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.Biological .. _!,~sting Methods

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PART A.

BIOLOGICAL TESTING METHODS

STATIC ACUTE FISH TOXICITY TEST

•

PART B• ACUTE ORAL RAT TOXICITY TEST

Prepared by

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. Hazardous· Waste Section

Washington State

Department of Ecology

Revised June 1991
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PART A.

TABLE OF CONTENTS

STATIC ACUTE FISH TOXICITY TEST

I. INTRODUCTION 1

1.

2.

3.

General

Terminology

Health and Safety

1

2

• 2

II. METHODS AND .MATERIALS . · . • . 4

1.

2.

3.

4.

5.

Holding Facilities . • . •

Test Chambers . . •

Cleaning and Disinfection .

Dilution Vater • . • •

Vater Quality . e. • •

4

4

5

6

• • 7

III. TEST ORGANISMS 9

IV: SAMPLE PREPARATION

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1.

2.

3.

1.

2.

. 3.

4.

5.

Care and Handling

Holding". . .

Quality . . . .

General . . ..

Particle Size Reduction .

Sample Extraction: Vrist Action Shaker Method

Sample Extraction: Rotary Agitation Method .

Rotary Agitation Apparatus

10

11

11

12

12

12'

13

13

14

V. TEST PROCEDURE · · · · · · · · · · · · · · . · . . 15

1. Conducting the Fish Toxicity Test · · · · · 15

2. Adjusting the pH · · · · 15

3. Replicates and Control · · · · · · · · · . 15

4. Dissolved Oxygen · · · · 16

5. Test Temperature · · · · · · · · · 16

6. Loading . · · · · · 16

7. . Dissolving Toxicant · · · · · 17

• 8. Feeding . · · · · · · · · · 17

9. Duration · · · · · 17

10. Biological Data . · 17

11. Chemical and Physical Data for Test Tanks 18

VI. QUALITY ASSURANCE/QUALITY CONTROL • · · · · · · 19

1. Procedures for Quality Assurance/Quality Control 19·

2. Reference Toxicants · · · · · · · · · · · · · · · . . · . . 19

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VII. CALCULATION OF RESULTS

1.

2.

3.

APPENDICES

Examples • • • •

Precision.

Reporting •

. ... . . . .. . .

20

20 - C

22

22

A. Neutralized Fish Toxicity Test Procedure

B. Effluent Toxicity Test Procedure

C. Good Laboratory Practice Standards

D. Recommended Fish Loading

E. Data Sheet for Static Basic Acute Fisb Toxicity Test

BIBLIOGRAPHY

PART B. ACUTE ORAL 'RAT TOXICITY TEST

1. INTRODUCTION

II. STANDARDS . . · .'

III. REPRESENTATIVE SAMPLING

IV. SAMPLE PREPARATION · · '.

V. TEST ORGANISMS · · · · VI. DOSING ..... . . · VII. TEST DURATION · · · · VIII. 'TEST PROCEDURE

IX. DATA REPORTING · · · · .

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27

32 '

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34

B-1

B-1

B-1 C

B-1

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· B-2

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B-2

B-2

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PART A

STATIC ACUTE FISH TOXICITY TEST

Revised June 1991
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PART A

STATIC ACUTE ~ISHTOXICITY TEST

I. INTRODUCTION

1. General

This acute fish toxicity test was developed specifically for

determining whether a waste meets the definition of dangerous waste

as found in Chapter 173-303 WAC. A modified test proced~re for

conducting a neutralized fish toxicity test is included in

Appendix A. A second modified test procedure for conducting

effluent toxicity testing is included in Appendix B.

The purpose of the acute fish toxicity test described in this

document. is to provide a simple, reliable, low-cost method for

testing the toxicity of a material against the requirements of the

Washington State. Hazardous Waste Regulation. This test method is

strictly a range-finding test and does not result in defining the

actual median lethal concentration (LCso) . The median lethal concentration is defined as the concentration of material that kills 50

percent of the population (fish) within a stated time period,

usual Iy 96 hours.

If the toxicity of a waste material is not known, the waste may be

tested at two concentrations, 100 mg/l and 1000 mg/l. The waste

concentrations of 100 mg/l and 1000 mg/l were selected to correspond

with the definition of dangerous waste (DW) and extremely hazardous.

waste (EHW) found in the state's Dangerous Waste Regulations,

Chapter 173-303 WAC. Wastes that kill fish at 100 mg/l are highly

toxic and are classified as extremely hazardous waste. Similarly,

wastes that kill fish at 1000 mg/l would be classified as dangerous

waste. Material so designated would be regulated and handled as

speo!fied in the regulation. The section "Calculation of Results"

g~es into greater detail on data interpretation.

Wastes being subjected to the acute fish toxicity test must be

sampled in accordance with the procedures referenced in WAC 173-303-

110, Sampling and Testing Methods.

Any person whose waste fails the acute fish toxicity test and who

believes that mortality resulted from effects other than acute

toxicity (e. g.", disease or physical stress) may (a) petition the

Department to exempt his waste from designation as a dangerous waste

(DW) under WAC 173-303-101, or (b) petition the Department to modify

the test procedure. Any person who believes that his waste fails

the acute fish toxicity test because of pH only, may choose to

demonstrate thiS fact by performing a neutralized fish bioassay test

as found in Appendix A of this manual. The purpose of such

demonstration would be for change of designation as provided in WAC

173-303-910 (5). Any pH wastes containing hazardous constituents

(such as ammonium hydroxide or hydrogen cyanide) which have inherent

toxicity (1. e . , ammonia and cyanide) will not qualify for such

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designation change. Petitions must be submitted in accordance with

WAC 173-303-910.

