe each treatment
(test concentration),  the  appropriate  statistical  procedure
(except in the case of probit analysis ). is- to conduct an "F
Test for Linearity."   The  comparable  test for probit
analysis is the Chi-square goodness-of-fit test.   If the
computed F value for  linearity  is large, then the  linear
regre^3sion does not adequately -describe- the *-.-ita- and -th-e •
EC50 value and confidence  interval  estimates are suspect.
If the computed F value  is acceptable  (i.e., P less  than or
equal to .05 or P less than or  equal  to .10), them there is
no reason to doubt that  the data have  been sufficiently
described with the regression function and it would  be
appropriate -to compute the- EC50- and., conf idenoe intervals
required by the test  guideline.
              c.  Bioconcentration Test
    The bioconcentration  data (tissue  test substance
concentration) should  be  determined and recorded  ?
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                                                         ES-3
                                                 August,  1982
bioconcentration test, duplicate .samples  are  a  necessity for
establishing whether steady-state  has  been  reached.   It is
not uncommon for bioconcentration  data  to vary  half  an  order
of magnitude from sample  to sample.  Therefore,  there should
be duplicate sample measurements for each sample  period.
Duplicate sample values are required for  computing whether
steady-state has bean reached and  for  accurate  computation
of uptake and depuration  rates, regardless  of  the
statistical methods used.  A minimum of four  or  more sample
values for each sample period is recommended.   The oysters
sampled at each period from each test  chamber should be
individually analyzed.  The control oysters en  be pooled
before analysis unless the chemical of  interest  or its
metabolites are found or  are expected  in  the  control oyster
                                   *
s amples ,--s inee the controls serve  only  to identify--
accidental and unknown contamination of test  oysters Ero.n
uncontrolled sources.
    The variance of each  sample period  is likely  to  increase
as the tissue concentrations increase,  thus for statistical
purposes multiple oyster  samples at each  sample  period  is
necessary for determining when stead-state  is reached for
calculating a suitable 95 percent  confidence  interval.
    An estimate of the time to steady state,  the  steady-
stato BC?, and the time to 95 percent  elimination should be
made for each compound tested.  If steady-state has  not been
observed during the maximum 23 day exposure period or if 95
percent elimination has not been achieved during  14  days
depuration, data generated during  these tests should be used
to es timate. .thes.e. values .- _ JThe. ,81OFAC -progr am. :d,e,vel oped by. -..~-
Blau and Agin (1978) uses nonlinear regression  techniques to
                                24

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                                                         ES-3
                                                 August, 1932
estimate the uptake  and  depuration rate constant, the
steady-state BCF,  the  time  to  reach 90 percent rf. steady-
state, the time  to reach  50  percent elimination and the
variability associated with  each  estimate.
    To date, there is  no  one specific method recommended for
identifying the  time  to  95 percent elimination of
accumulated residues.  However,  it is still of value to have
it reported.  The problems associated with  calculating this
95 percent point are:

    1)   identifying  the  shape  of  the depuration curve as to
         whether it  is linear or  curvilinear;
    2)   if it  is curvilinear,  what curve best fits the
         data; and
    3)   are the data, suf.f iciently good to  -allow -
         extrapolation to estimate the 95 percent point?

    Bioconcentration  data is best displayed as log or
natural log (In) of  tho  measured  residue concentration on
the vertical axis and  time (linear)  on the  horizontal
axis.  The uptake curve  will be  exponential and increasing
until leveling off at  steady-state.   This uptake curve is
well represented by  the standard  kinetic uptake §unction

         Residue = Cone.  * ^]_/&2  * (l-e^*1-)-

    This function has  been shown  to accurately represent
most uptake data and  has  been used to determine uptake rates
for oys ters..:. -.Howeve.r.,7;there is. ,a.o, y:ener,al,--£anc.tionr.th.at,.
consistently and adequately  represents the  depuration curve;
                                25

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                                                         ES-3
                                                 August,  1982
an appropriate choice should be  made  based  on  each data
set.  The common description of  the observed problem is that
chemicals partition within  the oyster into  different tissues
(compartments) that depurate the chemical differentially,
thus causing the depuration curve  to  be  more complex and  to

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                                                         ES-3
                                                 August,  1932
    method for computing the 95 percent depuration
    endpoint:  95% X In (500 ppm) =  .95 X  5.21  =  5.90;  and
    6.21 - 5.90 = .31.  Then the antilog of  .31 = e-31  =
    1.36 ppm is the endpoint.  The time required  for
    depuration to 1.36 ppm would be  reported.   The  last
    value, 1.36 ppm, is the actual 95 percent reduction
    (depuration) endpoint.  This is  because  we  are  dealing
    with a In-dose vs. time relationship and all
    computations and comparisons should be made on  the  In
    transformed data with final back transformation to  -
    normal units for reporting.  If  25 pprn were reported,
    the 25 ppm endpoint would  represent only 48 percent
    depuration (.43 X 6.21 = 2.93; 6.21 -  2.93  =  3.23;  and
    e3-23 = 25 ppm).

    In view of this example it is clear that the  exact
method of computing the 95 percent endpoint should  be
reported along withg the time  required for depuration  to
this point.
    In summary, the following  procedures should be  utilized
for analyzing oyster bioconcentration data:.

    1)   Accurately tabulate and quality assure the residue
         data, exposure concentrations, and sampling
         procedures and periods.
    2)   Compute the desired multi-comoar taient  kinetic
         and/or non-linear parameter statistical  equation
        .using log ra^5 vlue and linear time data.
    3)   Plot the resulting curve (s): and -da ta no i-nts .: -• "• -  -
                                27

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                                                         ES-3
                                                 August, 1982
    4)   Conduct a  lack-of-fit  test  to determine whether the
         resulting  equation(s)  satisfactorily describe the
         data.
    5)   If satisfied  that  extrapolation is reasonable,
         compute the steady-state  concentration,  BCF, and
         95 percent depuration  endpoint using log'
         transformed data.   Back  transform the endpoints to
         original concentration units  for reporting.
    The resulting constants  (K]_,  K2/ etc.) are required for
constructing  the computed curve ana  for estimating the time
to 95 percent 'lepuration.
              d.  Temperature Measurements
    In order  to substantiate that  temperature was maintained
within specified limits, it  will be  necessary to measure and
record temperature  throughout the- test.-  Requisite
instrumentation is  readily  available,  easy to maintain, and
should not increase complexity  or  costs of the test.
Temperatures should be  recorded hourly to prevent any severe
fluctuations  in temperature  that might affect growth
processes and/or chemical uptake.    '
    B .  Test Conditions
         1.  Test Species
              a.  Select ion
    The Eastern oyster,  Crassostrea  Virginia (Gmelin),
serves as a valuable indicator  of  biologically damaging
pollutants in estuaries  due  to  a  number of  important
characteristics.  First, the  oyster  is  a long-lived
sedentary filter feeder  that  is unable  to move away from
exposure, to e.n.vir.oninen±.al .c.ont.ami,n-an.ts-:no,r ;close'. i±B-~s-hel.l .
for excessively long periods  of tisae  to avoid  exposure.  It
                                28

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                                                         ES-3
                                                 August, 1982
accumulates organic  (both  biological  and  chemical)  and
inorganic substances  from  aquatic  ecosystems  in a manner
which accurately reflects  environmental  changes and quality
(GaltsofE 1964).
    Second, the oyster  is  economically  important as a
commercial and recreational  fishery resource  and as a human
food source.  For  example, 1977  commercial  landings were
valued at $52 million (Council on  Environmental Quality
1979).
    Third, the oyster occurs  naturally  over a wide
geographic range and  is locally  abundant  from Maine to the
Gulf of Mexico.  This wide geographic range allows
comparative studies  of  control and exposure organisms under
differing environmental conditions.
  .  Fourth, the. oyster  is  readily  cultured-throughout its
life cycle under controlled  conditions  (Maurei: aad  Price
1967, Epifanio and Mootz 1976, Loosanoff  and  Davis, 1963).
In a properly equipped  and maintained facility, the oyster
is a hardy species that can  be maintained for long  periods
of time with minimal  effort.
    Fifth, numerous  morphological, physiological and
pathological studies  have  been completed  on the oyster
(Wilbur and Yonge  1964, Galtsof f  1954 and Sindermann
1970).  It has been  claimed  that the  oyster is the  best
known, most studied  marine organism  (Galtsoff 1964).
    Sixth, Crassos trea  virginica has  been used extensively
as a bioassay organism  because of  its known sensitivity to a
wide variety of toxicants  (LaRoche et al. 1973).  It has
been shown tpvbe, a-a;. a5.fe.ct i.ve. bix>concen±T^atox/af^a:r:oma;ti.c. ::..-.
hydrocarbons (Lee  et al. 1978, Anderson  1978), the  inseo-
                                29

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                                                         ES-3
                                                 August, 1982
tic ides kepone  (Bahner et  al.  1977,  Hansen et al., 1976),
DDT (Lowe et al. 1971, Butler  1967)  and  chlordane (Parrish
et al. 1976), and various  heavy  metals  (Frasier 1975, 1976,
1979 a,b) .  In  addition, most  specific  responses of oysters
to their environment have  been studied  and .'juantitled
including shell deposition rate,  breeding  temperatures,
glycogen content, salinity requirements,  numbers of
reproductive cells, diseases and  predators, and soft/hard
tissue catios.
    Although no formal comparison of  bioconcentration
factors among bivalves has  been published, Butler (1967)
presented data  which show  that,  in general, oysters
bioconcentrate  insecticides  to a  greater:  rl^aree than most
other common bivalves.   Average  five-day  bioconcentration
factors cof. ^e-./e-n pes tic id-es -ran-ged  from 500--to--700 Cor the
hard clam, marsh clam, and  asiatic clam  to 1200 for the
oyster and 3000 for the  soft shell clam.   Butler; concluded
that, on the basis of  its  cjreator bioconcentration factors,
the large body  of knowledge  concerning  its biology, its
sessile nature  and its extensive  range,  the oyster makes an
excellent biological monitor.
    The voluminous literature  on  oyster  biology is scattered
through numerous scientific publications.   However, the
following references serve  as  suitable  entries into the
field: Galtsoff (1964),  La.Roche et al.  (1973), Sparks
(1972), and Loosanoff  and  Davis  (1963).
              b.  Sources
    Oysters may be cultured  in the laboratory, purchased
from cuIture f.acl-li ties , ,or^colle.ct'ed .from .arrva-tur.a-1-.,-.  .:•: .
population in an unpolluted  area,  free  from epizootic

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                                                          «~1 r^  T
                                                          5 o-3
                                                 August,  1982
disease.  Procedures  for  collecting,  transporting and
holding oysters  are  described  in APHA,  1975.  All oysters
used for a particular test  should be  from the same source.
T?j31 oysters should  not have  been used  in a previous test,
either in a treatment or  in a  control.
              c.   Size
    The test guidelines recommend using oysters between 30
and 50 mm in height.   This  range represents a synthesis of
the wide range  of  sizes reported in the literature.  Various
workers have used  oysters as  small as 29 mm (Parrish et al.
1976) and as large as 120 mm  (Scott and Middaugh 1978).
Typically, however,  experimental oysters have ranged from 40
to 60 mm.
    Butler and  Lowe  (1978)  and APHA (1975) recommend using
small (25-to 50  mm) ..oys-ters because they ..are -act ive over a  •-
wider range of  temperatures and because they need less
space.  The ASTM (1980) recommends 40 to 60 mm.  Therefore,
in light of past experience and current recommendations, a
size range of 30  to  50 mm is  justified.
              d .   Co .id i t io n
    Oysters should be in  a  prespawn condition of gonadal
development prior  to  and  during the test.   A test is
unacceptable if  oysters spawn  during  the test.  For this
reason, and in cons ideation  of test  temperature, it is
recommended th.it oysters  from  natural areas be collected and
tested in the spring  of the year.   Gonadal condition of
oysters should  be  more certain in stocks obtained from
culture facilities.   Prespawn  condition should be confirmed
by me as ur lag- the • cond-i t.ion~. and—go:nadal~- ind.ex •ofz,-a'^randomly.:-,.:.
selected representative sample of  oysters  to be used for
                                31

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                                                          SS-3
                                                 August,  1982
testing  by  the  method  of  Scott and Middaugh (1978) and by
preparing and  examining histological sections of tissues
from the same  oysters  by  the method of Tripp (1974) to
determine gonadal  condition and to additionally ensure the
population  is  not  diseased.
    Although several  authors utilized oysters from natural
populations for bioassay  tests (Schimmel et al. 1978, Rawls
1977), other investigators  utilized laboratory-reared
oysters  (Confer et al.  1978, Cunningham and Tripp 1973).  As
demonstrated by Scott  and Middaugh (1978), determining the
physiological  condition of  oysters is very important in '
minimizing  test variabilty.   The depletion of gametes and
glycogen that  occurs  during  spawning would certainly make
bioconcentration data  impossible to analyze, and thus should
.be avoided . by. .ensuring, test  oyste.rs.-rema in in a-prespawn— • -•
coivji t Ion.
    Oysters collected  from  a natural population should be
collected at those times  known to be free Eeo-n influences of
recent spawning, such  as  the spring of the year.  Gamete
production  can  be  monitored  by gross observation of
individual  oysters and  semiquanti iiative measurements -of . .
uonad development  can  be  made by the method of Tripp
(1974).  Oysters which  are  laboratory-reared should be
examined prior  to  use  to  determine iC their growth and shell
thickness is consistent with known population averages
(Pruder  and Bolton 1978).
    Oysters are susceptible  to a number of pathogens that
aay result  in  epizootics  and mass mortalities in both
a a tu r al.. and - cu 11 ur.al; :pop_ula tio.ns-.r (~3:ind e.rma.nrur, 19;ZO')y..":-J. t^ -is".
necessary to determine  that  purchased oysters do not
                                32

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                                                          E3-3
                                                 August,  1982
originate  from epizootic disease areas.
     In  addition to taking the physiological condition  of  the
oysters  into  account,  examinations for parasitism and
disease  should be conducted to ensure to the  investigators
that  the response of  the oysters to the toxicant is not
influenced  by such factors.  Common oyster diseases and
procedures  for the it:  assessment are outlined  in Cheng
(19'70),  Couch et al.  (1974), Galtsoff (1964), Sindermann
(1970)  and  Sparks (1972).
    Oysters with.shells  heavily infested with mud worms
(Polydora  websteri) should not be used.  the mudworm forms
black areas on the inner faces of oyster shells and make  the
shells  brittle (MacKenzie and Shearer 1961).  Heavily
Infested oysters may  become weakened" and eventually die'
. (Roughley.. 192.2,,, 192.5.). •  -- O.ys te.rs- can . be .protected-- f-r-om  - •   	
mudworms to some extent if they are reared OL c the bottom
(Loosanoff  and Engle  1943).
         2.   Maintenance of Test Species
               a.  Feeding
    The  test  guidelines  permit supplemental feeding if
natural  plankton concentr-H'cions are too low to support
oyster  growth, or if  artificial seawater systems are used.
This  statement, of course, leaves open the question of what
plankton concentrations  are adequate.  This question cannot,
at present, be answered  with any degree of accuracy.
Spifanio,  et  al. (1975)  discussed the relationship between
filtration  rate and algal densities and concluded that the
variability caused by  temperature, animal size, particle
compos i± ion,,mid- dens.i ty-.-preyents" an^accurate;:^pn-oi'S fccafion-'
of oyster  nutrition.   Their discussion presents information
                                 33

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                                                         ES-3
                                                 August, 1982
suggesting that at  20°C  oysters  feed  most efficiently at
algal densities around 2 x  105  cells  pec ral.   However, there
are apparently no studies of  the minimum necessary algal
densities at various  temperatures  for various sizes of
animal.
    In the absence  of adequate  information on oyster
nutrition in the wild, the  best  policy would  appear to be
test any source water suspected  to be inadequate to supply
growth. Juvenile oysters  should  be held in the testing
system to determine rates of  growth over an extended period
of time. This will  give  an  estimate of .the system's ability
to meet the demands  of the  flow-through bioassay.
    It should be pointed  out  that  most estuarine and
nearshore waters will contain adequate quantities of
phytoplank.too—during the. .period  -when- water- te-emperatur-es—are --
suitable for testing.  It is  particularly true if the
testing facility is  located near an.area which supports
natural oyster populations.  Therefore, it is unlikely that
food availability will be a major  factor when ambient water
of suitable quality is used.
    If supplemental feeding is  necessary, the methods-and
materials employed  by experimental aquacultace facilities
should be utilized.  Basically,  these consist of culturing
two or three algal  species  -  Isochrysis galbana, Monochrys is
lu then', and Thai las ios ira pseudonna have been used
successfully by the University  of  Delaware (Epifanio et al.
1975 Epifanio and Mootz  1976) -  to be Ced to  the oysters
either as the sole  ration,  or as a supplement to the natural
algal flora. .  Although--~.the.: actual  .al-.gal curt,ure~.-pr.e'5'.ents: no .
particular difficulties,  the  additional manpower and capital
                                34

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                                                          ES-3
                                                 August,  1982
costs  of  supplemental feeding make it an undesirable
strategy.   Ukeles  (1971)  has reviewed the nutritional
requirements  of  shellfish.
          3.   Facilities
               a.   General
     The  requirements for facilities as set forth in the  test
guidelines  are intended to  ensure that the conditions in the
test chambers  ace  as uniform as possible and that  the actual
concentrations of  test chemical in the test chambers are
s imilar  to  the intended concentrations.
     The  test  guidelines require that flowing seawater be
utilized.   Static  test design cannot be utilized due to
problems  in maintaining the oysters ia a state of good
health.   The  flow-through system more closely simulates  the
.natural -exoos.ure process, .eliminating- problems- -associated- •
with accumulation  of organic material (and associated
bacteria  v/hich could lower  dissolved oxygen) and toxic
metabolic products.   Test chemicals are more thoroughly
mixed  in  a  flow-through system and problems of sorution are
reduced .
     Galtsoff  (1964)  found that oysters held in flowing.
seawater  at Woods  Hole, Massachusetts, deposited a median of
1.4  milligrams of  shell material per centimeter squared of
shell  surface  per  day during the growing season.  With
sufficient, suitable phytoplankton food in tha dilution
water,  Epifanio  et al. (1975) found that a small oyster
between  30  and 50  millimeters in height may deposit as :auch
as 1.0 millimeter  of peripheral new shell per day.   Most
1 aboratory _sy.s .teirra.r~wh~ich  h.ave~.bee-n- des-ign-edi ±xr_-hoHba-ntl. • • :•
study  the  toxic  response  of oysters have employed a minimua
                                 35

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                                                          ES-3
                                                 August,  1982
flow-through volume  oE  (: I ye liters of water per oyster per
hour (Butler et  al.  1960).
              b.   Construction Materials
    Due to the  toxicity of  many heavy metals at low
concentrations  (USSPA 1976) and the ability of metal pipe,
galvanized sheeting, laboratory equipment, etc,.-  to  leacn
metals into water, no metal other than stainless steel
(preferably $316)  should  be used.  In the same manner, un-
aged plasticized plastic  (PVC) should not be used due to the
high toxicity of a main component, di-2-ethyl-hexyl
phthalate (Mayer'and Sanders 1973) and the ability of DEHP
to leach into aquaria systems  from these materials
(Carmignani and  Bennett 1976).  To avoid any possible stress
due to exposure  to low levels  of metals, ph thai at'-;*, and
other potential--contaminants..,.  #34-6 stainless -ste-e-1-, -g 1-ass- - .--
and per?; I no coca rbon  plastics should be used v\?henever
possible and economically  (feasible.  If other materials
should be used,  conditioning to a continuous flow OL heated
dilution water should be  performed for a minimum of 40
hours .
              c .   Test Substance Delivery System  	
    To maximize  the  accuracy and precision of test results
developed through  the use  of this test guideline, the
quantity of test chemical  introduced by the test chemical
delivery system should  be  as constant as possible from one
addition of test chemical  to the next.  Fluctuations in the
quantity of test chemical  introduced into the test chamber
may result in abnormally  high  or low response value  (e.g.
EC 5 0 ' s.) - -of the . t es-tv-o r,gan.i srms~aTid---rnj^ic.w td'e-e. ^ pr.e ad of.-.
response values  in replicate tests.  The greater  the
                                36

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                                                         ES-3
                                                 August, 1982
variation in the quantity  of  test chemical introduced, the
greater  the potential  for  abnormalities and spread of the
res ponse values.
    Variations  in  the  quantity of dilution water entering
the test chambers  during a given time interval may also
create undesirable differences in test conditions between
test chambers.  The  concentrations  of dissolved oxygen and
test chemical  in a test  chamber, for example, may decrease
more-rapidly in chambers having lower flow rates. •
Differences between  test chambers in the concentration of
dissolved oxygen,  test chemical, metabolic products  and
degradation products,  individually or in combination, may
result in response values  for the test organisms which are
in.Accurate.
    The  followi.ng-.»c-r.ite.ria -presented--by Hods-o-n (19-7-9) s-hou-ld
be considered  when selecting  or designing a toxicant
delivery system: 1)  if the delivery of dilution water stops,
so should delivery of  the  toxicant 2) consistency in
delivery amounts throughout the test periop 3) independence
from electrical failure  4)  independence from temperature and
humidity fluctuations  5) capacity to deliver, small   	.
quantities 6)  ease of  construction, with few moving  parts
and 7) ease of operation.
    Any  one of several toxicant devices can be used  as long
as it has been shown to be accurate and reliable throughout
the testing period.  The greater the variation in the
quantity of test chemical  introduced, the greater the spread
of response values measured during  testing.  Syringe
injector -^s.tems'^(.Barr..o\vs_et^::al..-^i-98Jl,^..3pe,lvar -:&£. .a£t .-.
iaetering pump  systems  (Veith  et al. 1979), and modified
                                37

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                                                         ES-3
                                                 August, 1982
proportional dilutotrs  (Macek et  al.  1975,  Neeley et al.
1974) have been  reported  to be successfully used.
    The solubility of  the  test compound  should also be taken
into account in  selecting  an appropriate delivery system.
If the compound  can be solubilized  in  water,  a device
capable of delivering  amounts of  test  solution greater than
1 milliliter (Til.) v/ill probably  be  needed.   If a carrier i.=
required, a system capable of accurately delivering small
amounts, less than 100 microliters  (ul), will probably be
required to minimize the  carrier concen-tration in the tost
solution.
    Each system  should be  calibrated prior to starting the
test to verify thai: the correct  proportion of test chemical
to dilution water is delivered to the  appropriate tes t
chambers =•                              ...
              d.  Test Chambers  and  Loading
    Flexibility  is allowed in the design of test chambers as
long as adequate space is  provided  I!or test oysters to meet
loading requirements.  As  a guideline  use  the US EPA Bioassay
Procedures for the Ocean  Disposal Permit Program Manual
(USEPA 1978), which recommends glass or  f iberglassed—wood
containers measuring 64 x  33 x 10 cm deep  (25 x 14x4 inches)
to provide adequate space  for 20 oysters.   Such containers
permit adequate  circulation of the  water,  while avoiding
physical agitation of  the  oysters  by the water current.
These containers hold  about 18 L at  75%  capacity and at a
flow rate of 100 L hour~l, will  provide  5  L of water hour ~1
oyster ~1.  Small oysters  were reported  to i:ee:.l and grow
readily-under these co.nd.i.tio-ns:^~"-j-^:.>...-.
    Silicone adl^3 we  is  the preferred bonding agent for
                                38

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                                                          ES-3
                                                 August,  1982
constructing  test  chambers.   It is  inert, and the solvent  it
generally contains  (acetic  acid)  is easily washed away or
volatilized £rom the  system.   A minimum amount of the
adhesive should contact  the  test solution because it may
absorb test materials.   If  large amounts of the adhesive are
needed for strength,  it  should be applied to the outs ides  of
chambers and  apparatus to minimize  contact.
              e.   Flow-through System
    The test  guidelines  require that flowing seawater be
utilized.  Static  test design cannot be utilized due to
problems in maintaining  the  oysters in a state of. good
health.  The  flow-through system more closely simulates the
natural exposure process, eliminating problems associated
with accumulation  oE  organic  material (and associated
bacterLa.-.vvhlci.^  could  lowe..r. dissolved- oxygen) .-a-nel- toxic
metabolic products.   Test chemicals are more thoroughly
mixed  in a flow-through  system and  problems of sorption are
reduced.
   • GaltsofE  (1964) found that oysters held in flowing
seawater at Woods  Hole,  Massachusetts, deposited a median  of
1.4 milligrams  of  shell  material  per centimeter squared of
shell surface per  day during  the  growing season.  With
sufficient, suitable  nhytoplankton  food in the dilution
water, Epifanio et  al. (1975) found that a small oyster
between 30 and  50  millimeters in height may deposit as much
as 1.0 millimeter  of  peripheral new shell per day.   Mos t
laboratory systems  which have been  designed to hold and
study  the toxic response of  oysters have employed a minimum
f 1 ow-1hr.ou-ghv.xolu-me -of_~fdve.rd.its-cs. of'.;wa±ex- ~pe;r:r-oy,s"-t.e:r.•£& c, :
hour (Butler et al. 1960).
                                39

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                                                         ES-3
                                                 August, 1982
              f.   Cleaning
    Before, use,  test  systems  should be cleaned to remove
dust, dirt, and  other debris  and  any residues that may
remain from previous  use  of  the system.  Any of these
substances may  affect the -results  of a test by sorption of
test materials  or  by  exerting an  adverse af!f!fjot on test
organisms.  New  chambers  should be cleaned to remove any
diet oc chemical residues remaining from manufacture or
accumulated during storage.   Detergent is used to remove
hydrophobia or  lipid-like substances.  Acetone is used for
the same purpose and  to .remove any detergent-residues.  It-
is important  to  use pesticide-frae acetone to prevent the
contamination of the  chambers with pesticides which
influence  the outcome of  the  test.  Nitric acid is used to
clean metal res idues— f ro
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                                                         ES-3
                                                 August, l'J82
large volumes required  to  maintain  the  flow and loading plus
the n^ed  to add  food  to  maintain growth makes use of
artificial seawater problematic.  Refer to Spotte (1979) for
methods to prepare  and  mix large volumes of artificial
seawater.
    The flow-through  system should  supply at least one liter
of water per oyster per  hour.   The  ASTM Proposed Standard
Practice  (ASTM 1980b) recommends  one liter per hour,
although this preliminary  recommendation has been questioned
in the review process,  and is  likely to be changed.
    Behind the ASTM recommendation  is a factor which has
only recently received  the attention it merits, namely the
problem of waste disposal.   Dilution water containing a
potentially hazardous test chemical cannot be discharged
directly into natural •wate-rs.-  -Some f o-rm=-off •-preliminary •  --•
treatment is necessary.  As  the amount  of dilution water
increases, treatment  facilities and costs go up.  These
considerations dictate  the use  of the  lowest flow rate which
will support oyster growth.
    The recommendation  that the flow rate be at least one
liter per oyster per  hour  is clearly at.the.lower .end--of the
range of  reported values.   Parrish  et al. (1976) exposed
oysters to chlordane  in  7.5  liters  of water per oyster per
hour.  Scott et  al. (1979)  used five liters per hour.
Schimmel et al.  (1973) supplied approximately six liters per
oyster per hour  to  test  sodium  pentachloro-phenate, but 11
liters to test lindane and 3HC  (Schimmel et al. 1977).
Scott and Middaugh  (1978)  used  at least five liters per
oyster rper/.hoarjrinr.vthe'l.r- acclrma.t.to.n- .tanks,.~v>;^S.ta.ndac.d-^—:-'.'- -
Methods (APHA 1975) advises  holding spawning stock in at
                                41

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                                                         ES-3
                                                 August, 1982
least seven  liters per  animal  per hour, but those are larger
oysters (7.5  to  15 cm)  than are used in the test
guidelines.   For  test oysters, "a minimum of five liters per
hour of seawater  per oyster provides adequate growing
conditions"  (APHA 1975).   However,  recent experience at the
US EPA Gulf Breeze Laboratory (Schimmel, personal
communication) and at private  facilitios (Parrish, personal
communication) has demonstrated the effectiveness of using
one liter.   Since that  flow rate is economical and has been
proven effective, its use  in the test guidelines is
justified.   The one liter  figure is a minimum; if a facility
can support  a higher flow  rate, it should by all means use
the higher flow.  It is  important that the .Qow of water be
constant.  If the flow  is  interrupt ed, the oysters will
quic-k.l-y deplete • the--food" supply-in  the--s-tagn-ated water - an-eh-
will increase the levels of metabolic wastes.   These factors
will c-mse the oysters  to  close their shells,  which will
invalidate the tests.
    Many previous tests  utilizing the oyster as a test
organism were conducted  at salinity regimes native to the
testing facility.  Thus, tests conducted by . Schimmel . et al.
(1970) were  conducted at a salinity range between 18 and 23
parts per  thousand, whereas those completed by Cunningham
(1976) were  run at salinities  close to full-strength
seawater (34 parts per  thousand).
    Salinity  is difficult  to control in a natural flow-
through system, and controlling it introduces  an element of
artificiality to  the test.   Therefore, the dilution water
should:be drawn  f.r.om:;-an7ar,ea-.r.wh:e.r-.e, tiie.r;expfi.cted".rang-e..of-—-.=• -
salinity is  within the  oyster's optimum.
                                42

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                                                         ES-3
                                                 August, 1982
    Regardless of  the  system used,  salinity should be
monitored daily during the  test.   If significant changes
occur, due  to severe weather changes or system malfuncion,
for example, the  validity of the  test is challenged and the
test should be repeated.
    To avoid possible  inconsistencies and inaccuracies in
test results, healthy  oysters  are needed for use in toxicity
taats.  There is  also  a need to determine thdt the dilution
water, whatever its source,  is  able to maintain the oysters
to be used  in a healthy condition for the duration of the
holding anJ testing periods.
    An appropriate way to make  that determination is to
place oysters in  the dilution  water for an extended period
of time and observe their behavior, growth and
development.....Ideally,,  those observations -should -be .made--by -
an experienced oyster  biologist familiar with certain stress
reactions which are difficult  for an untrained observer to
identify.
    Particulate matter and  gas  bubbles, if present in the
dilution water, nay clog the toxicant delivery system used
in flow-through tests.   Gas .bubbles._als-O.may-caus.e excessive
loss of volatile  test  chemicals.   Either circumstance may
alter the concentration of  test chemical to which the test
oysters are exposed.   To avoid  this problem an apparatus
capable of  removing particulate matter or gas bubbles from
the dilution water may be required,  If the dilution water
is heated prior to use,  it  may  also be necessary to de-
saturate the water from >100%  of  oxygen saturation.  Penrose
a nd Squi.res. -(±9;?.6) :.des'cr ibj&ca^sTiidtab J:e;.;appa r a.tu's>T.foir^_this;..— :.
                                43

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                                                         ES-3
                                                 August, 1982
    An adequate supply  of  dissolved  oxygen should be
available to  the oysters.   To  facilitate this,  the dilution
water or holding water  should  be  at  90-100% of  oxygen
saturation prior to deli vary  to  the  holding tanks or test
system.
              h.  Carriers
    Carriers  may be used  to aid  in the dissolution of test
compounds into dilution water  only after significant efforts
to dissolve it in dilution water  or  dilution water stocks
have failed.  Schoor  (1975) believes  that the use of a
carrier may interfere with the uptake  of the test compound
by l:iie test organism; if  the carrier  molecules  affect the
adsorption of the test  compound  at the gill surface, a
change in the rate of transport  into  the test organism may
result.  The. author also  s-tastes - thai™- i-.h-.-3 use of a-carrier • -
may increase  the concentration of  compound in the test
solution above solubility  by creating  a stable  water
emulsion.
    Since there is little  information available on the
effects of carriers on  oysters,  follow precautionary usage
procedures thai: have  been es tablished. wi Lh. fish.   When a-
carrier is used, triethylene glycol  (TEG), dimethyl
formamide (DMF), or acetone may  be used.  The solvents
should be tried in the  order stated  due to their  relative
toxicity to fathead minnows as reported by Cardwell et al.
(manuscript 1930).  The minimum  amount should be  used and
the concentration of  T3G  should  not  exceed 80 mg/1, the >1ATC
(maximum acceptable toxicant concentration)  value.
Concentrat-io:ns-~of"-DMF -and-, -ace.ton-e^-shouidi..n.ot..ex'ce^d.^-5-.fl:^, ". .
mg/1, the MATC for OMF.   Although  there is no MATC value foe
                                44

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                                                          ES-3
                                                 <\ugur, t,  1982
acetone, its acute  toxicity is  similar to that of DMF.
    Ethanol should  not  be used  due to its tendency  to
st.iraa.late the  excessive growth  of bacteria in the test
chambers .
         4.  Environmental Conditions
               a.   Dissolved Oxygen (See Section on
                                     Dilution Water)
               b.   Temperature
    It  is desirable to  standardize the range of test
temperatures to the extent possible so as to avoid
variability between ..diJLferent laboratories . in widespread
geographic areas.   Also,  tests  on some specific substances
will vary significantly at temperatures a:* ;auch as  10°C
apart,  a range commonly "experienced between, Cor instance,
New0 York..and -South  -Carolina,.... . ,WaLdichuk.-( 19..7.4~).--pr-e&ented
data showing such  a phenomenon  in the case of cadmium.
Gunter  (1957)  set  forth the relationship between oyster
growth  and temperature, as :3eterrnined by regional
location.  Based on Gunter's data and work conducted by
Butler  (1953), it  is  clear that regional temperature
differences should  be taken into account in establishing.
experimental bioassay conditions.  Most bioassay tests
utilizing the  oyster were conducted at ambient temperatures
of the  dilution water at the testing facility.  Tests
conducted by Schiramel et al. (1978) were conducted  at
ambient  temperatures  between- 7  and 9°C; those completed  by •
Cunningham (1976)  were run at times at temperatures as high
as 25°C.
    Within i'ts. ..natar.al - range.,-.Crassos>tr.ea-..v±rg.inica -..is-f o.uad
at great temperature extremes,  ranging lirora 1 °-C to  46°C
                                 45

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                                                         ES-3
                                                 August, 1982
(Epifanio et al.  1976).   However,  the  optimal temperature of
oyster growth has  been  reported  as between 15°C and 25°C in
the Gulf Coa*t  (Collier  1954)  and  between 13°C and 22°C in
Long Island Sound  (Loos anof f:  and Nomeiko 1949).  Although
oysters in the southern  range, such as  in the Gulf of
Mexico, have a greater  growth  per  year,  he maximum growth
per day occurs during the summer in oysters  located in the
mid-Atlantic region.  Loos an of £  (1958)  found that oysters
from Long Island  have maximum  ciliary  (feeding) activity at
approximately 25°C.  Cunningham  (1976)  and others observed
declining shell growth  during  cold- ambient .water temperature
periods.  Laboratory studies  conducted  by Epifanio and Mootz
(1976) utilized a  controlled  range between 16° and 26°C
throughout the year.  The ASTM  (1980b)  proposed .-standards
for tests with oysters ^c-a-tl -for- a  tea t-tempera t-ure- be twee n-—
3°C and 28°C. Butler and  Lowe  (1978)  recommend that the
source water be between  15°C and 30°C.   The  test guidelines
specify 20°C (with a permissable short  term deviation within
15°-25°C) because  it maximizes filtration, growth, and is
sufficiently low  that oysters  can  be  maintained in a
p/respawn condition.  In  addition,  i.t  will tend to diminis-h .
the influence of  disease  on test results because the fungus,
Dermocystidium marina,  causes  high levels of mortality above
25°C (Hewatt and  Andrews  1957).
    According to  Stickney (1979),  C.  viginica requires a
temperature range  of 21  to  27°C. for  spawning and can be
conditioned to spawn within about  6 weeks in the winter if
exposed to a temperature  range of  23  to  24°C,; even though
spawning.-naturatly,, joccurs.:rn: t:h.e "srpring£.. ~.v.~,  .
    The 20°C test  temperature  was  selected as a compromise,
                                46

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                                                          E3-3
                                                 August,  1982
striking a balance  between  the temperature that would
maximize the physiological  activities of the oyster  (i.e.
ciliary movement, water  transport,  metabolism, etc.) which
would thereby enhance  the uptake and exposure to the test
solution on the one  hand, and  on the other hand assuring
that the oysters  would not  spawn on the other hand.  The
Meal physiological  maximum temperature Cor the oyster is
approximately 25°C,  but  oysters  spawn above 20°C.  Since the
oyster approaches naximum physiological activities at 20°C,
and since this  temperature  does  not induce oysters to spawn,
it was selected.  It is  realized that temperature variation -
.may occur in controlling the volumes of water reduced in
flow-through systems.   Since prolonged exposure of oysters
to temperatures- above-  20°C  may-induce spawrving-, i~t~ is
preferred. that_.vari.a.tio-ns-.ia.. tes t_ temperature-be.. h.elcU~to-a.--
minimum or held to  temperatures  -below 20°C.  The effect of
temperature on  the  oyster is discussed in Galtsoff (1964).
    A standardized  temperature .is ordinarily desirable in
bioassay testing.   This  is  because  the toxicity of many
su'os tcuic'33 varies with temperature  (Tucker and .Leitzke 1979,
Frazier 1979a,  Waldichuk 1974).
    The effects of  sudden temperature changes on organisms
may range from  death to  temporary impairment of
physiological functions, depending  on the acclimation
temperature, the  magnitude  of  the temperature change, the
temperature tolerance  of the species, and the circumstances
and duration of the  exposure.   To avoid any undue stress,
accurate temperature control devices should be used  to both
ma in ta in-.. co:ns_t an.t,. tempera-t-ur.es- >•- "a nek to _gr adu-aMy.id.ucreas.ev..err
decrease the temperature during  acclimation procedures.
                                47

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                                                          ES-3
                                                 August,  19-82
Such mechanisms have  been  described by Defoe (1977) and
Lemke and Dawson  (1979),
              c.   Light
    The duration  and  intensity of  light are not important:
environmental variables  in oyster  tests.   Oysters ha\/e no
visual organ and  ace  not sensitive to light (Galtsoff,
1964).  Therefore,  no lighting regime is  required.
              d.   Salinity (See Section on Dilution Water)
    C.  Reporting
    The sponsor should submit to the Agency all data
developed during  the  test  that are suggestive or predictive
of oyster toxicity  and bioconcentration.   In addition,
information on water  quality,  experimental design,
equi
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                                                         ES-3
                                                 August,  1982
bioconcentration tests according  to  the  test  guidelines.
Two estimates were provided; a protocol  estimate and  a
laboratory price survey estimate,
    The protocol best estimate for  the oyster acute toxicity
test was $1480.  This estimate was prepared by separating
thy guideline into individual tasks  and  estimating  the hours
to accomplish each task.  Hourly  rates were then applied  to
yield a total direct labor charge.   An overhead rate  of  115
percent, other direct costs  (for  laboratory supplies  and
reagents) of $75.00, a general and administrative rate of 10
percent,- and a fee of 20 percent  were then  added to the
direct ' labor charge to yield the  final estimate.
    Snviro Control estimated that differences  in salaries,
equipment, overhead costs and other  factors between
laboratories would result in as much as  -. 0 percent  variation
from this estimate.  Consequently they estimated that test
costs could range from $740  to $2221.
    The laboratory price survey best estimate was $900.00
for the oyster acute toxicity test.  Two laboratories
supplied estimates of their  costs to perform  the tests
according to this guideline.  These costs ranged from
$700.00 to $1100.00.  The reported estimate is the  mean
value calculated from the individual costs.
    The protocol best estimate for  the oyster
bioconcentration test was $7680.  This estimate was prepared
by the same method of the oyster  acute toxicity test, with
the exception that the other direct costs totaled $250.   The
test c<:« t was estimated to range  from $3840 to $11,520.
    The laboratory..price survey bas-t. estimate, was-$8092- for.-
the oyster bioconcentration  test.   Four  laboratories
                                49

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                                                         ES-3
                                                 August, 1982
supplied estimates ranging  from  $4,000  to :?10..noo.   The
reported estimate  is  the  mean  value  calculated  from the
individual costs.
                                50

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                                                        ES-3
                                                August, 1982
IV.   REFERENCES
    AOAC.   1975.   Association of Official Analytical
    Chemists.   Official methods for analysis of the
    Association of Official Analytical Chemists.  12th
    Edition.  Washington, DC: Association of Official
    Analytical Chemists.

    APHA.   1975.   American Public Health Association, Water
    Pollution Control Federation.  Standard methods for the
    examination of water and wastewater.  14th Edition. New
    York:  American Public Health Association.

    Anderson RD and Anderson JW.  1976.  Oil bioassays with
    the American  oyster, Crassostrea virginica (Gmelin)
    Proc.   Nat. Shelfish Assoc. 65:38-42.

    Anderson RS.   1978.  Benzo(a) pyrene metabolism in the
    American oyster Crassostrea virginica, .Ecol. Res. Series
    U.S.  Environmental Protection Agency, Gulf Breeze, FL:
    25 pp.   tEPA-600/3-78-009.

    ASTM.   1979.   American Socle.ty for Testing and
    Material.   Annual book of ASTM standards.   Part 3.,
    Water.  Philadelphia, PA: American Society  for Testing
    and Material.

    ASTM.   1980a.   American Society for Testing and
    Materials.  Standard practice for conducting basic acute
    toxicity test with fishes,  macroinvertebrates, and
    amphibians.  Philadelphia,  PA: American Society for
    Testing and Materials. . E729-80.

    ASTM.   1980b.   American Society for Testing and
    Materials.  Proposed Standard practice for conducting
    bio-concentration tests with fishes and saltwater
    bivalve molluscs.  Draft No. 10  August 22, 1980.

    Banner  LH, Wilson AJ, Sheppard JM, Patrick JM, Goodman
    LR, Walsh GE.   1977.  Kepone accumulation, transfer and
    loss  through  estuarine food chains.  Ches. Sci.
    18(,3) :299-308.
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                                                     ES-3
                                             August, 1982
Barrows ME,  Petrocelli  SR,  Macek KJ.   1980.
Bioconcentration  and  elimination of  selected water
pollutants by bluegill  sunfish (Lepomis  macrochirus) .
In: Ann Arbor Science Pub.,  Inc.  Dynamics,  exposure and
hazard assessment  of  toxic  chemicals  Hague  R.   Ann
Arbor, MI.

Bishop WE and Maki  AW.   1980.   A critical comparison of
two bioconcentration  test methods.   In:  Aquatic
toxicology.  Eaton JG,  Parish  PR,  and Hend ricks AC, eds .
Philadelphia, PA:  American  Society  for Testing and
Materials.

Blau GE and  Agin GL.  1978.   A  users  manual  for BIOFAC: A
computer program  for  characterizing  the  ratio  of uptake
and clearance of  chemical  in aquatic  organisms.
Midland, MI: Dow  Chemical Co.

Bliss CI.  1935.   The calculation of  the dosage-
mortality curve.   Ann.  Appl. Biol.  22:134-307.

Branson DR,  Blau GE,  Alexander HC,  Neely WB.  1975.
Bio-concentrat.ion. of--2-, 2 ,--4,4  - tetrachlorobiphenyl in-  •
rainbow trout as measured by an accelerated  test.  Tran.
Am. Fish. Soc.  4:785-792.

Brodtmann NV.   1970.  Assimilation  of 1,1,1  trichloro-
2,2-bis (p-chlorophenyl) ethane (DDT) by Crassostrea
virginica.   Bull.  Environ.  Contain.  ToxicQl.  5:455-462.

Butler PA.   1953.   Oys'ter growth  as  affected by
latitudi-nal temperature gradients.   Com ml.  Fish. Rev.
352-7-12.

	.  1965.  Reaction of estuarine molluscs to
some environmental  factors.  Publ.  Health.  Serv. Publ.
999-WP-25.

	.  1967.  Pesticide  residues in estuarine
molluscs.  In:  Nat Symp. on  Estuarine Pollution,
Stanford Univ., 1967.   Gulf  Breeze,  FL:  U.S. Bureau
Commercial Fisheries  Biol.  Lab, 107-121.

Butler PA _and -Lowe . JL. .-197.8.  . --.Flowing. ..s,eawa.ter.- uto.xi.c i~ty-
test using oysters  (Crassostrea virginica);  In: Bioassay
procedures for  the Ocean Disposal  Permit Program Gulf
Breeze, FL:  US. Environmental  Protection Agency.  EPA-
600/9-73-010.
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                                                     ES-3
                                             August,  1982
Butler PA, Wilson AJ, Rick AJ.   1960.   Effects  of  pesti-
cides on oysters.  Proc. Nat.  Shellfish.  Assoc.  51:23-
32.

Cairns J and Dickson JKL, eds.  1973.   Biological  methods
for the assessment of water quality.   Philadelphia,  PA:
American Society for Testing  and Materials.   ASTM-STP
528.
Cardwell RP, Foreman DG, Payne  TR, Wilbur  OJ.   1980.
Acute and chronic toxicity of  four organic chemicals  to
fish. Manuscript.

Carmignani GM and Bennett JP.   1976.   Leaching  of
plastics used in closed aquaculture systems.
Aquaculture 7:89-91.

Cember H, Curtis EH, Blaylock  BG. 1978.   Mercury  biocon-
centration in fish: temperature -and concentration
effects.  Environ. Pollution  17:311-319.

Cheng TC.  1970.  Marine molluscs .as hosts for_
symbioses: with a review of known parasites  of
commercially. ..important s-pe.cies ... Vol..-5.—In: Russell. --
FS, ed. Advances of marine biology.  New  York:  Academic
Press.

Chiou CT, Freed UH, Schmedding. DW, Kohnert RL.   1977.
Partition coefficient and bioaccumulatioh  of selected
organic chemicals.  Env. Sci.  Tech. 11(5): 475-478.

Chipman WA, Rice TR, Price TJ.   1958.   Uptake and
accumu-lation of radioactive  zinc by marine  plankton,.
fish and shellfish.  U.S. Fish  Wildl.  Serv.  Fish.  Bull.
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                            53

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                                                     ES-3
                                             August, 1982
Couch J, Gardner G, Harshberger  JC,  Tripp  MR,  Yevich
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                                                     ES-3
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                                                     ES-3
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                            62

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EPfi

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                                         DRAFT
                              BG-7
                              August, 1982
   PENAEID  SHRIMP ACUTE TOXICITY TEST
       OFFICE OF TOXIC  SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC  SUBSTANCES
  U.S.  ENVIRONMENTAL PROTECTION AGENCY
         WASHINGTON, D.C. 20460

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Office of Toxic Substances                                  EG-7
Guidelines for Testing Chemicals          •         August,  1982


                PENAEID SHRIMP 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 tests using penaeid shrimp as test organisms to

develop data on the acute toxicity of chemicals.  The  United

States Environmental Protection Agency (EPA) will use  data from

these tests in assessing the hazard of a chemical to the aquatic

environment.

    (b)  Definitions.  The definitions in section 3 of the Toxic

Substances Control Act (TSCA), and in Part 792—Good Laboratory

Practice Standards apply to this test guideline.  The  following

definitions also apply to this guideline:

    (1)  "Death" means the lack of reaction of a test  organism to

gentle prodding.

    (2)  "Flow-through" means a continuous passage  of  test

solution or dilution water through a test chamber,  holding or

acclimation tank with no recycling.

    (3)  "LC50" means that experimentally derived concentration

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                                                           BG-7
                                                   August, 1982
of test substance that is calculated to have killed 50 percent of
a test population during continuous exposure over a specified
period of time.
    (4)  "Loading" means the ratio of test organism biomass
(grams, wet weight) to the volume (liters) of test solution in a
test chamber.
    (c)  Test procedures—(1)  Summary of the test.  Prior to
testing, the bottoms of the test chambers are covered with 2-3 cm
of sand and then filled with appropriate volumes of dilution
water.  The flow is adjusted to the rate desired to achieve
loading requirements.  Penaeid shrimp are introduced into the
test chambers according to the experimental design.  The shrimp
are maintained in the test chambers for a period of 3-7 days
prior to the beginning of the test.  The test begins when the
test substance is introduced into the test chambers.  The rate of
flow is adjusted to maintain the desired test substance
concentration in each chamber.  The shrimp are observed during
the test; dead shrimp are counted, removed, and the findings
recorded.  Dissolved oxygen concentration, pH, temperature,
salinity, test substance concentration and other water quality
characteristics are measured at specified intervals in selected
test chambers.  Data collected during the-test are used to
develop concentration-response curves and LC50 values for the

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                                                           BG-7
                                                   August, 1982
test substance.

    (2)   [Reserved]

    (3)  Range-finding test,  (i)  A range-finding test should be

conducted to determine the test substance concentrations to be

used for the definitive test.

    (ii)  The shrimp should be exposed to a series of widely

spaced concentrations of test substance (e.g. 1, 10, 100 mg/1,

etc.) .

    (iii)  A minimum of five penaeids should be exposed to

each concentration of test substance for up to 96 hours.  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

48- and 96- hour LC50 values with the minimum amount of testing

beyond the range-finding test.

    (ii)  A minimum of 20 shrimp per concentration should 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/1).  An equal number of shrimp

should be placed in two or more replicates.  If solvents,

solubilizing agents or.-.emulsif-iers have-to be used,- they shou-ld

be commonly used carriers and should not possess a synergistic or

antagonistic effect on the toxicity of the test substance.  The

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                                                           EG-7
                                                   August, 1982
concentration of solvent should not exceed 0.1 ml/1.  The
concentration ranges should be selected to determine the
requested concentration-response curves and LC50 values.  The
concentration of test substance in test solutions should be
analyzed for chemical concentration prior to use and at
designated times.
    (iii)  Every test should include controls consisting of the
same dilution water, conditions, procedures and shrimp from the
same population or culture container, except that none of the
chemical is added.
    (iv)  The dissolved oxygen concentration, temperature,
salinity and pH should be measured at the beginning of the test
and at 24, 48, 72 and 96 hours in each test chamber.
    (v)  The test duration is 96 hours.  The test is unacceptable
if more than 10 percent of the control organisms die or appear to
be stressed or diseased during the 96 hour test period.  Each
test chamber should be checked for dead shrimp at 3, 6, 12, 24,
48, 72 and 96 hours after the beginning of the test.
Concentration-response curves and 48- and 96- hour LC50 values
should be determined along with their 95 percent confidence
limits.

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                                                           BG-7
                                                   August, 1982
    (vi)  In addition to death, any abnormal behavior or
appearance should also be reported.
    (vii)  Distribution of shrimp among test chambers should be
randomized.  In addition, test chambers within the testing area
should 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 dissolved test substance (that
which passes through a 0.45 micron filter) in the test chambers
should be measured as often as is feasible during the test.  The
concentration of test substance should be measured:
    (A) in each chamber at the beginning of the test and at 48
and 96 hours after the start of the test;
    (B) in at least one chamber containing the next to the lowest
test substance concentration at least once every 24 hours during
the tes t;
    (C) 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 substance
should not vary more than 20 percent.
    (5)   [Reserved]-   • -   .

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                                                            EG-7
                                                   August,  1982
    (6)  Analytical measurements  (i)  Test chemical.  Deionized
water should be used in making stock solutions of the test
substance.  Standard analytical methods should be used whenever
available in performing the analyses.  The analytical method used
to measure the amount of test substance in a sample should be
validated before beginning the tes t by appropriate laboratory
practices.  An analytical method is not acceptable if likely
degradation products of the test substance, such as hydrolysis
and oxidation products, give positive or negative interferences
which cannot be systematically identified and corrected
mathematically.
    (ii)  Numerical  The number of dead shrimp should be counted
during each definitive test.  Appropriate statistical analyses
should provide a goodness-of-fit determination for the
concentration-response curves.  A 48- and 96- hour LC50 and
corresponding 95 percent intervals should be calculated.
    (d)  Test conditions — (1)  Test species — (i)  Selection.
This test should be conducted using one of three species of
penaeid shrimp: Penaeus aztecus (brown shrimp), Penaeus duorarum
(pink shrimp), or Penaeus setiferus (white shrimp).  Post-larval
juvenile or adult shrimp should be utilized.  Shrimp may be
reared from eggs-.-in--the labor-ato-ry or obtained -directly -as—  > -----
juveniles or adults.  Shrimp used in a particular test should be

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                                                            BG-7
                                                    August,  1982
of similar age and be of normal size  and  appearance.   Shrimp
should not be used for a test  if  they exhibit abnormal behavior
or if they have been used  in a previous test,  either  in a
treatment or in a control  group.
    (ii)  Acclimation.  During acclimation,  shrimp  should be
maintained in facilities with  background  colors  and light
intensities similar to those of the testing  areas.   In addition,
any change in the temperature  and  chemistry  of  the  dilution water
used for holding and acclimating  the  test organisms to those of
the test should be gradual.  Within a 24  hour  period,  changes in
water temperature should not exceed 1°C,  while salinity changes
should not exceed 2 °/oo.
    (iii)  Care and handling.  Upon arrival  at the  test facility,
the shrimp should be transferred  to water closely matching the
temperature and salinity of the transporting medium.   Shrimp
should be held in glass tanks  of-3JO liter capacity, or.larger..-.-.No
more than 22 to 24 shrimp  should  be placed  in  a  30  liter tank
unless the flow-through apparatus  can maintain dissolved oxygen
levels above 60 percent of saturation.  With species  of the genus
Penaeus, a minimum flow rate of 7.5 1/g body weight day should be
provided.  Larger flows, up to 22  1/g body weight day, may be
des irable toxinsur.e ,dissolv.ed^:axvy.geji.-corecentrations -'^bove- -e^Q*•*-••  <•*•
percent of saturation and  the  removal of  metabolic  products.  The

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                                                            EG-7
                                                    August/ 1982
period of acclimation to  ambient  laboratory  conditions  should be

at least 4-7 days.

    (iv)  Feeding.  Penaeid shrimp .should  not be fed during

testing.  Every two or three days during the acclimation period/

shrimp should be fed fish pieces  approximately 1 cm2.   Uneaten

food should be removed daily.

    (2)  Facilities—(i)  Apparatus.   (A)  Facilities  which may

be needed to perform this test  include:  flow-through tanks  for

holding and acclimating penaeid shrimp;  a  mechanism for

controlling and maintaining the water  temperature and  salinity

during the holding period; apparatus for straining particulate

matter, removing air bubbles, or  aerating  water when necessitated

by water quality requirements;  and an  apparatus providing a 12-

hour light and 12-hour dark photoperiod  with a 15-to-30 minute

transition period.  Facilities  should  be well ventilated, free of

fumes  and free of all other dis;tiarbanees~^that-"may *af f ect "test	

organisms.

    (B)  two to three centimeters of acid-washed sand,  free of

excess organic matter, should be  placed  in the bottom of test

chambers .

    (C)  Test chambers should be  loosely covered to reduce  the

loss of test—so^rtrion-'or-  dllu^tioTF wa=teir da^e^to *evapora-tiorr;-^"^--~~

mimimize entry of dust and other  particles and prevent escape of

the shrimp.


                                8

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                                                            BG-7

                                                    August, 1982
    (ii)  Cleaning.  Test substance delivery  systems  and test



chambers should be cleaned before  each  test following standard



laboratory practices.



    (iii)  Construction materials.  Materials  and  equipment that



contact test solutions should be chosen to  minimize  sorption of



test chemicals from dilution water and  should not  contain



substances that can be leached into aqueous solution  in



quantities that can affect test results.



    (iv)  Dilution water.  (A)  Natural or  artificial seawater is



acceptable as dilution water if shrimp  will survive  in it without



signs  of stress, such as. unusual behavior or  discoloration.
                                      o


Shrimp should be acclimated and tested  in dilution water from the



same origin.



    (B)  Natural seawater should be filtered  through  a five



micrometer filter with a pore size <  20 microns  prior to use in a



test.



    (C)  Artificial seawater can be prepared  by  adding



commercially available formulations or  by adding specific amounts



of reagent-grade chemicals to deionized water.   Deionized water



with a conductivity less than 1 u  ohm/cm at 12°C is  acceptable



for making artificial seawater.  When deionized  water is prepared



f r om a _g EJO u nd -;jo.r. ~s:ar£ac e:^wa tea^u* ao.uEc 6-7*1 ix2.oadu ct.i.vJutyr^a nd-^to fcai^-- -—



organic carbon (or chemical oxygen demand)  should  be  measured on



each batch.

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                                                            EG-7
                                                    August, 1982
    (v)  Test substance  delivery  system.   Proportional diluters,

metering pumps or  other  suitable  systems  should be used to

deliver test substance to  the  test chambers.   The system used

should be calibrated  before  each  test.   Calibration includes

determining the flow  rate  through each  chamber and the

concentration of the  test  substance in  each chamber.  The general

operation of the test substance delivery  system should be checked

twice daily during  a  test.   The 24-hour flow through a test

chamber should 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 among test chambers or  across time.

    (3)  Test parameters.  Environmental  parameters of the water

contained in test  chambers should be as specified below:

    (i)  Temperature  of  23 ± i°c.

    (ii)  Dissolved oxygen concentration  between 60 and 105

percent saturation...  Aeration, .. if -needed -to_ achieve— this^-level,

should be done before the  addition of the test substance.  All

treatment and control chambers should be  given the same aeration

treatment.

    (iii)  The number of shrimp placed  in a test solution should

not be so great as  to affect results of the test.  Loading

r eq u i r eme,n t s: x-wi l-'t-K-va^ry. ^d:ep.e~.nd i&ng*siOJi-::~teh e^f -l.ow-s.-r a-t e^of ~ *d-M u -t io a

water.  The loading should not cause the  dissolved oxygen

concentration to fall below  the recommended levels.
v*'_. jY-JjV:
                                10

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                                                            EG-7
                                                   August,  1982
    (iv)  Photoperiod of 12 hours light and 12 hours darkness,

with a 15-30 minute transition period.

    (v)  Salinity of 20 ± 3 Percent.

    (e)  Reporting.  The sponsor should submit to  the  EPA  all

data developed by the test that are suggestive or  predictive of

acute toxicity and all other toxicological manifestations.   In

addition to the general reporting requirements prescribed  in Part

792—Good Laboratory Practice Standards, the reporting of  test

data should include the following:

    (1)  The nature of the test, laboratory, name  of the

investigator, test substance and dates of test should  be

supplied.

    (2)  A detailed description of the test substances should be

provided.  This information should include the source, lot

number, composition, physical and chemical properties  and  any

carrier or addltives--us-ed.  ...,-.—

    (3)  Detailed information about the shrimp should  be

provided: common and scientific names, source of supply, age,

history, weight, acclimation procedure and feeding  history should

be reported.

    (4)  A description of the experimental design  including  the

number ,ofc4:es-t^xxtotd.ajis-:z5Qrrc^
                                11

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                                                             BG-7
                                                    August/  1982
number of shrimp per  replicate should be provided.

    (5)  The source of  the dilution water, its chemical

characteristics  (e.g./  salinity)  and a description of any

pretreatment.

    (6)  A description  of  the test chambers/ the depth and  volume

of solution in the chamber,  the number of organisms per

treatment, the number of  replicates/ the loading, the lighting,

the test substance delivery  system and flow rate expressed, as

volume additions per  24 hours.

    (7)  The concentration of the test substance in each test

chamber before the start .of  the test and at the end.

    (8)  The number of  dead  shrimp and measurements of water

temperature, salinity,  and dissolved oxygen concentration  in each

test chamber should be  recorded at the designated times.

    (9)  Methods and  data  records of all chemical analyses  of

water qual ity._and. _tes-t.-s.ubs-tance^conce.ntr-atio.ns ,—incl-udi-ng. -me thod

validations and reagent blanks.

    (10)  Recorded data for the holding and acclimation periods

(temperature, salinity,  etc.).

    (11)  Concentration-response  curves should be fitted to

mortality data collected at  24, 48, 72 and 96 hours.  A

s tat is t.ical.--tes;.t.^of-^~goodn.essi=xDf-=-J-J;.t rs^ou:ld^±iei'jpexf-arrae^^r^siJXo.-- .

    (12)  For each set  of  mortality data, the 48- and 96- hour


                                12

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                                                           EG-7
                                                   August, 1982
LC50 and 95 percent confidence limits should be calculated on the

basis of the average measured concentration of the test

substance.  When data permits, LC50 values with 95 percent

confidence limits should be computed for 24 and 72 hour

observations.

    (13)  The methods used in calculating the concentration-

response curves and the LC50 values should be fully described.
                                13

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                                        DRAFT
                               ES-4
                               August,  1982
       TECHNICAL  SUPPORT DOCUMENT

                  FOR

   PENAEID SHRIMP ACUTE TOXICITY  TEST
       OFFICE OF TOXIC SUBSTANCES
OFFICE  OF"PESTTCrDES'-ftND:"TOXICT"SUBSTANCE^"
  U.S.  ENVIRONMENTAL PROTECTION  A3ENCY
         WASHINGTON, D.C. 20460

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                        TABLE OF CONTENTS

       Subject                                          Page
I.      Purpose                                           1
II.     Scientific Aspects                                1
       Test Procedures                                   1
       General                                           1
       Range-Finding Test                                2
       Definitive Test                                   2
       Test Conditions                                   3
       Test Species                                      3
       Selection                                         3
       Sources       "                                    6
       Maintenance of Test Species                       7
       Handling and Acclimation                          7
       Feeding                                           9
       Facilities                                        12
       General                                           12
       Construction Materials                            13
       Test Substance Delivery System                    14
       Test Chambers                                     16
       Cleaning of Test System                           17
       Dilution Water                                    17
       Controls                                          19
       Carriers                                          20
       Randomization                                     20
       Environmental Conditions                          21
       Dissolved Oxygen                                  21
       Light                                             21
       Temperature and Salinity                          22
       Reporting                                         23
III.   Economic Aspects^   . . .                             26
IV.     References                                        28

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Office of Toxic Substances                               ES-4
                                                 August,  1982

 Technical Support Document for Penaeid Acute  Toxicity Test

I.  Purpose
    The purpose of this document  is  to provide the
scientific background and rationale  used  in the  development
of Test Guideline EG-7 which uses Penaeid shrimp to  evaluate
the toxicity of chemical substances.  The Document provides
an account of the scientific evidence and an explanation of
the logic used in the selection of the test methodology,
procedures and conditions prescribed in the Test
Guideline.  Technical issues and  practical considerations
relevant to,the Test Guideline are discussed.   In addition,
estimates of the cost of conducting  the test are provided.
II.  Scientific Aspects
    A.  Test Procedures
         1.  General      •
    A flow-through bioassay technique was  chosen  because  of
several distinct advantages over static exposure  methods.
Continuously flowing seawater not only simulates  the  natural
exposure process but, when used as a laboratory tool,
eliminates problems associated with the accumulation  of
organic matter-and,.toxic metabolic,.=products-.^--F.low^thr-ough." -
techniques should be used with materials which have a high
oxygen demand, are highly volatile, are unstable  in aqueous
solution, are readily biodegradable, or are  removed from
test solutions in significant amounts by the test
organisms.  Toxicants flowing through this system are more
thoroughly mixed and loss due to sorption  to sediments and
feces is minimized.  Flow-through techniques for  holding  and
ac c 1 ima-tion :.
testing.

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                                                         ES-4
                                                 August,  1982
    For acute tests, 96 hours  is a convenient  interval  of
time for starting and completing a test  in  a  normal  five-day
work week, and is better than  shorter periods  for  estimating
cumulative and other chronic effects.  Because set-up is  the
most expensive portion of a test, a 96-hour test is  only
slightly more expensive than 24 or 48 hour  tests.  Yet
additional data on the LCSO's  over time  and the observation
of other abnormal effects that do not appear  in shorter
tests are gained for this slight increase in cost.   Although
the 48 hour test can reduce costs, the 96-hour toxicity test
was selected for the penaeid test guidelines because of
greater probability for determining the  incipient  LC50
(threshold limit for acute toxicity) through extension  of
the toxicity curve.-	
         2.  Range-Finding Test	
    The concentration range for the definitive tests is
normally chosen based on the results of  a range-finding
test.  Range-finding tests with penaeid  shrimp are usually
short-term (24-96 hour) flow-through bioassays  which utilize
fewer organisms per test substance concentration, than	
required for the definitive test.  In all cases, the range-
finding test is conducted to reduce the  expense involved
without having to repeat a definitive test due to
inappropriate test substance concentrations.
         3.  Definitive Test
    The concentration range for the definitive test  is
chosen based on results on the range-finding test.   By  using
a minimum of five test substance concentrations, partial
kills both above and ..below ..the—median .5.0—per.ceni~.mort allty..-,..
level are probable and will help define  the concentration-

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                                                         ES-4
                                                August,  1982
response curve.  The more partial kills, the better the
definition of the concentration-response curve.  The slope
and shape of the concentration-response curve can allow
insight into the mode of action of a chemical and will allow
estimation of the effects of lower concentrations upon the
test organisms.  In addition, by having partial kill data, a
greater array of statistical methods can be used to
determine an LC50 value.
    A sample size of 20 shrimp permits several combinations
of replicates and sample sizes to be used.  The use of
replicate samples allows an analysis of variance to be
performed on the results.
    Measurements of test substance concentrations at
des ignated periods - during- the~~f low-^through • tes fc-~aliows~ = ~
documentation of real test concentrations at appropriate.
periods under acute conditions.
    Chemical and physical parameters (temperature, pH,
dissolved oxygen, and salinity) are recorded at specified
times to permit evaluation of the biological conditions
present for shrimp survival in test water.
    Specified observations on mortality characteristics  are
designed to allow an adequate evaluation- of dose-response
effects in acute penaeid tests.  In addition, these defined
observation times allow greater comparability of dose-
response data between different chemicals and laboratories.
    B.  Test Conditions
         1.  Test Species                         :
              a.  Selection
    The prime cons idera.tions_in. the_s elect ions—of	test——-~
organisms for toxicity tests are: (a) their sensitivity  to a

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                                                         ES-4
                                                 August, 1982
wide spectrum of test substances;  (b)  their geographical
distribution and abundance;  (c)  their  recreational, economic
and ecological  importance;  (d)  their availability as test
organisms, and  existence  of  established culture. Penaeid
shrimp have become  the  most  valuable marine species
harvested from  U.S.  Coastal  waters  by  commercial fisherman
(Temple, 1973).  In 1972, an estimated 190.6 million pounds
of shrimp were  harvested  from coastal  waters; 87 percent of
this harvest was from the Gulf.   During 1974, the Louisiana
brown shrimp catch  alone  was 27.4 million pounds, and was
valued at 18 million dollars (Temple,  1973; Knudsen et al.,
1976).
    Perhaps the most important quality of penaeid shrimp for
tox ici ty -t.es.tl ng—is—th.e-i.r-- cons is-t en-t ly: h4gh-sens-i- t-i-vi-ty - to--•<---
test substances.   In virtually all  comparative toxicological
studies in the  laboratory, penaeid  shrimp proved the most
sensitive marine organism to a variety of toxins.  Pink
shrimp have been used repeatedly in the last 10 years (Lowe
1971, Tagatz 1975;  Schimmel  1979; Parrish 1976; Nimmo and
Bahner 1976); white shrimp and  brown shrimp have also been
used successfully  in toxicological  tests (Nimmo and Bahner
1974; Curtis 1979).
    Penaeid shrimp  are  also  sensitive  at sub lethal doses of
toxins; this allows  the maximum amount of information to be
gleened from each  test.   For example,  pink shrimp were one
of a selected group of  estuarine animals used to assess the
effects of mirex leaching into  the  environment; toxicity was
latent and became more  apparent with increasing length of
exposure.. . _Unde.r _s-tr.es.s—they— s.howed,-.da.rke-r,_»colx3.ra^tio.n, -_l
of equilibrium  and  a cessation  of burrowing behavior,

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                                                        ES-4
                                                August, 1982
measures which provided valuable sub-lethal effects data
(Tagatz et al. 1975).
    In juvenile pink shrimp exposed to Mirex, Lowe (1971)
observed the first case of delayed toxicity.  Mortality at
the end of seven days was 25 percent, but increased to 100
percent by day 11 even after the shrimp were removed to
mirex-free water.  In a flow-through acute  toxicity test,
Schimmel,..et al. (1979) found pink shrimp especially
sensitive to the insecticides EPN and leptophos; in this
case there was 20 percent mortality at non-detectable
(nominal) concentrations of these insecticides in test
water.
    An additional point to consider is the  suitability of
the species for cultivation, since cultured shrimp are
preferable for use in toxicity testing.  Previous history of
an organism is a major variable affecting the potential
response to a test substance.  While the tropical species P.
monodon and P. orientalis have been shown to grow most
rapidly under high-density cultivation, these species are
not representative of those organisms residing in U.S.
coastal waters.  Penaeus aztecus (brown shrimp) and P.
setiferus (white shrimp) grew significantly longer in low
densities (25m2) than high densities (166m2).  However, of
the 9 shrimp species studied, brown shrimp  had the second
highest survival rate at high densities (Forster and Beard
1974).  In the same study, penaeid shrimp were shown to be
less variable in individual growth rates than Machrobracium
spp., the freshwater prawn.
    Some limitations in the use of penaeid  shrimp have been
reported.  High mortalities due to cannibalism, cramped test

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                                                        ES-4
                                                August, 1982
conditions, or control mortality can be expected in culture
(Curtis et al. 1979; Tagatz et al. 1976).
    In view of the continued successful use of penaeid
shrimp for toxicity testing in many laboratories, and their
sensitive and varied response to sublethal toxic concen-
trations, they are the species of choice for this flow-
through bioassay.  In fact, because of their suitability as
a test organism and their value as an economic resource, a
wealth of literature is available for reference in
developing culture and testing techniques, as well as a
comparative toxicology data base.
              b.  Sources
    Whether collecting organisms for testing or for
culturing purposes, a great deal of care should be taken to
avoid stress and insure survival in transport to the
laboratory. Shrimp should be collected from unpolluted
sources and measurements of water temperature, salinity and
pH should be taken at capture time.  This allows for
successful acclimation to laboratory conditions.  Taking
organisms from areas of known high levels of parasitism,
disease, pollution, or where deformed individuals are found
should be avoided to insure valid results.
    The following salient points are emphasized by APHA
(1975) when collecting organisms for bioassay purposes; in
general, great care should be taken to insure the shrimp are
not damaged in the collection, transfer and transporting
process:
    (1)  When seining or using trawls, make short hauls;
    keep gravel, sand and other debris out of net.

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                                                        ES-4
                                                August, 1982
    (2)  Do not expose delicate, easily damaged stages to
    the air; juvenile shrimp can be transferred with dip
    nets.
    (3)  Do not collect too many animals at one time.
    (4)  Do not crowd the organisms during transportation
    and watch for signs of stress; observe animals in the
    laboratory for additional signs of stress.
    Juvenile arid adult shrimp are most easily collected by
hand-held seines or boat trawls.  For test organisms, one
should select shrimp of uniform size and in the post-larval
stages.  Do not mix stages within the test.  For culture
purposes, the preferred method is to collect gravid females
and allow them to spawn in the laboratory. It may not yet be
feasible to breed penaeid shrimp prawns in captivity (Walker
1975).  As a guide for distinguishing life stages in
penaeids, Rose et al. (1975) has suggested these total
length criteria: juvenile (25 mm); subadult (90 mm); and
adult (140 mm).  Shrimp have been success-fully transported
by motor vehicle in plastic bags or buckets filled; with
oxygenated seawater (Mock 1974).  It is recommended that
test organisms come from a controlled environment such as a
laboratory maintenance system.  This insures that shrimp are
uniform in age, size and experi-mental history.
         2.  Maintenance of Test Species
              a.  Handling and Acclimation
    Tanks for holding and acclimation should be identical to
those used for testing, which eliminates further stressing
of shrimp by an additional transfer.  Water should be of the
same temperature and salinity as water from the collection
site; a gradual change in water quality parameters should be

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                                                        ES-4
                                                August, 1982
made to acclimate the shrimp to the test conditions.  This
gradual period of acclimation should be at least 7 days; and
up to 2 weeks has been suggested (APHA 1975).  It has been
shown that activity responses to tidal rhythms do not fade
until 7 days of captivity have passed.  It is important that
shrimp be similar in their activity cycles before testing
begins (Subrahmanyam 1976).
    Following the initial holding period, shrimp should be
randomly assigned to their respective test chambers and held
there until testing begins, again eliminating further
handling.  However, as will often be the case, extra shrimp
will need to be maintained in separate tanks.  No more than
22 to 24 shrimp should be kept in a 30 liter tank with a
flow-through mechanism to allow maintenance of dissolved
oxygen (DO) levels above 60 percent of saturation.  Flow
rates should be great enough to remove metabolic products
and food build-up which have been demonstrated to cause high
mortality (Mock 1974).  A minimum flow of 7 1/g day shrimp
should be maintained, while flows up to 22 1/g day may be
needed.  Holding tanks should attain preliminary test
condition within 7 days; gradually acclimate shrimp to
salinity and temperature conditions required by the test so
as to minimize stress.
    Inspection for parasitism and disease should be made
during the acclimation period; diseased shrimp should never
be used in tests.  Methods for detecting and treating the
following prawn diseases are given in Delves-Broughton
(1976) and should be referreed to when needed; shell
disease, black module, vibriosis, hasplosporidian infection,
filamentous gill growth, filamentous bacteria on eggs, and

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                                                        ES-4
                                                August, 1982
systematic fungus disease.  Spontaneous muscle necrosis is
the result of abrupt changes in temperature and salinity;
treatment is discussed by Lakshmi (1978).  When antibiotic
use is necessary, oxytetracycline and oleandonycin are
suggested.  They are 99 percent bactericidal at high doses
and do not significantly depress respiration in shrimp (Chan
and Lawrence, 1974).  If antibiotics are used in the water
of test chambers (during acclimation), they should be
removed before testing begins.  This is possible when the
chelator EDTA is substituted at a concentration of 10 mg/1
of seawater (APHA 1975).
              b.  Feeding
    During holding and acclimation period juvenile or adult
penaeids may be fed cut-up fish.  Fillet from mullet,
grouper, or other abundant species should be cut into pieces
about 1 cm 2 and fed, one per shrimp, every 2 or 4 days.
Uneaten food should be removed every 24 hours to reduce
fouling.  Protozoal stages of shrimp are generally fed
algae, chiefly the diatoms Thalassios ira and Skeletonema
(Cook & Murphy, 1969, Cook 1967, Mock 1974).  Techniques for
culturing the diatom, and mechanisms for maintaining them in
shrimp rearing tanks are discussed in detail by Mock
(1974).  Equipment and procedures for the continuous mass
culture of algae as a food source are also found in APHA
(1975).  Add algae as a concentrate either fresh
(centrifuges) or frozen.  Do not add algal culture medium 'to
acclimation or test water since it is toxic to shrimp (Mock
1974).  The number of algal cells necessary to rear a
population of larval shrimp during the pro.tozoal-s.tages .are -
as follows (APHA 1975):

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                                                        ES-4
                                                August, 1982
    Protozoel I              Skeletonema    50,000' cells/ml
    Protozoel II             Skeletonema   150,000 cells/ml
    Protozoel III            Tetraselmis    20,000 cells/ml

    It was found that the addition of several algal foods
insure higher rates than additions of a single algal species
at comparable concentrations (Mock 1974).  When only one
species is used for larval shrimp, it should be Skeletonema
cos tatum.
    Brine shrimp (Artemia sp.) nauplii have been used
extensively as food for the mysi stage through the fourth
post-larval stage.  In tests of food preferenc in brown and
white shrimp, both species demonstrated a preference for
nauplii of brine shrimp (Artemia) when given a choice of
diets.  Brown shrimp were more flexible, but still preferred
Artemia.  Karim and Aldrich (1976) tested various commercial
foods and brown shrimp preferred Vio Bio Fish Flour and
white shrimp preferred Silvray Fish Feed.  It was stressed
that these prepared foods not be recommended for general use
until their effect on survival and growth are demonstrated
to be favorable.
    Therefore, Artemia should be used for post-larval
stages.  The quantities required are:

    Mys is                    Artemia nauplii          3/ml
    Mys is                    Artemia nauplii          3/ml
    Mys is                    Artemia nauplii          3/ml
    Post-larval I-IV         Artemia nauplii          3/ml
                                10

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                                                        ES-4
                                                August, 1982
    A recent study by Johns and Walton (1979) reported that
adult Mysidopsis bahia fed Artemia spp. from San Pablo Bay/
California exhibited increased mortality, did not reproduce
and showed reduced growth rates.  In contrast, both juvenile
and adult mys id shrimp fed Artemia spp. strains collected
from Brazil, Australia, Italy and Utah maintained high
survival and growth rates.  These results imply that
nutritional quality of Artemia, possibly associated with
pesticide or heavy metal contamination, can significnatly
influence test results and, therefore, should be considered.
    There are several basic methods of crustacean
aquaculture: extensive culture (using large outdoor
enclosure); intensive culture (small outdoor tanks); and
indoor intensive (high-density flow through tanks in the
laboratory).  The indoor intensive system is most practical
for use with bioassay techniques because of the relative
ease of controlling the aquaculture environment. Tanks are
stocked at high densities (for this guideline, not more than
22-24 adults per 30 liter tank).  In order to prevent       >
fouling of the system, it is necessary to circulate water
through the tank to maintain high DO levels.  The overflow
test water may overflow to a drain or be recycled through a
biological filter (Walker 1975).
    A temperature range of 28°-30°C and a salinity range of
27-35°/oo are most satisfactory for shrimp larval culture.
Since the shrimp will be later used in acute toxicity tests,
they should be acclimated to the presceibed test conditions
by post-larval stages.  The specific requirements of each
species should be considered.
                                11

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                                                  ;       ES-4
                                                August,  1982
    Penaeid shrimp can be reared regularly from the  egg  to
post-larval stage in the laboratory.  Cook and Murphy  (1969)
have described, in detail, equipment and techniques  for
conditioning, spawning and rearing large numbers of  shrimp
larvae from eggs.  Methods of rearing shrimp larvae  for
experimental studies have been described by Cook (1967).
These references should be followed closely.
         3.  Facilities
              a.  General
    The delivery of constant concentrations of test
substances is required to reduce variability in test
results.  Large fluctuations in test substance concentration
will give abnormally high or low responses, depending upon
the mechanism of toxic action.  Proportional diluters and
metering pumps  (Mount and Brungs 1967) have been found to
provide constant concentrations and are widely used.
    Proportional diluters operate on a sequential filling
and emptying of water chambers.  The water chambers  are
cali-brated to contain a measured amount of water.   Separate
water chambers can be provided for toxicant and diluent
waters.  Diluent and toxicant waters are mixed in siphon
tubes and delivered to the replicate test chambers.  The
cyclic action of the diluent is regulated by a solenoid
valve connected to the inflow dilution.  The system  is
subject to electrical power failure, so an alternate
emergency power source is recommended.
    The proportional diluter is probably the best for
routine use.  It is accurate over extended periods of time,
nearly trouble free, and has fail-safe provisions (Lemke et
al. 1978).  A small chamber to promote mixing of toxicant-
                                12

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                                                August, 1982
bearing water and dilution water should be used between the
diluter and the test chamber for each concentration. Since
replicate chambers are used in this test, separate delivery
tubes can be run from the mixing chamber to .each replicate
test chamber.
    Calibration of the toxicant delivery system should be
checked carefully before and after each test.  This should
include determining the flow rate and toxicant concentra-
tion through each test chamber.  The general operation of
the system should be checked daily.
    Alterations in the design of the proportional diluter,
such as the use of six or more concentrations have been
useful in some situations (Benoit and Puglisi 1973).
              b.  Construction Materials
    In an excellent review of potential sources for chemical
contamination in the culture system and laboratory, Bernhard
and Zattera (1970), stress the importance of avoiding
chemical contamination in culturing marine organisms.
Therefore, choice of laboratory equipment on toxicant
testing is critical.
    Several materials such as rubber and polyvinyl chlorides
have been found highly toxic; and should never -be used in
culture or testing of marine organisms.  Teflon ( algof Ion),
Perspex, Polyethylene, Tygon, Polypropylene, Polycarbonates
(Makrolor) and Polyester (Gabraster) have been shown to be
non-toxic and suitable for experiments with marine
organisms.
    All pipes, tanks, holding chambers, mixing chambers,
metering devices, and test chambers should be made of
materials that minimize the release of chemical contaminants
                                13

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                                                August, 1982
into the dilution water or the adsorption of the test
substances.  Chemicals that leach from cons true-1 ion
materials can stress test organisms/ or possibly act
synergistically or antagonistically with test substances to
give inaccurate results.  Generally, undesirable substances
are not leached from perfluorocarbon plastic, titanium, and
borosilicate glass; in addition, the tendency of these
materials to adsorb substances, is minimal.  Rubber, copper,
brass, galvanized metal, lead and epoxy resins should not
come in contact with dilution water, stock solution, or test
solutions because of the toxic substances they contain
(USEPA 1975).  All containers and pipe need to be
conditioned before use in order to leach and wash away any
undesirable residues that may be present.
              c.  Test Substance Delivery System
    Flow-through systems should have the capability to vary
and maintain water temperature, dissolved oxygen, and
salinity at desired levels during holding, acclimation and
testing. Penaeid shrimp are extremely sensitive to
fluctuations in these parameters, which affect test
validity.  Tagatz et al. (1975) reported that a slight (3-
4°C) change in water temperature resulted in significant
increases in the mortality rates of juvenile Peneaus
duorarum exposed to mi rex.  These mortality increases were
greater than those due to longer (3x) exposure times (Lowe
et al. 1971).  Similarly, salinity decreases have been shown
to cause significant increase in mortalities of P.  aztecus
exposed to Aroclor (Nimmo and Bahner 1975).  Combined, or
synergistic effects of dissolved oxygen (DO), temperature
and salinity on the toxicity of toxaphene on pink shrimp
                                14

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                                                August, 1982
were identified and are discussed in detail by Courtenay and
Roberts (1973).
    Shrimp health and survival are directly affected by
water quality and handling.  Physically stressed organisms
are not valid test subjects.  Attention to husbandry and
routine water quality monitoring are of paramount importance
in prevention of disease (Delves-B rough ton and Poupard
1976).  Spontaneous muscle necrosis (exhibited as white foci
on the 4th, 5th and 6th abdominal segments) in brown shrimp
(P. aztecus) was induced in healthy shrimp by over-crowding,
lowering dissolved oxygen (DO) levels, or changing physico-
chemical conditions (Lakshmi et al. 1978).  High mortality
in adult brown shrimp from gas bubble disease was caused by
supersaturation of dissolved oxygen (DO) in water (Supplee
and Lightner 1976).  This occurred with dissolved oxygen
(DO) levels exceeding 250 percent saturation.  Morbidity and
mortality not only hinder the progress of testing, but alter
those toxic effects of concern, thus invalidating tests.
Salinity and temperature also affect burrowing behavior,
metabolic rate, and cause increased aggression; such
aberrations cause distorted test results.  For example,
hyperactivity, was shown in brown shrimp within 30 minutes
of a change from optimum conditions (Lakshmi et al. 1978).
    The dilution water should be filtered through a twenty
micrometer filter (or smaller) to sufficiently reduce the
amount of suspended sediments, organic material and
biological organisms (phytoplankton, zooplankton, fungi,
bacteria, etc).  This will minimize the confounding of
results associated with the differential sorption of the
test substance on cell walls, clay particles, etc. which in
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                                                August,  1982
turn may enhance or reduce the availability of the test
substance to the shrimp.
    Accumulation of gases can cause adverse effects;
therefore, a device for removing air bubbles may be
necessary (Penrose and Squires 1976).  When the dissolved
oxygen (DO) in the dilution water is less than 60 percent,
aeration is suggested.  Culturing techniques recommend 70-
100 percent saturation for penaeid shrimp (Forster and Beard
1974; Supplee and Lightner 1976).  A device for simulating
natural photoperiod with transitions from light to dark is
suggested so that conditions can be optimized for shrimp
(Drummond and Dawson 1970).
              d.  Test Chambers
    Choice of test chamber size should consider both the
needs of the test organism and the requirements of the
test.  Chamber size should reflect the appropriate loading
require-ment using the number of organisms specified in the
experimental design.  P. aztecus and P. setiferus showed a
significant difference in length attained when grown in low
(25/m2) and high 166/m2) densities (Forster and Beard
1974).  Stress caused by crowding has been shown to induce
latent viral infections in healthy pink shrimp (Couch 1974).
    Penaeid shrimp are large organisms.  Juvenile
individuals range from 0.4 mm up to approximately 25 mm in
length (Rose 1975),  In the 60x30x30 cm high container
recommended, 20-22 juvenile shrimp may be housed, as long as
the total live weight is no more than 50 g per chamber. A
substrate of two to three centimeters of organic free sand,
permits the shrimp to burrow.  Screens for chamber tops are
recommended (APHA, 1975) to prevent the escape of shrimp
from the test chambers.
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                                                August,  1982
              e.  Cleaning of Test System
    Before use, test systems are cleaned to remove dust,
dirt, other debris, and residues that may remain from the
previous use of the system.  New chambers should be cleaned
to remove any chemical or dirt residues remaining from
manufacture or accumulated during storage and
construction.  Detergent is used to remove hydrophobic or
lipid-like substances.  Acetone is used for the same purpose
and to remove any detergent residues.  It is important to
use pesticide-free acetone to prevent the contamination of
the chambers with pesticides.  Nitric acid can be used to
clean metal residues from the system.
    At the end of a test, test systems should be washed in
preparation for the next test or storage.  This will prevent
chemical residues and organic matter from becoming embedded
or absorbed into the equipment.
    Priming the system with dilution water before use allows
equilibrium to be reached between the chemicals in the water
and the materials of the testing system.  The testing system
may sorb or react with substances in the dilution water.
Allowing this equilibrium to be established before exposure
of the test shrimp to the test substance lessens the chances
of water chemistry changes during a test.
              f.  Dilution Water
    A constant supply of dilution water is required to
maintain consistent experimental conditions.  An
interruption in flow or changes in water quality parameters
can change the chemistry of the test system and possibly
affect the response of the test population.  Therefore, the
results of a test with variable dilution water quality are
not comparable to tests run under constant conditions and
                                17

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                                                August, 1982
the results are more difficult to interpret.
    For acute toxicity tests, a minimum criterion for an
acceptable dilution water is that healthy test organisms
will survive for the duration of the acclimation period
without showing signs of stress.  Signs of stress in penaeid
shrimp include darkened coloration, cessation of burrowing
behavior, loss of equilibrium, and antennae-chewing (Tagatz
1976; Forster and Beard 1974).  Investi-gators should be
familiar with normal shrimp behavior patterns, as well as
gross physical changes which may occur during testing.
    Since shrimp have both estuarine and marine phases
during their life cycle, the salinity of dilution water is
of prime importance.  Determination of the desired salinity
was made by considering the natural habitat characteristics,
laboratory results, and individual species preferences.  The
most important test requirement will be to maintain a
constant salinity level for the entire holding and testing
period.  It is important also to monitor dissolved oxygen
(DO) levels; they should be kept above 60 percnt of
saturation.  The pH of the test solution appears to be less
important to the health of shrimp.  Some studies have
suggested a pH of 8.3 to 8.7 for white shrimp (Curtis et al.
1979) and 8.0 for euryhaline species in general (Kester et
al. 1967; Zaroogian et al. 1969).
    Natural seawater, obtained from a point source with
similar characteristics to those designated for the test
species, or water from an area where the test organisms were
obtained, is preferrable to artificial sea water.  Dilution
water should be of constant quality and should be
uncontaminated.  Contaminants may affect the results
directly and indirectly.  For example, low levels of
                                18

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                                                August, 1982
organochlorine chemicals have been shown to increase the
prevalence of latent viral infections in pink shrimp (Couch,
1975).  This is as important during holding as during
testing.
   , If alternatives to reconstituted seawater are used, they
should meet the following specifications for contaminant
levels (USEPA 1975).
    Suspended Solids                             < 20 mg/1
    TOG                                          < 10 mg/1
    Un-ionized ammonia                           < 20 ug/1
    Residual Chlorine                            <  3 ug/1
    Total organophosphorus pesticides          <   40 ng/1
    Total organochlorine
    pesticides plus PCB's                        < 50 ng/1

    Maintaining the desired salinity level in natural waters
often poses a problem.  When possible, obtain water from an
area of high salinity and obtain low salinities by adding
either deionized or glass distilled water of a satisfactory
quality.  To increase salinity, use a strong, natural brine,
which can be obtained by freezing and then partially thawing
seawater.  This procedure can be used if limited amounts of
seawater are needed.  However, it is recommended that
artificial seawater be used when large quantities of
dilution water are needed (APHA, 1975).
              g.  Controls
    Controls are required for every test to assure that any
effects which are observed are due to the test substance and
not to other factors.  These may include effects from
construction materials, environmental factors, vapors,
stressed test organisms, etc.
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                                                 August,  1982
    Ten percent morta." . ty may be anticipated  due  to  inherent
biological factors.   I i a test  chamber  of  20  organisms,  this
amounts to two deaths.  Any increase  above  this may  be
attributed to conditic is of the  test.   The  ten percent
mortality figure is re >resentative of a  wide  variety of
organisms including be .h fish and  invertebrates captured
from the wild.  Captur  tends to stress  organisms  so there
is more likelihood of  tress related  death.   In addition,
invertebrates are gene ally more vulnerable to handling
injury.  If penaeid si 'imp are  raised under controlled
conditions, they are  c nerally  more .healthy than  are
captured organisms, tl Before,  fewer  should die during a
test because of inhere .t biological factors.
              h.  Car) .ers
    Carriers can effec . test organisms  and  can possibly
alter the form of the  .est substance  in  water.  Therefore,
it is preferable to av dd the use  of  carriers in  toxicity
tests unless required  .o dissolve  the test substance.  Since
carriers can stress 01 adversely effect  test  organisms,  the
amount of carrier shoi d be kept to a minimum.  A
recommended maximum is 0.1 ml/L  (APHA 1975).
    Triethylene glycol and dimethylformamide  have  been shown
to exert the least inf uence on  test  organisms and test
substances of several  arriers  that have been used in
testing marine organic .s.  Acetone and  ethanol have  a
stronger tendency to  r duce the surface  tension of the water
and therefore decrease oxygen saturation (Veith and  Cornstock
1975; Krugel et al.,  ] 78; APHA  1975).
              i .  Ranc imization	
    The positions of  t st chambers are  randomized  to prevent
conscious or unconscic s biases from  being  introduced.
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                                                August, 1982
These biases can be in environmental conditions such as
temperature and lighting, shrimp selection and distribution,
diluter system function, etc.
         4.  Environmental Conditions
              a.  Dissolved Oxygen
    Large variations in flow rates to chambers will result
in environmental differences between chambers.  Parameters
such as dissolved oxygen (DO) and test substance concen-
tration can decrease more rapidly in chambers with low
salinities.  High salinities can be decreased by adding
either deionized or glass distilled water of a satisfactory
quality.  To increase salinity, use a strong, natural brine,
which can be obtained by freezing and then partially thawing
seawater.  This procedure can be used if limited amounts of
seawater are needed.  However, it is recommended that
artificial seawater be used when large quantities of
dilution water are needed (APHA, 1975).
              b.  Light
    The three species of penaeid shrimp have been shown to
have a nocturnal peak in activity when held in captive
laboratory conditions.  Burrowing frequencies and durations
were highest during bright light hours, and shrimp were more
active above the substrate during dark hours.  Of the three
species tested, P. setiferus (white shrimp) was least
influenced by the light schedules and was more active than
either pink or brown shrimp, being exposed on the substrate
day and night (Wickham and Minkler, 1975).  This is
supported by catch data for shrimp which show higher catch
levels in daylight for white shrimp than the other species.
                                21

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                                                        ES-4
                                                August, 1982
    Cool white fluorescent lighting should be used.  White
light has been determined necessary for maintaining a
circadian burrowing pattern.  A 15-30 minute transition
period between light and dark cycles is suggested.  It
appears that shrimp initiate activity rhythm changes during
this transition period (Bishop and Herrnkind 1976). Thus,
the 12:12 light-dark schedule using white light not only
mimics environmental conditions, but also allows for
equalizing the time organisms spent exposed to test
substances in the water and substrate.  Furthermore this
standardization facilitates comparison between tests using
different species.
              c.  Temperature and Salinity
    Penaeid shrimp occur naturally in estuarine waters where
temperature and salinity vary over a wider range than in
oceanic waters.  A review of the literature of toxicity
testing demonstrates the broad range of conditions over
which penaeid shrimp have been maintained.
    Brown shrimp (Penaeus aztecus) occur in waters ranging
from 15°-35°C in temperature and 9-40 °/oo in salinity
(Copeland and Bechtel, 1974).  In culture, growth was found
to be optimum between 15-20°C (Temple, 1973).
    Pink shrimp Peneaus duorarum) occur naturally in waters
where temperature and salinity vary from 5-38°C and 20-
35°/oo respectively (Copeland and Bechtel, 1974).  Optimal
temperatures for growth are in excess of 20°C (Copeland and
Bechtel, 1974).                                    ;
    White shrimp (Penaeus setiferus) are found in water
ranging from 10-40°C and 0-33 °/oo in salinity (Copeland and
Bechtel, 1974).  Optimum temperatures for growth have been
                                22

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                                                August, 1982
shown to be between 15 and 20°C (Temple, 1973).
    Thus there is considerable overlap in temperature and
salinity requirements of the three penaeid species.
Therefore, it is reasonable to select a single temperature
and salinity level for testing purposes; tests should be
conducted at a temperature of 23 +_ 1°C and a salinity level
of 20 +_ 2 °/oo to minimize the difficulty in obtain-ing a
suitable source of dilution water.
    Furthermore, minimizing variability in testing
conditions by specifying temperature and salinity conditions
allows greater comparability of inter-laboratory test
results and for the development of a comparative toxicology
data base.  An acceptable method for maintaining desired
temperature and salinity ranges in flow-through bioassays
with marine organisms is described in Bahner and Nimmo
d975).                                            ;
    C.  Reporting
    A coherent theory of the dose-response relationship was
introduced by Bliss (1935), and is widely accepted : today.
This theory is based on four assumptions:
    (1)  Response is a positive function of dosage, i.e., it
    is expected that increasing exposure should produce
    increasing responses.
    (2)  Randomly selected animals are normally distributed
    with respect to their response to a toxicant.
    (3)  Due to homeostasis, response magnitudes are
    proportional to the logarithm of the dosage, i.e., it
    takes geometrically increasing dosages (stresses) to
    produce arithmetically increasing responses in test
    animal populations.
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                                                August, 1982
    (4)  In the case of direct dosage of animals, their
    resistance to effects is proportional to body mass.
    Stated another way/ the treatment needed to produce a
    given response is proportional to the size of the
    animals treated.                              [
    The concentration-response curve, where percent
mortality is plotted as a function of the logarithm of test
solution concentration, can be interpreted as a cumulative
distribution of tolerance within the test population
(Hewlett and Plackett 1979).  Experiments designed to
measure tolerance directly (Bliss 1944) have shown that in
most cases tolerance is lognormally distributed within an
experimental population in most cases.  Departures from the
lognormal pattern of distribution are generally associated
with mixtures of very susceptible and very resistant
individuals within a population (Hewlett and Plackett
1979).  In addition, mixtures of toxicants can produce
tolerance curves which deviate significantly from the
lognormal pattern (Finney 1971).
    If tolerances are lognormally distributed within the
experimental population, the resulting concentration-
response curve will be sigmoidal in shape, resembling a
logistic population curve (Hewlett and Plackett 1979).
While estimates for the mean lethal dose can be made
directly from the dose response curves, a linear trans-
formation often is possible, using probit (Bliss 1934;
Finney 1971) or logit (Hewlett and Plackett 1979) trans-
formations.
    Once the mortality data have been transformed, a
straight line can be fitted to the data points.  This line
is more often fitted by eye (APHA 1975), but a least square
                                24

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                                                         ES-4
                                                 August,  1982
linear regression procedure is strongly recommended  (Steel
and Torrie 1960).  From the regression equation,  confidence
limits can be determined for predicted mortality  values.  An
additional advantage  is that the significance  of  the slope
of the regression line can be. determined  (Draper  and Smith
1966).  By using replicate experimental chambers,  an
analysis of variance  can also be performed  to  determine
whether deviations of data points from the  regression line
are random fluctuations and indicate whether a linear model
is an appropriate representation of the data points  (Draper
and Smith 1976).
    While values for  the mean lethal dose,  LC50,  can be
estimated graphically from the linearized concentration-
response curve  ( APHA  19-7.5.)-, ~o.ther~.technique.S-...ar.e~pr.ef er.able-..
since the graphical method does not permit  the calculation
of confidence limits.
    The probit method (Finney 1971) uses  the probit  trans-
formation and the maximum likelihood curve  fitting
technique.  The Litchfield and Wilcoxon method (19'49)  is  a
modified probit method which does not require  partial kills,
as does the unmodified probit method.  The  log it  method
(Ashton 1972) utilizes either the maximum likeli-hood or  the
minimum chi-square method (Berkson 1949)  to estimate LC50.
The moving average (Thompson 1947) is simple to apply but
depends on the symmetry of the tolerance  distributions to
provide accurate estimates.
    The moving average method can only be utilized' to
calculate the LC50.   An additional disadvantage of this
method is that confidence limits for LC50 cannot  be
calculated if partial kills are not available.
                                25

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                                                         ES-4
                                                August,  1982
    The lack of partial kills seriously  impairs the utility
of the probit, logit, and moving average methods.  : In
situations where there are no partial kills, the binomial
test (Siegal 1956) can be used to estimate  the confidence
limits around the LC50 value (Stephan, 1977).  The  LC50
value can be calculated from the relation:

                     LC50  =  [(A)  x  (B)]/2
    where

         A = concentration at which no organisms die
         B = concentration where all organisms die

    A and B are the confidence limits .of . the - es tima.te_and	
are significant above the 95 percent level since more than
six test organisms are exposed at each concentration level
(Stephan 1977).
    If dose-response data is plotted for each 24 hour
interval throughout the test, the LC50 determined  from each
curve can be plotted as a function of time, yielding an
acute toxicity curve (APHA 1975).  This curve approaches the
time axis asymptotically, indicating the final or  threshold
value for LC50.  The absence of a threshold LC50 may
indicate the need for an acute test of longer duration.
III.  Economic Aspects
    The agency awarded a contract to Enviro Control, Inc. to
provide us with an estimate of the cost for performing a
flow-through acute toxicity test.  Enviro Control supplied
us with two estimates; a protocol estimate and a laboratory
survey estimate.
                                26

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                                                         ES-4
                                                August,  1982
                      Protocol Estimate
                                                  i
                         range                 mean
    Acute             $510-$1529              $1019

    This estimate was prepared by separating the guidelines
into individual tasks and estimating the hours used to
accomplish each task.  Hourly rates were then applied to
yield a total direct labor charge.  An overhead rate of 115
percent, other direct costs of $105, a general and
administrative rate of 10 percent and a fee of 20 percent
were then added to the direct labor charge to yield the
final estimate.

                 Laboratory Survey Estimate
                         range
    Acute             $1000-$1450
    The laboratory survey estimates were based on two
laboratory estimates.
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                                                August, 1982
IV.   REFERENCES
    Ashton WD.  1972.  The logit transformation.  New York:
    Hafner Publishing Co.

    Benoit DA and Puglisi FA.   1973.  A simplified flow-splitting
    chamber and siphon for proportional diluters.   Water Res.
    7:1915-1916.

    Berkson J.  1949.  The minimum Chi-square and  maximum
    likelihood solution in terms of a linear transform, with
    particular reference to bioassay.  J. Amer. Stat. Assoc.
    44:273-278.

    Bernhard M and Zattera A.   1970.  The importance of avoiding
    chemical contamination for a successful cultivation of marine
    organisms.  Helgo. Weiss.  Meres. 20:655-675.

    Biship JM and Herrnkind WF.  1976.   Burying and molting of
    pink shrimp, Penaeus duorarum (Crustacea: Penaeidae) under
    selected photoperiods  of white light and ultraviolet light.
    Biol. Bull. 150(2):163-182.

    Bliss CI.  1935.  The calculation of the dosage-mortality
    curve. Ann. Appl. Biol. 22:134-307.

    Chan E and Laurence A.  1974.  Effect of antibiotics on the
    respiration of the post-larval brown shrimp, Penaeus aztecus
    Texas. Sci. 25:134.

    Clark SH and Caillouet CW.  1975.  Diel fluctuations in
    catches of juvenile brown  and white shrimp in  a Texas
    esturine canal.  Contri. Marine Sci. 19:119-124.

    Cook HL and Murphy MA.  1969.  The  culture of  larval Penaeid
    shrimp. Trans. Amer. Fish. Soc. 98:751.

    Copeland BJ and Bechtel TJ.  1974.   Some environmental limits
    of six Gulf Coast estuarine organisms. Contri. Mar. Sci.
    18:169-204.

    Couch JA.  1974.  An enzootic nuclear polyhedrosis virus of
    pink shrimp: ultrastructure, prevalence, and enhancement.  J.
    Invert. Path. 24:311-331.
                                28

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                                                    ES-4
                                            August, 1982
Courtenay WR and Roberts MH.  1973.  Environmental effects on
toxaphene toxicity to selected fishes and crustaceans.
Ecological Research Series No. EPA-R3-73-035.  April  1973,
73pp.

Curtis MW, Copeland TL, Ward CH.  1979.  Acute toxicity of 12
industrial chemicals to freshwater and saltwater organisms.
Water Res. 13:137-141.

DeFoe DL.  1975.  Multichannel toxicant injection system for
flow-through bioassays.  J. Fish Res. Bd. Canada 32:544-546.

Delves-Broughton J and Poupard CW.  1976.  Disease problems
of prawns in recirculation systems in the U.K.  Aquaculture
7:201-217.

Draper NR and Smith H.  1966.  Applied regression analysis.
Ne York: John Wiley and Sons.

Drummond RA and Dawson WF.  1970.  An inexpensive method for
simulating diel pattern of lighting in the laboratory. Trans.
Amer. Fish Soc. 99:434-435.

Forster JRM and Beard TW.  1974.  Experiments to assess the
suitability of nine species of prawns for intensive
cultivation. Aquaculture. 3:355-368.

Hansen DJ, Schimmel SE, Matthews E.  1974.  Avoidance of
Aroclor 1254 by shrimp and fishes. Bull. Environ. Contam. and
Tox. 12(2):253-256.

Hewlett PS and Plackett RL.  1979.  The interpretation of
quantal responses in biology.  Baltimore, MD: University Park
Press.

Karim M and Aldrich DW.  1976.  Laboratory study of the food
preferenc of post-larval brown shrimp, Penaeus aztecus (Ives)
and White shrimp JP. setiferus (Linneaus). Balgladesh  J. Zool.
4(1):1-11.

Kester DR, Dredall IW, Connors DN, Pytokowicz RM.  1967.
Preparation of artificial seawater. Limnol. Oceanog.  12:176-
179.
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                                            August, 1982
Knudsen EE, Herke WH, Mackler JM.  1976.  The growth rate of
marked juvenile brown shrimp, Penaeus aztecus, in a semi-
impounded Louisiana coastal marsh. Proc Gulf. Caribbean Fish
Inst. 29:144-159.

Krugel J, Jenkins D, Klein SA.  1978.  Apparatus for the
continuous dissolution of poorly water-soluble compounds for
bioassays.  Water Res. 12:269-272.

Lakshmi GH, Venkataramiah A, Howse HD.  1978.  Effects of
salinity and temperature changes on spontaneous muscle
necrosis in Penaeus aztecus Ives. Aquaculture. 13:35-43.
Lasker R and Theilacker GH.  1965.  Maintenance of euphausid
shrimp in the laboratory. Limnol. Oceanogr. 10:287-288.

Lemke AE, Brungs WA, Halligan BJ.  1978.  Manual for
construction and operation of toxicity-testing proportional
diluters. EPA Report No. 600/3-78-072.

Lowe JI, Parrish RR, Wilson AJ, Wilson PD, Duke AT.  1971.
Effects of Mirex on selected estuarine organisms. Trans. 36th
N. Amer. Wildlife and Nat. Res. Conf., Gulf Breeze. Contrb.
No. 124.

Martosubroto P.  1974.  Fecundity of pink shrimp, Penaeus
duorarum Burloenroad.  Bull. Mar. Sci. 24(3):606-627.

Mock CR.  1974.  Larval culture of penaeid shrimp at the
Galveston Biological Laboratory. NOAA Tech. Rep. NMFS
Circular. 388:33-40.

Mount DI and Brungs WA.  1967.  A simplified dosing apparatus
for fish toxicology studies.  Water Res. 1:21-29.

Nimmo DR and Bahner LH.  1974.  Some physiological conse-
quences with polychlorinated biphenols and salinity stress in
Penaeid shrimp. In: Pollution and Physiology of Marine
Organisms.  New York: Academic Press.

Nimmo DWR and Bahner LH.  1976.  Metals, pesticides and
PCB's; toxicity to shrimp singly and in combination.  In:
Estuarine Processes. Vol. I. New York: Academic Press, pp.
523-532.
                            30

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                                                     ES-4
                                             August,  1982
Parrish PR, Couch JA, Forester J,  Patrick  JM,  Cook GH.
1973.  Dieldrin: effects on several  estuarine  organisms.
Proc. 27th Ann. Conf. of S.E. Assoc. Game  Fish Commissions.
pp.  427-434.

Parrish PR, Schimmel SC, Hansen  DJ,  Patrick  JM,  Forester J.
1976.  Chlordane: effects on several estuarine organisms.   J,
Tox. Environ. Health. 1:485-494.

Penrose WR and Squires WR.  1976.  Two devices for removing
supersaturated gases in aquarium systems.  Trans  Am.  Fish
Soc. 105(1):116-118.

Rose CD.  1975.  Extensive culture of Penaeid  shrimp  in
Louisiana salt-marsh impoundments. Trans Am. Fish Soc.  2:296-
307.
Schimmel SC, Patrick JM,  Eaas LF.   1978.   Effects  of  sodium
pentachlorophenate on several estuarine animals: Toxicity,
uptake and depuration...  In:. Rao..RK,_ed —..Pen.tachlorp-henol,.	
New York: Plenum Publishing Co.

Schimmel SC, Hamaker TL,  Forester J.   1979.   Toxicity and
bioconcentration of EPN  and leptophosin estuarine  animals
USEPA/ERL Gulf Breeze. Contribution No. 354.

Siegel.  1956.  Nonparametric statistics  for  the behavioral
sciences.  New York: McGraw-Hill, Publ. Co.

Subrahmanyam CB.  1976.  .Tidal and  diurnal rhythms  of
locomotory__activity-..and. _oxy-gen_cons-umption—in—the.pink  	
shrimp, Penaeus duoranum.  Contrib. Marine Sci. 20:123-132.

Supplee VC and Lightner,  DV.  1976.  Gas-bubble disease due
to oxygen supersaturation in raceway - reared California
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Tagatz ME, Borthwick PW,  Forester J.   1975.   Seasonal effects
of leached mirex on selected estuarine animals. Arch. Environ
Contam. Tox. 3:371-382.

Tagatz ME, Borthwick PW,  Invey JM,  Knight J.   1976.   Effects
of leached..mir.ex. on:experimen-t.al-~c:ommunr±:i:es.v-ofc--es-tuarrn.e.-,:. . ^
animals.  Arch. Environ  Contam. Tox. 14:435-442.
                            31

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                                                    ES-4
                                            August, 1982
Temple RF.  1973.  Shrimp research at the Calves ton
Laboratory of the Gulf Coast Fisheries Center.; U.S. Marine
Fish Ser. Mar. Fish. Rev. 35(3-4), 16-20.

Thompson WR.  1947.  Use of moving averages and interpolation
to estimate median effective dose.  I.  Fundamental formulae,
estimation and error, and relation to other methods.
Bacteriol. Rev. 11:115-145.

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Environmental Protection Agncy.

Veith GD and Comstock VM.  1975.  Apparatus for continuously
saturing water with hydrophobic organic chemicals.  J. Fish
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Walker A.  1975.  Crustacean aquaculture. Proc. Nutr. Soc.
34:65-73.


Wickham DA and Minkler. FC...  197S.  Laboratory observations on
daily patterns of burrowing and locomotor activity of pink
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43.

Zaroogian GE, Pesch G, Morrison G.  1976.  Formulation of an
artificial seawater media suitable for oyster larvae
development.  Am. Zool. 9:11-41.
                            32

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                                       DRAFT
                              BG-8
                              August/ 1982
       ALGAL ACUTE TOXICITY TEST
       OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND  TOXIC SUBSTANCES
  U.S.  ENVIRONMENTAL PROTECTION  A3ENCY
         WASHINGTON, D.C. 20460

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                     TABLE OF CONTENTS
         Contents                                     Page
I.     Purpose                                         1
II.    Scientific Aspects                              2
       Test Procedures                                 3
       General                                         3
                                                  i
       Range-finding Test                              6
       Definitive Test                                 7
       Analytical Measurements                         9
       Test Conditions                                 10
       Test Species                                    10
       Facilitites                                     14
       Test Containers                                 15
       Cleaning and Sterilization                      15
       Conditioning                                    16
       Nutrient Medium                                 16
       Environmental Conditions                        17
       Reporting                                       20
III.   Economic Aspects                                20
IV.    References                                      22

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Office of Toxic Substances                             ES-5
                                               August, 1982
  TECHNICAL SUPPORT DOCUMENT FOR ALGAL ACUTE TOXICITY TEST
    Purpos e
    The purpose of this document is to provide the
scientific background and rationale used in the development
of Test Guideline EG-8 which uses freshwater and marine
algae to evaluate the acute toxicity of chemical
substances.  The Document provides an account of the
scientific evidence and an explanation of the logic used in
the selection of the test methodology, procedures and
conditions prescribed in the Test Guideline.  Technical
issues and practical considerations relevant to the Test
Guideline are discussed.  In addition, estimates of the cost
of conducting the tests are provided.
II. Scientific Aspects
    A.  Test Procedures
         1.  General.  A balanced growth of algae in the
aquatic environment is essential, but extremes in
productivity may be detrimental to other organisms.  Some
algae are able to inhibit or stimulate the growth of other
algae, for example Selenastrum can inhibit Microcystis
growth in eutrophic water (Toerien et al. 1974).  Inhibition
of algal growth would alter the food web and reduce the
productivity of ecosystems.  The toxic effect of a chemical
or other inhibitor may increase the susceptibility of algae
to other environmental stresses (Fisher and Wurster 1973).
Stimulation of algal growth may cause an algal bloom which
may have negative aesthetic effects; may adversely affect
commercial sport fisheries (Lightner 1978, Lovell 1979) and
recreation; may -impart unpleasant taste- to- drinking---wa-te-r;  -
may release substances deleterious to aquatic animals,

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                                                       ES-5
                                               August, 1982
and/or may indirectly kill aquatic organisms by creating
anoxic conditions (Shilo 1964, Schwimmer and Schwimmer
1967).  Stimulation of algal growth, while primarily a
problem in eutrophic freshwaters, has created serious
ecological problems in the open ocean as well.  In the
spring of 1976 and extending into the fall, there was an
extensive algal bloom, dominated by Ceratium tripos, located
off the New Jersey coast.  The bloom, together with a dearth
of storm activity, anomalous surface wind conditions, and
unusually warm sea surface temperatures resulted in a huge
anoxic area, 100 miles long and 40 miles wide which had a
severe impact on the finfish and shellfish populations  in
the area.  The immediate effects on commercial and sport
fishes, lobsters, and shellfish were not entirely .known.
However, an estimated 59,000 metric tons of .surf, clams  were
killed (representing twice the annual U.S. harvest), and up
to 50% of other shellfish populations sampled were killed.
One commercial trawler reported up to 75% of fish collected
were dead.  It was predicted that these mortalities would
affect recruitment, population size and harvests for years
to come (Sharp 1976).
    Another more commonly known phenomenon is the adverse
effect caused by stimulated growth of toxigenic marine
algae.  Frequently explosive mass development of these
organisms in the form of blooms and tides occur, resulting
in fish kills, contaminated shellfish, and outbreaks of
paralytic shellfish poisonings in humans.  (Shilo 1964,
Taylor and Seliger 1979).
    Even when toxigenic organisms, are not present....in	 	
sufficient concentrations to affect human health, red tides

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                                                       ES-5
                                               August, 1982
may reduce the market for shellfish because of adverse
publicity (Council on Environmental Quality, 1979).
Furthermore, the high concentrations of phytoplankton that
occur during blooms can be harmful to shellfish because the
rate of water transport by molluscs is reduced and feeding
ceases (Galtsoff 1964).
    Algal growth was selected to measure phytotoxicity for
the following reasons:
    o    The selection of phytoplanktonic algae for toxicity
         testing is based upon their importance in aquatic
         ecosystems.  Algae were one of the first cellular
         life forms, dating as far back as 3.1 billion years
         in the fossil record (Bold and Wynne 1978) and are
         numerous today.  Because phytoplankton are
         ubiquitous., it is usually the case that,most marine
       ,  and freshwater ecosystems are based upon the
         primary production of phytoplankton (Stern and
         Stickle 1978).  Primary production is of prime
         significance to estuarine energetics since the
         primary producers are at the base of the food
         web.  In estuaries phytoplankton are the main
         primary producers in the water (Vernberg 1977).
    o    Algae convert inorganic carbon to organic carbon
         and liberate oxygen during photosynthesis.  Thus,
         they are primary producers of food and energy for
         the lower trophic-level herbivores which in turn
         provide food for the upper trophic-level
         carnivores, generally fishes (Vance and Maki
         1976).  Some species fix nitrogen, required for .the
         growth of vascular plants.  Therefore, much of the

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                                               ES-5
                                      August,  1982
food people eat and the oxygen they breathe are the
result of algal productivity.
Inferences may be drawn from laboratory tests for
inhibition or stimulation of algal growth  as  to the
extent to which a chemical substance can interfere
with primary productivity and nutrient cycling  in
lakes, streams, estuaries, and oceans.  Further
inferences may be drawn from algal bioconcentration
data as to the potential of a chemical substance to
bioaccumulate  in food chains.  However, in the
natural environment there are too many factors
acting to regulate algal populations which cannot
be simulated in a simple laboratory test.   The  real
value of--the -test guideline -is -to" determine	
thres hold toxici.ty.-va.lues..-and—to .-ev.aluate^-th.e- _		
relative  toxicity of test substances to one another
under rigidly controlled conditions.
Algal testing has been well established in the
literature.  In 1967, the EPA began developing
algal assays for evaluating the ecological effects
of pollution to the environment.  Initially
designed  for considering problems associated  with
eutrophication (Maloney and Miller 1975),  algal
assays have also been used to define the toxic
effects of heavy metals (Davies 1978), pesticides
(Schauberger and Wildman 1977, Walsh and Alexander
1980), oil spills (Corner 1978, Fisher and Wurster
1973, O'Brien and Dixon 1976, Vandermeulen and
Ahern 1976), chemical substances  (USEPA 197.8 -a,b,c,
Harding and Phillips 1978), dyes  (Little and

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                                                        ES-5
                                                August, 1982
         Chillingworth  1976),  complex industrial wastes
         (USEPA  1978d,  Walsh and Alexander 1980, Walsh et
         al.  1980)  and  natural organic components of fresh
         and  marine water (Prakash and Rashid 1968).  Over
         the  years,  extensive  use of  this test has
         sufficiently refined  it to qualify as a standard
         method  to  measure water quality.  Algal assays are
         recommended for use by the APHA (1975) USEPA (1977,
         1978 a,b,c,d)  and are currently under review by the
         American Society for Testing and Materials.
         Further discussion on the validity of applying
         algal assays in water quality assessment is found
         in  Fitzgerald  (1975);  Joint Indus try/Government
         Task Force on—Eutrophl-cat-ion--(-19&S)-;'~Le-ischman'e't  •
    	°  al  (1979);  .USEPA UaT-abUMiller-B-t-al._.(_l9-Z8)-;		
         Murray  et  al.  (1971);  Reynolds  et al. (1974);  and
         USEPA (1971, 1975a).
    o    The  algal  growth method is 1) relatively .rapid, 2)
         inexpensive, 3)  capable of being performed by
         persons with minimal. technical  training and 4)
         reproducible,  using large numbers  of organisms with
         sufficient replication and precision.
    The test procedure  involves  assessment  of algal growth
in test chambers relative to controls by requiring! a ~
quantitative determination of  algal cell numbers, and by
recommending  a)  a qualitative  appraisal  of  algal numbers and
size by means of microscopic observation, and b) a
determination of viability of  growth-inhibited alglae by
means of -mortal-s-ta-i-ning-. coupled-wi-th- microscopic---!?---••_-..- --.•-
observation  and/or  subcultur ing.  The test  procedure is

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                                                        ES-5
                                                August,  1982
simple because it requires only  the combination  of  set
amounts of test substance, nutrient medium and  algae,  and
then monitoring the growth response 96  hours  later.   At the
end of 96 hours a further  assessment  of growth  and  viability
is recommended.
    In the test the following  procedures are required:
    o    Algal growth should be  logarithmic at  the  beginning
         of  the test and algal number should be determined.
    o    The number of  algae should be  determined at the end
         of  the test.
    o    The concentration of  chemical  in the test  solution
         should be determined  at the  beginning  and  end of
         the test and  the  concentration of chemical
         associated"wi-th~the~alga±" cel'ls "srhou±d~al"s'o""tie"
         determined.          .     .  . .                  .
    o    growth and bioconcentration  data should be
         subjected to statistical analyses.
    These requirements  will  ensure consistency  and  will
minimize variability of  the  test results.   The  test also
recommends testing of  algicidal  and/or algistatic chemical
effects.
         2.  Range-Finding Test
    It is recommended  that a range-finding test be  conducted
prior to the definitive test irr those Instances'where no
information  is available or  can  be elucidated on the
phototoxicity of the test  chemical.   This  approach  should
minimize the possibility that  an inappropriate  concentration
series will  be utilized in the definitive test  and  under
c e rt a in -c i rcums-tanees -ffla-y-^ve^'pr-eerl-ude—t-he-_*need^ttos.'condu:c-ti:~
the definitive test.   In order to minimize the  cost and time

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                                                   :     ES-5
                                                August, 1982
required to obtain  the  requisite data nominal concentrations
are permitted,  test duration may be shortened, replicates
are not required and other  test procedures and conditions
are relaxed.
    If test results indicate that the chemical is non-toxic
or very toxic to algae  and  if  definitive testing is not
conducted, it is necessary  to  ascertain that the control
algae have attained a logarithmic growth rate by 96 hours
and that the test was conducted at the specified incubation
temperature.  These verifications establish-.that the-.algae
tested were viable  and  that the test was properly .conducted.
    In some situations  there may be enough information
available on toxicity to  select the appropriate concen-
tration -wf t hou t~ a—ra-rrge^f-rrrdl-ng-- tes tv—The—range-f-trrdtn-g	
test (or other  available..J.iif-O.rma-tioji.)._jxeeds, ,.to-_be. accurate—
enough to ensure  that dose  levels  in the definitive test are
spaced to result  in  concentrations above and below the EC-10
and EC-50 values  for algal  growth  and mortality.  If the
chemical has no measurable  effect  at the saturation
concentration  (at-least  1000 mg/1),  it is considered
relatively nontoxic  to algal growth and definitive testing
for effects on these processes  is  deemed unnecessary.  In
all cases, the range-finding test  is conducted to reduce the
expense involved with- -having -to--repeat *a~definitive~tes t -
because of inappropriate tes t .chemical concentrations.
         3.  Definitive  Test
    The specific  requirements  of  the definitive test are the
analytical determinations of chemical concentrations, the
unbiased select i©.n-_of„-aLgaie-£ai^p:eae--h-.^tEea-tme^fcfi&t^
controls, the assessment of  test validity,  and the

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                                                         ES-5
                                                August,  1982
recording, analysis,  and  presentation of data.  These
requirements  assure  that  the chemical concentration -  algae
response relationship is  accurately known, that chemical
effects are not  confounded by differential algal growth  and
that the relationships  are clearly present.  Reporting the
occurrence of such abnormal effects as irregular cell  size
or shape, clumping,  loss  of chlorophyll, cell mortality,  or
other unusual effects provides qualitative data that further
assist the assessment of  phytotoxicity.
    The purpose  of the definitive, .test.. is~.to determine- the
EC-10,. EC-50  and concentration-response- curves-for-.algal	
growth for each  species tested with a minimum of testing
beyond the range-finding  test.  The concentration range  for
t he- -de f -i n-i ti-ve—t es"t~i-s~t>as ed~upo-n—trhe~ res-ai t'S"~of ~~the^rancpe-""'
f inding for .that speci.es..	I.t_is,.pxob^bLe-_tha.t.^ea.ch-.JDf---th-e--M._.
species tested may have a different estimated EC-50 based on
the range-finding test and that more than five
concentrations of a  test  substance in a geometric series  may
be needed to  properly describe the dose-response
relationship..for- either- species being tested-.— By test-ing -a
minimum of five  concentrations in a series per.species,	
the dose-response relationship will be. bet ter~ defined .   The -
slope and shape  of the dose-response curve can give an
indication -of --the-mode-of— action~of" the -chemical and will
allow estimatiion of the  effects of lower concentrations  on
the algae.
    The primary  observations - number of algae per  chemical
and determination of the  actual chemical concentrations
employed,_in^the^ef-ini±»i.v-e~^tes-tv,—a-re^needed -to?;?accu:r-afeel-.y.'^i=:;i
describe the  dose-response curve from which the EC-10  and
EC-50 are calculated.

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                                                        ES-5
                                                August,  1982
    The recommended experimental design  is  the  randomized
complete block.  As discussed  by Hammer  and Urquhart (1979),
it is essential that the  investigator  randomly  assign test
containers to  treatments  to  assure  that  each aliquot of
algae has the same chance of receiving any  of the  treatments
(exposure level of test chemical).   To account  for variation
within the growth chamber and  to increase the sensitivity
for detecting  treatment differences, small  square  blocks
should be delineated in the  growth  chamber  with
randomization of treatment within blocks.. —. Replica-tion	...
should occur over growth  chambers (of  the same  type.)  as,-in...
many cases, a within-growth  chamber estimate of residual
variance badly underestimates  the between chamber  estimate
(Hamme r -a-nd—Urq-u-h^rt^l-S^tv^Th-is^
between growth..chambers are.  of ten .greater.,-±han;,,dif f.erences	
between growth and environmental conditions  within chambers.
         4.  Analytical Measurements
    The actual chemical concentration  used -in-the_definitive
test should be determined with the  best  available  analytical
precision.  Analys is-~of- stock  solutions-  and-test-solutions •
just prior to use will minimize problems with storage (e.g.,
formation of degradation products,  adsorption,
transformation, etc.).  Nominal concentrations  are  adequate
for the -purposes-of- the—range-f- irnding- test:  "If "definitive "  "
testing is not required because the  chemical elicits  an
insufficient response at  the 1000 mg/1 level in the range-
finding test, the concentration of  chemical  in  the  test
solution should be determined  to confirm the actual exposure
level.		.--   	

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                                                       ES-5
                                               August, 1982
    The pH of the test solution should be measured prior to
testing to determine if it lies outside of the species
optimal range.  While it is recognized that algae may grow
over a broad range of hydrogen-ion concentrations and
typically exhibit a pH optima for logarithmic growth, this
test guideline does not include pH adjustment for the
following reasons: the use of acid or base may chemically
alter the test substance making it more or less toxic, the
amount of acid or base needed to adjust the pH may vary from
one test solution concentration to the next, and the effect
the test chemical has on pH may indirectly affect growth and
development of the algae.  Therefore, the pH of each test
solution should be determined and compared to the acceptable
range for growth and development of the test algae.
    The data obtained in bioassays are usually expressed as
standard response curves in which growth response of the
test species is plotted against the concentration of the
test chemical.  The manner of expressing algal growth
response varies considerably.  For this guideline algal
growth responses are expressed as direct measurements of
number of algae per ml of solution.  The statistical
analysis (goodness-of-fit determination) facilitates
accurate calculations of EC-10 and EC-50 as well as
providing confidence limits for the concentration (dose)-
response curve.
    B.  Test Conditions
         1.  Test Species
    Both Salenastrum capricornutum and Skeletonema cos tatum
have a number of useful characteristics as listed below,
                                10

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                                                       ES-5
                                               August, 1982
which are necessary for an algal species to be used in
bioassays (Toerien et al. 1971):
     (a)       broad nutrient response  (grows both in
              oligotrophic and euthropic waters).
     (b)       distinct shape
     (c)       uniform size
     (d)       divide distinctly
     (e)       do not attach to glass or surface
     (f)       stay in suspension with slight agitation
     (g)       cells do not clump (aggregate)
     (h)       grow at a maximum rate in a short time in a
              medium simple to constitute
     (i)       do not excrete autotoxins
     (j)       cells are easy to count by both direct or
              indirect methods.
     Selenastrum capricornutum is an excellent laboratory
freshwater organism, easy to culture and count, and is both
sensitive and consistent in its response to a wide range of
nutrient levels (Payne and Hall 1979).
    When included in multispecies toxicity screening tests,
Selenastrum has been found to be a comparably sensitive
species.  Maki and Macek (1978) found this to be true in an
environmental safety assessment for a nonphosphate detergent
builder.  Selenastrum was as sensitive to trinitrotoluene as
the copepod, Trigriopus californicus, and was twice as
sensitive as oyster larvae (Smock et al. 1976).  Selenastrum
was as sensitive as Daphnia and the fathead minnow to eight
preparations of synfuels (Greene, personal communication).
In a study of the toxicity of 56 dyes to Selenastrum and
fish (fathead minnows), basic dyes do not markedly inhibit
                                11

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                                                       ES-5
                                               August, 1982
algal growth, and "of special significance, however, is the
rather startling correlation between results of algal assays
and the results of fish bioassays" (Little and Chillingworth
1974).  Greene (personal communiation) analyzed the results
of this study and found the algae appear more sensitive than
fish to 35 of the dyes tested while the fish were only more
sensitive to seven of the dyes tested.  In a recent test
conducted on 35 chemicals on the EPA priority pollutant list
by EG & G Bionomics  (Parrish, personal communication), there
were no significant differences in the EC-O's between
Selenastrum and Skeletonema, Daphnia and bluegill fish,
Lepomis macrochirus.  Selenastrum was significantly more
sensitive than sheepshead minnow.  In another 2 tests EG & G
performed for Monsanto Industrial Chemical Co. (1979a,b)
evaluating two phthalate esters (Santicizer 60 and 711),
Selenastrum was as sensitive as Microcystis aerugenosa,
Navicula pelliculosa, Skeletonema costatum and Dunaliella
tertiolecta.  Palmer (1969) has extensively reviewed the
algal literature and has ranked the 60 most pollution
tolerant genera as reported by 165 authors.  In comparing
two green algae often used in algal toxicity testing,
Chiorella and Scenedesmus to Selenastrum, great variation  is
found.  Of the 60 genera, Scenedesmus  was the fourth most
tolerant, Chlorella  was the fifth most tolerant, but
Selenastrum was the fifty-seventh most tolerant.  This
analysis is borne out by recent results obtained by Green
(personal communication) in testing effluent toxicity to
algae.  He found that Chlorella and Scenedesmus are
generally more resistant to industrial effluents .and both-
were naturally present in 100% effluents (eight submitted  by
                                12

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                                                        ES-5
                                               August,  1982
the US EPA  Industrial Environmental Laboratory,  Research
Triangle Park, Raleigh, North Carolina).   Selenastrum  only
grew when  the effluents were diluted to 1-10% of the
original concentration  (which supported Chlorella  and
Scenedesmus growth).  This was also the case in another
effluent which contained 1.7 rag/1 cyanide.  Both Chlorella
and Scenedesraus grew in it, but Selenastrum grew only  when
the effluent was diluted to 1% or less.   Chlorella has also
recently been shown to be much less sensitive to toxics than
Daphnia or fish (Kenaga and Molenaar, 1979).
    While  it is recognized that numerous  marine algae are
sensitive  to toxicants (North et al. 1972); heavy metals
(Davies 1978), simple organics (benzene,  cresol, hexane,
phenol and toluene), various inorganics (Cl, CN, Hg) and
complex wastes (industrial sewage, sulfite waste liquor,.- ...
detergent), and petroleum compounds (Corner 1978),
Skeletonema costaturn was selected for use  in the toxicity
test guideline.  This species has been frequently reported
on in the  bioassay  literature (US Army 1978), and is a
recommended bioassay organism (APH 1975,  USEPA 1977a, b,
1978, Gentile and Johnson 1974).
    The testing procedure for Skeletonema  has recently
proven useful for the evaluation of the relative potential
hazards of a compound or a complex waste  by providing data
for the calculation of the EC-50 or SC-20  (Walsh and
Alexander  1980, Walsh et al. 1980).  Skeletonema was as
sensitive  to the 35 priority pollutants and two phthalate
esters as  Selenastrum in multi-species toxicity screening
tests, as  in the previously described studies.-  --
                                13

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                                                       ES-5
                                               August, 1982
    Skeletonema was found to be more sensitive (at lOppb) to
growth inhibition effects induced by PCB's'than two
freshwater algae (Euglena gracilis and Chlamydomonas
reinhardtii) and two other marine algae (Thaiassios ira
pseudonana, and Dunaliella tertiolecta) (Mosser et al.
1972).
    Skeletonema costatum was also more sensitive  (growth
inhibited) at lower concentrations of wastewater
chlorination products (3-chlorobenzoic acid, 5-chlorouracil,
4-chlororesorcinol, 3-chlorophenol and Captan) than
Dunaliella tertiolecta and Porphyridium sp.  (Sikka and
Butler 1977).
    Skeletonema and Selenastrum are specified for testing
toxicity of pest-icides (-Subpart -J-,--Pesticide-Regrs-trafion	
Guidelines).  Additional justification .for. seLection. _of_ -	_..
these test species is provided in these guidelines (see  PR
45 (214): 72948-72978).
    Other species may be substituted for either of these two
species when appropriate.  Some freshwater or marine species
which are of concern or have a significant ecological role
may constitute a more crucial risk population.  If so, those
species of particular ecological or economic value should be
selected.  The rationale for selection of alternative
species should be discussed with the Agency  and/or supported
in the report of findings.
         2.  Facilities
              a.  General                         :
    The test requires a growth chamber or  temperature
controlled- enclosure-capable =of maintaining :-a-uniform .-•  —. -
temperature of 24° _+ 1°C if Selenastrum is tested or
                                14

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                                                        ES-5
                                                August, 1982
20° +_ 1°C if  Skeletonema  is  tested.   Other facilities
typically needed  include  standard laboratory glassware,
culture flasks, work  areas  to clean  and prepare equipment
and to measure  chemical concentrations and algal growth and
proper disposal facilities.   Without these facilities, the
testing cannot  be  adequately conducted.
              b.   Test Containers
    Sterile Erlenmeyer flasks are recommended as test and
culture containers.   Any  flask volume may be used between
125-500 ml.   However, it  is  imperative that flasks of the
same volume be used throughout the test.   Hannon and
Patouillet (1979)  found a marked  difference (2.6x)in mercury
toxicity for  marine algae,  Phaeodactylum tricornutum,
depending -on-the -s-urf-ace—r~volume "ra-tio~of—the"cu~rture~	
vessel. . Flasks -S-hould...be.-S±oppfired_wi.tn_^.terJJ.e.^iiu.gs_ .(.s-uch.
as foam rubber or  cotton  stoppers) which will prevent
possible bacterial contamination  yet allow air flow.
              c.   Cleaning  and Sterilization
    Standard  good  laboratory practices are recommended to
remove dust,  dirt, other  debris,^ and organic and inorganic
residues from the  test containers and other glassware and
supplies should be washed and sterilized to prevent
contamination.
    Algal cells are discarded at  the end of a test.  Algae
are capable of considerable  adaptation to the toxic effects
of antimetabolites and antibiotics,  such as streptomycin,
penicillin, chloramphenicol,  sulfanilimide and sodium
selenate (Kumar 1964).
    It is impo.r.tan.t,-to--av-oid-contam-ina.tion -ofit-alga-1 cultures-,
by bacteria.  Bacteria may metabolize high molecular weight
                                15

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                                                        ES-5
                                                August, 1982
organic compounds to produce  carbon dioxide and/or cofactors
that stimulate growth of  Selenastrum (Tison and Lingg 1977,
Sachdev and Clesceri 1978).   Consequently axenic cultures of
algae should be maintained  by proper sterile culture
techniques as well as growing and  testing algae in sterile
containers and nutrient medium.
              d.  Conditioning
    Test containers are to  be rinsed with appropriate test
solutions prior to the beginning  of the toxicity tests.
This method should allow  for  sorption of the test substance
to the test container, thereby saturating the container
surface so that no further  interactions of test substance
will take place when new  test solution is added and the test
begins .-- Hannan—and -Pa-toa-il~let-~(-19"7'9') "found" th'atrup" to"50%
of mercury could be .Lost  _to.-adso-rp.tiaa-_tOL--vessjel_wal.ls-. in~~a....
two-day toxicity test.  Therefore, with proper conditioning
all the test substance in the test solution should be
available to test algae and any  results will reflect an
accurate concentration response.
              e.  Nutrient  Medium
    The nutrient medium recommended in-the test guideline,
are those currently recommended  by the-US EPA for-use in
bioassays (USEPA 1977, 1978a,b,c,  Walsh and Alexander 1980,
Walsh et al. 1980).
    Use of the nutrient media under the test conditions will
ensure maximum growth rates (i.e., logarithmic) in test
algae and controls.  Selenastrum and Skeletonema will divide
2-3 times per day (Nielsen  1978,  Lewin and Guillard 1963,
US EPA 19 71 b~)	Th is s houl-d--e.nhance^exposu&e--of--4^es-ti-^aiga>er to:
the test substance because  algal  cells in this growth phase
                                16

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                                                        ES-5
                                                August, 1982
absorb and metabolize substances  at a rapid rate (Fogg
1965).  Shiroyama  et al.  (1973)  found maximum phosphorus and
nitrogen uptake occurred  in  the  first five days of growth.
    Many media used- for  culturing algae contain a chela ting
agent, usually EDTA,  to  keep micronutrients in solution.
However, a medium  containing a chelating agent is :less than
ideal for testing  toxicants  because chelators can increase
or decrease  toxicity and  can add  uncertainty to the test
results (Payne 1975,  Fogg 1965,  Prakash and Rashid 1968,
Bender 1970, Giesy 1974,  Lin .and. S.ch.elske. 1979.,. Barber...and- ...
Ryther 1969, Johnston 1964,  Droop 1960, 1962; Eyater-196.8,._._
Erickson et  al. 1970).
         3.  Environmental Conditions
    S e 1 e n as-t-ru-m- -a-ncH- Ske-le-tcm-ema— wr±±~ g r X3w"o verr—a— withe --------
temperature, range,, JLrom,Jes.S— than-S^C. .to,3 5JLC_-(.Claes son_-and —
Forsberg 1978), and between  13°C and  30°C (Fogg 1965),
respectively.  The temperature  selected for toxicity testing
using Selenastrum -was  24°C because  luxury uptake of ammonia
nitrogen, maximum specific growth rate, and sensitivity to
phenol occur at that temperature (Reynolds  et al. 1974,
1975a, 1975b 1976).  The  test temperature 20 °C .selected -for. _.
Skeletonema is recommended in other toxicity testing manuals
(USEPA 1978a,c) and in recent publications  (Walsh and
Alexander 1980, (Walsh and Alexander- 1980,  Walsh et al .
1980).
    Algae require light for  photosynthesis  and growth.
Fitzgerald (1975) and Miller et al.  (1978)  have shown that
light intensity will affect  the rate  of growth of
                          M^
procedure (Joint Industry/Government Task Force 1969)
                                17

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                                                        ES-5
                                                August, 1982
development work was done on  Selenastrum at 400 ft-c/  it was
not seen as necessary  to make  a  change  (USEPA 1978b).
Continuous lighting of algal  cultures is required  for
Selenastrum in the test guideline.   While this does not
reflect environmental conditions, it does maximize testing
for toxicity.  Practically  all toxicity tests using
Skeletonema have recommended split  day/night lighting (USEPA
1978a, 1978c, Walsh and Alexender 1980, Walsh et al.
1980).  For the sake of consistency, it was not seen  as
necessary to make a change  in the procedure.
    The test guideline requires  a test  solution pH of 7.5
for Selenastrum because  it  maximizes growth.  Selenastrum
grows between pH 4 and 10  (Brezonik et  al. 1975) and
max imal- 1-y- be twe en—pH—7~-a-nd-9^6—(-eia'es-s'om-and-FoTstrerg	—
1978).  Maximum adenosine_tr.iphos.phate^-tAIP_X—C-i—e*,~ene.r.gy	
production) occurs in  Selenastrum cultured between pH 7.5
and 8 (Brezonik et al. 1975).  The  pH selected., for-testing
with  Skeletonema, 8.1, was  selected because it is
recommended by other toxicity  testing manuals (USEPA 1978a)
and in recent- publications  (Walsh and-Alexander -1980:> Walsh
et al. 1980) and approximates  the natural oceanic-.pH.	The,-
pH should be adjusted  as exactly as_ possible—to-Jthe—test-pH
because fluctuations in pH  affects  toxicity.
    The purposes of bs ell la ting  the--cultures' are" to enhance
exposure of algal cells -to  test  substances and to enhance
dissolution and solubilization of test  subs tances >in the
test  solution.  Turbulence  created  by shaking algal cultures
is important to enhance  the transfer of dissolved substances
between-the~media andv~khe^celrls..:i™.Mu^^
showed that this transfer  is  faster if  nutrients are
                                18

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                                                          ES-5
                                                 August,  1982
 continually renewed adjacent  to  the cell by movement of the
 medium.
     Oscillating test containers  is also analogous to wind
 and wave induced mixing  of  natural waters.  This agitation
 and mixing serves to maximize algal exposure to the test
 substance.
     Temperature, light  intensity,  pH and oscillation rate
 are all recorded as specified in the test guideline to
 ensure that the environmental conditions of the test are
 met.
     Temperature should, be . recorded- at- leas t hourly ..-to. ensure   •-
 that  it does not exceed  the specified limits.   Inexpensive
 growth chambers are available which are equipped with
 adequate-recorx}rng~ins-tTorments"-cn:—ch~amtre~rs~~may "be'"equipped""	"  '
.with  .ones  at mi.nimal~cos.±..—Se.vjaxe~EluatuaJtians—in—~—_.	.-. .-
 temperature may affect  algal  growth and/or subsequent
 chemical uptake or metabolism.
     Light intensity readings  at  the surface of the solutions
 may be made manually and ensure  that all containers are
 receiving  equal light.   Light variations:::wi-lJ.-. aff.e-Ct~al.ga-l.-.:.-..- . .
 growth so daily recordings,  are .nec.essary_±o_maintai.n_.uniform	. ..
 and constant radiation* . -The- pH ._is._ me as.ur-ed_a-t--the-J3eg.i.nn-i.ng---. 	
 and end of the test as an indication of effects of test
 chemical additions and "subsequent  algal metabolism oh the
 hydrogen-ion concentration.-  This  will  indicate if the test
 solution is outside of  the  algal pH optima for growth as
 well  as show what pH variations  may exist between chemical
 concentrations.
                                 19

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                                                         ES-5
                                                 August, 1982
    C.  Reporting
                                                   i
    The sponser should submit to the Agency  all data
developed during the  test that are suggestive  or predictive
of phy to toxic ity.   If testing specifications are followed,
the sponsor should  report that specified  procedures were
followed and  present the results.  If  alternative•procedures
were used instead  of  those recommended  in the  test
guideline, then the protocol used should  be  fully described
and justified.
    Test temperature, chemical concentrations, _tes_t..da..ta^. ..,-..
concentration-response curves,-._and._sta.tlstioal^aiLalyses.;	~,.
should all be  reported.  The justification for this body of
information is  contained in this support  document.   If algal
s pe cies othei: -tfraTV-the-^tw^                        \ the ~
rat ion ale- -for--th e—s.ele,ct -ion—of-.-the -at her,- s-pe e-i-es—&fe-oulcUJie.-r- -
provided.
III.  Economic  Aspects   .                         :
    The Agency  awarded a contract to Enviro Control, Inc.  to
provide an estimate of the cost for performing aniacute
toxicity tes t us-i-ng fr-es-hwa±er :-a-lgae ac cord rng tor the ----- ----- -•
Guideline.  Enviro Control -supplied-two.,estimate s.;.~a	
protocol estimate  and -a labora-toxy-.survey_es-timate^—	-•
    The protocol estimate was ?1760.   This estimate was
prepared by identifying~the~major tasks needed to;do a test
and estimating  the  hours to accomplish-each  task-.-  -- -
Appropriate hourly rates were then applied to  yield a total
direct labor  charge.   An estimated average overhead rate of
115%, other direct costs of $400, a general  and  :
ad mi n is-1 ra t i ve._ r.-a t e-^of-^-.iUQ^^^iandr^a—sfiejar.--of --.2:2;0.%i;we.rB
to the direct labor charge to yield the final  estimate.
                                 20

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                                                        ES-5
                                                August,  1982
    Environ Control estimated that  differences  in  salaries,
equipment, overhead costs  and other factors  between
laboratories could result  in as much as  50%  variation  from
this estimate.  Consequently, they  estimated  that  test costs
could range from $878 to $2636.
    The laboratory survey  estimate  was  $1465,  the  mean of
the estimates received from eight laboratories.  The
estimates ranged from $430 to #3600 and  were based on  the
costs to perform the test  according  to  the Guideline.
    Although a cost analysis was -not_perf.ormed~.for-a. test-- —
using marine algae, the .procedures. .us.ed_ar.e-similar _ta_the	
freshwater algal test and  the costs  should be  similar.
                                21

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                                                        ES-5
                                               August,  1982
IV.  References
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Barber RT, Ryther JH.  1969.  Organic chelators  factors
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Bender ME.  1970.  On the significance of metal  complexing
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Bold HC and Wynne MJ.  1978.  Introduction to the  algae.
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Brezonik -PL,- Browne-FX7"Fox~~JL7  1975.   Application of "ATP
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Claesson A and Forsberg A.   1978.  Algal assay procedures
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Corner EDS.  1978.  Pollution studies with marine
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Council on Enviromental_.Quality.	19.7-9-  -Ecology-and-living
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Davies AG.  1974.  The growth kinetics of Isochrysis galbana
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Davis AG.  1978.  Pollution  studies with marine  plankton.
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                                                        ES-5
                                                August, 1982
Droop Mr.   1960.   Some  chemical considerations in the design
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Erickson  SJ,  Lackie N,  Maloney TE.   1970.  A screening
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Eyster  C.   1968.   Microorganic and  microinorganic
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Fisher  NS  and Wurs ter CR.   1973.   Individual and combined
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Fogg GE.   1965.   Algal  cultures and phytoplankton ecology.
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Galtsoff  PS.   1964.   The  American Oyster, Crassos trea
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Giesy JP.   1974.   The ef fects'~of "humic acids on the growth	
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Hammer  PA  and Urquhart NS.   1979.   Precision and
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Hannan  PJ  and Patouillet  C.   1979.   An algal toxicity test
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                                23

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                                                        ES-5
                                                August, 1982
Harding LW and Phillips JH.   1978.   Polychlorinated biphenyl
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Johnston R.  1964.  Seawater,  the  natural  medium of
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Joint Industry/Government -Task Force  on Eutrophication.
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Kenega EE and Molenaar RJ.   1979.   Fish and Daphnia toxicity
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Leischman A A,* Green JC," Mil'ler WE.   1979.   Bibliography of
literature pertaining to the genus  Selenastrum.   Corvallis,
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021.

Lewin JC and Guillard R.  1963.  Diatoms.  Ann Rev.
MicrobioTv--7-r3-7-3-414v ..........  •"   •            '     ' ,

Lightner DV,  1978.   Possible  toxic effects of  the marine
blue-green alga Spirulina subsalsa,  on the blue  shrimp/
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Lin KG and Schelske CL. .-197-9.  -Effects, of .nutrient
enrichment, light intensity  and  temperature-on...gr-owth,.of. -
phytoplankton from Lake Huron.   Duluth, MN: U.S.
Envi ronmentai ^Proteet'iow^'ge-ffeyf^EP-A— 6 0 07*3^7 6 -"OTS.*^ -•*••*=-•-• - •-
Little LW and Chillingworth  MA.   1974.   Effect of 56
selected dyes on  growth  of  the  green alga Selenastrum
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Lovell RT.   1979.  Fish  culture in the  U.S.  Science
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Maki AW and  Macek KJ.  1978.  Aquatic environmental safety
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Sci. Technol.--l»12r5;73--58{K >----•-  -~         •       :
                                24

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                                                         ES-5
                                                 August, 1982


Maloney TE and Miller WE.  1975.  Algal assays:  development
and application.  STP 573.  Philadelphia, PA:  American
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Miller WE,  Greene JC, Merwin EA, Shiroyama T.   1978.   Algal
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_ .   1979b.i .TS.CA s.e.c .- 8:(.d--) = s ubmiss i
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                                25

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                                                  :      ES-5
                                               August,  1982
Payne AG and Hall RH  1979.  A method for measuring  algal
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                                26

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                                                        ES-5
                                                August,  1982


 Shiroyama  T,  Miller  WF,  Greene  JC.   1973.   Effect of
 nitrogen and  phosphorus  on the  growth of  Selenastrum
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 Sika HC and Butler GL.   1977.   Effects  of  selected
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 Tison DL and "Lingg" A3.   1977.   Algal bacterial  mutualism  in
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 Toerien  DF,  Huang CH,  Radimsky  J,  Pearson  EA,  Scherf ig J.
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 Springfield,  VA: National  Technical  Information Service.

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 The  interlaboratory precision test:  an eight laboratory
 evaluation of the provisional algal  assay procedure bottle
 test.  CorvallisT' ORY U.SY' Environmental Protection Agency.
                                27

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                                                        ES-5
                                                August, 1982
                   1973.  U.S. Environmental Protection
 Agency.  Biological field and laboratory methods for
 measuring the quality of surface waters and effluents.
 Cincinnati, OH:  U.S. Environmental Protection Agency.  EPA
 670/4-73-001.                                     ;

 _ .  1974.  U.S. Environmental .Protection
 Agency.  Marine algay assay procedure: bottle test.
 Corvallis, OR:  U.S. Environmental Protection Agency.

 _ .   1975.   U.S. Environmental Protection
 Agency.  DDT: a review' of scientific and economic aspects of
 the decision to ban its  use as a pesticide.  Washington,
 D.C.  U.S. Environmental  Protection Agency.  EPA-540/1-75-
 022.

 _ .   1977.  U.S. Environmental Protection
 Agency/Corps of Engineers Technical Committee on criteria
 for dredged and fill material.  Ecological evaluation of
 proposed discharge of dredged material into ocean waters.
 Vicksburg, MS: U.S. Environmental Protection Agency.

 _ _ __._  197 8a.  U.S.  Environmental Protection
.Agency Ocean Dispos al -Bioassay_Jto.rJcing,_GrjDup.. — Bio-assay— — .......
 procedures for the ocean disposal permit program.  Gulf
 Breeze, FL: U.S.  Environmental Protection Agency.

 _ .   1978b.  U.S.  Environmental Protection
 Agency.  The Selenastrum capricornutum Printz algal assay
 bottle test:  Experimentalal design,  application, and data
 interpretation protocol.  Corvallis,  OR: U.S. Environmental
 Protection Agency, EPA-600/9-78-018.

 _ .  1978c.  U.S. Environmental Protection
 Agency.  IERL-RTP  Procedures Manual:  Level 1 Environmental
 assessment, 2nd ed . Research Triangle Park, NC : U.S.
 Environmental Protection Agency.   EPA-600/7-78-201.

 _ .  1978d.   U.S. Environmental Protection
 Agency.  Environmental Assessment: source test and
 evaluation report — Chapman low-Btu gasification. EPA-600/8-
 78-202, Research Triangle Park,  NC:  U.S. Environmental
 Protection Agency. •
Vance  BD  and -Maki .-...AW.-*:- -193-6.
Stigeoclonium  pachydermum.   Bull.   Environ.   Contam.
Toxicol.  15:601-607.
                                28

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                                                       ES-5
                                               August, 1982
Vandermeulen JH and Ahern TP.  1976.  Effect of petroleum
hydrocarbons on algal physiology: review and progress
report.  INL Lockwood APM, ed.  Effects of pollution on
aquatic organisms.  Cambridge, Eng: Cambridge University
Press, PP. 107-125.

Vernberg FJ.  1977.  Characterization of the natural estuary
in terms of energy flow and pollution impact.  In: US EPA.
Proc. Conf. on estuarine pollution control and assessment.
Vol. I & II.  Springfield, VA:  NTIS, pps. 29-39.  EPA 44/1-
77-007.

Walsh GE and Alexander SV.  1980.  A marine algal bioassay
method results with pesticides and industrial wastes.  Water
Air Soil Pollut. 13:5-55.

Walsh GE, Bahner LH, Horning WB.  1980.  Toxicity of textile
mill effluents to freshwater and estuarine algae,
crustacean, and fishes.  Environ. Poll. (Ser A) 21:169-179.
                                29

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                                         DRAFT
                               ES-5
                               August, 1982
       TECHNICAL SUPPORT DOCUMENT

                  FOR

        ALGAL ACUTE TOXICITY TEST
       OFFICE  OF .TOXIC SUBSTANCES
OFFICE  OF PESTICrDES^AND~TOXICSUBSTANCES	'— '  ' -
  U.S.  ENVIRONMENTAL PROTECTION AGENCY
         WASHINGTON, D.C.  20460

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Office of Toxic Substances                        ,          EG-8
Guideline for Testing Chemicals                    August,  1982


                    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) (P.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

(USEPA) will use data from these tests in assessing  the hazard of

a chemical to the environment.
    (b)  Def in it ions .  The definitions in Section 3  of the Toxic

Substances Control Act (TSCA) and the definitions in Part 792—

Good Laboratory Practice Standards 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

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                                                            EG-8
                                                    August,  1982
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.  (A)  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

chambers are established at predetermined limits.

    (B)  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 EC-10,  EC-50, and

EC-90 values.

    (2)  [Reserved]

    (3)  Range-finding test,   (i)   A range-finding test should- be

conducted to determine if:

    (A)  definitive testing is  necessary

    (B)  tes t chemical-concervtrat-ions--^or^ the—d-eJrin'itu-ve •t/es-tv " ='

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                                                             EG-8
                                                    August,  1982
     (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 104 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-concen-tra-t-ion-or—

 1000 mg/1) 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 EC-

. 10' s, EC--SO.1 s~,_:-and =Ec-^Oj'-S!»vfor. al.gai- grrowtti- -Bor*ieadhjas-pe
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                                                             EG-8
                                                     August, 1982
 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, 6, 8, 16,  32 and  64 mg/1).

 Algae  should 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.-determina-tion of -tes~t~subs tanee •

 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"1 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 lowest concentrations of  test substance compared to  controls.

     (iii)  Every test should  include a control consisting- of the

_s ame nu tr.ient ,medJ..UITU,-~condi tio,ns-/~.-pru3eedur es7~-andw al-gae - firom---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 seven  to ten-day-old

 stock  cultures  are placed in  the test chambers containing test

 solutions* .havarng 5>t

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                                                            EG-8
                                                    August, 1982
substance.  Algal  growth  in  controls  should reach the logarithmic
growth phase by  96 hours  (at which time the number of algal cells
should be approximately 1.5  x 106/ml  for Skeletonema or 3.5 x
10°/ml for Selenastrum).   If growth in controls does not reach
this logarithmic phase within this 96-hour period, the test is
invalidated and  should be repeated.  At the end of 96 hours the
algal growth response  (number or  weight of algal cells/ml) in all
test containers  and  controls should be determined by an indirect
(spectrophotometry^uelectr.cmic,„cell,-counter.sr..dry_weigh.t^..etc.4
or a direct (actual  microscopic  cell  count) method.  Indirect
methods should be  calibrated by  a direct microscopic count.  The
percentage inhibition or  stimulation  of growth for each
concentration, EC-10, EC-50, EC-90 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 should-be performed.:
    (1).  De t e rm ine— whe,the.r^t^.er^al ter^dr^g.rrOWth^Ees^pans e™be:twee-n----.- *•«
controls  and test  algae was  due  to a  change in relative cell
numbers,  cell sizes  or both.  Also note any unusual cell shapes,
color differences,  flocculations, adherence of algae to test
containers, or aggregation of algal cells.
    (2)  In test concentrations  where growth is maximally
inh ibit.ed ,^ .algisJiaiii-Gcsefjfie

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                                                             EG-8
                                                     August,  1982
 algicidal effects by the following two methods:

     (A)   Add 0.5 ml of a 0.1 percent solution  (weight/volume)  of

 Evans  blue stain to a one milliliter aliquot of algae  from  a

 control  container and to a one 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 -chemieal—wkijdi-inhibited .,algai.>gcowth.)^.^--.Wait.-ten^tOt.-***^.,, ,

 thirty minutes,  examine microscopically, and determine the

 percent  of the cells which stain blue (indicating  cell

 mortality).  A staining control should be performed concurrently

 using  heat-killed or formaldehyde-preserved algal  cells;  100

 percent  of these cells should stain blue.

     (B)   Remove  0.5 ml aliquots of test solution containing

 g r owt h-i nh ib i ted algae from..each .replicate- test -container having —

-the  concentration^of^-tesi^-svubs-t-ance^evaluat?ed-'-i;H--<-^)-|^) ^above-. ^~^-=

 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 nine  days, and

                            ^

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                                                             EG-8
                                                     August, 1982
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 should be used  in the preparation of

the nutrient medium.  The pH of the test solution  should 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,-±es±_con£ainejs^S:h.auld>J}e~,detfi^                  -..

beginning and  end of the definitive test by  standard analytical

methods  which  have been validated prior to the tes.t.  An

analytical method is unacceptable i-f 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. .determinaiio.ns.,..mi.cj:.os..cLopj..c.. __..

examina.ti.on ,-
containers  for  each chemical concentration may  be pooled into one

sample.  An  aliquot- of the -poaited- sample- -may-- -then^b«^feaJcen -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

f r om - the~^tes-t2.sOiu't ion^byi..c:e4i:tr rfaa-gi rig^or^fakl.teri .iwpsthe? -.E amain*! n'g>*«-'

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                                                             EG-8
                                                     August, 1982
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-f it determination for the  concentration
response , x3ur-ves-.v,.^Jlbfi~cu3acjei^r,avti0B^^                                -..
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 _obiained._fr-om_.commerclal..-sour-ces-^nd-^_- —
s ubs equenil-y «.- eu l.tu&ed- us^ng^e^e^i-leM^ehn i^ae^^'-P^x-i e-i -ty <^tes tifl-g*9**
should not be performed until algal cultures  are  shown  to be
act ively growing*" (~ive-.  •eapabl-e-^o&^l-ogari t-hmic-gr-owth^-within^t-he' tr~
test period) in  at least two subcultures lasting  seven  days each
prior to the start of the definitive test.  All algae used for a
particular test  should  be from the same source and  the  same stock
culture .- "~Tes=t algae- sAffcai-id~=rio4r;fi ave~

                                 8

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                                                              EG-8
                                                     August,  1982
 test,  either in a treatment or  a  control.

     (2)   Facilities — (i)  General.   (A)   Facilities needed  to

 perform  this test includes:   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)   Dis^pos al -£ac i.li t i.es-~s-h.ould ^he^adequa .fce^ta-sacc cam-moida.^^^

 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 .flas.k  	

 volume ...,--

     (iii)  Cleaning and sterilization.  New test containers may

 contain - s.ubs tances^wnvir-ch: -i»rah iJsi fe=r>g>r owteh*s-of3«a-J?gaperi'--v*Ttoey-"S hou^d•« ^t,v

 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 should be  cleaned and sterilized

..prior  tor u s:e -ac coasd imgastos'sfcantlarrd^go'bdiul abxw-va tossy-rspractei C'

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                                                             BG-8
                                                    August,  1982
    ( 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 appro-

priate 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 .,s
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 should be 7.5 for Selenastrum and  8.1

for Skeletonema  at  the  start of the test and may be -ad justed- ..... --

prior to test chemical  addition with 0.1N NaOH or HC1.

    (B)  Dilutioji_-wa-t.e.r_ used_fojr_pr£p.ara-tio.n-.JDf-_-nuir-ient-- medium .....

and test-solutions^s-hou-ld^be^f.ilter.ed7^-dei=0n4=z-ed--or -gdass -••--—-•• - -

distilled.  Saltwater for marine algal nutrient medium and test

solutions - s houid^bei-prepar-ed^by^-addrimg^a1' e:mmer;e^^                *

sea salt formulation or a modified synthetic seawater formulation

to distilled/deionized  water to a concentration of 30 parts  per

thousand .

    (vi )  (2aj:rjj2r^T-rSNu^rl"en^^
                                10

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                                                              BG-8
                                                     August,  1982
 stock solutions of the  test  chemical.   If a carrier other  than
 nutrient medium is absolutely  necessary to dissolve the  chemical/
 the volume used should  not exceed  the  minimum volume necessary to
 dissolve or suspend  the chemical  in the test solution.
     (3)   Tes t par ame te rs .  (A)  The test temperature should be
 maintained at 24°±i°c for Selenastrum  and 20°±1°C for
 Skeletonema.  Temperature should be recorded hourly during the -
 test.
     ( B) _ Test ch amb e cs— contain ing^ Sele.nas^ruin.^s hoal cL Jae~ .**»*. .>&~~- .
 illuminated continuously and those containing Skeletonema should
 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^ sec (approximately 400 ft-c) measured adjacent to
 the test chambers -at -the -level of -test solution.- >- -
     (C)   Stock algal cultures  should be shaken twice daily by
 hand.  Test containers- should  be placed -on a rotary shaking.... -_
. apparatus- .and-jos c Jdlaied-.a^appEaxAma^ly^tlvOvQ*^^
 Selenastrum and at approximately 60 cycles/min for Skeletonema
 dur ing ..-.the. test^_.~.Th.e*_ra;fce^of -iOS^c^J;a*i.GLn^shoxJi-d2£:b^-u3e-t^nnd^n-ed-s£aiti
 least once daily during testing-.--  ~~
     (D)   The pH of nutrient  medium in  which algae are subcultured
 should be 7.5 for Selenastrum  and  8.1  for Skeletonema, and is  not
 ad jus ted4 ~af ten. fehe^ad diifciom-cof^itfae^l^

                                 11

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                                                             EG-8
                                                     August, 1982
solutions  and  controls should be measured  at  the beginning and

end of  the test.

    (E)  Light intensity should be monitored  at least daily

during  the test at the level of the test solution.

    (e)  Reporting.  The sponsor should 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., .±he..J£allQW.Lng---sJaQuJLd-J3e...j^epo.r.ted^;~.**>.*»*-*•....  —

    (i) Detailed  information about the test organisms, including

the scientific name,  method of verification,  and source;

    (ii)   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, osc il la t ion -rates, _and.._type-_of_-ap-par-atu.s-;-- _ -. _ _-_ . . .

    - ( i i i)   T.h.e .concentr.aTtioji.rxjfe-^t.he^teSit^chenvieal; **in -fehe ^control- •

and in each treatment at the end of the test  and the pH of the

solutions ;   --  ^ -

    (iv)   The  number  of algal cells in each treatment and control

and the method  used to derive these values at the beginning and

end of  the test;  the  percentage wof inhibition or stimulation of
                                 12

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                                                             EG-8
                                                    August/  1982
growth relative  to  controls;  and other adverse effect in  the

control and  in each treatment;

    (v)  The 96-hour EC-10,  EC-50 and EC-90 values and  their  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;

    (vi)  Methods and data  records of all chemical analyses of

water quality and test substance concentrations, including method

validations .and ..re agent-blanks ;<. — ~~	~- .

    (vii)  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 celis, the-

concentration of  test, substance-associated .with algae and test

solution supernate-or filtrate?   -  -

    (viii)   If the  range-finding test showed that the highest

concentration ..of—the -chemical tested- (not- less than 1000 -mg/1 or

s atur ation. concentra;tion^ih.ad-,,-no^ef-f ect ...on^the^al-gae^ ,,-xep.orti t&e ••-

results and concentration and a statement that the chemical is of

minimum .phyto.toxic ..concern.;---:---L-.L? .

    (ix)  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,,

c o n c e n trataron^?^ndis a- si-feat emeri.t*Efcfa;atettlie .sch emic afe&irs^fryt o>t o>x i d &?"~

below the analytical detection limit.
                                13

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                                       DRAFT
                              BG-9
                              August,  1982
        FISH ACUTE TOXICITY TEST
       OFFICE OF TOXIC SUBSTANCES
OFFICE ~0Er:
-------
Office of Toxic Substances                                  EG-9
Guideline for Testing Chemicals                     August,  1982


                     FISH ACUTE TOXICITY TEST



        (a)  Purpose.  This guideline will  be used  in  developing

data on the acute toxicity of  chemical  substances and  mixtures

("chemicals") to fish 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. seg.).  The  United

States Environmental Protection Agency  (EPA) will. use_data.. frx>m.- .

these tests in assessing -the hazard of'  a chemical to the

environment.

        (b)  Definitions-.— The definitions ~ rn"sectron~3~of "the""  '

Toxic Substances Control Act (TSCA) and'~in Tart 79"2—-Good'  ~  " ""

Laboratory Pracice Standards apply to this  test guideline.  The

following definitions also apply:

        (1)  "Acute toxicity"  is the discernible  adverse  effects-

induced in an organism within  a short period of time  (days) of

exposure to a chemical. _ For. aquatic animals . this-usually-refers

to continuous exposure to the  chemical  in water for a  period  of

up to four days.  The effects  (lethal or sub-lethal) occurring

may usually be observed within the period of exposure  with

aquatic organisms.

        (2)  "Acute lethal toxicity" is the lethal  effect

produced on an organism within a short  period of  time  of  exposure

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                                                            EG-9
                                                    August, 1982
to a chemical.

         (3)   "Confidence  limits"  are the limits within which, at

some specified level  of probability, the true value of a result

lies.

         (4)   "LC50"  is  the  median lethal concentration, i.e.,

that concentration of a chemical  in air or water killing 50

percent  of a  test batch of  organisms- within a partrcu±ar period

of exposure (which should be stated).

         (5)   "Static  test"  is  a toxicity test with aquatic

organisms in  which no flow  of  test solution occurs.	(Solutions  r

may remain unchanged  throughout: the duration of the test) .

         (6)   "Semi-static test" is a test without flow of

solution, but with occasional  batchwise "renewal of" test solutions

after prolonged periods (e.g.,  24 hours).

         (7)   "Flow-through  test"  is--a toxicity test in which

water is renewed'-constarrt'*ly—inr^h-e- •'tes-t- "chamber sv^lre-^cfremrcal1*-*"--"-

under test being transported with the water used to renew the

test medium.

         (8)   "Time-response curve" is the curve relating

cumulative percentage response  of a test batch of organisms,

exposed  to a  single dose or single concentration of a chemical,

to a period of~expos'Tarev"~	v	•"" -^ .-.-.

         (9)   "Toxicity curve"  is  the curve produced from toxicity

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                                                            EG-9
                                                    August, 1982
tests when  LC50  values  are  plotted against duration of
exposure.   (This  term  is  also used in aquatic toxicology, but in
a less precise sense,  to  describe the curve produced when the
median period of  survival is  plotted against test
concentrations.).
         (10)  "Units"   all  concentrations .are...jgi.ven-m.-weight..-pe-r
volume (e.g., in  rag/liter).
         (c)  Test procedures — (1)  Summary of the test.  ( i)  The
aqueous -sol-ubili-ty-and "the—vapor-press'ure"~of"th'e~t"est~ chemical
should be known  prior  to  testing.  The structural" formula' of the
test chemical, its purity,  stability in water and light,
_n-octanol/water  partition coeffecient, and pKa value should be
known.   The results  of  a  biodegradability test and the method of
analysis for the  quantification of the chemical in water should
also be  known.
         (ii)  The fish  are  exposed to a range of test substance
concentrations preferably for_a period of up to 96 hours.
Mortalities are  recorded  at 24,  48,  72 and 96 hours and the
concentrations which kill 50  percent of the fish (LC50) are
determined  where  possible.
         (iii)  The maximum  concentration tested producing no

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                                                            EG-9
                                                    August, 1982
mortality and the minimum  concentration tested producing total

mortality should be  recorded.

         (iv)  For chemicals  with limited solubility under the

test conditions, it  may  not  be  possible to determine an LC50.

         (2) •  [Reserved]

         (3)  Range-finding test.  It may be necessary to perform

a range-finding test prior to a definitive test. "It provides

information about the—range- of^coneentrat-ions^to-be used- in- the •*

definitive test.

         (4)  Definitive  test, ~(i)   Fish should be: exposed~toif at

least five concentrations  spaced by a constant factor not

exceeding 1.8.  A control  and solvent control, when appropriate,

should also be tested.

         (ii)  Stock  solutions of the required strength are

prepared by dissolving- the -appropriate---amount-of --the—tes t— ~- -

substance in~ the*required-volmne--of—drlution water.— Tire pH"value

of the stock solution should be adjusted to the pH value of the

dilution water unless there  are specific reasons not to do so.

The test should be carried out  without adjustment of pH if there

is evidence of marked change in the pH of the solution, and it is

advised that the test be repeated  with pH adjustment and the

results-"reportedv —Th-is pH~-adjus'tmefTit—strou-rd"~be~made~~irrs'uch' "a" ™

way that the stock solution  concentration is not changed to any

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                                                              EG-9
                                                      August, 1982
significant extent and that no  chemical reaction or physical

precipitation of the test compound is caused.  NCI or NaOH should

be used  to  adjust the pH.

     (iii)   Stock solutions of substances of low aqueous

solubility  may be prepared by ultrasonic dispersion or,  if

necessary,  by use of organic solvents,  emulsifiers or dispersants

of low toxicity-to f ishr -When  such"auxiliary'substances~are	

used , the 'eont:Tol^->f-iSih'i:S'hoQi»d^be».expos!ed^^oG?:^'esyam'e:^^^'^^-'*~ •--<--*-•

concentration of the auxiliary  substance as that used in the

h ighes t  concentratio.n-of^the^es±-soabs-tancer. ~ zTke~-co:rccen:tr-a-tio.n.--r_-

of such  auxiliaries should not  exceed 0.1 ml/1.

     (iv)  The chosen test concentrations are prepared by dilution

of the stock solution.

     (v)  For test to be valid,  the following criteria apply:

     (A)  If • it is obse:rved= ±ha-t~tn:er s^tabi 1-ity.-or.r homogeneity~.o:f .--r--

the  tes t^subs-tance^ cannot^be~ina-i^±^'ined7''-thein-Tcare"-s>hocrldi-' be—*-**-•—-

taken in the interpretation of  the results and a note made that

these results -may_no.t~be..-reproducible..,.-~		

     (B)  The mortality in the controls  should not exceed 10

percent  at  the end of the test.

     (C)  The dissolved oxygen concentration should have  been >60

percent-of.-air-iS-a&uratian^tii^                                 ...

     CD)  There should be evidence  that  the concentration of the

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                                                            EG-9
                                                    August, 1982
substance being tested has  been  satisfactorily maintained (e.g.,

within 80 percent  of  the  nominal concentration) over the test

period.

    (5)  Test  results,   (i)   The fish are inspected after 24, 48,

72 and 96 hours.   Fish are  considered dead if touching of the

caudal peduncle produces  no reaction.  Dead fish are removed when

observed, and  mortalites  are recorded.   Observations. after the..- .

f irs t three hours,  and_s ix—hours  are ,.des.irab le..		 	

    (ii)  Records  are kept  of  visible abnormalities (e.g., loss

of equilibrium, swimming  behavior,  respirtory function,.	_	

pigmentation,  etc.).

    (iii)  The cumulative percentage  mortality for each

recommended exposure period  should-be plotted.-agains t_ -  	  ..

concentration  on logarithmic-probability paper.  A line is then

fitted by eye  to these points  and_the concentration_corresponding

to the -50 .pef c?ertf-.. rggPOnse_pQ.LnJ-,.. ,i .g. read, -of f -•-— This—is^JJie—L.C.SO-—..

for the appropriate exposure period.   Median lethal

concentrations

given in any of the references cited  in section (f).  Confidence

limits (p=0.95) for the calculated  LC50 values can be determined

using the standard procedures.   The LC50 value should be  rounded

off to ~tw;o
    (iv)  Where  the data  obtained  are inadequate for the use of

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                                                             EG-9
                                                   .  August, 1982
standard methods  of  calculating the LC50  (because most of the

results are  for either no deaths or total mortality,  where a

dilution ratio  of 1.8 has been used) then the  highest

concentration  causing no deaths and the lowest concentration

producing 100 percent deaths should be used  to determine the LC50

(this being  taken as being the geometric mean  of  these two

concentrations).

     (6)   [Reserved]	

     (d)  Test  conditions — (1)  Test species — (i)   Selection.

(A)  One or  more  species may be used, the selection-being at-the--

discretion. of _ the_ test ing-laboratory. — -It is—sugges ted-- that -t-he-

species used be. selected ..on.. the...bas.is of-.s.uch- important-practical-

criteria as:   their  ready availability- throughout-the.--year-, their

ease of maintenance,  their convenience for  testing, and any

economic, biological or ecological factors  which  have-bearing.	

The fis.h.should be._i.n ^ood-^health-^and-_.f r.ee~fr.om.^a.ny—^appar-en^t_-~=r-.~,

malformation;   If other species fulfilling  the.-thei-above criteria-

are used, the..tes..t _method-.S7hould^be^adap.ted-i:n--;.&uch-;aai,way. as -to. ~

provide suitable  test conditions.

     (B)  Examples of  fish recommended for testing and their size

are given in Table 1.

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                                                             BG-9
                                                     August, 1982
          TABLE 1—RECOMMENDED  SPECIES FOR ACUTE TESTING

Recommended  species                                Recommended
                                                   total length
                                                   (cm)

Brachydanio  rerio  (Teleostei,2.0 > 1.0

Cyprinidae)  (Hamilton-Buchanan)

Zebra-fish

Pimephales promelas  (Teleostei,                   2.0 _+ 1.0

Cyprinidae)  Fathead-minnow

Cyprinus  carpio  (Teleostei,                       3.0 +_ 1.0

Cyprinidae)  (Linne 1758)

Common carp

Oryzias latipes  (Teleostei,                       2.0 +_ 1.0

Poeciliidae)  (Schlegel 1850)

Red killifish

Poecilis  reticulata  (Teleostei,                   2.0 _+ 1.0

Poeciliidae) . (Peters. 1859)	..

Guppy

Lepomis macrochirus-_.(J.el.eostel.,-_ -.	  ......       2.0 .+_ 1..0 -.—.

Centrarchidae)  (Linnaeus  1758-)

Bluegill

Salmo gairdneri  (Teleostei,                       5.0 _+ 1.0

Salmonidae.).i;(..Ri:ehards.QBii8i3&)~ .=r^™;,-.-..-,      -    .  •

Rainbow trout
                                 8

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                                                            EG-9
                                                    August, 1982
    (ii)  Collection or acquisition.   The  fish mentioned above
are easy to rear or are widely  available  throughout the year.
They are capable of being bred  and  cultivated  either in fish
farms or in the laboratory under  disease-  and  parasite-controlled
conditions so that the test animal  will be healthy and of known
parentage .,
    (iii)  Holding and acclimation.   (A)   Fish should be held for
at least 12 to 15 days before testing.  All fish  should be
maintained in water of the quality  to  be  used  in  the test for at
least seven days before they are  used.
    (B)  Coldwater fish should  be held in  tanks containing at
least 300 1 of water while warmwater fish  should  be held in tanks
containing at least 100 1.
    (C)  The temperature of the holding water  should be the same
as that used for testing.  The  dissolved oxygen concentrations
should be maintained above 80%  of the  air  saturation value.  A 12
to 16 hour photoperiod should be  used.
    (D)  All fish should be fed three  times per week or daily
until 24 hours before the test  is started.
    (E)  A batch of fish is acceptable for testing if the
percentage mortality over the seven day period prior to testing
is less.;than-f iy.e-^-.:J£.-the. mortali±y_j.si.-.be;twejen--i5T:ya-ndrild) iper-cejit-™
acclimation should continue for seven  additional  days.   If the

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                                                            EG-9
                                                    August,  1982

mortality is greater than  10 percent,  the  entire  batch of  fish
should be rejected.
    (2)  Test facilities^—(i)  Apparatus .   An  oxygen meter,
equipment for determination of water hardness,  adequate appartus
for temperature control, test  tanks made of  chemically inert
materials and other normal laboratory  equipment are needed.
    (ii)  Dilution water.  (A)  Drinking water (dechlorinated  if
necessary), good quality natural water, or reconstituted water,
with a total hardness of between 50 and 250 mg/1  (as CaCO)3  and
with a pH of 6.0 - 8.5 are preferred.
    (B)  Reconstituted water should be prepared from deionize.d
water or distilled- .water- wi.th_a conducti-v-i-ty-.^-10-  Scm~l.  One   -  -
hundred liters of reconstituted water  can  be prepared by adding
2.5 1 of the following solutions  to a tank  and bringing the
solution to volume with deionized water:
    11.76g CaCl2 ' 2H20/1
    4.93g MgS04 • 7H20/1
    2.59g NaHC03/l
    2.59g KC1/1
The sum of the calcium and magnesium ions  in this  solution  is  2.5
mmol/1.  The proportion of Ca:Mg-ions  is 4.13  and  of Na:K-ions is
10:1.  The acid capacity of this solution  is 0.8 mmol/1.
    (C)  The dilution water should be  aerated  until oxygen

                                10

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                                                            E3G-9

                                                    August, 1982
saturation is achieved  and  then  stored  for about two days without


further aeration before use.
     i

     (3)  Test parameters,   (i)   Constant conditions should be


maintained as far as possible  throughout the test and,  if


necessary, semi-static  or flow-through  procedures should be used.


     (ii)  The preparation and  storage of the test material, the


holding of the fish, and all operations and tes=ts should be


carried out in an environment  free  from harmful concentrations of


dust, vapors, and gases  and in.such\a.way as .to avoid cross-	.


contamination.  Any disturbances  that may change the behaviour of


the  fish should be avoided..	..  ...


   .  (iii.X.. The. fol.l.awi.ng...pac,ametej:s—a.r.e-^-impor.tan.t-:.-.-~—-..-	


     (A)  Dissolved oxygen.  The  dissolved oxygen concentrations


should be at least 60 percent  of  the air saturation value.


     (B)  Light.  A 12 to 16 hour  photoperiod should be  used.


     (C)  Loading.  A maximum loading of 1.0 g/1 for static and


semi-static tests is recommended; for flow-through systems a


higher loading can be acceptable.


     (D)  Temperature.   Test temperatures of 15 _+ 2°C for rainbow


trout and  22 _^ 2°C for  carp are  recommended.   The other


recommended species should  be  tested at 23 _+ 2°C.  The


temperature should be maintained  within +_ 1°C of the selected


test temperature throughout the  test period.




                                11

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                                                            EG-9
                                                    August, 1982
    (E)  Feeding.  The fish should  not  be  fed during the test.

    (e)  Reporting.   (1)  The  sponsor should submit to the EPA

all data developed by the test that are suggestive or predictive

of toxicity.

    (2)  In addition  to  the reporting requirements prescribed in

Part 792—Good Laboratory Practice  Standards the  reported test

data should include the  following:(

    (i)  Details of the  test procedures used (e.g. static, semi-

static., .flow-through.,.- aera.ted.,..etc..) .	, .,„.,.-

    (ii)  Information about the  test  organism (scientific name,

strain, supplier, any. pre.trea.tment,~.e±c.)..		

    (lii)  .The. concentrations—-tes,ted...~.-^,.—._.	      ........

    (iv)  The number  of  fish in  each  test  chamber and the loading

rate.

    (v)  The methods  of  preparation of  stock and  test solutions.

    (vi)  The dissolved  oxygen concentrations,  pH values,

temperature, total hardness of  the  test solutions  measured each

24 hours and any other available information on water quality.

    (vii)  Any available information  on the  concentrations of the

test chemical in the  test solutions.

    (viii)   The maximum  concentration causing no  mortality within

the period  of the test.

    (ix)  The minimum concentration causing  100 percent mortality


                                12

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                                                             BG-9
                                                     August,  1982
within the period  of  the test.

     (x)  The  cumulative  mortality in each concentration  according

to the recommended  observation times.

     (xi)  The  LC50  values (based on nominal concentrations)  at

each of the recommended  observation times (with 95 percent

confidence limits,  if possible).

     (xii)  A  graph  of the concentration-mortality curve  at  the

end of the test.

     (xiii)  The statistic.al.-pr-ocedur.es--uaed-_for...dete.rjnining..-the	

LC50 values.

     (xiv)  .The mortality..-.of.-the—control, -animals..	.———	._  -

     (x v)  Any  i n aide n.ts~~i.n~the^-cours-e—a£-4:h e~ -tes-t - vhA-eh  m-i gh-t   - • •-•

have influenced the results.

     (xvi)  Any abnormal  responses of the fish.

     (xvii)  A  statement  that  the test was carried out  in

agreement with the  prescriptions of the Test Guideline given

above (otherwise  a  description of any deviations occuring).

     (f)  References.

     (1)  APHA.  1975.  American Public Health Association,

American Water Works  Association, Water Pollution Control

Federation.   Standard methods  for the examination of water  and

wastewater, 14th ed.  New York:  American Public Health

Association.


                                 13

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                                                             EG-9
                                                    August,  1982
    (2)  Committee  on  Methods  for Toxicity Tests with Aquatic

Organisms.   1975.   Methods  for acute toxicity tests with  fish,

macroinvertebrates  and amphibians.   Corvallis, Oregon:  U.S.

Environmental Protection Agency.   EPA-660/3-75-009.

    (3)  Finney AJ.  1978.   Statistical methods in biological

assay.  Weycombe:   U.K.   Griffin  Ltd.

    (4)  Litchfield  JT,  Wilcoxon  F.   1947.  A simplified  method

of evaluating dose-effect experiments.   J. Pharm.  Exp. Ther.

96:  99-1113.

    (5)  Peltier W.  1978.   Methods  for measuring the acute

toxicity of  effluents  to aquatic .organisms.. . Cincinnati., ..Ohioj ......

U,. S.  Env,i.ronmen.tal . P.r.o±je.ct-ioji-Ag.eJi.cy....—EP-A-.6 0O/&=-J.B.-J3£r2.:-_- „ ^	 ...

    (6)  Sprague JB.   1969.  Measurement of pollutant toxicity to

fish.  I:  Bioassay  Methods  for Acute Toxicity.  Water Research

3:  794-821.

    (7)  Stephan CE.   1977.  Methods for calculating an LC50.

In:  Mayer FL,c Hamelink  JL.  eds.   Aquatic Toxicology and  Hazard

Evaluation.  ASTM STP  634.   American Society for Testing  and

Materials,  pp. 65-84.

    (8)  Tabata K.   1972.   Quality  control of  Japanese rice fish

for TLm-test.  Water and Effluent 14:  1297-1303.     :
                                14

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                                           DRAFT
                               ES-6
                               August, 1982
       TECHNICAL  SUPPORT DOCUMENT

                  FOR

        FISH ACUTE TOXICITY TEST
       OFFICE OF TOXIC SUBSTANCES
OFFICE  OF PESTICIDES AND TOXIC SUBSTANCES
  U.S.  ENVIRONMENTAL PROTECTION AGENCY
         WASHINGTON, D'.C." 204'6"0 ' - "	- "

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                     Table of  Contents
        Subject                                         Page
           i
I.       Purpose                                          1
II.      Scientific Aspects                       '        1
        Test Procedures                                  1
        Range Finding Test    "                           1
        Definitive Test                                  3
        Time-dependent vs. Time-independent Test         4
        Static vs. Flow-through Test                     6
        Length of Exposure                               8
        Test Results                                     9
        Analytical Measurements                          10
        Water Quality Analysis                           10
        Collection of Test Solution Samples              11
        Test Substance Measurement                       12
        Test Conditions                                  14
        Test Species	                                  14
        Selection                                        14
        Sources                                          16
        Maintenance of  Test Species              ,        18
        Age and Condition                                18
        Care and Handling                                20
        Acclimation                                      21
        Facilities              "                         22
        General                                          22
        Construction Materials                           25
        Test Substance Delivery System                   26
        Test Chambers                                    28
        Cleaning of Test System                          29
        Dilution Water                                   30
        Carriers .•-.-.-.....-.-  ...                      37
        .Environmental Conditions                         38

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        Subject                                         Page
        Loading                                          38
        Temperature                                    .  41
        Light                                            42
        Reporting                                        42
III.     Economic Aspects                                 46
IV.     References                                      ,55
                                11

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Office of Toxic Substances                               ES-6
                                                August,  1982
   Technical  Support Document for Fish Acute Toxicity Test

I.  Purpose
    The purpose of  this document  is  to provide  the
scientific background and rationale used in the development
of Test Guideline EG-9 which uses rainbow  trout, bluegill  or
fathead minnow to evaluate the acute  toxicity of chemical
substances to fish.  The Document provides  an account  of the
scientific evidence and an explanation of  the logic used in
the selecion of the test methodology, procedures and
conditions prescribed in the Test Guideline.  Technical
issues and practical considerations are discussed.   In
addition, estimates of the cost of conductiing  the test are
provided.
II.  Scientific Aspects 	
    A.  Test Procedures     	
         1.  Range  Finding Test
    A range finding test is recommended for determining  the
appropriate concentrations of test substance to use in
performing a definitive acute toxicity test. In the  range
finding test, groups of five or more  test  fish are exposed
to a broad range of concentrations of the  substance.   Enough
concentrations should be tested such  that  concentration
lethal to approximately 50% of the organisms can be
ascertained.   The number of concentrations ..will normally
range from 3-6 depending upon the shape of  the  toxicity
curve for that chemical and prior knowledge of  its
approximate toxicity.  Only concentrations  less than the
solubility limit in water are tested.  The exposure period
used in the range finding test can be as short  as..24 hours
or as long as 96 hours.  If an exposure period  less than 96
hours is used, the  test substance concentration range

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                                                          ES-6
                                                  August, 1982
selected  for the definitive test  may  have  to be adjusted to
make  allowances for a greater potential  toxicity after 24-
hours  of  exposure.  If no mortalities  at a test
concentration equal to the solubility limit are produced, no
additional higher concentrations  need to be tested.
          2»  Definitive Test
              .a.  General
    The  results of a definitive test  are used to .calculate
the 96-hour LC50 and the incipient  LC50  when appropriate,
and the  concentr.ation-res-ponse~rel.ationship- of-the-test
substance and. the, .test-fish.	If—the._concen-tr.atio.ns—of -tes-fe-
substance which produce no effect,  a  partial kill, and 100
percent  mortality have been determined during the range
finding  test, -then five~~or-~six™"test" substance* concentrations"
.should be. saf.f.i.c.ieji.t.-.to.-es-t.ijnate—the..appropriate--LC5-0 values
in a  definitive test. In some cases however, to obtain two
partial  kills bracketing the 50%  mortality level, it may be
necessary to test 8-10 concentrations.
    The  slope of the concentration-response curve provides
an indication of the range of • sensitivity of - the "test -f ish" '
to the .test .substance_-ajid_jnay_r_all.ow_.es-tiraa.t.ians~of-.-loweC'—-—
concentrations .-that will-affect the test organisms. For
example  if the  slope of the concentration-response curve is
very  steep, than a slight increase  in concentration of the
test  substance  will affect a much greater  portion of the
test  fish than would a similar increase  if the slope of the
curve  was very  shallow.  The slope  of  the  concentration-
response  curve  reveals the extent of  sensitivity of the test
fish  over.:a-.range; ofcconcjenita^a±33jnsrsjear^3,^-i~~~::z..:...•;
    The  exposure of a minimum of  20 fish,  divided into two

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                                                           ES-6
                                                  August,  1982
or more  replicate groups  ,  to each  test substance
concentration is required  in  the  guideline. That minimum is
based on an  optimum number  of test  fish needed for
statistical  confidence, equipment requirements, and
practical considerations  of handling the test organisms.
    At least two replicates should  be included in order to
demonstrate  the level of  pecision in the data and indicate
the significance of variations.   Test chambers holding
replicate groups should have no water connections between  -
them.  The. dis.tribat-io.o..of —tes-t- frish—to—the--tes t- ch-ambers—	
should- be_ randr)nLLzed__to_pr-event-.Jsias,-,f.r.onu b.eing-J,a.tr-oduce4—-.- -
into the test results.
    Fish should not be fed.-dur.ing-the-tes-t-for-two. .reasons ...
First, fecal matter which -may—acrcumu'la'te" can result  in  a
decrease,ln~fehe.-d.issolv4ed^.oxy§en' -Goneentr'afe-iorH-i-n-the - tes t— •*—--
chamber.   Second, some test substances can physically  bind
to the uneaten food or fecal matter, thus making a portion
of the test  substance unavailable for uptake by the  fish.
An occurrence of either of  these  conditions could produce
unreliable test data-. -•--::
               b.  T ime - d epejid.e jut—Ms.. ~Xi mea-.ijid.ep e jid£-n.t-- ^^.-.-^ ^ _
    In time-rdependen-t-.tes-ts-,-=f-iB-h- a-re—continuously- ex-posed- —— -
to a series  of concentrations for a specified period of
time, usually 96-hours, at  which  time the LC50 is'
calculated.   In time-independent-tes-ts -(-T-r's )>— f rsh  are--  -~-
continously  exposed to a  series of  test concentrations  until
such a time  when no additional mortalities  are expected  to
result from  continued exposure.   The LC50 calculated at this
t ime is. ±te rraed~t'hesdrrtci^:i;e:n'1rri^^^
been termed  the ultimate  median tolerance limit, the lethal

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                                                          ES-6
                                                  August, 1982
threshold  concentration or the asymptotic LC50 by many
researchers.   Sprague (1969) has stated  that the incipient
LC50  is  the  "most useful single criterion of toxicity."  In
a review of  375 toxicity tests by  the  author,  a lethal
threshold  clearly had not been reached  in 42 tests  while in
122 tests  the  threshold was reached  in  four days or
longer.  Eaton (1970) proposed the use  of TI's as the
resulting  50%  effect concentration is dependent upon the  '
response of  the test fish and not based  on an  arbitrary time
period.
    Use  of TI's greatly: increases-one Vs  ability-to-evaluate	^,
the chronicity of compounds as discussed by Tucker  and
Leitzke  (1979).  The chronicity of a  compound, or the degree
to wh i ch a ~ c omp ou nd—eirre-cts—ad-dirt±on-a~l~mo r ta"l rt res ~ o ve r~ a ~ ~' '
pro longed ..period ».of  t ime-^-is—as^.eas.ed—b.y,_aompa,r-ing,^.LC.5.0J-s_	„..,_.
over  time, e.g. 96/48 hour LC50 or 10 day incipient LC50/96
hour  LC50.   Those compounds-with relatively-high ratios
(i.e. >0.5)  would not be expected to cause chronic
effects.   If only 96-hour toxicity tests were  performed, the
toxicity - of  thos-e comf>ou-ndsr:-whose-:mode-,-of-^-act-ion—r-eefuires^atr:;-;
1 eas t 3-4  days - to-begin to..,-expr.es^__tox.ic.i ty_.would_be_.gr.os-s.l-y-_.,_
underestimated..	.— ..
    It should  be remembered that for compounds that do not
express  chronicity,  i.e. there is little additional
mortality  during-the -last-48-hours of the--test,- the-test-	—
will be  a  96-hour toxicity test.  Consequently, the more
costly testing for the estimation of an  incipient LC50 will
be performed only for compounds for which additional testing
will prabaJ3ly^e^pe;r.f-Gcrn^dr^^
assessment process.

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                                                           ES-6
                                                  August,  1982
               c.  Static vs.  Flow-through
     In the static system,  the test substance and dilution
 water are not changed during  the  96-hour exposure period.
 In promulgating test rules,  the Agency will prescribe  the
 use of a static or flow-through system, or both, for fish
 acute toxicity tests for developing,  cost effectively,  those
 data essential to assessing  the risk  to the environment of  a
 given chemical.
     Each of these methods .of .expos ing . fish- to _a.. test-  	
 s ubs tance,.of.£ers -ce-r-tain~»advaatages..«and-presents ^certain— - - -
 d isad vantages -not-shared -by—the ~ot her. ~.*- The - static-expos ur-e —
 system requires less equipment and set  up time, and
 therefore is a less expensive, test* -On .the .other hand, in..a-
 flow-through system, -loss  of  the  test substance due-to     '"
.uptake by-.-the -f-ish^-deg-r-ada^fe-ion^ror-- to--vola>tiMza-tion' -is- •——•.-
 minimized, and metabolic products  toxic to the test
 organisms (e.g. ammonia) do  not build up.- The concentration
 of  dissolved oxygen in the test chamber can also be
 maintained above the level that might stress the fish.
     Beeause .-ofrrcthesfiTf eatares5^tlre~Agen-cy^wdi-irvspeerf y~'the~ ~~
 use of the f low,- thr.ou-gh._me-i:had-^i-n^tes.ting-^ihe_ toxic ity-- of ~.......
 chemicals which volatil-i-ze~or~degr-ade—rap.-idlyr-which -r-educe—
 the dissolved oxygen concentration within the test chamber,
 or  which are taken up by the  test  organisms at a rate that
 s ign if icantly—towers"the—concentration~'of ~the~ tes t subs tance
 within the test chamber.   In  additon  to developing data
 needed to determine the 96-hour LC50  and the concentration-
 response curve for such test  substances, a flow-through
 e xpos ur--err.mayjxbe^
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                                                            ES-6
                                                   August,  1982
 tests can not be  continued past 96 hours  and still yield
 reliable results.
     In a static system/ since the test  substance is not
 renewed, the concentration of the chemical  to which test
 fish are exposed  does  not remain constant if the chemical
 adheres to the test chamber walls, vaporizes readily,
 degrades rapidly,  or is readily taken up  by the test fish.
     The static method  of exposure however,  can be used to
 develop toxicity  data  for those substances  which ,a.re_not- .-	.
 subject_±o.a...s.igniiLLcan£_,r^uction~i^
 the exposure_perJLod..	S.tatic—toxicity_data-in— combination—	— ~
 with data developed through the use of  a  flow-through test
 can also be used  to detect and evaluate the toxicity of
 metabolities and— degrB-datron~~productsT""If "for' instance, the1
,.9 6-hou.r, JLC,5,(LJ:r_om...a.,.s-ta.t ic^tasJ^is-~les,s~~>than .=.th-a.t~,f r--onu,--a..-9 6»» - -
 hour flow-through  test, it can be assumed that more toxic
 metabolites or degradation products were  formed during the
 static test.
     The Agency forsees a need for both  flow-through and
 s tat ic_- tes t-;me-fchods.-; and :^ea eh—me thodrrasdrl-fc:be-;cons±de,red~.i-n~rt:: ^r-
 the dev.elop.ement_-of._a_±es±_.rul£^.--.The_ch.emic.al^-natuxe-.of—.the.—.™-...
 test substance.,-J.ts-.~use-~.and-,the~nat.ure.-of-its-rele-ase-into-  -~-~.
 the environment (e.g.Continuous or intermittent) will be
 considered.  The  Agency does not, however,  assume that data
 developed through -the- use-of- one—of---thes-e tes-t—methods—can—	
 substitute for data developed through the use of the other,
 since evidence exists  in the literature to  show that the
 toxicity of some  test  substances for test organisms may be
 10 times. gr.ea±er^a-jasfe±ow^-hr^G^^
 (Mauck et al. 1976).

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                                                           ES-6
                                                  August,  1982
               d.   Length of  Exposure
    The  minimium exposure time  of  96 hours in the  fish  acute
toxicity test standard is specified  in order to permit  a
comparison of data developed  through the use of this  test
guideline  with the large base of acute toxicity data  in the
published  literature.  The 96-hour exposure period  is
advocated  by various groups concerned with establishing
uniformity in testing methodology  (APHA 1975, ASTM  1980,
Committee  on Methods... 1975).  Most  of the recently	 ._
p ub 1 is hed  da t a_ OIL, the_acuJte- -ioxi-c i t-y~~o£ -.ckemi cals*«to>*»~ -—~—... -
freshwater -fish- werfi_.dav.el.oped_ajsj..ng_^a96=hour».exposure—-»..—.-«
period (Brugns et al. 1977, McKim  et al. 1976, Spehar et al.
19.79, 1980).   The use of the 96-hour exposure period was
p r opos ed - i n i t i atiy ~rn "IS'S'l-' by ~~a n"^aqua tic "b-i o as's ay '-•c om mi 11 e e -
(Doudorotf^e-t^al..—19-5-1.).<^ajid-jwas-=»s-eJ.-e.cted:,^in=^la.r.ge~rpa*r&r as*. •
a matter of  convenience since it is  easily scheduled within
the five-day work week. Only when  there are indications  of
chronicity during a 96-hour  test will the test-per-iod-be
extended.   The previously cited studies indicate that this
is not a frequent—ooeur-rencei.r—---.= .-••-
          3.   Test Results	.
    Wh il e  de aJth .ls_the_pr-imary_.e.ndpo-i.n-t—in-~thesje^ tes-ts, —any- -
behavioral or physiological changes  in the fish such as
erratic  swimming, lost of reflexes,increased excitability,
lethargy,  diseolora-t"idn-,~excess-ive—mueous—production-/	
hyperventilation, opaque eyes,  curved spine, hemorrhaging or
any other  observed effects should  be recorded.
Quantification of such observations  at test substance
c o nc e n t r at io,ns. -^ot2^ausd;ng^^:t hs±i^yr~^:ne^                 - -.. •
identifying  and  assessing potential  chronic lethal  effects

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                                                         ES-6
                                                 August, 1982
at these lower test concentrations.
    Mortality data at  24,  48,  72  and  96 hours  and at
termination if the test  exceeds 96 hours,  should be
presented for each time  interval. Whenever sufficient data
exists at these time intervals,an LC50  and 95% confidence
interval should be calculated  and a graph  of percentage
mortality - concentration  prepared.   When  more than one
LCSO's at various time  intervals  should be prepared.
    The recommended methods  of LC50 calculation include the
probit, logit, binominal and moving average angle methods.
The data obtained -from :each.....tes.t—will ..de-texmijie- .which, method
is the most appropriate  for  that  data se't.
         4.  Analytical  Measurements
              a.  Water  Quality Analysis
    Measurement of certain water  quality parameters of the
dilution water such as hardness,  particulate matter,
alkalinity, acidity, conductivity, TOC,  and pH is important.
Quantification of these parameters at the  beginning and end
of the exposure period of  flow-through  tests  is necessary in
order to determine if the  water quality-varied duri-ng the --
test.  If significant variation occurs,  the resulting, data  .
should be -interpreted~Tn-"tight of--the -estimated~toxicity~ "'
values.
    In Static Systems the  dissolved oxygen concentration and
pH should be measured in each  test chamber at  the beginning
of the test just prior to  addition of the  fish and then as
often as needed to document any subsequent changes from the
initial levels.
    In Flow-through the  dissolved oxygen concetration (DO)
and pH of the tes't "solution" "in" each' chamber 'should be

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                                                            ES-6
                                                    August, 1982
 determined at the beginning  of  the test and every  48  hours
 thereafter until the end of  the test.  A decrease  in  DO
 indicates  that the flow rate should be increased.
                b.  Collection of Test Solution Samples
      The objective of the recommended sampling procedure is
 to obtain  a representative sample of the test solution for
 use  in measuring the concentration of the test substance.
 Although there is mixing in  the test chamber, especially in
 flow-through tests, .material -,can-,.concen.trate.njear-the-sides
 and  bot.tom-of~the_jch-ambej^.xiue~^^p,hysdcal.^r^^
 properties . of —the .substance.,—ojr~to~i.nte.r.ac=tions~wi*th^ organdc^-
 material associated with the test animals.  For this  reason,
 water  samples should be taken  near the . eeoter_of _the~tes.t	
 chamber. ~  The" handling "and s torage- of the s amples~ requires"
,°care to-4>r.ev~e-nt^the-Xcs^~of^the.-..*tesJ:-.^
 sample before analysis.
                c.  Test Substance measurement- .
      In Static Systems  the concentration of dissolved  test
 substance  should be measured in-each test clramber  a~t  least
 a-t the • begintvi-ng-;-and—end™ of—eacfar :tes"t7- r.^f-~the^r-eda-c±loTi-.-inr^:~
 tes t s ubs tance_ concentr,atiaa~jexce£.ds~ 5.Q.&.,"' the^tesut.,s hxHild. b-e--
 repeated- at-a~ lower- l-oading-^a4:e^- o.r-a--f4.ow^thr-ough --teS't-  - -------
 should be  performed.
      In Flow-through Systems  the test substance concentration
 should be -dete-rmi-ned—in-e-ach-test-ch-amber—at~0~and-96-hours	
 and  if the test continues past  96 hours, at least  every four
 days up to and including the day of termination.   To  further
 assess and quantify any possible changes in test substance
- concentrait>i*o:n^whe^;eiversasmavlfaan-c^                          ." -•
 delivery is detected, all potentially affected test chambers

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                                                            ES-6
                                                    August, 1982
should be sampled  at that time.
     If the measured concentrations  of dissolved  test
substance are  50 percent more or  less than the nominal
concentrations, steps should be  taken to determine the cause
for this deviation.  A sample of  the stock solution as well
as  samples of  the  influent to various test chambers should
be  analyzed to determine if the reduction in test  substance
occurs prior to delivery of the  test solution to  the
aquaria.  If results of these analyses indicate that, the-.. _.,
prope r. amounts_.of--.tfis.±_.s.ubs tanee.. axe_en±ex.ing~~the.-tes.±—-—~~	
chambers , then, th.e_-.tot.al^±esJt^s-uhs±anc.e;- .co.ncentra£ion~.s--hould-~i^~—.
be  measured in at  least the chambers containing  the highest
test substance concentration.  These data will give
i nd i c a t io ns - i-f -~the~drf •£e r encre";be-twe-eTi-r~nom-rnal^a-nd8" me~as'ur^d " ~~ - *'-v"
..tes t cone en ,tr,a.tians~is_due:w±o~..vo.la.t. ill za.tiorwo.r,^d egr.ada,t-io-a~«-«—. • -
of  the test substance, or to insolubility of the  test
substance in the dilution water.
     If the toxicant delivery sys tern-has-been-pr-operly -
calibrated 'and the  fish randomly  introduced into- e-ax:h test  • - "
c h amb e r, --t he~ -me-asiir--ed^d-i^dfer.eiiQesr^e±wfi:enraiep^                   :
concentra±ion_s.ho.uld...b.e.._Less,_.than_.2-O.S.,.	Jf—Jthe—di.f Terences	--
exceed-this., Jihe-±es-t..-shou-l.d-.be—r.ep.e^-ted-.	.—.... . —•  -
     The  concentrations of test substance  measured  after
initiation should  be within 30% of" the concentrations
measured- prior -to-introduction of -the -f is-h-; - -If—the - —  -
difference exceeds  this, the test should be repeated using a
higher flow rate.
     Use  of reliable and validated analytical techniques and
methods,;v:tei;:fi&s:ent~i^feto,^tehe;ais:e^                                   •
assessing the environmental hazard  of the chemical.
                                  10

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                                                            ES-6
                                                   August,  1982
Significant variation  in  the measured  concentrations  lessens
the  value of the toxicity data generated.
     B.   Test Conditions                       s
          1.  Test Species
               a.  Selection
     The  species of fish selected for use  in this test
guideline are the rainbow trout/ bluegill  and fathead
minnow.   Adelman and Smith (1976) listed  the following  as
the  important cri.teria_ta_us.e~-in—the—s.election-.of-.a	-
"s tand a rd—fisJai-fjOJ^-.bixDass ays*;*=«>«l'}—eel a-feiv-e-iy-* cons-tea n-t-"*-- •-«-*-—
res pons.e_.~to~.a^rjO,ad^Ea^ge-.4Df*.vtQ.x^                 3 a-nde-r* -^^.-^ •-
similar  conditions 2)  available in large  quantities with
close quality control  3).  eas ily_handled__fo.r..bio.as.say		
purposes  4) --easily-transported =5^continuous avail abti-ity-of"' ~
the  des-ired-S-i-ze.-.and^fr) neap abie-a-.of=is-ucoess-f-ul-7reomp-le t i-on —of?- •> ••-
a life cycle in 1 year or less.  These  species meet all
criteria  but .the-last; only-the-fathead-minnow .can-complete
a full life cycle in less than one year.-
     The  main reason why these three species -were selected  is  -
becau s e--there--- is™anven^nlaxge^tax±c:i^y^a33
and.  alL  three, are^re.adJ.ly.;<-a.vaJJcabJ.-ft,-rand^&equ^r-e.^
expe rt ise in -ma inta in ing -heal-fchy-^Qp u-l-a-t4-o-ns-.- - -Al-1 - thr-ee -ar-e
widely distributed in  the United States, and are either
ecologically or economically important  (Scott and Grossman
1973, Ki-tchel'1-et-al.-1979)." -	
     Studies on relative sensitivity of  the three species
have been performed and indicate that rainbow trout are
generally the most sensitive and fathead minnow the least
s e ns i t i.we:stora"7?va^de:ty.v-iof ^*;esstoSHate5tanc^^
compiled  LC50 data on  20  pesticides with  all three
                                  11

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                                                         ES-6
                                                 August, 1982
species.   With  12  of  these compounds, the trout was the most
sensitive  and  the  fathead  minnow the least.  In five cases
the bluegill was the  most  sensitive and in one case the
fathead minnow  was the most sensitive.  The LCSO's were
similar between the three  species  for the remaining two
compounds.  The LCSO's for the fathead minnows were
generally  6x those for the trout and 2x those for
bluegill.  .As  it was  not stated if the tests were performed
under comparable conditions  these  values only approximate
relative sensitives of the species.
    Nevins and  Johnson-'(.19-78.) .tested .thr.ee. -phos.p.ha.te..,es.ter ....
mixtures with  all  three species ofs fish'under "identical
static conditions  and two  compounds under flow-through
conditions.  In all five cases, rainbow trout were the most
sensitive, but  only in 2 cases  were the fathead minnows the
least sensitive.                                         :
    Folmar et  al.  (1979) performed static acute toxicity
tests with technical  grade glyphosate, the formulated
pesticide Roundup® surfactant  with the above 3 fish
species.   In these tes-t& however,-the -fathead mi-nnow was- the
most sensitive.
    From these  data-it-is  clear that it can "not-be-assumed ••-
-that rainbow trout will be the  most sensitive species.
Although fathead minnows are generally the least sensitive
of the three species,  their  small  size, ease of culture, and
short life cycle make them the  easiest to work with.  Their
extensive use in early life  stage  testing and full-chronic
testing (Macek  and Sleight 1977, McKim 1977) adds to the
importance of their role is  aquatic toxicology programs.
Although" rainbow trout can also be "used in early life" stage
                                12

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                                                          ES-6
                                                 August,  1982
and full or potential  chronic toxicity testing, the expense
is only occasionally warranted.
              b.   Sources
    All three species  are  readily available at the
appropriate test sizes  from commercial fish suppliers.
There are some suppliers  that now specialize in culturing
fish under controlled  conditions  just for toxicity
testing.  Rainbow  trout can be purchased and readily shipped
to researchers as  either eyed eggs or as finger lings already
the appropriate size for  testing.  Trout should be purchased
only from supplLexs_±ha±, havje_heen,,-sJta±.e^cex£ifl.ed.~..ta~.rxaiSve *
disease-free fish.
    There are many suppliers  of  bluegill throughout the
country that will  readily- air-freight fish.  As these fis-h--
are not amenable to artificial fertilization or spawning in
the laboratory, -f-igerli rigs  are "normally shipped'.
Researchers need to carefully select their suppliers, as
many are known to  have  little concern for providing disease
and parasite-free  fish.
    Fathead- minnows- can—be-pur-chased -as--eg-gs-- or- juve-nil-es-,- •
or cultured in the laboratory in  a brood unit (U.S. EPA
1971). ; In light of-past" problems •vwi7tfrr the~-heal-thr of' f ish~-
received from some, suppliers,-lt_.;is recommended^tha±.  -..•-.-.
researchers rear their  own  fatheads for toxicity testing.
    Whenever fish  are  to be  used  for a test or a set of
tests, all fish used for that test should be from the same
source and held under similar conditions prior to testing to
minimize variability.   Alexander  and Clarke (1978) performed
toxicity tests with 2 strains of  rainbow trout exposed to 13
mg/1 dodecyls'ddium sulf ate'.'* "The "median survival" time "of "the
                                13

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                                                          ES-6
                                                 August,  1982
Idaho strain exposed  to phenol was  significantly less than
the median survival  time  of  the Nisqually strain.  Response
times for the other compounds  were  similar between
strains.  In a second  set of  tests  with just phenol, no
difference in LC50 values  was  found between Idaho and
Nisqually strains, but a  significant difference was found
between Idaho and Manx strains.
         2.  Maintenance  of  Test Species
              a.  Age  and  Condition
    The age, and consequently  the size of rainbow trout,
bluegill, and fathead  minnows  was selected based on the ease
of handling -and—test-ing ""fish" of-this"size?' ~Ail fish"used"" in
the same test should  be as similar  in  size as possible to
limit the effects due _:to.  size, differences.	 -
    The health-..and_cojidl±ion._bf.._£is-h_.us.ed^.i.n~.a.cu.te .=£axi-city—
tests is an important  consideration. .Diseased or stressed
fish may increase the  sensitivity of the fish to the
toxicant.  Iwama and Greer (1980) performed 96-hour acute
toxicity tests with Coho  Salmon (Oncorhynchus kisutch) that
had been exposed to,  and  contracted, a mild state of
bacterial kidney disease  .(Conynebacterium-salmon.inus-)..- .-When
diseased fish were exposed  to  pentachlorophenate, the
estimated LC50 was 39  ug/1, s igriificantly -lower than the
LC50 of 65 ug/1 for healthy  fish.
    Prior expos ure„ to~contaminaixts^^ay^also-~ef~£e.ct-~the--~. .--* =.
response of test fish  to  a toxicant.  Bills  et at. (1977)
performed acute toxicity  tests  with several compounds using
rainbow trout that had previously been exposed to PCB's
(Aroclor 1254).  Previously  exposed trout that had body
lowe r LC 5 0' s... wh en_ .expos ed_ to_. two ...of ..t he_tes-t_compounds-,	
                                14

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                                                          ES-6
                                                 August,  1982
cyanide and  chromium.   Trout with body burdens of only  0.46
ug/1 of PCB's  were more sensitive to cyanide with a LC50  of
66 ug/1 versus  90  ug/1  for "clean fish."
    Alexander  and  Clarke (1978)  performed static acute
toxicity tests  with rainbow trout that had been exposed to
40 ug/1 of  total  residual chlorine for 24 hours immediately
prior to testing  with phenol.   Trout previously exposed to
chlorine had a 48  hour  LC50 of  7.7 mg/1 for phenol,
significantly  lower than the LC50 of 10.1 mg/1 for non
chlorine- exposed  trout.
    Based on these data it is  recommended that all fish used
in aquatic 'toxicity^tes ting -contain" no'more'than- 0; 5 ug/l~
PCB, and not be exposed to any  contaminants during holding.
               b.   Care  and Handling
    Upon arrival—a.t,^t:he,~lah.oEaiojqE^-^tis;h—or^eg^gs^s^houid^---**--^
immediately be  cared  for to prevent additional stress from
crowding during transport.  The test organisms should be
gradually transferred to the holding water at the testing
facility as soon  as possible.
    Alexander  and  Clarke (1978)  performed-acute toxicity
tests with  rainbow .-trout and five different ^potential.---;----.-
reference toxicants to  determine what effects^starvation,
changes in  temperature, and crowding would have on the
median survival time  (MST) for  each toxicant.  Trout that
were starv.ed -£or=i5-=21^days^before^5sepa^ate;^exp0ffui?«s-^o^'?s«i'1
phenol and  sodium  pentachlorophenate had s ignif icantly_lower
MST's than  fed  fish.  There were no differences in MST's
among fish  exposed to sodium azide, copper sulfate, or
dodecylsodium  sulfate.   When trout that had been held at
10 °C were s ub.je ct ed to._ temper at.ure_dec.r.eas-es_^of__L=.5 .?_C~.a.ve.r
                                15

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                                                          ES-6
                                                 August,  1982
24 hours and then  immediately transferred to test chambers
at 15°C, only  fish exposed  to phenol had a significantly
lowe MST than  fish gradually acclimated to 15°C.
Temperature stress did  not  alter the MST for the remaining
four compounds.
    To determine  the  potential effects of crowding, one
group of trout was  held  at  a high density of 3.3 g/1 day,
compared to the normal  holding density of 0.6 g/1 day.  Only
fish exposed to sodium  azide and sodium pentachlorophenate
had significantly  lower MST's due to crowding.
    In subsequent  toxicity  tests with just phenol, the
authors determi^ed"^hmt'troat''wrth*holding™mcirtal-r'ties' as	
high as 7-9% and  18%  had LCSO's similar to those generated
with trout with- only-a- lr?2%~holding mortality.._._,—
    Althou-gh—the^abo^€^j^s~e,ar.:cJaa*djaes^nQt^p:r.es.eji
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                                                          ES-6
                                                 August,  1982
to testing chambers.
    Although  the  importance of  temperature acclimation  to
the test temperature  is  still  unclear/ a maximum gradual
change of 3°C/day  is  recommended at this time.  Peterson and
Anderson (1969) concluded  that  complete acclimation to
temperature,  based  on changes  in locomotor activity and
oxygen consumption, requires  approximately two weeks before
metabolism is  back  to normal.   They also determined that the
rate of change was  more- important.than the amount- of change.
    Changes in the  hardness of  water to which the fish  are
exposed should also be controlled.   Lloyd (1965) determined
that t r ou t tr ans f e r red ~f rowfrard-" to 'soft" wa t e r ire ed ed ~ a t~
least 5 days  of acclimation to  the  soft water before their
response to.a., toxic metal  was-the same as the response  of
f ish -continuall.y_-h.el.d--»inii.s..of-t«.waiter-.--^,-^..— ——— —
         3.   Facilities	
               a.  General
    Facilities needed to perform this test include:  (1)
flow-through  tanks  for holding  and  acclimating fish, (2) a
mechanism for  controlling  and  maintaining the water
temperature-during. the_hol.ding..,-acclimation.,-and test-   -
periods, (3)  apparatus"for straining'particulate'matter, ~
removing gas  bubbles, or aerating the water when water
supplies contain-particulate" matter, gas bubbles,  or
insufficdent-dis-so Lv-edra o x y.§e n^*i*e&pe.£ fcriv e 1 y y ^4^a n t -ars. < 3 &
apparatus for  providing  a  16-hour light and 8-hour dark
photoperiod with a  15- to  30-minute transition period,  (5)
chambers for  exposing test fish to  the test substance,  and
(6) a test substance  delivery system.
                                11

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                                                         ES-6
                                                 August,  1982
    Flow-through tanks, into which a continuous or
intermittant flow of water occurs, should  be  used for
holding and acclimating test fish.  The renewal of  the  water
in flow-through tanks minimizes  the accumulation of
metabolic products such as ammonia.  The build up of organic
matter within the tanks might provide  a nutrient source for
bacteria present in the water.   Bacteria using oxygen to
metabolize and decompose  the organic matter  in the  tank
could then reduce the dissolved  oxygen concentration of
water.  Decreased dissolved oxygen concentrations as well  as
the accumulation of ammonia could increase the likelihood  of
disease in the test fish  (Brauhn and Schoettger 1975).   The
use of diseased fish in acute toxicity tests  could  result  in
the development of inaccurate and unreliable  data.
    The effects of sudden. temperatur.e._,changes__ozL_f.is.h ..may	
range from death to temporary impairment of physiological
functions, depending on the acclimation temperature, the
magnitude of the temperature change, the temperature
tolerance of the species, and the circumstances and duration
of the exposure.  To avoid any undue stress,  accurate
temperature control devices should be used to both  maintain
constant temperatures, and to gradually increase or decrease
the temperature during acclimation procedures.  Such
mechanisms have been described by DeFoe (1977) and  Lemke and
Dawson (1970).
    Particulate matter and gas bubbles, if present  in the
dilution water, may clog the toxicant delivery system used
in flow-through tests.  Gas bubbles also may  cause  excessive
loss of volatile test substances.  Either circumstance  may
alter the concentration of test  substance  to-which  the  test
                                18

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                                                         ES-6
                                                 August,  1982
fish are exposed.  To avoid this problem  an  apparatus
capable of removing particulate matter  or gas  bubbles  from
the dilution water may be required.   If the  dilution water
is heated prior to use,it may also  be necessary  to  de-
saturate the water from >100% of oxygen saturation.  Penrose
and Squires (1976) describe a suitable  apparatus  for this.
    An adequate supply of dissolved oxygen should be
available to the fish.  To facilitate this,  the  dilution
water or holding water should be at 90- 100% of  oxygen
saturation prior to delivery to the holding  tanks or test
system.
    The duration, and intensity of  light  are environmental
variables which could possibly influence  the results of
acute toxicity tests.  Any possible variations  in test data
due to - dif f erences--in~ Light, .conditions, can -be - mi-n i-mized - by -—••
using uniform light conditions during testing.   A device  °
capable of regulating photoperiods and the transitions from
light to darkness and darkness to light has  been described
by Drummond and Dawson (1979).
              b.  Construction Materials
    Due to the toxicity of many heavy metals  at  low
concentrations (U.S. EPA 1976) and the ability of  metal
pipe, galvanized sheeting, laboratory equipment, etc.  to
leach metals into water, no metal other than  stainless steel
(preferably #316) should be used.  In the  same manner, un-
aged plasticized plastic (Tygon® tubing) should  not  be used
due to the high toxicity of a main component, di-2-ethyl
hexyl phthalate (Mayer and Sanders 1973) and  the abilitiy of
DEHP to leach into aquaria systems from plastics (Carmignani
and Bennett 1976)	To avoid any,~pos.s,ib.le_s-tres.s...due—to	
                                19

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                                                          ES-6
                                                 August,  1982
exposure to low  levels  of  metals,  phthalates, and other
potential contaminants,  #316  stainless steel, glass and
perfluorocarbon  plastics  (e.g.  Teflon®) should be used
whenever possible  and  economically feasible.  If other
materials should be used,  conditioning to a continuous flow
of heated dilution water  should be performed for a minimum
of 48 hours.
              c.   Test Substance Delivery System
    To maximize  the accuracy  and precision of test results
developed through  the  use  of  this  test guideline, the
quantity of test substance introduced by the test substance
delivery system  should  be  as  constant as possible from one
addition of test substance to the next.  Fluctuations in the
quantity of test substance introduced into the test chamber
may result .in .ab.normaJ..ly.—higJi-»or-1J.ow...r-es.po.ns.e-^va.lae--(-e-^g-.- .—~
LCSO's) of the test organisms and  in a wider spread of
response values  in replicate  tests.   The greater the
variation in the quantity  of  test substance introduced, the
greater the potential  for abnormalities and spread of the
response values.
    Variations in  the  quantity  of  dilution water entering
the test chambers  during  a given time interval may also
create undersirable differences in test conditions between
test chambers..   The concentrations of dissolved oxyen and
test substance in  a test -chamber-,  ~for example/ -'may decrease ~
more rapidly in  chambers  having lower flow rates.
Differences between test  chambers  in the concentration of
dissolved oxygen,  test substance,  metabolic products and
degradation products,  individually or in combination may
result .in -respons.e_-values  .for__the-._tes-t,..or.gaaisras—whi.cn.-.a.r-e—_-
inaccurate.
                                20

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                                                         ES-6
                                                 August, 1982
    Any one of several  toxicant  delivery devices can be used
as long as  it has been  shown  to  be accurate and reliable.
Various modifications of  proportional diluters have been
used by Auwarter  (1977),  DeFoe (1975),  Mount and Brungs
(1976), and Ozburn and  Smith  (1977).   A manual for their
construction and  operation  has been prepared by Lemke et al.
(1978).  A metering pump  system .has also been used by
Chandler and Partridge  (1975), as  have  saturator systems
(Krugel et al. 1978, Veith  and Corns tock 1975).
    The following criteria  presented  by Hodson (1979) should
be considered when selecting  or  designing a toxicant
delivery system;  1) the delivery of the toxicant should stop
if delivery of the dilution water  stops, 2) it should be
consistent  in delivery  amounts throughout the test period,
3 ) i ndepe nden.t .jDJL~e:le.c.tr.ic-al-_f ^.i.lur.e-^~4J«-i-ndepe.ade*it-=of.*~—..—~
temperature and humidity  fluctuations,  5) capable of
delivering small quantities,  6)  easy  to construct with few
moving parts and  7) easy  to operate.
    The solubility of the test compound should also be taken
into account in selecting an  appropriate delivery system.
If the compound can be  solubilized in water, a device
capable of delivering amounts of test solution greater - than
1 millilter (ml)  will probably be  needed.  If a carrier
should be used, a system  capable of accurately delivering
small amounts.,—less,-,than^lO.Q-.microli-ters.4ul):,> .will -probably
be required to minimize the carrier concentration in the
test solution.
    Each system should  be calibrated  prior to starting the
test to verify that the correct  proportion of test substance
to dilution water is delivered to  the .appropriate. tes.t_  	
chambers.
                                21

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                                                          ES-6
                                                  August,  1982
               d.  Test Chambers
     Test chambers should be constructed of stainless  steel,
 perfluorocarbon plastic, or glass.  Stainless steel should
 be welded,  and the glass bonded with silicone adhesive.   If
 adhesive is used, the amount exposed to the  test  solution
 should be minimized to limit the sorption of test
 materials.   The size, shape and depth of the test chambers
 are not imporant, as long as the volume accomodates  the
 loading requirements.  The chamber however, should be
 sufficiently large and contain enough water  such  that the
 fish are not stressed due to crowding.
               e.  Cleaning of the Test System
     Before use, test systems should be cleaned  to remove
..dus.t, ..dirt., -and^anv.~jath£:r^>debrFas>ie*5^;i^s^
 from previous use of the system.  Any of these  substances
 may affect  the results of a test by sorption of test
 materials or by exerting an adverse effect on test
 organisms.   New chambers should be cleaned to remove  a-ny
 dirt or chemical residues remaining from manufacture  or
 accumulated during storage.  Detergent is used  to remove
 hydrophobic or lipid-like substances.  Acetone  is used for
 the same purpose and to remove any detergent residues.   It
 is important to use pesticide-free acetone to prevent the
 contaraination*rof.i"tehe.sch:arabe:rs«^                            >~
 influence the outcome of the test.  Nitric acid is used  to
 clean  metal residues from the system.  A final  thorough
 rinse  with water washes away the nitric acid residues.   At
 the end of  a test, test systems should be washed  in
 preparation for the. next-.tes-t.»....It—is .e.as.i.er-_to.~cle.an.-th.e	
 equipment before chemical residues and organic  matter become
                                 22

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                                                          ES-6
                                                  August/  1982
embedded or  absorbed  into the equipment.
    Conditioning  the  flow-through system with  dilution water
before it  is used  in  a test allows an equilibrium  to  be
established  between any substance in the water and  the
materials  of the  test system.  Since the test  system  may
sorb or react  with substances in the dilution  water,
allowing this  equilibrium to become -established  before the
test begins  lessens the chances of changes  in  water
chemistry  occurring during at test. Even after extensive
washing, new facilities still may contain toxic  residues.
The best way to determine if'toxic res idues~ remain"rs~ to "~
test for their presence by maintaining or rearing  the test
fish species in-the facility for a period of time  equal to
or exceeding.™£he^^.ime^x.eqoidxjedw^to
               f.   Bilution Water	
    A constant supply of good quality dilution water  is
needed to  maintain consistent experimental conditions during
testing.   A  change in water quality during a test  may alter
the response of the test fish to the test solution.   Most
research on  the effects of water quality-have  centered--	
around the ef-f-e-cts~ of~Jcha-rrges~±n~p~H~B7id~"total~  ha"rdnes's~~6Yr'
the acute  and  subacute toxicity of compounds.   Mauck  et al.
(1977) performed static, acute tests with bluegill  and
Mex a c a r ba t e.*-a&s.va r^iQus3rpft*s:»^sriF.h^
was 38 times more  toxic at a pH of 9.5 and 5 times  more.   	
toxic at a pH  of  8.5  than at a pH 7.5.  They ascertained
however that these large increases in toxicity were mostly -
caused by  the  rapid hydrolization of the parent  compound to
more toxic br.eakdo.wn-_produc.ts.,.,-and_no.t-.to._an._iiicrjaas,e.-La	,
sensitivity  of the fish at the higher pH levels.
                                 23

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                                                         ES-6
                                                 August,  1982
    In another study of the effects of pH, Mauck et  al.
(1976) found that the acute toxicity of  five pyrethroids  did
not change as the pH of the dilution water was  increased
from 7.5 to 9.5.  The toxicity of pyrethrum extract  however
decreased as the LC50 increased from 41  mg/1 at a pH of 6.5
to 87 mg/1 at a pH of 9.5.
 i
    In a study with the formulated herbicide,  Roundup®,
Folmar et al. (1979) determined that the herbicide was five
times more toxic to rainbow trout as the pH increased from
6.5 to 7.5.  Additional increases to pH's of 8.5 and 9.5  did
not further increase the toxicity.  When bluegill were
similarly tested, there was only a 2 fold increase in
toxicity between a pH of 6.5 and 7.5.
    When the toxicity of nitrite was tested at  different  pH
levels, an inverse trend relationship _was observed; -toxicity
decreased with increasing pH (Wedemeyer  and Yasutake
1978).  In static tests with steelhead trout (Salm'o
gairdneri) the toxicity decreased 8-fold for 5  g fish and 3-
fold for lOg fish when the pH was increased from 6.0  to 8.0.
    To estimate the potential chronic effects  of reduced  pH
on freshwater fishes, chronic toxicity tests were performed
with the fathead minnow by Mount (1973)  and with the  brook
trout (Salvelinus fontinalis) by Menendez (1976).  The
results of. both studies were similar; hatchability of eggs
was reduc.ed..,at p.H levels <6.5.,...-Both authors recommended
that the pH of water should be above 6.5 to fully support
the growth and reproduction of these fishes.
    Much work has been performed studying the  ameliorating
effects of increased hardness on the toxicity  of heavy
metals to freshwater fish (Carrol et al. 1979,  Holcombe and
                                24

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                                                         ES-6
                                                 August,  1982
Andrew 1978, Howarth and Sprague et  al.  1976),  but  much  less
has been done with organic compounds.   In  the study by Mauck
et al. (1977) the toxicity of Mexacarbate  to brown  trout
(Salmo trutta) sac fry and Coho salmon  (Oncorhynchus
kisutch) fingerlings did not change  when the hardness  of the
dilution water was increased from  40-48 mg/1 to 160-180
mg/1.
    Mauck et al. (1976) found only slight  variations in  LC50
values when they performed static, acute,  toxicity  tests
with bluegill and five pyrethroids at hardnesses  of 10-13,
40-48, 160-180, and 280-320 mg/1.  The  LC50 of  pyrethrum was
however significantly reduced from 62 to 46.5 mg/1  as  the
hardness was increased from 10-13  to 280-320 mg/1.
Wedemeyer and Yasutake (1978) found  that the toxicity  of
nitrite to 5g steelhead trout decreased  24-t4mes- as the —
hardness was increased from 25 to  300 mg/1.
    Although the reported data demonstrate that relatively
large differences in the pH and hardness of the dilution
water (> 2x) can effect the toxicity of a  compound, it is
not known what role,if any, small  changes  or even large
gradual changes (> 2x) will have on  the acute toxicity of
compounds.
    Brungs et al. (1976) performed a fathead minnow chronic
toxicity test with copper using water collected downstream
from a sewage-treatment plant as the "dilution water. •*• '-~
Throughout testing, the hardness varied  from 88-352 .mg/1,
akalinity from 50 to 248 mg/1/ pH  from  7.5 to 8.5,  .DO  from
5.0 to 13.0 and temperature from 0 to 30°C.  When results
from this test were compared to the  results of  a similar
chron ic tes t -performed- .with- .cons.tan-t.-q.uali-ty-.~d.ilu.tion. .water
                                25

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                                                         ES-6
                                                August,  1982
(Mount 1978), it was shown that the variations  in water
quality had little or no effect on the  toxicity of copper.
    McLeay et al. (1979) performed static acute toxicity
tests with rainbow trout and a pulp and paper mill effluent
using 10 different dilution waters.  Dilution water hardness
ranged from 5 to 400 mg/1, pH from 6.4  to 8.4,  conductivity
from 15 to 778 umhos/cm, and alkalinity from 11 to 392
mg/1.  The 24 hour LC50 values using the 10 waters ranged
from 4.4 to 15.6% effluent and was pH related.  After
adjusting the pH of each dilution water to 6.5, the
variation in the LCSO's was reduced to a range  of 4.4 to
6.9% effluent, indicating little effect due to  the other
measured and non-measured dilution water characteristics.
    Mattson et al. (1976) performed.static-, _acute toxicity--
tests with five organic compounds and fathead minnows in
Lake Superior water and in reconstituted soft water and
found no differences.  Although no data on measured water
quality paramaters of each dilution water were  presented, the
similarity of data from tests done in two obviously
different dilution waters is noteworthy.
    Although there is little data demonstrating that changes
in the quality of the dilution water during testing will
affect the test results, the dilution water should be kept
as constant as-.poss.ible,.during tes.ting -to minimize such, a---
risk.
    A dependable source of clean surface or well water
usually will provide water having greater consistency in its
chemical makeup than water from a municipal water supply.
Municipal water may originate .from several sources, which
differ in chemical makeup.  In addition, municipal water
                                26

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                                                        ES-6
                                                August, 1982
frequently is treated chemically as part of a purification
process.  Since the proportions in which water from
different sources are mixed, and since the chemical
treatment given water during the purification process may be
different from time to time, the chemical makeup of
municipal water may vary considerably.  Reconstituted water,
while theoretically more consistent from batch to batch than
either surface or ground water or municipal water, may  in
some instances lack trace minerals required by some species
of fish.  Cairns (1969) performed many acute toxicity tests
on some compounds with both reconstituted water and natural
water and found that the data generated from the tests  in
natural water were not consistent or reproducible whereas
the results from the tests with reconstituted water were
cons4s-tent.  Of more concern, however, is the prohibitive
expense of continuously preparing reconstituted water for
use in fish-holding and flow-through toxicity tests.
    Fish culturists do not know all of the conditions
required to maintain healthy fish, nor do they know all of
the components and combination of components in water that
adversely affect the health of fish (Brauhn and Schoettger
1975).  Nevertheless, to avoid possible inconsistencies and
inaccuracies in test results, healthy fish are needed for
use in toxicity tests.  There is, therefore, .a need >to
determine that the dilution water, whatever its source, is
able to support the fish species to be used in a healthy
condition for the duration of the holding and testing
periods.
    An appropriate way to make that determination is to
place young fish of a sensitive species, preferably the one
                                27

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                                                         ES-6
                                                August,  1982
to be used in subsequent tests/ in the dilution water  for an
extended period of time and observe their behavior, growth
and development. Ideally, those observations should be made
by an experienced fish culturist familiar with certain
stress reactions which are difficult for an untrained
observer to identify.
    Surface and ground water may vary considerably  in  their
chemistry depending upon the season of the year and
precipitation patterns.  Variations in the chemistry of
surface water may involve the quantity of particulate
matter, dissolved organic and inorganic chemicals, un-
ionized ammonia, residual chlorine and various other
contaminants.  As an indication of uniformity of the
dilution water used in the toxicity tests, it is recommended
in the guideline that certain water chemistry parameters be
measured at least twice a year, or more frequently if it is
suspected that one or more of those parameters has changed
significantly.  The water chemistry parameters singled out
and the maximum acceptable concentrations listed for these
parameters are among those generally accepted as substances
and concentrations which do not adversely affect freshwater
fish (APHA 1975, ASTM 1980).  Recognizing that some
variation in water chemistry is normal in natural surface
waters, a 10 percent fluctuation from month to month in
water hardness, akalinity, and conductivity, and a variance
of 0.4 pH units is accepted as suitable.
              g.  Carriers
    A carrier may be used to aid in the dissolution of a
tes t compound -into- dilation-water-only-af ter_s-ignif ican.t	
efforts to dissolve it in dilution water of dilution water
                                28

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                                                         ES-6
                                                 August,  1982
stocks have failed.  Schoor  (1975) believes  that  the use  of
a carrier may interfere with  the uptake of  the  test  compound
by the test organisms; if the carrier molecules affect  the
adsorption of the  test compound at the gill  surface, there
will be a resultant change in the rate of transport  into  the
test organism.  The author also states that  the use  of  a
carrier may increase the concentration of compound in the
test solution above solubility by creating  a stable  water
emulsion.
    When a carrier is required, triethylene  glycol (TEG),
dimethyl formamide (DMF) or acetone may be used.  The
solvents should be tried in the order stated due  to  their
relative toxicity  to fathead minnows as reported  by  Cardwell
et al. (manuscript 1980).  The minimum amount should -be used
and the concentration of TEG should not exceed 80 mg/1, the
MATC (maximum acceptable toxicant concentration)  value.
Concentrations of  DMF and acetone should not exceed  5.0
mg/1, the MATC for DMF.  Although there is no MATC value  for
acetone, its acute toxicity is similar to that of DMF.
    Ethanol should not be used due to its tendancy to
stimulate the excessive growth of bacteria in the test
chambers .
         4.  Environmental Conditions
              a.   Loading
    In the static  tests, the  loading should  not be so high
to deplete the dissolved oxygen or result in significant
depletion of the toxicant due to uptake of  the  chemical by
the fish.  A maximum loading of 0.5 g/1 will generally  be
sufficient. fox-_comp.ounds	that-. do._not- -have	a. high-  		  	
bioconcentration potential, or are not likely to  reduce the
                                29

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                                                         ES-6
                                                 August,  1982
dissolved oxygen concentration due  to degradation.   For
other chemicals, flow-through tests should  be  performed.
    A maximum loading rate of 0.5 g/1 day flow-through  tests
should be sufficient to maintain proper  dissolved  oxygen  and
ammonia concentrations, and the proper concentration of the
test chemical in the test solution.
    In a flow-through study by Blanchard et al.  (1977)  a
loading of 1.9 g/1 day was not sufficient to prevent loss of
14 C-sec-butyl-4-chlorodiphenyloxide from the  test  water.
The concentration of test substance decreased  more  than 50%
during the first 12 hours of exposure and did  not return  to
the expected concentration until after 72 hours.
    In flow-through studies with 2 strains  of  rainbow trout,
Alexander and Clarke -(.19.7.8.) .t.es ted-phenol._a.t. _thr-ee_diifej:eot-
loading rates of 0.7, 1.4, and 2.6 g/1 day  and found no
significant differences in MST's between the three  rates  for
each strain.  These data indicate that at least  for phenol
loading up to 2.6 g/1 day is not an important  factor.
              b.  Dissolved Oxygen
    The level of dissolved oxygen maintained in  a  test
chamber can influence the sensitivity of tesf  organisms to a
test substance.  Increased acute toxicity of hydrogen
cyanide was observed in various fish species with the
dissolved oxyge.n ..concentr-a.tion-was, below -5-mg/1 ~or-:~    ~~
approximatly 60% saturation at 25°C (Smith  et  al.  1978).
Fathead minnow growth was inhibited at a dissolved  oxygen
concentration between 5.0 mg/1 and 7.3 mg/1 at a temperature
range of 15-25°C, equivalent to approximately  65% saturation
(Brungs 1971).  .
                                30

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                                                          ES-6
                                                 August,  1982
    In order  to  provide  a minimum margin of safety  in  acute
toxicity tests,  a  dissolved oxygen concentration greater
than 50% of saturation  is recommended as a minimum.
              c.   Temperature
    Since fish are poikilothermic, nearly all their
biochemical processes are affected by the water temperature
to which they are  exposed.  Prosser (1973) states that for
every 10°C rise  in temperature,  the metabolism of fish
normally increases by a factor of two.  Therefore,  it  is
likely that the  toxic effects of chemicals can be
temperature dependent.
    During 96-hour tests with mercuric chloride and rainbow
trout, MacLeod and Pessah (1973) noted that increased
toxicity.~was .di^ectly-^-r..ela4:ed~"to-an-inc-j-ease -i-n-~—  --•  - -
temperature.  Similar results were seen for the herbicide
Roundup® (Folmar et al.  1979) and quinaldine sulfate
(Marking and Olson 1975).  It should be recognized,
however,that not all chemicals exhibit a temperature related
variance to acute  toxicity (Smith and Heath 1979).
    The optimal  temperature, at which acu-te toxicity tests
were conducted has  yet  to be identified, but there  are some
temperatures which have  undergone wide spread use and
acceptance.   In  accordance with  these practices the Agency
recommends --• tha^-acu^-*tO'x*fcei"ty<*-tes bs * with-3-the" fa thread'*mi:nnow"
and bluegill be .performed at. 22± l°C.and tes_ts wi.th-rainbow-
trout be performed at 12° ± 1°C.
              d.   Light
    Although light is recognized as a potentially important
e n vi r o nme n.t al. _v.ar.Lab Le..,,_vjs.ry__f -ew..,.s t.udies. Jxa v.e_ be.en_pe.r-f ojrjned
evaluating its potential effects.  McLeay and Gordon (1978)
                                31

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                                                         ES-6
                                                 August, 1982
 found no difference in the toxicity of a pulpmill effluent
 between tests performed with 8/16 and 16/8 hour light/dark
 photoperiods.  To avoid any possible effects from extraneous
 light sources in the laboratory, the recommended photoperiod
 is  16-hours  light,  8-hours dark, with a 15-30 minute
 transition period.
     C.   Reporting
     A coherent theory of the concentration-response
 relationship was introduced by Bliss (1935), and is widely
 accepted today.   This theory is based on four assumptions:
 (a)  response is  a positive function of dosage, i.e., it is
 expected that increasing treatment rates should produce
 increasing responses, (b)  randomly selected animals are
 normally distributed with respect to., their sensitivi.ty _to~ a	
 toxicant, (c) due to homeostasis,response magnitudes are
 proportional to  the logarithm of the dosage (stresses) to
 produce arithemtically increasing responses (strains) in
 test animals populations,  (d) in the case of direct dosage
 of  animals,  their resistance to effects is proportional to
 body mass.  Stated  another way, the treatment needed to
 produce a given  response is proportional to the size of the
 animals treated.
     The concentration-response curve,  in which percent
^mor.t al i ty^is-^JLo tted^as^a^f-uactiorwof ^£
 substance concentration, can be interpreted as a cumulative
 distribution of  tolerance  within an experimental population
 (Hewlett and Plackett 1979).  Experiments designed to
 measure tolerance directly (Bliss 1944) have shown that
 tolerance, in most cases,  is lognormally distributed_within
 a population. Departures  from the lognormal pattern of
                                32

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                                                          ES-6
                                                 August,  1982
distribution are  generally associated with mixtures of very
susceptible and very  resistant individuals (Hewlett and
Plackett 1979).   In addition/  mixtures of toxicants can
produce tolerance curves  which deviate significantly from
the lognormal pattern (Finney  1971).
    If tolerance  is lognormally distributed within the test
population, the resulting concentration-response curve will
be signmoidal in  shape, resembling a logistic population
curve  (Hewlett and Plackett 1979).  While estimates for the
median lethal dose can be made directly from the
concentration-response curves,a linear transformation often
is possible, using probit (Bliss 1934, Finney 1971) or logit
(Hewlett and Plackett 1979) transformations.
  . Once the.JOQorLtal.Lty_^a±a.^hayje-JDe1ej^-^tr^j^»formed»^^c^r *.~-^~.-,
straight line can be  fitted to the data points.  Although
this line  is most often fitted by eye (APHA 1975), a least
squares linear regression procedure is strongly recommended
for this purpose  (Steel and Torrie 1960).  From the
regression equation,  confidence limits can be determined for
predicted  mortality values. An additional advantage is that
the s ign if icance  of--the • -slope- of—the-~r egress"i~orr~ilne~can~be ~*
determined (Draper and Smith 1966).  By using replicate
tests, and analysis of variance can be performed to
d e termi ne --whre^fetee r^e3^ad:Hi^ire->iiofegdafca^^^                rssr>s. .--.-
regression line are random fluctuations or indications that
a linear model is an  inappropriate representation of the
data points (Draper and Smith  1966).
    While  the values  for  the median lethal dose, LC50, can
be estimated graphically__.fr.om_linearl.i.z.ed__conaen-trat.io.n=	
response curve, other techniques are preferable since the
                                33

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                                                         ES-6
                                                August,  1982
graphical method does not permit the calculation of a
confidence limit (APHA 1975).  The probit method uses probit
transformation and the minimum likelihood curve fitting
technique (Finney 1971).  The Litchfield and Wilcoxon method
is a modified probit methods which does not require partial
kills, as does the unmodified probit method (Litchfield  and
Wilcoxon 1949).  The logit method utilizes either the
maximum likelihood or the minimum Chi square method to
estimate the LC50 (Ashton 1972, Berkson 1949).  The moving
average method is simple to apply but depends on the
symmetry of the tolerance distribution to provide accurate
estimates (Thompson 1947).  It cannot be utilized to
calculate any concentration level other than the LC50.   An
additional disadvantage is that confide nee -limLts -far -the	
LC50 cannot be calculated if no partial kills are available.
    The lack of partial kills seriously impairs the utility
of the probit, logit, and moving average methods.  In
situations where there are no partial kills the binomial
test (Siegel 1956) can be used to estimate the confidence
limits around the LC50 value (Stephan 1977).  The LC50 value
can be calculated .from, the-.relation

              LC50 = (A B) 1/2

    Where

    A = concentration at which no organisms die
    B = concentration where all organisms die

    A and B are the confidence limits of the estimate and
                                34

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                                                        ES-6
                                                August, 1982
are significant above the 95 percent level if six or more
tests organisms are exposed at each concentration level.
    If dose-response data are plotted for each 24 hour
interval throughout the test, the LC50 determined from each
curve can be plotted as a function of time, yielding an
acute toxicity curve (APHA 1975).  This curve may approach
the time axis asymptotically, indicating the final or
threshold value of the LC50.  The absence of a threshold
LC50 may indicate the need for a test of longer duration.
    The LC50 value has limited utility, since a number of
substances with entirely different toxicity characteristics
can produce identical LC50 numbers.  The difference will
therefore be in the slope of the concentration-response
curve (Casarett and Doull 1975).
    The majority of response data will produced a near-
linear regress-ion: liner" Yet"very valuable informa'tron is
gained when the regression line is found to deviate
significantly from a straight line.  For example, in fish
bioassays, the concentration-response line can appear
straight from the one percent to the-40 percent, effect- level
and then bend abruptly to the horizontal. Above a certain" "
level of test subs tance-concentrafrionrno"further "mortality""
of fish occurs; '~Further increments of test substance simply
precipitate from solution and become unavailable to fish.  A
low slope or broken regression line can occur when the
experimenter has inadvertently mixed two populations of
experimental animals (markedly different in their
susceptibility) together at each treatment level.
                                35

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                                                         ES-6
                                                 August,  1982
III.  Economic Aspects
    The Agency awarded a contract  to Enviro Control,  Inc.  to
provide us with an estimate of the cost for performing
static and flow-through acute  toxicity tests  according  to
this Guideline.  Enviro Control supplied us with  two
estimates; a protocol estimate and a laboratory survey
estimate.
    The protocol estimate was  $621 for a static test  and
$743 for a flow-through test.  These estimates were prepared
by separating the Guideline into individual tasks  and
estimating the hours to accomplish each task.  Hourly rates
were then applied to yield a total direct  labor charge.  An  -
overhead rate of 115%, other direct costs  of  $40  for static
and $50 for flow-through tests, a  general  and  administrative
rate of 10%, and a fee of 20%  were then added  to  the  direct
labor charge-to-yield the=- final- estimates "	~~ ~""~
    .Enviro Control estimated .tha.t. .differences--in~s.alar.ies.^—
equipment, overhead costs and  other factors between
laboratories could result in as much as 50% variation from
this estimate.  Consequently they  estimated that  test costs
could range from $310 to $931  for static tests and $372 to
$1115 for flow-through tests
    The laboratory survey estimate was $471 for static.tests
and $795 for flow-through tests._-Five laboratories-supplied-
estimates of their costs to perform the tests  according to
this Guideline;' These costs ranged •from>$300""td*$625~f6iri"~  J
static tests and-$550 to $1250 for flow-through tests.  -The
reported estimate is the mean  value calculated from the
individual costs.
                                36

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                                                         ES-6
                                                 August, 1982
IV.   REFERENCES
    Adelman IR, Smith LL Jr.  1976.   Fathead minnows
    (Pimephales promelas  and  goldfish (Carassius auratus)
    as standard fish in bioassays  and  their reaction to
    potential reference toxicants.   J.  Fish.  Res. Board Can.
    33:  209-214.

    Alexander DG, Clarke R  Me  V.  1978.   The selection and
    limitations of phenol as a reference toxicant to detect
    differences in sensitivity among groups of rainbow trout
    (Salmo gairdneri).  Water  Reseach  12:   1085-1090.

    APHA.  1975.  American .Public  Health Association,
    American Water Works Association/  and Water Pollution
    Control Federation.,  .J3t,a.ndard  Methods  for Examination of
    Water and-Was tewa-ter, 14th ed.  New York.   American-    •- -•
    Public Health Association.

    Aston WD.  1972.  The Logit Transformation.  New York:
    Hafner _Eub.li.shi.ng^.Co..—	.•>	-
    ASTM .   -19 80-. - -Amer-ican--Soci-e*y'"6o"r--Tes-ting'-a-nd —  -	 •• ••-
    Materials.  New Standard  Practice  for Conducting Basic
    Acute Toxicity Test with  Fishes,  Macroinvertebrates, and
    Amphibians.  E 729-80.

    Auwarter AG.  1977.  A flow-through system for study
    interactions of two toxicants  on  aquatic organisms.
    In:  Mayer FL, Hamelink JL,  eds.   Aquatic Toxicology and
    Hazard Evaulation.  ASTM  STP 634."  American.Society-for_
    Testing and .Materials-:- ~pp,. ,- 9 0^-98. ---

    Berkson J.  1949.  The minimum Chi-square and maximum
    likelihood solution in terms of  a  linear transform, with
   . particu-lar.^r.e£erea©e^to^bi'aass.ay»'a^JU^-Am...y~--Sfea?t-;- As's=0G .-*
    44:  273-278.

    Bills  TD, Marking LL, Olson  LE 1977.  Effects of the
    residues of the polychlorinated  biphenyl:  Aroclor 1254
    on the sensitivity of rainbow  trout to selected
    environmental contaminants.  Prog.  Fish-Cult.  39:  150.
                                37

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                                                     ES-6
                                             August, 1982
Blanchard FA, Takahashi  IT,  Alexander HC,  Bartlett EA.
1977.  Uptake, clearance and  bioconcentration of  14C-
Sec-butyl-4-chlorodiphenyl oxide  in  rainbow trout.
In:  Mayer FL, Hamelink  JL,  eds.  Aquatic  Toxicology and
Hazard Evaluation.  ASTM STP  634.  American Society for
Testing and Materials:   pp.  162-177.

Bliss CI.  1934.  The method  of probits.   Science 79:
38-39.

Blish CI.  1935.  The calculation of  the  dosage-
mortality curve.  Ann. Biol.   22:  134-307.

Bliss CI.  1944.  The U.S.P.  collaboratove rat assay for
digitalis.  J. Amer. Pharm.  Ass.   33:   25-245.

Braughn JL, Schoettger-RA.	1975.  Aquisition and
culture or research fish:  rainbow trout,-, fathead	  ..
minnow, channel catfish,  and  bluegill.  Corvallis,
Oregon:  U.S. Environmental  Protection Agency.   EPA-
660/3-75-011.

Brungs WA.  1971.  Chronic effects of  low dissolved
oxygen ,concentr,a-tions- .on^thfi—faiAa.ad—jninnow ( Plmephales	.
promelas).  J. Fish. Res. Board Can.  28(8):  1119-1123.

Brugns WA, Geckler JR, Gast M.. 1976.  Acute and  chronic
toxicity of copper to the fathead  minnow  in a surface
water of variable quality.  Water Research 10:   37-43.

Brungs WA, McCormick JH,  Neiheisel TW,  Spehar RL,
Stephan CE~, - Stokes- GN.—-1977.' -.Effects:" of -pollution on: .—.
freshwater fish.  J. Water Pollut. Control. .Fed._ 	..
51(6):  1425-1493.

Cairns J Jr.  1969.  Fish bioassays  -  reproducibility
and rating.	Rev-ista..de~Bi.olQ.gLa..,J7:,!•=!3. = , —^.-.	

Carmignani- GM, Bennett-JP.-   1976.  Leaching -of  plastics --
used in closed aquaculture systems.   Aquaculture   7:
89-91.

Cardwell RP, Foremn DG,  Panye TR,  Wilbur  0J.   1980.
Acute and chronic toxicity of four organic chemicals  to
fish.  Manuscript..._ - „__,. ._ -.
                            38

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                                                     ES-6
                                             August, 1982
Carrol JJ,  Ells  SJ,  Oliver  WS.   1979.   Influences of
hardness constituents  on the  acute toxicity of cadmium
to brook trout  (Saluvelinus fontinalis).   Bull.
Environm. Contam.  Toxicol.  22:   575-581.

Casarett LJ, Doull J.   1975.   Toxicology, the Basic
Science of  Poisons.  New York:   MacMillan Publishing Co.

Chandler JH Jr,  Partridge SK.   1975.   A solenoid acuated
chemical-metering  apparatus for use in flow-through
toxicity tests.  Prog.  Fish-Cult.   37(2):  93-95.

Committee on Methods for Toxicity  Tests with Aquatic
Organisms.  1975.   Methods for  acute toxicity tests with
fish, macroinvertebrates, and  amphibians.  Corvallis,
Oregon:  U.S. Environmental Protection Agency.  EPA-
660/3-75-009.

DeFoe DL.   1975.   Multichannel  toxicant injection system
for flow-through bioassays.   J.  Fish.  Res.  Board Cananda
32:  544-546.

DeFoe FL.   1977.   Temperature  safety  device for aquatic
laboratory systems.  Prog.  Fish-Cult.  _39:_  131-  	

Doudoroff P, Anderson  BG, Burdick  GE,  Galtsof PS, Hart
WB, Patrick R,  Strong  ER, Suber EW, Van Horn WM.
1951.  Bioassay  Methods  for the evaluation of acute
toxicity of industrial wastes  to fish.  Sewage Ind.
Wates 23:   1380-97.

Draper NR>-- -Sm-i-th H--.- —19-66-. -—Applied-Regress-ion-'-"	
Analysis.   New  York:   John  Wiley and  Sons.

Drummond RA>~ Dawson -WFv  1970.-"  An" inexpensive" method"
for simulating  diel  patterns  of  lighting  in the
1 ab or a tory... -JTr,ans-_~Amex,. Elsiu- ..So.c....  ,9,94.  434r- 4-3-5- .-*•—*•.. _,*

Eaton JG....  197-0. .Chronic malath-ion toxieity-to the
bluegill (Lepomis  macrochirus  Rafiesque).  Trans. Amer.
Fish. Soc.  103:   729-735.

Finney AJ.  1971.  Probit Analysis.  London:  Cambridge
University  Press.
                            39

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                                                      ES-6
                                             August,  1982
Folmar  LC,  Sanders  HO,  Julin AM.  1979.  Toxicity  of  the
herbicide  glyphosphate  and several of its formulations
to fish and  aquatic invertebrates.  Arch. Environm.
Contain. Toxicol.   8:   269-278.

Hewlett PS,  Plackett  RL.   1979. The interpretation of
quantal responses  in  biology. .Baltimore:  University
Park Press.

Hodson  PV.   1979.   Metering device for toxicants used in
bioassays  with  aquatic  organisms.  Prog. Fish-Cult.
41:  129-131.

Holcomb GW,  Andrew  RW.   1978.  The acute toxicity  of
zinc to rainbow and brook trout.  Duluth, Minnesota:
U.S. Environmental  Protection Agency.  EPA-600/3-78-094.

Howarth RS,  Sprague JB.   1978.  Copper lethality to
rainbow trout  in waters  of various hardness and pH.
Water Research  12:  455-462.

Iwama GK,  Greer GL.  1980.  Effect of bacterial-  .-.:..
infection  on the toxicity of sodium pentachlorophenate
to juvenile  coho salmon.   .Trans . -Amex.. ^Eis-h^ Sac;._	
109:  290-292.

Kenaga  EE.   1979.   Acute and chronic toxicity of. 75
pesticides  to  various animal species.  Down to Earth
35:  25-31.

Kitchell JF, O'Neill  RV,  Webb D, Gallepp GW, Bartell  SM,
Koonce JF-f -Ausmus -B-S-.—— 1979.-- -Consummer- Regxi±ation"~of-	
Nutrient cycling.   Bioscience 29:  28-34.

Krugel  S^ 'Jenkins "D;- Klein "SA.'' 1978. : Apparatus" for  the"
continuous  dissolution  of poorly water-soluble
c omponents _ f or—bioassays,-—JHaJter, Res£ar.ch~1.2z,~ -26.9 =-.27 2,~~

Lemke AE, -Dawson -WF. - 1979. - Temperature mon-i-tor-ing--and-
safety control  device.   Prog. Fish-Cult.  41:  165-166.

Lemke AE,  Brungs WA,  Halligan BJ.  1978.  Manual for
construction and operation of toxicity testing
propotional diluters. Duluth, Minnesota:  U.S.
En vi ro nme-aal -.P-Eafcect-ioa-Ageivey/.=. ^-i-EEA-^.6.0^/-3r-7;8j-sO 7;2- ..-^•= _-.; = -.
                            40

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                                                      ES-6
                                              August, 1982
 Litchfield JT Jr, Wilcoxon F.  1949.   A simplified
 method of evaluating dose-effect experiments.  J.  Pharm.
 Exp.  Ther.  96:  99-1113.

 Macek KJ, Sleight BH III.  1977.   Utility  of  toxicity
 tests with embryos and fry of fish  in  evaluating  hazards
 associated with the chronic toxicity of chemicals to
 fishes.   In:  Mayer FL, Hamelink JLf eds.  Aquatic
 Toxicology and Hazard Evaluation.   ASTM STP  634.
 American Society for Testing and Materials:   pp.  23-34.

 MacLeod  JC, Pessah E.  1973.  Temperature  effects on
 mercury  accumulation, toxicity and  metabolic  rate in
 rainbow  trout, (Salmo gairderi).   J. Fish. Res.  Board
 Can.  30:  485-492.

^Marking  LL, Olson LE.  1975.  Toxicity of. .the -lampr icide
~3-trif luorome.thyl-_4-nitr.op.h.enal— (-TF.M-). -to. -non target -fish
 in static tests.  Investigations in fish control  No.
 60.   Washington, D.C.:  U.S. Fish  and  Wildlife Service,
 Department of the Interior.

 Mattson  VR, Arthur JW, Walbridge CT. 1976.   Acute
 toxicity -,of-.sele.c.ted.-orga>ni-G-Gompou-nds-"to-f a-thead- -  ->= — —
 minnows.  Duluth, Minnesota:  U.S.  Environmental
 Protection Agency.  EPA-600/3-76-097.

 Mauck WL, Olson LE, Hogan JW.  1977.   Effects  of  water
 quality  on deactivation and toxicity of Mexacarbate
 (Zectran®) to Fish. Arch. Environm. Contain.  Toxicol.
 6:  385-393.

 Mauck WL, .Olson LE^._MarJcing..,_LL*. ._19r7.&.  JToxi city—of, . -•- -.,-.
 natural  pyrethrins and five pyrethroids to fish.   Arch.
 Environm. Contain. Toxicol.  .4:. 18-29.  ......
 Mayer
 esters  in aquatic organisms.  Environmental  Health
 Prespecti-ve- 3: - -15 3— 157. — -  -  -

 McCarthy LS, Henry JAC, Houston AH.   1978.   Toxicity of
 cadmium to goldfish ( Carass ius auratus )  in hard  and soft
 water.   J. Fish Res. Board Can. 35.   35:   42.
                             41

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                                                     ES-6
                                             August/ 1982
McKira JM.  1977.   Evaluation  of  tests  with early-life
stages of fish  for predicting long-term toxicity.  J.
Fish. Res. Board Can.   34:  1148-1154.

McKim JM, Anderson RL,  Benoit DA,  Spehar RL,  Stokes
GN.  1976.   Effects  of  pollution of  freshwater fish.  J.
Water Pollut. Control Fed.  48:   1544-1620.

McLeay DJf Gordon  MR.   1978.   Effect of seasonal
photoperiod  on  acute toxic  responses of juvenile rainbow
trout (Salmo garidneri)  to  pulpmill  effluent.   J. Fish.
Res. Board Can.  35:  1388-1392.

McLeay DJ, Walden  CC, Munro JR.   1979.   Influence of
dilution water  on  the toxicity of  kraft pulp  and paper
mill effluent including  mechanisms of  effect.   Water
Research 13:  151-158.

Menendez R.  1976.   Chronic effects  of  reduced pH on
brook trout  (Salvelinus  fontinalis).  J.  Fish. Res.
Board Can.   33:  118-123.

Mount DI.  1966.   The effect  of  total  hardness and- pH o.n
acute toxicity _of:_zinc_to_f.ish<.—:Air_and_Wat.e.r.-JPxxLlat.ian	
Int. J.  10:  49-56.

Mount DI.  1968.   Chronic  toxicity of  copper  to fathead
minnows (Pimephales  promelas  Rafinesque).   Water
Research 2:  215-223.

Mount DI.  1973.   Chronic  effect of  low pH on fathead
minnow survival^ growth—and-reproduction;"-" Water' -	-~
Research 7:  987-993.

Mount DI, Bruhgs~WA.  1967.   A s implif ied  dos ing ' .
apparatus for fish toxicology studies.   Water Research
1:  21-29	-._..   -

Nevins MJ, Johnson WW.   1-978.  Acute toxieity-of
phosphate ester mixtures to invertebrates  and  fish.
Bull. Environm. Contam.  Toxicol.  19:   250-255.

Ozburn GW, Smith AD.  1977.   A mechanical  toxicant
injector for flow-through  toxicity tests.   In:  Mayer
FL, Hame 1 i nk:.rJ:L>-.-eds,w-  Acfuat ic-sToxicoliog^ja-ndnrHa^zacd~~^-r:::
Evaluation.  ASTM  STP 634.  American Society  for Testing
and Materials,  pp.  85-89.
                            42

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                                                      ES-6
                                             August,  1982
Penrose WR, Squires  WR.   1976.   Two devices for removing
supersaturating  gases  in aquarium systems.  Trans.  Amer.
Fish Soc. 105(1):   116-118.

Peterson RH,  Anderson  JM.  1969.  Influence of
temperature change  on  spontaneous locomotor activity and
oxygen consumption  of  atlantic  salmon (Salmo solar)
acclimated to two temperatures.   J. Fish Res. Board Can.
26:  93-109.

Prosser CL.   1973.   Comparative  Animal Physiology.
Philadelphia:  W.B.  Saunders  Co.

Sauter S, Buxton K,  Macek KJ, Petrocelli SR.  1976.
Effects of exposure  to heavy  metals on selected
freshwater fish.  Duluth, Minnesota:  U.S. Environmental
Protect ion -Agency.-- -EPA- 60 0/3- 7 6 -10 5.

Schoor WP.  1975.   Problems  associated with low-
solubility compounds in  aquatic  toxicity tests:
theoretical model and -solubility "characteristics of
Arolor® 1254  in  water.   Water Research-9: — 937-^944." ..:.:
Scott. WB.,. Cros.smaji. EJ.. ____ 19JL3..~ -JlresJiwaJ:ej:».£-isiies-.of— .- ..... ......
Canada.   Bulletin  184,  Fisheries Research Board of
Canada.

Siegel S.   1956.   Nonparametric  Statistics for the
Behavioral  Sciences.  New York:   McGraw-Hill.

Smith HT, Schreck  CB, Maugham OE.   1978.  Effect of
population- -dens-i-ty-and -freedrng -ra±e— on—the -fathead -- .""-"
minnow ( Pimephales  promelas ) .  J .  Fish. Biql.  12:   449--
455.

Smith MJ, Heath AG .   1979.   Acute toxicity of copper,
chroma te.,. ..zinc.^ .ayao-ide. ^to* .*fjs:es>h-wa.t.ej:« .XisJi t. ^efc£ects^«.o.fi .. —
different termperatures.   Bull.  Environm. Contam.
Toxicol.- -22:-. -113-119.

Spehar RC,  Holcombe GW,  Carlson RW, Drummond RA, Yount
JD, Pickering QH.   1979.   Effects  of pollution on
freshwater  fish.   J.  Water . Pollut. Control. Fed.
51(6):  1616-1694.
                            43

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                                                     ES-6
                                             August, 1982
Spehar RC, Carlson  RW, Lemke AE,  Mount DI,  Pickering QH,
Snarski VM.  1980.   Effects of  pollution on freshwa-ter
fish.  J. Water Pollut. Control.  Fed.   52:   1703-1768.

Sprague JB.  1969.   Measurement of  pollutant toxicity to
fish.  I. Bioassay  methods for  acute  toxicity.   Water
Research 3:  794-821.

Steele RGD, Torrie  JH.  1960.   Principles and Procedures
of Statistics.  New York:  McGraw Hill.

Stephan CE.  1977.   Methods for calculating an LC50.
In:  Mayer FL, Hamelink -JL, eds.  Aquatic Toxicology and
Hazard Evaluation.   ASTM  STP 634.   American Society for
Testing and Materials,  pp. 65-84.

Thompson -WR-r >"19*47.-- -Use  of ~movrng "averages-"• and"- •""—
interpolation to estimation and error,  and  relation to
other methods.  Bacterial Rev.   12:   115-145.

Tucker RK, Leitzke- JS* —19-7-9.   Gomparat-ive- toxicology -of
insecticides for vertebrate--wiidlife  and--f ish;-  -Pharmac;-
Ther. 6:   167-220.

USEPA.  1971.  U.S.  Environmental Protection Agency.
Tentative plans for  the design  and  operation of  a
fathead minnow stock culture unit.  Natl. Water Quality
Lab., Duluth, Minn.

USEPA.  1976.  U.S.  Environmental Protection Agency.
Quality Criteria for"water.

USEPA.  1979.  U.S.  Environmental Protection Agency.
Toxic substances control.  Discussion  of  premanufacture
tes ting palrcy -and-technical" issue's'; "request ~for	"
comment.   Fed. Regist.  March 16, 1979.   44:  16240-
16292.
USEPA..- -198.0..  JU..S— -Env.ir.onmental~-.Pr.otection-Agency.— --
Office of Pesticide Programs.  Guidelines  for
registering pesticides  in  the United  States.   Subpart
E.  Hazard evaluation:  wildlife and  aquatic
organisms.  Draft, November  1980.
                            44

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                                                    ES-6
                                            August, 1982
Wedemeyer GA, Yasutake WT.  1978.  Prevention and
treatment of nirite toxicity in juvenile steelhead 'trout
(Salmo gairdneri).  J. Fish. Res. Board Can.  35:  822-
827.

Veith GD, Comstock VM.  1975.  Apparatus for
continuously saturating water with hyrophobic organic
chemicals.  J. Fish. Res. Board Can.  32:  1394-1851.
                            45

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IJVHQ
                                              EG-10  ,
                                              August, 1982
                   FISH BIOCONCENTRATION TOXICITY TEST
                       OFFICE OF TOXIC SUBSTANCES
                OFFICE OF PESTICIDES AND  TOXIC SUBSTANCES
                  U.S.  ENVIRONMENTAL  PROTECTION  A3ENCY
                        WASHINGTON,  D.C. 20460        :

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Office of Toxic  Substances                                  EG-10
Guideline for Testing  Chemicals                     August,  1982


                    FISH BIOCONCENTRATION  TEST
    (a)  Purpose.   This  guideline is intended to be used for

assessing the propensity of  chemical substances to bioconcentrate

in freshwater fish.   This guideline describes a bioconceritration

test procedure  for  the continuous exposure of fathead minnows

(Pimephales promelas) to a test substance in a flow-through

system.  The United  States Environmental Protection Agency  (EPA)

will use data from  this  test in assessing -the haza.rd a chemical

may present to  the  environment.

    (b)  Definitions..- . Jrhe.-def-in.it.io.ns-J..n.-sac.tion..-3^~of. .the-Toxic. -

Substanc.es. ,Co.n.t.roL.~Aat_-(T.SCA)- and-..the.-definitions-i-n-Par-t- -79-2—

Good Laboratory Practice Standards)  are applicable to this  test

guideline.  The following definitions also apply:

    (1)  "Acclimation" is the physiological compensation by test

organisms to new environmental conditions (e.g. temperature,

hardness, pH) .

    (2)  "Bioconcentration"  is the net accumulation of a

substance directly  from  water into and  onto aquatic organisms.

    (3)  "Bioconcentration factor (BCF)" is the quotient of the

concentration of a  test  substance in aquatic organisms at or over

a discrete time period of exposure divided by the concentration

-------
                                                            BG-10
                                                    August,  1982
in the test water at  or  during  the same time period.

    (4)  "Carrier"  is a  solvent used to dissolve a test substance

prior to delivery of  the test substance to the test chamber.

    (5)  "Depuration"  is the elimination of a test substance  from

 a test organism.

    (6)  "Depuration  phase"  is  the portion of a bioconcentration

test after the uptake  phase  during which the .organisms-are in

flowing water to which no test  substance is added.

    (7)  "Dilution  water"  is the water to which the test

substance is added  and in which the organisms undergo exposure.

    (8)  "Loading"  is  the  ratio of fish biomass (grams,, wet

weight) to the volume  (liters)  of test solution passing through

the test chamber during  a  24-hr, period.

    (9)  "Organic chlorine"  is  the chlorine associated with all

chlorine-containing compounds that elute just before lindane  to

just after mirex dur ing ....gas- chr-omotog-r-aph-ic-anal-ys is -using- -a—.-.  -

halogen detector.

    (10)  "Organochlorine.*.pes.ticides"^.are^.thos.e-pesvticides twh-ich

contain carbon and  chlorine  such as aldrin, ODD, DDE, DDT,

dieldrin, endrin, and  heptachlor.

    (11)  "Steady-state" is  the time period during which the
                                                   i
amou n t sinof :• ;;t es tizs ubs^taracssbe^i-TigEitallceiEE 'up&iandr

test organisms are  equal,  i.e., equilibrium.

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                                                           BG-10




                                                    August,  1982
    (12)  "Steady-state bioconcentration factor"  is  the  mean



concentration of the  test substance  in  test  organisms  during



steady-state divided  by the mean concentration  in the  test



solution during the same period.



    (13)  "Stock solution" is the concentrated  solution  of the



test substance which  is dissolved and introduced  into  the



dilution water.



    (14)  "Test chamber" is the container  in which the test
    ( 15 )  "Tes t solution?-, is- -dilation -.water- eonfeaijii-ng—the -=••• - • —



dissolved test substance to which test organisms are  exposed.



    (16)  "Uptake" is the sorption of a  test substance  into  and



onto aquatic organisms during exposure.



    (17).  "Uptake phase" is the  initial  portion of  a



bioconcentration test during which the organisms are  exposed to



the test solution.



    (c)  Test procedures — (1)  Summary of  the  test,   (i)  Fathead



minnows are continuously exposed to at least one constant



sublethal concentration of a test substance  under flow-through



conditions for a maximum of 28 days.  During this time, test



solution and fish are periodically sampled and analyzed using

-------
                                                             EG-10
                                                     August,  1982
 appropriate methods to quantify  the test substance
 concentration.  If prior  to  day  28, the tissue concentrations  of
 the substance sampled over three consecutive sampling periods
 have been shown to be statistically similar (i.e., steady-state
 has.been reached), the uptake phase of  the test may be terminated
 and the remaining fish transfe.rred  to., untreated -flowing water
 until 95 percent of the accumulated res idues. h.ave. been.	....  _...
 eliminated, or for a maximum depuration period of. .1-4 days.
     (ii)  The mean test substance concentration in the fish  at
 steady-state is divided by the mean test solution concentration
 at the same time to estimate the--bioconcentration factor  (BCF) .
     (iii)  If steady-state is not reached during 28 days  of
 uptake, the steady-state  BCF is  calculated using non-linear
 parameter estimation methods.
     (2)  [Reserved]
     (3)  [Reserv.edJ	
     (4)  Definitive test—(i)  Background information.. -..The
. .f o 11 owi-jag.-- da t su.on«.ike^fcesdk3rSteo~**--•
 testing:
     (A)  Its solubility in water.
     (B)  Its stability in water.
     (C)  Its ;octano£^waiter^pa:ct±&ian~coer^^^                      " .
     (D)  Its acute toxicity  to fathead  minnows.

-------
                                                            EG-10
                                                     August,  1982
    (E)  The  validity accuracy and minimum detection  limits  of
the proposed  analytical methods.
    (ii)  Selection  of test concentration.   (A)  At least  one
concentration should be tested to assess the propensity  of the
compound to bioconcentrate.  The concentration selected  should
not stress or-adversely affect-the fish and shoul-d be less-than
one-tenth the -96^hr  or incipient--LC50 -determined from a-flow-  	
through test  with-fathead.-minnows-.,—~Thfi~ tes-t~CQncentr.atian-&hould-»	
be less than  the  solubility limit of the compound in  water and
close to the  potential or expected environmental- concentration.
The limiting  factqr  of how low one can test is-based  on-the   	
detection limit of the analytical methods.  The concentration of
the test material in the test solution-should-be-.at least  3  times
greater than  the  detection limit in water.
    (B)  If it is  desired to document that the potential to-.._ _..
b io concen tr.a te —is - ..i-ndepe-ndje^t-^ofe^fehe—fees^t^icoaee^ntr*a-fciQ3n.^=*=a^t~.>-leas t *• -•-- - *•<=
two concentrations .should.-be~tested—that...are -at -least a  factor of
10 apart. .  • .- -
    (iii)  Es t ima t ion' of • t es t~ dur a t ion." ~ (A)'  An estimate  of  the
length of the uptake and depuration phases should be  made  prior
to testing.   This will allow the most effective sampling schedule
to be .deite^rmlTEedte^saa^^
state has been reached,  but need not be longer than 28 days.   The

-------
                                                              EG-10
                                                      August,  1982
 test should continue  for at least 4 days.

     (B)   The depuration phase should  continue until at  least 95

 percent  of the accumulated test substance  and metabolites  have

 been eliminated, but  no longer than 14  days.

     (iv)   Test initiation.  (A)  The  test  should not be started

 until  the tes.t. subs tance-delivery sys tern has  been observed-to-foe

 f unct ion ing .proper ly—f or. .at—leas t -48 -.hours...-. —This, r-t.ime— s-hou4d-.be—

 sufficient to-allow .the_..tes..t_subs-tance-.concentr-a.tion.^,to.-become-^— -.

 equilibrated with the test exposure system.   Analyses of  two sets

 of  test  solution .samples -taken prior  to-test-initiation-should-	--

 document, this equilibrium -(-i.e., the- concentrations do not vary  --

 more than 20% from  each other).  At initiation (time 0),  test

 solution  samples should -be-:eollected_-.Timmedia-tel-y- prior - tor.the

 addition of fish to  the test chambers.

     (B)   The appropriate number--of—fathead minnows^s-hould- be

,impa rt.i al ly ,d is-tr i±>u±ed-_Jux.:,each=—tssJt^ch amb-err^-^u^^to^.£«iv.e.^a4tf*>a«rfeimea^

 until  the -appropr.iate-Jiumbers._Jiay.e^JDe.en_disir.ibuJ:ed^^,.4rhe-exa.c^t -,-»->.--

 numb e r .*O£L^ t es ^oi=gaa*i^mssdjepe5mdsia^^                                   ' ~---

 testing,  sample size, and the-number  of- additional-specialized" ' ~ '

 analyses  to be performed at termination.

     (v)   Feeding.   (A)   Fish should be  fed once a day throughout

 the uptake ~ands£d€p;ui£a:t&aa22plxas'es?;rja^^

 just after sampling  to  minimize the effects of the test substance

-------
                                                             EG-10
                                                      August, 1982
present  in  the gut when sampling.   Fish should  be  fed the same

food  at  a similar quantity  as  they received during holding and

acclimation.

    (B)   Uneaten food and fecal  material should be removed from

the test aquaria within 30  minutes after feeding  to minimize

uptake of test substance, by the  food or feces.    ,

    (vi)  Qbservatlons..	(..Aj - * Obs-erva-tions—on-fj.sb-~appearance-and.—..—-..- ..

behavior should. be_ made..and-.records d~da.ily._. Any. abnormal—	.._,-.  	

behavior such  as erratic swimming/ lethargy,  increased

excitability,  or any changes in  appearances or- phys.iol.og.y~ such  as--

discoloration.,..hyperventilat.ion-or- opaque eyes  should be	-

recorded.

    (B)   Observations-on. compound  solubi-lity-should-also be-.:--.:  -----  -

recorded.   These include the appearance of surface slicks,

precipitates,  or material adsorbing—to...the—tes-t— chamber..	

  . _ (vii)   Water .q.u.a.,11 ty-.me.as ur-emenjbs~>-;~Jrhg-~wat.e-r»-tampera-fcuge*. aadm^^w^.-

dissolved oxygen._co.nx:entr.a±.ioa.-s.hou.ld-..be-xecorded~-ai-J,eas4:..dadly- -—-..--

and _.the .pH, tw.i.c.e . week-iy-^niEea.<^^es-^^^

depuration.

    (viii)   Sampling procedures.   (A)   At each  of  the designated

sampling times,  triplicate  water samples and  enough fish should

be collec.t:ed.:;£r:om^thB:^xpasi^

four  fish tissue analyses.   A  similar number  of  control fish

-------
                                                             EG-10
                                                      August, 1982
should also be collected  at each sample point,  but only fish
collected at the first  sampling period and weekly thereafter
should be analyzed.   Triplicate control water samples will be
collected at the time of  test initiation and weekly thereafter.
Test  solution samples should be removed from the  approximate
center of the water  column.
     (B)   At each sampling period, . the -appr.opr.ia.te- namb.er.-of-f.is.h-.  	
is  netted and- removed 'from:-eax:lr"testr chamber;.~~'eare should "be  :: "
taken not to sample  the weakest and consequently  usually the
smallest fish,  especially'during the first few  sampling periods,
to  prevent biasing the  test, results .--Each f is tu is-.pithed , . ~.
blotted  dry and,.then  fr.o.zen._ at.. <-10°.C -if- -not -analyzed...within- four
hours .
     (C)   At termination,  an extra set of fish should be sampled
and eviscerated for quantifying the residues in the viscera.and. .  .
.carcass. . If .a .radio-lab.el.led-_ies,t_compound._.is~us.ed-f_~a».s4a£fici.eat-—,.
number of f ish. should, be..sampled-.a-t_terminatio.n...to_permit.	...  -
ide n t if ica.tio n^and ^uainttl,tafeioji^oi^any^ma^QE^^i^O^ «®f".vpa.r.e-n.t$«!n: .w^ .;**=•
metabolites presenti—I-b  is--crucial;-.to-determi-ne  how~much~of- the *
activity present in  the fish is directly attributable to the
parent compound.
     (.5)   Tes:t. res:U^tS55s(^r)'j=grfri^±og&                               K;^.:;
allowable mortality  of  fish is  10 percent per week.  If more than

-------
                                                              EG-10
                                                      August,  1982
 10  percent of the fish  in the control or  test chamber(s) die

 during any week of  testing, the test should  be repeated.

     (B)   Steady-state has been reached when  the mean

 concentrations of test  substance in whole fish tissue taken on

 three consecutive sampling periods are statistically similar  (F

 test, P=0.05).  A BCF Is -then -calculated .by  div id ing -.- the -mean - ~

 tissue .res idue ..concen-tratian._dur.ing~s-te.adysrs~tate-.by~.th.e~jne.an~tes.t-. - . -.*,,

 solution concentration-dur-ing ith is. ,same...ipe.r_iod«.-.-. _A~9.5~.percent-.,_._	

 confidence interval should also be derived for the BCF.  This  can

 be  done  by calculating  the mean fish tissue .concentration-at_..	

 steady state (Xg) and its 97.5-percent confidence -interval, _+-t-

 (S.E.),  where t is-the  t  statistic    =0.025 and.S.E.  is one    . _   .

 standard error of the mean.  This calculation-would yield tower   - -

 and  upper confidence limits (Lf arid Uf).   The same procedure  can

 be  used  to calculate-the_mean_and -3.7-.5~percent-conf-idence- _	-	

.lnterva.l-.-frcm~the.-.tes-t~saJai.kio.n.^caLnce^



 1 imits—(Jjg- -a'nd -^Sg-^-sas-^Jhe^S -percenfeaeonf :idrem3e^-iflit?eir^a!i»0f^*th'e^*-'1- **••-• "-1""

 BCF  would then~be "between"Lf/Us" and Uf/Ls .

     (C)   If  steady-state  was  not reached  during the 28  day uptake

 period,  the  maximum BCF should be calculated  using the  mean

 tissue ^eora:ettt^:atri^Tir2f*r^^^                             vwsrra«sa\- .i^r~*™- •--<,

 concentration from that and the previous  sampling day.   An uptake

-------
                                                             EG-10
                                                     August,  1982
rate  constant should then be calculated  using appropriate

techniques,  such as the BIOFAC program developed by Blau and Agin

(1978).   This rate constant will  allow the estimation of a steady

state BCF and the estimated time  to  steady-state.

     (D)   If  95 percent elimination has not been observed after  14

days  depuration,-then a depuration^ rate, .cons tant;should-be	-  -.

calculated.   This  rate_constant.-will-allow es.timat.ion-.of,~.the-time	-

to  95% elimination.

     (ii)   Analytical.  (A)  All samples  should be analyzed using

EPA methods  and guidelines whenever  feasible.  The specific   .  . .

methodology-used should_be- validated -before-the- test is

initiated. _  The. accuracy-of.-the-method -should-be-measured by the	

method of known additions.- This  involves-adding a.~known amount-

of  the test  substance to three water samples taken from an

aquarium  containing... dilution- water -and- a number-of—fish-equal  to	•-

.that, to.Jae. us.ed-in^the^tes.t*-.^The,-nomiaal^.coneen-tr^^c^^.o&«tfees«e.--.««--^ -

s amples -s.ho.ald-.be—the^same_as^^the^concen.tr;ation-.to-.be^used -in^t-he ----- -'.-•*•

.test..	S:amplesi^feta&efl«oi*s^^o-asiepsr^te

The accuracy and precision of the -analytical method-should be

checked using reference or split  samples or suitable

corroborative methods of analysis.   The  accuracy of standard

so lu t ions i .Siheoal^v£b:e:schB:ck^d3^                                         - :>*

whenever  possible.


                                 10

-------
                                                             EG-10
                                                     August,  1982
     (B)  An  analytical method is not  acceptable if likely
degradation  products of the test substance, such as hydrolysis
and oxidation  products, give positive or negative interferences,
unless  it  is shown that such degradation products are  not  present
in the  test  chambers during the test.  Atomic absorption
spectrophotome trie: me-thods—^foir-mentals" and gas "chroma tog rap hie
methods .for jD.r.ganlc_c.ompjounds=.-are^pr.eier,ab.le-.to-color?imet-r-ic-—- -
me thods .
     (C)  In  addition to analyzing  samples of test solution,  at
least one. re.ageni. blank .should—also- be~ analyzed- when-a- reagent is
used in the  analysis.
     (D)  When  radiolabe-1-led -test- compounds---are -used ••> total  -----
radioactivity  should be measured, in all samples.  At the end of
the uptake phase, water and tissue samples should be analyzed
us ing appropriate_jtiethodology,-.to.-identify-.and- es-t.ima.te~: the--amount- ~ -
of any,major 4^r
-------
                                                             EG-10
                                                      August,  1982

as possible  to reduce variability.   The standard  deviation  of  the

weight  should be less than  20 percent of the mean (N= 30).

     (C)   Fish used in the same test should be  from the same

supplier or  culture unit and  from the same holding and

acclimation  tank(s).

     (D)   Fathead minnows should not be used if they appear

diseased -or- otherwise -s tressed -or vi-f : more -than~5~percent~- die '•'•' -'•"

during  the 48 -hours prior, to -testing. .,- -Diseas-ed— £is h-s-hou Id. -be— ------

discarded or treated- and -held—for _a--minimunu,o£.-.44. : days, -before— .--.,- -

testing.

     (ii)  Care and handling ... (-A) -  Fish pur chased _fr-om- a ---------

commercial -source -should -be atte-nded to immediately upon

arrival,.-- -Trans fer.-of— the-f -ish- from -the-HS^ippi-ng • to the holding .....

water should -be gradual--- to-reducei-stress^ e'aused."-by=r^rf ferences •* in~-*- •- " ••"•

water quality characteristics  and temperature.  Fish should be

quarantined  and observed, format-least -14~,days.. prior to .-testing.-. „,. r. ,

    .( B )   Dur ing ,-

dis solved _oxyjgen;~.conceatea.tiQn£:s&ouid^                         '<* '• --'•

s a tu r.a t io Q i .£ sHailzdi.ngj€st3iaJcs^sjho4^d:3fee^                  ©e^-of •^--isff.a.ts.

debris.   Fish should-be—f ed ~at - teas t "once -a 'day with~'a-"food which

will support their survival and growth.

     (C)   Fish should be handled as  little as possible.  When

h a nd 1 i ng= ris. .ree
                                   ^^
12

-------
                                                                EG-10
                                                        August, 1982
  and quickly as possible using dip nets made  of small  mesh nylon,

 silk, bolting cloth,  plankton netting, or  other similar knotless

 materials.   Handling  equipment should be sterilized between uses

 by autoclaving, treating with an  iodophor  or  with 200  mg

 hypochlorite/liter.

     (iii)   Acclimation.   If the holding water is not from the

 same source .as: -the ---tes'-t "dilution :wate-r, acclimation '• to the

 d ilu t ion. _wa t e.r,.s.ho.u-Ld— be— done— gradual-ly— ov-e-r- -a -4 8--"hou-r---pe-roid .— — —

 The f is h .s hould -thejx ~be~ h.eld,^_aa,^adx3i.td.on,al.~L4~days. =in* ^fc&e..-— .-..-~=-. .

 dilution water prior  to  testing.   Any changes  in water

 temperature should .not _exce.ed _3-°C .per— day _ .. Eis.h- should -be— he-Id-- --

 for a minimum -of- 7 days  at the test- -temperature prior-  to testing .--

     ( iv)   Lo a d i ng-. - - <•• -Th e- - number -of -Ms-h^ placed --iiv-.e-ach--tes-t-.- --^~--.

 chamber and the--\flow---rate^throu:g-hr-^th

 that the uptake of the test substance by fish  upon introduction

 into the .tes-t. .salutd.an_does--^o,t-.-reduae., the -me assured

. of the tes.t .salut.ioju_ by. ^rao.r.e^th.a^---20»^per-c^ix^a;of^^feh«-^3on;Gen'tr^4^^^^

 me as ur ed vbe£oxe..^tii'6-r£is±[^were^^ati?Qdu^ed.^«^Th^

-exce.ed 0-^lg:.f.isfc^exsM~t'e^^ofc^^e^-t^-s'QskPt^

 hour period,"-aTid~"theTnTnimum'"turTi'ove"r"ra'te""Bhoul""d b'e "6 aquaria

 volumes per 24 hours.   For some compounds,  loading rates  less

 than O.lg/1 may be needed to prevent a substantial loss of test

 s ubs tan;ce,>asaasir:essu-l£^ofe;f r
                                   13

-------
                                                               EG-10
                                                       August, 1982
     (2)   Facilities — (i)  Dilution water.   (A)  A constant supply
of good  quality  water should  be available  throughout  the holding,
acclimation and  testing periods.   Although  unadulterated well
water  is recommended, de-chlorinated tap water or reconstituted
soft water may be used.  A dilution water  is  acceptable  if
fathead  minnows  will survive  and  grow normally for 60  days
wi t h ou t  exh i bi-ting.-s igns _of ~s±res s->-.ri.i.ie..., -^discolor-at.io-rr,~-laTzk--of ~ ~
feeding, poor, res,ponse_.ta_£Xternal..s±.irauli^..~oji.-lethargy^	,,_...„. ..
     (B)   The total  hardness'r.'alka.liart.yvL.pJJ:,:.spe.cif:i:c-ii._-—..   . .
conductance, temperature and  dissolved oxygen concentration of
the dilution water should be  determined weekly.   The. pHlshould	_
not vary more 0..-4-units .and—the~other. .pa.r.ame£e.rs~more—than-1-0 —
percent  on a monthly basis.
     (C)   Recons.tiiat.ed- sof-t~ wa ter-,—i-f - -used-, -s-hould^be=-^pr ep-ared-?-by
adding 4.8 g NaHC03, 3.0 g CaS04    2H20, 3.0  g MgS04/  and  200 mg
KC1  to each 100  1 of deionized.  o.r_.glass. di.S-til_L.ed.1-wa±er,-.o.r—to --
dechlorina.t.ed__tap—waJter. ,jd.th_ a-_toial-_res-id.aal-.clilorJ.na.-	—-,...- .~.
concen.tratioa rl.as.s-.: thaiv l-.ug/l~.—..In..^alL_gas£s .JLhe4-S-pexxL.f j.c^.^.^~.-^-
conductance^ a;ta*2-5-°^£~of^ t h e^^wattjed?. ^ssj-ucc eu-'Srbo.ttl dfisbe^jkessa; itoaaia-.l^sa&t..
micromho/cm.
     (D)   All water  should be  extensively aerated prior to use  if
the dissolved oxygen concentration is less  than 90 percent of
                                  14

-------
                                                          EG-10
                                                    August, 1982
saturation.  If the concentration  of  dissolved  gases  exceeds 110

percent of saturation,  the  excess  gases  should  be removed using

appropriate apparatus.

    (E)  The quality of  the dilution  water should be  constant and

should meet the following specifications  measured at  least twice

a year.



    Subs tance  -—,-.-                    Maximum-Concentration -



Particulate matter                            20  mg/liter

Total organic  carbon     ..    .                  2  mg/liter

          or

Chemical oxygen demand                          5  mg/liter

Un-ionized ammonia- ---•-•                       1  ug/liter   	

Residual chlorine                               1  ug/liter

Total organophosphorus, pesticides--	 50  ng/lite-r	-..•—.



polychlorinat-ediJDiphenyis-4£CBs)^ .*,^.-• -,...=   50  ng/ldter <•*- .-•-.-  -•

                or      -

Organic chlorine -----                         25  ng/liter

Copper, cadmium or zinc          .             10  ug/liter
                                15

-------
                                                              EG-10
                                                      August,  1982
     (ii)  Construction materials.  Materials  and equipment  that

 contact dilution water, stock solutions  or test solutions should

 not leach or absorb substances.  Glass,  #316  stainless steel  and

 perf luorocarbon plastics  (e.g. Teflon ®)  should be used whenever

 possible.  Concrete, unplasticized plastics  and fiberglass  may  be

 used for :holdrng™and.-^accldmation~±anks: and- in---the~ water-supply-"-—"

 sys tern, but -they, .should^be —thoroughly^ condition ed«be.£Q^e-use~by« —

 r is ing with . a. continuous— f low_of-_wa.ter_.>~25f C~ for. 48-Jaours-.^,.— The.

 use of flexible tubing should be avoided as phthalate esters

 leach from these materials.. Cast  iron .pipe., may ~be_used. -but —

 filters will .be -needed- to -remove rust  particles.   Rubber, copper-,

 brass, galvanized metal, and epoxy glue  should not come in

 contact with dilution water, -stock -solutions  or- -test- soiut ions *

     (iii)  Fish holding and acclimation.   (A)   Tanks are needed

 for holding and acclimating_.f athe.ad._minn.ows._pr Lor. Jto -test ing ... __

. Th e. nu mb e r a nd._ s. i z.e_of . -tanks. -,njefieLed_de.pe nds~.upoji^4iie— amoaa.t~«o£. — . •«

 testing .to .be  performed_:and=d:he™availab;idi±

, ;.ag e . .A ?e o ns fc an t-^s up-pliy^oftsigo od^qo-aibsfety^jd i-i=u;t.io msr.
 supplied to a-1-1 -tanks. - -The voltirae"requ-i-red-depends—upon""-the"

 holding temperature and the number of  fish being held, but  the

 flow should be great enough to maintain  a  dissolved oxygen

         :at±on:i>;^&OMp^^                                     -  ..... •


                                  16

-------
                            EG-10
                     August, 1982
     (B)   Temperature control  apparatus are needed  to maintain the
desired  holding and acclimation temperatures.   Apparatus controls
should be able to maintain  temperatures within  1°C of the
appropriate temperature.   If  the water is heated,  care should be
taken to avoid supersaturation of gases in the  water.
     (iv)   Testing apparatus.   (A)  Test chambers  can be made from
welded. #316- stainless: -Steel ..or^£rcm^doui3iers-ti:ettgtrh7rgdas-s;rgx>ined'-rrrrr-r.-
with clear silicone— adhes-iv.e,_ — Th.e— s,ize-,—s hape.  and-dept±i~of -Uie-- -.-=
test chambers—are .^ol^impaEtarLt^asr^ong^a&-JJa,e^
loading  requirements.
     (B)   The test subs tance_deliv-ery_sys tenuused-should __________
accommodate -the phys ical~and  chemical properties-  of  the- tes t--
subs-tance- and -the -s elected • -expos ure.-e.oncen-t-ra-t-ion-.—-_. The. apparatus---- -• ••
used should -aC'Curateiyr-a*id^ precis eiyrdeiiver^thevapprQpr.-iatev^--' ?-'^-  •
amount of  stock solution  and  dilution water  to  the test
chambers .-  The, introduction ..of ,.ihe^tes±_subs±ancew.sh.ould^.iDe^done _____ .-__
in such =a..way as^-to. maximiie^.fe^e^omo,g.ei*e4^s
test, subs tancec.1:J3EOughoutz,the^t;es~t^chamber^.^
   ---(-C)   The .'dilurfeioniwa-tec-s^o^^d^be^desMve^ed^tio
headbox  from'whrich rt-can~-f"l*ow-by-gravrty~to--the™t'es-t"sabstance '
delivery system.  Use of  a  headbox facilitates  a  constant
delivery rate and heating or  cooling of the water  to the
                 «.-
17

-------
                                                           BG-10

                                                    August, 1982
headbox may also be easily aerated or degassed  as  the situation


dictates.


    (v)  Cleaning of test apparatus.  Delivery  systems and test


chambers should be cleaned before and after  each use.  If there


is obvious absorption of a test substance  by the silicone


adhesive, those applicable parts of the  delivery system should be


discarded.


    ( 3)  Tes t par.ame.ters^-=.(.l)—„Dissolved-oxygen,.-  The-dissolved*.^


oxygen concentration"in-each  chamber" should  be  greater than 5.3


mg/1 (60 percent of sea-level saturation at  22°C)  throughout


testing.


    (ii)  T em pe ra'tu rev •' %Th e tes't tempera ture~shou-ld" tie 22 "il°C.


Temporary excursions (< 8 hours) to 20 or  24°C  are permissible.


    (iii)  Lighting.  A photoperiod of 12  hours light and 12-


hours dark with a 15-30 minute transition  period is recommended.-


    (iv)  Test substance-.  The name- and—purity-of  the~test" -—-


substance to be tested will be specified in  the test  rule.


Radio-labelled compounds should not be used  unless -there are no


suitable, validated,.analytical techniques to measure unlabelled
                                           \

test substance in fish, or the costs of  these analytical


techniques are very high.


    (v)  Carrier use.  Whenever gps s iblei,  t he _._ tes t s ubs t ance


should be added directly to the dilution water  or  from a water



                                18

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                                                          EG-10
                                                    August, 1982
stock solution.  With  compounds  having a low water solubility, it

may be necessary to prepare test solutions using a carrier.

The carriers to be used,  in order of  preference are:  triethylene

glycol (TEG), dimethyl formamide (DMF) and acetone.  The amount

used should be kept to a  minimum and  should not exceed 80 mg/1 in

the test solution for  TEE and  5.0 mg/1 for DMF and .acetone. .	

    (e)  Reporting.  In addition to the information required in

Part 792.^:Good Labor>atoxy^EEa&ice^Sfeand-a•Eds^^-•»-•the^•E•epo^t•>s-hou-ld^•-;''•-

contain the following:

    (1)  The source.of_the-dilution.water,-its mean monthly

chemical characteristics  (total  hardness,  "alkalinity, ~pH,

specific condu-ct-anc'ey- temperature and D.O.) and a'description of

any pretreatment.

    (2)  Detailed information  abou-t the-fathead -minnows us-edv

including age,-^mean -and—standa-rd-d^viati-ow^ wet—weigh t~<-bl-ot tedJ —

dry) and standard-length,-source/ history  of disease, parasites

and treatment, acclimation procedures, and food used.

    (3)  The number of  organisms ..tested , -loading -rate ..and...volume

additions per 24 hours.

    (4)  The percentage mortality of  control fish and fish in

each exposure chamber  and any  observed abnormal behavioral or

physiological effects.  .  ..   .     -

    (5)  The method of  stock solution preparation including
                                19

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                                                            EG-10
                                                     August, 1982
nominal and  measured concentrations and  solvent used.

     (6)  The mean,  standard deviation  and  range of the

temperature, dissolved oxygen concentration  and pH during the

test period.

     (7)  Photoperiod length and light  intensity.

     (8)  Description of sampling and analytical methods for water

and  tissue analyses.

     (9 )  The .mean,. s±andard,-deviation.!_and~-.r.angre-of^.fefeeoii-.»-^--^-- -—-

concentration -of - testr compound~rn"the::.t.es±-solution—and—f ish—- —

tissue at each  sampling period.

     (10)  The -time—to ~s~te ady-s'tatev	" ~"
                                        o
 ""   (11)  The "steady^sfate or"maxiinuiTrBCF  and" "the~95%":"corif"iderhce" '

limits.

     (12)  The time-to 95 percent elimination-of-accumulated

res idues......                               .

     (f )  Ref erenees•-. — Bl-au~ GE-7-Agin CL.-	-A  users-  mararar-fOTr- •  "'J'

BIOFAC:  A computer program for characterizing the ratio of

uptake and clearance of chemicals in aquatic organisms.  Dow

Chemical Co.  March  15, 1978.
                                 20

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                                           DRAFT
                              ES-7
                              August,  1982
       TECHNICAL SUPPORT,DOCUMENT^.

                  FOR

       FISH  BIOCONCENTRATION TEST
       OFFIC E-OF ATOXIC,
OFFICE  OF PESTICIDES  AND TOXIC SUBSTANCES
  U.S.  ENVIRONMENTAL PROTECTION AGENCY
         WASHINGTON, D.C.  20460

-------
                      TABLE OF CONTENTS

                                                         Page
I.     Purpose                                             1
II.   Scientific Aspects                                  1
      General                                             1
      Test Procedures                                     1
      Test Substance Concentrations                       4
      Duration of Test                                    5
      Test Conditions                                     8
      Test Species                                        8
      Selection                                           8
      Maintenance of Test  Species                         10
      Acclimation                                         10
      Facilities                                          10
      Dilution Water                                      10
      Cons truGtJ.Q.n-.Ma.ter-ials	                       13
      Testing Apparatus                                   13
      Cleaning                                            14
      Carriers                                            15
      Environmental Conditions                            16
      Loading                                             16
      Dissolved Oxygen                                    17
      Temperature -  •  -                                   17
      Light                                               19
      Reporting                                           19
III.Economic -Aspects.-.^--.—x. -   ...                          19
IV.   References                                          21

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Office of Toxic  Substances                      ES-7
                                                  August,  1982

  Technical Support Document  for  Fish  Bioconcentration Test

I.  Purpose
    The purpose  of  this support document  is  to  provide  the
scientific background  and rationale used  in  the development
of Test Guideline  EG-10 to evaluate the bioconcentration
potential of  chemical  substances  in fish.  The  Document
provides an account of the scientific evidence  and an
explanation of the  logic used in the selection  of the test
methodology,  procedures 'and conditions  prescribed  in the
Test Gu.ide.line-__Te.chnic.al—issues—and.-prac.tica]—~~~-—
cons iderations _relevant_to—the-Tes-t Guideline are	
discussed.  In addition,  estimates of the cost  of conducting
the test are  provided.-	-
II.  Scientific  Aspects
    A.  Tes t  Procedures - - --'-—' •
         1.   General
    Fish are  nearly unbiquitous inhabitants  of  freshwater
and marine environments.   In  addition to  their  economic  and
recreational—value r *fJLs.h .QGcu>py-xD.n~essen-tial--posl*taon~in --—
aquatic -food -chains7-feeding"on~ various' forms of~~plant and
animal -llf-e -in'-the 'aq(ja*tic~env-rronm'ent-~and;-~rn~~turrr>~'berng
eaten by. _s.ome .other.. aq.uatic_^oJi.-tarr.estr.taL-cons:amer-r™ --;::"	
Through these  trophic  or feeding interactions,  nutrient  and
energy exchanges occur which  are needed to maintain  the
ecological stability  of the aquatic environment and  sustain
the food chains  of  commercially and recreationally valuable
fish.  Because they are critical links in these food chains,
certain species  of  fish which have no direct commercial  or
recreational  value"1 rn^'themsel^&s-^aT^^es-s-e'rtfiBl^^to"the'"wel"l-J
being of economically  important fisheries.   A chemical  which

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                                                          ES-7
                                                  August, 1982
is highly cumulative  could destroy an economically  or
ecologically  important fish population through  secondary
toxicity or through contamination via food chain  transfer or
magnification (e.g. Kepone/ PCB.).
    In view of  the economic and ecological importance of
fish/ and since man-made chemicals may be released  directly
or transported  into aquatic environments, the tendency of
some of those chemicals—to~accumula.te:~in-£-rsh~.is -ofc --concern--—
to us as we assess the risk they may pose to the
environment.   For this reason, reliable and adequate data on
the bioconcen-tration-poterrt±'ai"of"-chemicals-~±n"f±s'h~shbuTd"~~"
be available  when the effects'of those chemicals  on the
environment are assessed..
    Data from _a_f ish~bioconcen,tration~tesJ:.~caia^.be^used~iaav.~_-.:
conjunction with .data..on_.the_ acute - .to.xicity-.ajid-on- the	
trans po rt and., f ate_of --the.~ch.emi.cal~.Ln~.aii- aqua tic- h-ab i-tatr- in- •
assessing the risk resulting from the release of  that
chemical into the environment.
    Although  water solubility and octanol-water partitioning
data have been  shown  to be useful tools in predicting
bioconcentr-ationc£ae*ars.^-B<3E-^
estimates only  within one  order of magnitude of the degree
to which most-compounds-~'mayi"accumulate~fn"fish"tissue'  "	
(Kenaga .,and- .GoE-L-ng^l9&Q/^N&eiy. ^t^                          -ajk.
1979, 1980).   This  information is .also. not. useful for-some
heavy metals  and inorganics that might bioconcentrate in
noh-lipid tissues such as  bone.  For some compounds  such as
hexachlorocyclopentadiene  and  chlorinated ecosane,  however,
the partition coefficient  grossly, overestimates the BCF.... 	
(Veith et al.  1979).   Such data, if  used exclusively, could

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                                                           ES-7
                                                  August,  1982
result  in  over-regulation of compounds.
     If  data on water solubility,  octanol-water partitioning,
biodegradability, or structure-activity relationships
suggest that a compound may substantially bioconcentrate  in
fish, a bioconcentration test with  fathead minnows should  be
performed  to quantitate empirically the degree to which such
a compound  may be accumulated in  fish  tissue.    >
     There  arer -two-- -.mecthodalog- ies. -usecfc- -to^ay~:to"'es t^ima-tee" ------ ~
bioconcentration potential; the kinetic approach and the
steady-state approach.   Bishop and Maki (1980) and Hamelink
(1977)  review-froth; Busing "the-  kinetic -appro a-chv"Bi"S""hop~ 'arid -
Maki(1980),  Branson et al . (1975),  Cember et al . (1978) and
Krzeminsky  .(-19Z7
e xpr.es s ions » -f 3£jcrau~&alA£ii&eJi^s^JSt^                               •
and depuration ..periods .to- calcula.te-,-Ajp.take..and..depura-tion . -
rate cons tan-ts-..— -These -rate ^^ consrtanfe& -ar.e.- --?t-heiv • u-s-,ed? to-t— .i.-
estimate  the BCF at the time of apparent steady-state, and
the time  to  50%  elimination.  The  steady-state me:thod, in
more wide-spread use, exposes fish for  a longer period of
time until steady-state _in_ the. _tiss.ue— is. -.expeximen tally. ..... . ...
observed-  (-Barrows-^t->»al-r''=i~19^0T"^t^-h-op^an-d^^aMw"^           -i-—
et al . 1979) and continues with a  depuration phase until 50
or 95% eliminatl-orr -h-as~t>eerr o-bserved.   The" estimation of
b io c o n c en.t r-aJ^^oii
and adjust  for changes in the rates  of -uptake ,andi depuration
such as those  observed by Barrows et al.  (1980) and Melancon
and Lech  (1979).   The use of the kinetic  approach; also
requires  access to a sophisticated  computer system,
apparatus not. ..readily aval lab le—to—many-. labora..tori.es... ____ -.» ,  .

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                                                           ES-7
                                                  August,  1982
    Although  Bishop and Maki  (1980)  and Branson et al.
(1975) have shown excellent agreement between estimates of
bioconcentration factors for  some compounds  us ing'both
approaches, we recommend a modified  steady-state method for
determination of bioconcentration potential.   The empirical
nature of  the data, the relative ease with which the test
can be performed, and the number of  researchers and
1 aboratori-es:--that^haver-pecformedrsuch ~ tests  make-this- tes t~~
more appropriate at this time.  As the data  base for
comparisons of BCF's between  the two methods  grows, the
kinetic -appro-ach^may?be-come~moreT"-us'-ef ul 'and-'valuable; • 'Under
the Toxic  Substances Control  Act, we are required to review
all tes t _gu ide.lines-_aanoally:v—and.-in~the_-f uture-we—w-ill- —	
c ons id e r-.ad-.Qp t ing.iJthei«Ici-.nja;t i.^-~a^i&oa(^^*^s&*i*~^
          2.   Test Substance. Concentrations ....   ,	
    Although  vi-rtu a ll-y--all--rese a rckers-, invol ved-.-i-n ------  ...
bioconcentration testing state that the  exposure
concentration should be below toxic effect  levels>  there are
few data  supporting this recommendation.  Tests  determining
bioconcentratio.n_fa.cto-rs--wiJth-fathead-~minnows.^and-i..PCBs-  .--...
(DeFoe et -al.—1978) / - toxapheTre~tMayer"et~-al.--1977)," three'"
chlorinated cyclodiene intermediates  (Spehar et al. 1979),
and acrolein'(Macek eft ~al. "1976 ) shdwed  that there  was

exposure  concentrations-up to and -including  at least one
concentration that caused a reduction in survival or growth.
    Mayer (1976) however performed a  study  with di-2-
ethylhexyl phthalate and fathead minnows  and found  that the-
BCF's increased_sixfold^.,from.l5,5.,..to_8.86f~_as—th.e..,exp.os.-urje..~-~,	
concentration decreased from 62 to 1.9 ug/1.  Bishop and

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                                                           ES-7
                                                   Augus.t, 1982
Maki  (1980)  tested four compounds with bluegills  at two
concentrations-a factor of -10 apart.  In-the  tests with DDT;
tetradecylheptaethoxylate  (AE),  and EDTA, similar BCF's were
observed  at both-concentrations-tested.  Elimination of
accumulated  residues however  was  considerably slower at the
lower EDTA concentration.   In the test with LAS however, the
BCF at  the low concentration  of  0.064 mg/1 was  260, more
than  twi.ee the^&CEiabaerve,  - •  ,
        3.  Dura t ion.-of -Tes t -	- -_ _-	
    The  expos.ure-period..should be-long enough  to—demonstrate
that steady-state has been  reached  in the fathead minnow
tissue.   Most compounds will reach  steady state within the
recommended  28 day -maximum ...up-take..period ..(-Barrows.^et ..al... ..  ^.
1980, Bishop and Maki 1980, Macek et al. 1975, Veith et al.

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                                                           ES-7
                                                   August, 1982
1979).   The depuration period  should continue  until  at least
95% of  the -accumulated residues have been eliminated.  This
will normally occur within  the 14 day maximum  depuration
period.
    Most of the 33 chemicals  tested by Barrows  et al.
(1980),  reached steady-state  in 3-10 days, and  50%
depuration was usually reached in less than  1  day.
Cons equen tly-: i t~ is—clear :that~th:e-~re-lat-i-vely-- I'o^ng'-uptFake" and- -~: "-•
depuration periods (>_ 28 days  uptake, 14 days  depuration)
used by  many researchers are usually not required.
    Before s tart ing-- -a~ M-ocorfcen'tratiotr t*es±> 'air es timatioir • ~ " "
of the BCF and the time to  steady-state should ~be  made. 	
Kenaga - and - Goring-..-(19-8-0)	pr-esen-t—data- a-nd- me t-hods- -to	 —
es t imate..-th-e^BC£.i^^!ibe^"
determined empirically in the  lab.oraiory-or.,-in.-some-cases,	
taken from the literature (Chiou  et al. 1977,  Kenaga and
Goring 1980).   Octanol-water partition coefficients  can be
de t e rmi n.ed _ empir i c al 1-y-f — esiomat ed .-by.- r-e ve rs-e^phas e -h i-.g.h-  -^ ^ .^ - -
pr es s ur e—1 iqaid* ch'roma-tograp'hy^ac ct3rdtiimg^±d*:J^-i'thT^a'nd^ Mo^t r i-g"——-
(1978),  calculated according to Leo et al. (1971)  or taken
from the" literature (Chiou1  et' al. 1977, Hahsch ""et""al"." 197"2,     "
Kenaga and .Gor:iaigo:19;80^ .^«ais- ----"•.- 	
    The  time to steady-state  (S in hours.) can  be. estimated-.
from the  water solubility or the  octanol-water- partition
coefficient using the equations  developed by ASTM  (1980b):

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                                                          ES-7
                                                 August,  1982
       S=3.0/antilog  (0.431  log W-2.11) or
       S=3.0/antilog  (-0.414 log P + 0.122)
       where
       W=water solubility  (mg/1) and
       P=octanol-water  partition coefficient

    Presented below  is  a summary of data correlating various
exposure times to~ the corresponding estimates of partition
and BFC

                           Log P   Log BCFa     BCF
2
4'
7
12
18
22
fit •£•
28
1,585
8',710 ~ — -
33,113
120,226
316,228
524.807
_ ^ t+ ^ j \j \j,.i~ - .. - ~
933,254
3.2
3.9'4
4.52
5.08
5.5
5.72
«/ • 1 £•
5.97
2.02
" 2.65
3.14
4.62
4.0
4.16
• — - - ~ • A v
4.37
105
446
1 387
4150
10,000
14, 521 	
--• ... i*S^^^ii — -
23,686 	
    aLog BCF was~"es t.imated_us,ing  the.^eq.ua..tio.n_i.of ,-V.ei.th :e.t_ -
    al. (1979) where log BCF=0.85 log P - 0.70.
    Based on the estimate  of  the  time to steady state, one
of the following sampling  schemes may be used to generate
appropriate data.

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                                                            ES-7
                                                   August,  1982
Time to Steady-State

Test Period/ Sa<4

Exposure lb
6b
1
2
3
4

Depuration 1
6b
12b
1

S4-14
Sampling
4b
1
3
7
10
12
14
1
2
4
6

> S15-21
Days
1
3
7
10
14
, 18
22
1
3
7
10

S>21

1 '.
3
7
10
14
21
28
1
3
7
10
14
    alength of estimated" time to steady  state in days".
     hours.
    B.   Test Conditions
          1.   Test Species                           :
               a.  Selection                        |
    The  most common fish species used  to  determine  the
bioconcen tr.a.tioj\_..p£it£,n.t i-aJL--Qf_c.ompounds..-.unjder, .fXovjfc through--
c o nd i t io ns •'
(Mayer  1976,  Spehar et  al .  1979, Veith  et al .  1979); rainbow
t rou t , -Sal mo •garrrdneTrr "tBl'an'chfaifd' ~e t "al". "T977, Brans o rT e it
al . 197^. Reiancon^a^d^€«;Gte£»19i7^>.-.tNe3erlLyrf eifessasb. idj9,!7a4s,.-T^*^^.. ..
Reinert et  al .  1974);  and  bluegill Lepomis macrochirus
(Barrows  et al . 1980, Gonz  et al . 1975,  Macek  et al. 1975).
    The fathead minnow  has  been selected  as the test species
for use.  It  can be easily  cultured in  the laboratory  (U.S.
EPA 1971),  thus insuring  an almost cons tant s,upply -of .  .  ....
healthy fish  of the proper  size throughout the year.   It  has

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                                                           ES-7
                                                  August,  1982
been used  extensively in life-cycle  chronic toxicity tests
and early-life  stage tests as summarized  by Macek and
Sleight  (1977)  and McKim (1977).  A  definitive study on
measuring  and  estimating the bioconcentration factor of
chemicals  in fish has also been performed using the fathead
minnow as  the  test species (Veith et al .  1979).  Results of
this study  clearly demonstrate the suitability of this
species. - ..lni--t.es tsuwi±it: hexa.chl-orobenze-rre---and --Iy2-r4- -  ~ •-—
trichlorobenzene, the authors found  that  fathead minnows
accumulated  these compounds to the same  extent as green
sunf ish  ( Lepomis ° cyan el lus-t ^and-" appro x imately '"twice* as"much ~
as rainbow  trout.
    In a sep.ar.ate_s.et._of -tes-ts-wi.th-hexa.chlorobenzene-, --the---
au t ho rs,
had little  effect -on,.bioconcentr-ation., ..Tests with-newly  --
hatched -f ry,,>30 )and^90-,day— o-l-d-rju-ven-iles-y^aad-^appr-oxima-te-ly-^
180-day  old  adults yielded similar BCF's.
    The  source  of the test fish was  also  found by Veith et
al. not  to  be  a significant source of variation in the
bioconcentratio.n._of— a_PCB..raixture~(.Ar.oclar^l.ai6(S.) * - -Tests -.- -
with three  drf-§eTentHHrs^pop:u^l-a't^
brood culture  and with two populations from ponds yielded
s imilar  BCF's .
    Stud i es? by-^Be-F.a© ^eterafe^(d957j'8^«and^Ne^^
demonstrated that gravid fathead minnows. .bioconcentrated. -PCB
mixtures twice  as much as males duri-ng laboratory tests.
This increase  was due . to the increased lip id content of the .
females  compared  to the males.  Consequently we recommend
that
used.  Fish  older than 120 days should  not be used as by the

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                                                           ES-7
                                                   August,  1982
end of  testing they may be sexually mature.  The  detection
limits  of  the analytical procedures used to measure  tissue
residues may preclude the use  of  very small fish  (eg.  <30
days old)  with compounds that  do  not bioconcentrate  *   ~
appreciably.
         2.   Maintenance of Test  Species
               a.  Acclimation
    Brauhn --and~Schoettge-r-- tl^S) : found tha-t- :f is-h: that had - - :
become accustomed to unrestricted  swimming in rearing  ponds
underwent  intense competition  for food and swimming  space
when placed  in-conf jrred~ holding" tanks-; — These "a'athors -----------------
recommended  that fish be maintained  in confined holding
tanks with -color backgrounds- a-nd— 1-igh-fe-i-nfee-nsi-t-ies—s imila-r - —
to thos.e  in^the-^esJri^ng-TTa.r.eatft<^]ai^v.e.n:fea^
when transferred to test chambers. —
    Al t hou gh - the- ?:imp£Hrt aa*ee^-o£^ae cUma t-icxn^-to-- £he ^ es t^- ~ •"••— r •
temperature  is  still unclear,  a  maximum gradual- change -of ~ ._
3°C/day is recommended at this  time.   Peterson and Anderson
(1969) concluded that complete acclimation to temperature, ..... -
based on_ changes— .i-n^J.ocomotor-^activ-i-ty-.-and -oxygen*- -;— .^^-.-, .-
c o ns u mp t io n TT r equsrr es-'^ap pr-o xiTnart^^y wtwO'^we'eksis'-b'etfQ'r 0?™****^-^ --- =
metabolism is  back to normal.  They  also determined  that  the
rate of change  was more important  than the amount of  change.
         3.   Facil i t'ies..^ x rsas: - .-—-- .•--.. -  -  .- -
               a.  Dilution Water
    A constant  supply of good  quality  dilution water  is
needed to maintain consistent  experimental conditions  during
testing.  A  change in water quality  during -a test may  alter
the res pons e . of ..the . tes±_ f .isJi~ to—the ~tes-t~ solution.. ---------
Although there  is substantial  information on the effects  of
                                 10

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                          ES-7
                 August,  1982
pH (Folmar  et  al .  1979, Mauck et al .  1976 ,  1977, Menendez
1976, Mount 1-973)  and hardness  (Carroll et al .  1979,
Holcombe and Andrew 1978, Howarth and Sprague 1978, Sauter
et al . 1976) on the acute and chronic toxicity  of compounds,
there is no apparent data describing  the effects of these
water quality  parameters on bioconcentration potential.
    A dependable source of clean surface or ground water
usually- wil lr prQyid'fcvwa±fir--thavitig~gre:ate:^^
chemical makeup than that from  a municipal  water supply.
Municipal water may have originated from several sources
wh i ch <3-i f -£. e r^ire*ch enri-c^^^ta^up^^^ManitC'iiyal-iwa?fe-e-r - ^^
is also treated chemically as part of  a purification
process*  Since the proportions, in. whi.ch waters -from.
different- s our c es- jnay~i) e—mi x& d .,».aj3d- sJjac>e~..t he~.cn .em-Leal
treatment given water .during.. ,the.pur.if ica£.ion_-proc.ess.. .may_.be — ;
different- from . tlme-.to-Ju.me-,— ±Jae— ch.atoa.cal^.makeup~x>f ----- — —
municipal water may vary considerably.   Reconstituted water,
while theoretically more consistent from batch  to batch than
either surface or  ground water  or municipal water, may in
some  instances lack trace minerals required-.-by—some. spe-des^ ------
of f isb. , Cadrns^4'i£'6i9i)^site©weve33^^
toxicity tests on some compounds with both  reconstituted
water and-nat-ural~wa'ter and~"f otmd~ that" the~data~~generate~d
f rom  the i-tes,ts ^ten&tw£a^wa±ex^e&&tt\ote^or&&s&&^
reproducible, - whereas- -the.- results -from  the., tes-ts with  -  .
reconstituted- water -were, consistent. --
    Fish culturists do not know all of  the  conditions
required to maintain fish health, nor do they know all of
the components  and combinations of components in water that
adversely affect the health of  fish (Brauhn and Schoettger
11
                                * -----

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                                                           ES-7
                                                   August, 1982
1975).   Nevertheless,  to  avoid  possible inconsistencies and
inaccuracies "in- test results,  healthy fish are  needed for
use  in  bioconcentration tests.   There is, therefore,  a need
to determine that the -dilution water, whatever  rts -source,  -
is able to support the fathead  minnow in a healthy  condition
for  the duration of the test period.
     An  appropriate way to  make  that determination  is  to
place young- f a-thead^mr-nnows^i-n^fce— driert ion"- water- u-nde-r  -------
flow-through conditions for  60  days and observe  their
behavior,  growth and development.   Ideally, those
familiar with certain stress  reactions which are-difficult
for an _un tr.aiae4~obs.e«r.vec^to--iden.fci.f-v^4*BKauhn-»and -Schoe t-fege*!?-'
1975) ......
    . Surf ace -and- ground --wa^te-r- may vary considerably— in- their
chemis try depe«di-ng«rup0n".?fch«*.-seas©ri~-0f=:-ithe^year-^and-i^-: -=•«•* . -  -
precipitation patterns.  Variations in the chemistry of
surface  water may involve the  quantity of particulate
matter,  dissolved organic and  inorganic ch.emicals.,.. un-_ ... ____
ion i zed  ammon.ia_and_y.ar-ious _jo.ther.._con±aminan,ts_ — As. ..an ------ —
i nd i c a ti-o n -of ^re^un^rf-e'-rmirty^ of "-th'e^dl-la-tro n^wa'te r;~-l t~ is- ^~ •"-""-"
recommended in the guideline  that certain substances be
quantif red ~at~±east" twrc'e- ~s "year~ or~ more~f reque~ntl'y  if " it 'is
s us pe c t ed >>tha-t^th'eaeonc:eja±-x2a*i®^
substances have- changed significantly. -The maximum  •
acceptable- concentrations -listed -for -these subs-feances  are--  -
among . those general ly. accepted as concentrations which do
not adversely affect freshwater  fish  (APHA 1975, ASTM
1980a) .   Concentrations . in. excess -.of ,..-the..J7alue,s.~ci±ed . in«±h.e
guideline may affect the data  developed from the
                                 12

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                                                           ES-7
                                                  August,  1982
bioconcentration test.
    Re cognizing" that-some 'variation in water chemistry is
normal in natural surface or ground waters/ a 20 percent
fluctuation  from month to month  in water hardness; ......
alkalinity and  conductivity and  a  variance of 0.4 pH  units
is acceptable.
              b.  Construction materials
    Due to-the u.
                                                       --  -==.-..-
concentrations (U.S. EPA  1976)  and the tendency-of. metal
pipe, galvanized sheeting,  laboratory equipment,  etc.  to
leach me:ta±s—-i:nto~wfffei-rT=no^instal-~affeh6Tr~ thanks tainless-"s-teel
(preferably #316) should  be-used.   For the same- reason, --------
plasticized-plas,tics_should-.not-.be~us.ed_due.—to-the—high—
t ox i c i ty j> of.. .v.the -»iina-i.nv!=j,G^snponejife^*«sd'i=a2 =ue4i hyirnti.ej&y.-l
(Mayer, ajvd. Sanders—197.3..)  which  may—lea-ch into, aqaaria
s ys t ems -4 C a^rn ig-aa-n-i ~-and -Be nn-e t £^ 19 7 6 -). *. *• To / av o id  a ny- -s.-
possible stress from exposure to low levels of metals,
phthalate  esters, and other potential contaminants, .#316
stainless  steel, glass and  perfluorocarbon plastics  (e.g.
Tef 1 on®)_. s hou 1 d_be_us£d_wh-enev-e-r-,poss.ib-le^and -economical-l-y^ ^
f eas ible r~ ^ If Bother ^pias;trt^-3shou4d^be^as-'ed^'eo-R"dirtion"iing-1CT^^J
with a continuous flow of dilution water > 25°C should be
performed "for ~a min'imuirrof ~48 hours.
               c.  Te s-1 i ng aapp-aa; a tursa^r ^ art.«- ^-^^ -....-..
    The size and "shape of ^t-he ~tes t-ch-ambers-are-not  -   .-  -
important  as long-as---they-aecomodate—t-he- l-oading-	
requirements.  The chambers should however, be large enough
and contain enough water such that the fish are not  stressed
by c rowd i ng . .. _.
    The following criteria  presented by Hodson (1979)  should
be considered when selecting or designing.a toxicant
                                 13

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                                                             ES-7
                                                    August,  1982
delivery system:   1)  capable of  stopping the  delivery of  the
toxicant if delivery  of the dilution water  stops, 2)
consistent in delivery amounts throughout the test period/
3)  independent of- electrical failure-,-- -4)- -indepe-ndent~of'~ •  —
temperature and humidity fluctuations, 5) capable of
delivering small quantities, 6)  easy to construct with  few
moving  parts and 7) easy to operate.
    Any one-.-ofr-sev-enral^vty^esirof^px                           - —
can be  used as long as it has been shown to be accurate and
reliable throughout the testing  period.  The  greater the
variation inT=1rtie!^quain1:i**ty**of" 1res"lr"sii'bs^tan'c'e*'itn-troduc;ex3',"'tti'e~"" "
greater the spread -of- response value s"me-asxirTed"during	
testing.., .. Syringe,lji.jectcar~s^J^ms~4£a^
Spe har_.,e± i-al-.^-lS^^^meiexi^u^^
1979) and mo.dif ied,.px£)por.t.io.nal--1.dllu,te.rsJ-i4^c-ek. e>t ..-al.- -19.7.5-,
Neely -e-t-.al..^. ,19JA.) -Jiave^ baan^.-u-aed--.-.siiaejess.faili^t»^--»L-- -.*•«• ^  -..
    The solubility of  the test compound should also  be taken
into account when  selecting a delivery system.  If the
compound can be dissolved in water,  a device  capable of
d el ive ring amou n ts_ .of_ .tes_t._so-luJbj.on_gr_ea.teji _than_-1__ml—wl 1J
probably -be-ne&de&v*^M'^^.^c&K^ex^sfa(^^
capable of accurately  delivering very small amounts  (< 100
ul)- will be required—to—ra±n"rmrze- t'he~^;arrTeirx:oTic'ent:ratioTi
in  the., tes t_so:!utio.n,v^__^*.».^_	-—.»	
               d.   Cleaning	.	
    Before use,- the, tes.-t_ system-should-be.--cleaned -to—remove—
dust, dirt and other debris and  residue.that  may remain from
the previous use of the system.   Any of these substances  may
affect  the res.ults...of. a test. by,..sorp.tioji_,of^,±es.t. ma-teria.ls	,
or by exerting an  adverse effect on test organisms.   If any
                                                            ES-7
                                                    August, 1982
                                  14

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test chambers  or  parts  of  the testing apparatus have
obviously absorbed  test compound, those chambers or parts
should be discarded.   New  chambers should also be..cleaned-to
remove any chemical or  dirt res idues that have accumulated
during construction or  storage.   Detergent is .used . to  remove
hydrophobic or lipid-like  substances.  Acetone is used  for
the same purpose  and  to remove any detergent  residues.   It
is important  to use pesticide-free acetone to prevent  the
contamination of  the  chambers with pesticides which may  be	
toxic to the  test organisms or which might otherwise
influence the  outcome of  the test.  Nitric acid is used  to
clean metal, r^s Ldu^Suf;E_oin-^^be^s-vsfcpntjn•- Th^-^^i In-ril-----ri'ns-Q- wi'tjhh*-
water washes  away the nitric acid.  At the end of-a test?
the system should be  washed in preparation for the next
test.    .  '
    Conditioning  the  test  system with dilution water before
it is used allows an  equilibrium "to be "established between
the chemicals  in  the  water and the materials  of the test
system.  Since a  test system may sorb or react with
substances in  the dilution water, allowing this equibibrium
to become established before -the- tes t-beg-ins—:less ens •••-the—--*"
chances of additional changes__in.water .chemis-try_ojccur-ri-Qg__
during a test;    -
    Even after. ex£.eflsiHe.:.wasiiing.^.^newsa.£aai-.liti
contain toxic res.idues.._A-good .way^to determine-if-toxic. ..-
residues remain  is  to  test for their presence by maintaining
fathead minnows  in  those  facilities for a period of time
equal to or exceeding  the time required to complete a  test.
              e.  Carriers
Carriers may be  used  to aid  in the dissolution of test
                                                         ES-7
                                                 August, 1982.
                                15

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compounds  into dilution water only after significant efforts
to dissolve it in dilution water stocks  or  test solutions
have  failed.  Schoor  (1975)  believes that the use of a
carrier  may interfere  with the uptake of  the  test compound
by the  test organism;  if the carrier molecules affect the
adsorption of the test compound at the gill surface, there
will  be  a  resultant change in the rate of  transport into the
test  organism.  The author also states that the use of a
carrier  may increase  the concentration of  a compound in the
test  solution above solubility by creating  a  stable water
emulsion.
    When a carrier -is- jieces.sa.igL/i3tE-.isthyl.ejifi~~9Lyjc,al 4-1,03.,)^,- ^ ^ +
dimethyl formamide -(-J3MJ1-)- or: acetone ;-may ~be  us'ed-s7 '-The--
solvents should be tried in the order stated  due to their
re lat ive toxici ty -ta-fathead:r-rainn;ows;T;as-~Jr-e-po-r-ted... by Ca-rdwel 1-	
et al.  (manuscript,.-19-80-).. - .-The minimum  amoun-t should -be
used, and  the" concentration—of- 'TEG "sfiould not^exceed ":80
mg/1, the  MATC (maximum acceptable toxicant concentration)
value.   Concentrations  of DMF and acetone should not exceed
5.0 mg/1,  the MATC for DMF.   Although there is no MATC value
.for ace..tone.,—J..ts. -Acu-te^tojcie.i^t-y*»>is...*s.4tm4>lar~~t0^fcfea-t~o§*-DMF"i>—•.•«»*-
          4.   Env i r onme'ntralrzCo'nd-i.t ions-:"- >"'-'.:-  . ;
               a.  Loading ~  " • "
    The  flow rate --thr:Qu,gh..^:a-:-tes»t^.ch:amb:e.c:~siiou±ddDjecJaig.h^^^ i;i-v_
enough to  maintain the  dissolved oxygen  concentration
greater  than 60% of saturation (5.3 mg/1  at sea level and
22°C) , minimize buildup  of  ammonia, and  limit to 30% the
loss  of  compound by adsorption onto the  walls of the test
apparatus  and by absorption  by the fish.
                                  16

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                                                            ES-7
                                                   August,. 1982
     Different researchers  have used widely different  flow
and  loading rates.   Analysis of the data reported by  Veith
et al.  (1979) indicated  that they used  a loading rate of
approximately 0.1 g/l/day  and a turnover rate of 12.   Spehar
et al.  (1979) used a loading rate of  0.125 g/l/day and  had
27 aquaria turnovers per day.  Barrows  et al. (1980)  used a
loading  rate that ranged from 0.4 to  1.0 g/l/day and  a
considerably slower  turnover rate of  6-7."  With many  of  the
compounds  s tudied. by. Barrows_Jiowej/er.,-_-the_compoand	„ -
c on ce ntr a t io n_. i n... wa t e r -dropped^SiAibsJ: an±ial, ly.Uaeljaw.-wfeai^ i*
was  be fore the. f is.h- were— introdiLC.ed....and_us.ual Ly«J*xok. l^to- 3- .. --—»
days to  recover to pre-test levels (Personnel communication,
Barrows')-.--  In"a study  by Blanclrafd-el:"al.-11977) "a'Toadirig" ""
of 1.5~g/l/day and ^a~ tu:r nover- xate"x>f r ;6- we-re~no-t~Stff •ficrefrt	
to prevfij£t^oas..,..o£^lj4C^s.e;CrJDU±^l:r;4^^
the  test water.—The concentration-of- -the—test- substance
decreased  more-than-,.5JO.%-.duri-ng_.thfi^f irst,12_-hours...of~^ ui_  ..
exposure and did-not- return- to ^t-he--expected--concen-tT?-a-t-ion- ---------
until after 72 hours.  Such a phenomenon did not occur  in
t he  s tu d y  :by - V-e i±h ~(--pe.rs:oiT^±Tam:mun:ic^^                hs:rz:r~i-~r.
flow rate  was... used... 	  _
     We recommend a maximum loading rate of  0.1 g/l/day  and a
minimum  turnover rate  of 6.  An even  lower loading may  be
needed • if  the" compound—rs'-STispected-toTeadily degrade,  is'
highly volatile or is  expected-—to- ^accumui.-ate^qui'C-kl-y"and-•- •=-'--•»  -
substantially in fish.
               b.  Dissolved Oxygen (See-Loading-) "t	
               c.  Temperature
     S i nee  feis.'h-:'raT.e^po;iki?l:p:t-he:nii'i^^
activities  are affected  by the water  temperature to which
                                  17

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                                                             ES-7
                                                    Augus t,  19 8 2
they  are exposed.   Although there  are some  exceptions,
Prosser (19 73) states that there "is" •approxima~tely~~a~ 2 fold
increase in fish metabolism for  each 10°C rise  in water
temperature.
    During 96-hour  studies with  rainbow., trout and methyl
mercuric chloride HgCl, MacLeod  and Pessah  (1973) found that
accumulation of mercury increased  with temperature and that
this  incr:eas:e-:-was^-due-=to^atr^ncr«as^                         =v-
    Re inert et al .  (1974) exposed  rainbow trout to HgCl and
DDT separately, at  5, 10 and 15°C  and found  that
accumula t ion--
and DDT accumulation -increased- 140% over- the-same- --- ••~=
temperature range- . Thsy s.t a£ed- .tha t this— lac r^as.e-.was-~Jic4^ «~-,~.
due to . .t h e— i nA.rJ.ns.ig~ ja-afcoge^of ^..bfoec^acbema. .ca,l-STipbutt==dueu^to«4^h.ev^^-^.
increased metabolism  of- the- -fish- _______ •..-     _
    In  the— s tudy,Jiy_-V.e ith -e4u.al .^ .19-7-9 )*,~,£a£he ad=.,-mi-nflows.-. «--*--/:*.«
were  exposed to Aroclor 1254® at  5, 10,  15, 20  and 25°C.
The log BCF's increased substantially between  5,  10 and  15°C
and slightly between  20 and 25°C.   There was little
difference between  log BCF.'s. -a±. 1.5  .and. 2 0_°-C. _________ ...
    Al t hou g h - r ese^a rcteesrs1" 4i;av-e!t'pe*E-6 ^;rme€-' ^^p^pa^r ^eirtiy^^'1^ «* fc" •-s*rr*" '~~~ '
successful tests at 16°C (Barrows et al . 1980),  20°C (Macek
e t al . -  1 9 7 5 ) and" 2 5«V -( Ve±th: • e-f Aai . ' T9T9 ) /' ' a JfiT t'h'e r e are
s ome  in d.i e afrxo-r&'^t&ate.'q&as^vssdtt^^^                           :
increased temperatures-, -we recommend^ testing" af '22 r± l°C.r ": . "'
As testing -at- 25 ^-C- may -induce- sexual— matura-tion- -(-U-.-Si -EPA-   -------
1971),  the test temperature- should- -be-J.es s- than. .25 ?.C. -.A- —
test  temperature of 22°C is also consistent with  the test
t empe r a tur e. recomme nd ed.. .in. .a. samil a r. JT.SCA.. -tesJ:_.gulddeline^-£o,r., ____
acute toxicity tests  with fathead minnows.   Having identical
                                  18

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                                                             ES-7
                                                    August,  1982
 test temperatures will limit  the number  of  separate fish
 populations -required-to be  he Id-and acclimated" at~the~— ~
 testing laboratory.
                d.   Light
     Although  many researchers have used  a  16  hour light -  8
 hour dark photoperiod  (Neely  et al. 1974,  Spehar et al.
 1979, Veith  et al. 1979) and  an ASTM task-group has
 rec ommendedidsta-^us e^iit:^Dra£fc----l:0irofr r t h e~Propos*ed?^Er-actiree~~ for -~:~ -~^T— -
 Conducting Bioconcentration Tests with Fishes and Saltwater	-	
 Bivalve Molluscs  (1980b), there is no scientific
 j u s t i f re at io n-g,rven~foT-Hr ts^vrs'eT"' •—~ ~~ ~" ~•••''
     To retard gamete-maturation,--a-photoper4od—ef-i-2 -hours • — -*---	
 light-12 .hours .dark. wi±h._.a .15-3 0.. mi.nu.te_transJ.iiaa_.pe.r-iod~-is	
 recommended.		
     C.  Reporting ._	
     An -es tima.ta—of—-khe~J^i.rae~.to.^ateady-^staJte,^.-the^s-teady= _-^-^i. f*~- - -.—--,
 state BCF, and  the time to  50% or 95% elimination should be
 made for each compound tested.  If steady-state has not been
 observed during the maximum 28 day exposure period or if 95%
 elimination  has not .been..achieve.-duning-_.14-days..-.depur-atipay	—-—	
 data generated.sdur^g^fch«sre«tesHfes-~s-h«uid^
 these values.   The BIOFAC program developed by Blau and Agin
-(1978 )- us-es-non-iT:near™'regress~ro7r-t'ex:hnlqti'es~ to—es'tTmalre^ttre	•""—"	
.uptake -andc%dep^e-a:feioa£,Eate^co^
 the t ime . to .reach .9.0.%_,of .-.-s±e.ad.y.!rs tate^-the.-1 ime- to..^r.each-. 5-0%-- ------
 elimination,  .and	the-»var-iability^asso,ci.a.ted—wLt-h-.each-^.•--**...•- •.-
 estimate.
 III.  Economic  Aspects
          The Agency awarded^ a .contract..to _Enviro-Con.trol,_.,. -~
 Inc.  to provide-us--with/an-estimate-of "the; cos t for;
                                  19

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                                                            ES-7
                                                   August,  1982
performing a bioconcentration test using  the fathead minnow
according  to- this -Guideline— Enviro~"Control"supplied"us"'"
with  two estimates; a protocol estimate and a laboratory
survey  estimate.
    The protocol estimate was $8,274.   This estimate was
prepared by identifying  the major tasks needed to do a  test
and estimating the hours  to accomplish  each task.
Appr opria te vhouriy-rates-we-re-; then"appiued^o^yi'ejbd-• a^-;to£ai~ -~-~ --v-
direct  labor charge.  An  estimated average  overhead- rate- of.-.._	
115%, other direct costs  of $300, a general and
admin is-tr a t ive^- ra t^-of-^ 3:0%7~ a~n&°*~s~ f e^ - of ~ 2 0% ~we r S* the n-ad-ded- - - "
to the  direct-labor charge.-to-yield the—final es't-ima't-ev—"r-s    -
    Enviro Control. es.timated-~tlia.t_.di.fie-rences^-in-salar-i.es.,—--—
equ ipmen-t^- o-ve rhe ad~,cos fes -*-ajid^Qfch.ej?_^a<^ors.^be^/eea^^^«gi.-=a=.-J---«t-..
laboratories -could result  in,.as. much-as^50%-variation- from:. ::
this -est-imate. - -.Coas-egueaA4yv^^th^y^-es^tA-mafaed^.t»kafc-.Jc.esvt?-coatt-s- —£^
could range from $4,137  to $12,411.
    The laboratory survey  estimate was  $10,938, the mean of
the estimates received from four laboratories.  The
estimates  ranged, from. $..6,,0.00--to_-J$15-,7-5-0-and_wer.e~bas.,ed-on.	
the cos ts  to^perf-ormsthB^es^                                    -
Enviro  Control listed the  following as possible sources of
variation  in-'tfte~-eos^~est-imatesji—"—"•	~"
         o  understandimg.s.ttt.eri'aGuideAiinTeise.-ri.^^ir'-r. , . -.
         o  overhead rates
         o  salary-rates
         o  in-house expertise
         o  worker productivity and efficiency
         o  degree of., automation ~	
         o  accuracy of protocol costing  procedures
                                  20

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                                                           ES-7
                                                  August,  1982
IV. REFERENCES
    APHA.  -1975.   America-n— Public-'Associa't-ion; 'Ameriocan — - —
    Water Works Association, and Water-Pollution Control
    Federation.   Standard Methods for  Examination of Water
    and Was tewater, -1 4th ed. -New- York:   American ^Public  -
    Health Association                            '

    ASTM.  1980a.   American Society  for  Testing and
    Materials.  Standard Practice for  Conducting Basic Acute
    Toxicity Tests With Fishes, Macroinvertebrates , and
    Amphibians.  -E7 29-80 < --—--   •      -

    ASTM.  1980b.   AmericaniSociety , for -Test ing _and~— ;. .._ ....... •-.
    Materials.  Proposed Standard Practice  for Conducting
    Bioconcentrationrlests^HiitteF^sft^^
    Mol lus cs . __ Draf t .. No...- 10 , ...... August. .2 2-^. -19 .8.0.--. ~.— ........ =~.~

    Barrows ME, Petrocelli SR, Macek KJ.   1980.
    Bioconcentration-'~and™ellmfnatlmr-of~B^±ect«d"' water — •*"-•-
     .o 1 lu t a n t s~. by
    In: Hague R,  ed . -.Dynamics ,- Expos ure-andrHazard -. - -  -  ~
    Ass;es.sment™ofsJEoj{tc~C-h,eTiii.calsr.,;;-^Ann,J^rbor.v~^
    Arbor Science~.Pub.. , . Inc. .- - ..... ™.
    Bishop WE.,^ Make -= AWr.^^ -19.5.0.. ^A_criti.cal i-comparrdsoniofiivtwo' JF— -
    bioconcentration test  methods.   In:  Eaton JG, Parrish
    PR, Hendricks  AC,  eds .  Aquatic  Toxicology.   ASTM STP
    707.  American Society., for.. Testing and -Materials : pp.
    61-73.
    Ca rm ig ri an i- GM ,,. Be nne tt_. J.P-.	1917-6..~-.ILe.ach i.ng -,o£.-pJL as±.ics	
    used in closed  aquaculture system.   Aquaculture 7:89-91.

    Carroll JJ,  Ells  SJ, Oliver WS.   1979.   Influences of
    hardness. ~cx>i^£itue-ats.^.B7it&es.%ax2a&
    to brook trout  (Salvelinus fontinalis).   Bull. Environm.
    Contain. Toxicol. .2-2:575-581. --.•••-

    Cember H, Curtis  EH, Blaylock BG.   19.78.  -Mercury
    bioconcentration—in-fish-:—tempera tar e~and" concentration		
    effects.  Environm.  Pollution 17: 311-319 ./
                                 21

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                                                        ES-7
                                                August, 1982
Chiou  CT,  Freed VH,  Schmedding  DWf  Kohnert  RL.   1977.
Partition coefficient and bioaccumulation of  selected
organic- -ch-emicals—'- EnvironvSxrr.  Techno"rl'l-: 475-47 8'-   * '

DeFoe  DL,  Veith GD,  Carlson RW.   1978.  Effects of

Aroclor®  1248 and  1260"on" the fathead minnow  •( Pimephales
promelas). J. Fish.  Res. Board  Can.  35:997-1002.

Folmar LC, Sanders HO, Julin AM.   1979.  Toxicity of the
herbicide  glyphosate-and -several-of  -its..'.-formula td-o-ns- to-
fish and  aquatic invertebrates.   Arch. Environm.  Contain.
Toxicol;  8:269-278. 	
Blanchard- F Ay-Takahaslri- IT7 ~ Alexa'nde-r""HC",- Ba'ttlett"Ea~.*
1977.   Up take.,- clearance-and™ bioconceja,tra,tJ..Q£u-jofvJ.4Ca-.

FL,  Hane 1 i nfe*'3.L;,r-nedsm •".'-•ficju-a.t lc~3o'xico'iogyr^a-nd^^laizaT-d'*-3.v:3;.-
Evaluation.  ASTM  STP 63~47" American Society  for Testing
and  Materials: pp.  16.2-177.
B lau G.E)..r-Agiin., .Gfe*
computer-_progr-am-for-"characterrzrng" the" ratio  of; .uptake..- .
and cd-erarance-^of^chemrcrais'^rn="aqu'atl'e^organljs*rns"r"^       ^"~
Chemicals-Co-;^ Middand-,-Michigan;	--

Branson DR-,~BlauGEr;--A-l=elc-ander-HC-,-"Neely WBV~ 19"75. ""
Bioconcentration of _2, 2.,j4,_4.,_-. tetrachlorobiphejiyl-in-
rainbow trout as measured by an  accelerated  test.  Tran.
Am. Fish.  So.c.. 4:7.35-792.-
Brauhn^JL^,.-^:Scfeo6-tdtgiej^S^i»w«1^^5-.^.^ifiiqiuisi'*ta-£X^                  .™
of resear;c^->fis^:3^^:ad
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                                                      ES-7
                                              August, 1982

Ganz CR. ,  Schulze J, Stensby PS, Lyman  PL,  Macek KJ.
1975.   Accumulation and elimination  studies of four
detergent  fluorescent whitening agents  in  bluegill
(Lepomis macrochirus ) .  Environment.  Sci.  Techno.
8:738-744   -

Hamelink JL.   1977.  Current bioconcentration test
methods and-. theory..-- -Inr Mayer FLf-Hamel-ink—JLi"eds-. — -
Aquatic Toxicology and Hazard Evaluation.   ASTM STP
634.  American Society for Testing and  Materials,  pp.
149-161.
                                              /
Hansch  C,  Leo A,  Nikaitani DJ.  1972.   On  the additive-
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