Terminology

The words "must," "would," "may," ·can," and "might" have very

specific meanings in this guide. '"Must" is used to express an

absolute requirement, that is, to state thae ehe eese oughe to be'

designed to satisfy the specified condition, unless the purpose of

the test requires a different design. "Must" is only used in

connection with the factors that direcdy relate eo the

acceptability of the test. "Should" is used to state that the

specified condition is recommended and ought to be met if possible.

Although a violation of, one "should" is rarely a serious maeeer,

violation of several will often render the results questionable.

Terms such as "is desirable," "is often desirable," and "might be

desirable" are used in connection with less important factors.

"Kay" is used to mean "is(are) allowed to," "can" is used to mean

"is(are) able to," and "might" is used to mean "could possibly."

Thus, the distinction between "may" and "can" is preserved, and

"might" is never used as a synonym for either "may" or "carr."

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3. Health and Safety

Toxicity testing-may involve significant risks to personal safety

and health. Personnel conducting toxicity tests should protect

themselves by taking all safety precautions necessary to prevent

bodily injury. Procedures to prevent inhalation or skin absorption

of test materials should be observed. Each laboratory should be ~) equipped with all necessary safety equipment prior to conducting

toxicity tests.

Personal Safety Gear. Personnel should use safety equipment as

required, such as rubber aprons, lab coats, respirators, gloves,

safety glasses, and safety shoes.

Laboratory Safety Equipment. Each laboratory should have safety

equipment such as first ,aid kits, fire extingUishers, and fire

blankets.

Included below is a summary of recommended laboratory procedures

which, if followed, will promote a safe working environment and

minimize health risks to persons in the lab. While this list

contains good suggestions regarding health and safety in the lab, it

is by no means exhaustive. Testing laboratories are encouraged to

implement these, and other, recommendations which promote the health

and safety of laboratory persons.

a. Testing of industrial wastes should be performed in compliance

with accepted rules pertaining to the handling of hazardous

materials. It is recommended that personnel carrying out

~oxicity tests should not work alone.

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b . Because the chemical composition of industrial wastes is

generally only poorly defined, such wastes should be

considered potential health hazards, and exposure to them·

should be minimized.

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c. It is advisable to immediately wash with soap· and water any

body parts which may contact the waste.

d. All containers should be adequately labeled to indicate· their

contents.

e. Good housekeeping contributes to safety and reliable results.

f. Only electrical equipment having Underwriters Laboratories

approval should be used. Ground-fault interrupters should be

installed in all "wet" labs where ·electrical equipment is

used.

g. Staff training in basic first aid and cardio-pulmonary

resuscitation is strongly recommended.

Disease Prevention. Personnel handling samples which are known or

. suspected . to contain hUDian wastes should be immunized against

tetanus,. typhoid fever, and polio.

Safety Manuals. For further guidance on safe practices when:

conducting toxicity tests, see permittee and general industrial

safety manuals, including USEPA, 1977, and Walters and Jameson,

1984 .

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II. -METHODS AND MATERIALS

The methods and materials used iJl this procedure were taken from numerous 0

-sources (see Bibliography).· This method is an attempt to balance

scientific principles against practical considerations without

compromising the accuracy or precision of the test. Although more

stringent test procedures may be used (1.e., using more fish, longer

acclimation times), the requirements presented will usually be sufficient.

The purpose for establishing a standard laboratory method is to ensure

that results obtained-from different testing laboratories are comparable.

1. Boldine Facilities

The facilities should include tanks for holding and acclimating test

fish and a constant-temperature room or recirculating water bath for

the test chambers. - Holding tanks must be operated on a flow- through

basis. Holding, acclimation, and dilution-water tanks and headboxes

should be equipped for temperature control and aeration. Air used

for aeration should be free of oil and fumes; filters to remove oil

and water may be used. During holding, acclimation, and testing,

test fish should be shielded from disturbances. The test facility

should be well ventilated and free- of fumes. A photoperiod of 8-16

hours of light per 24 hours should be provided during the test. The

light iritensityshould be in the range of natural outdoor light.

High intensity and extreme lighting conditions should be avoided.

Tanks find utility in the lab as holding tanks, stock solution

tanks, or test solution tanks. Whether fabricated in the lab or

purchased commercially, tanks should not be constructed of -0,

materials, or have surface coatings, which will leach or dissolve

into aqueous solutions. In addition, tanks and other equipment that

contact stock solutions or test solutions should be chosen to

minimize sorption of toxicants from water. Glass, stainless steel,

and fluoroplastics should be used whenever possible to minimize

leaching, dissolution, and sorption. Concrete and rigid

(~lasticized) plastics may be used for-holding and acclimation

tanks and in the water supply system. Cast iron pipe may be used in

freshwater supply systems, but colloidal iron will be added to the

dilution water and strainers will be needed to remove rust

particles. Natural rubber, copper, brass, galvanized metal, solder,

and lead should not come in contact with dilution water, stock solutions, or test solutions before or during exposure of test fish.

2. Test Chambers

The test chambers most commonly used in static tests for fish are

wide-mouth, 0.95 L (1 qt), 3.8 L (1 gal) or 19.0 L (5 gal) softglass bottles or aquaria. Special glass or stainless steel test

chambers can be constructed to accommodate test fish requiring

particular physical conditions (large surface-area-to-volume ratio).

Test chambers may be constructed of 3-mm (0.125-in.), double

strength, or 6-mm (0.25-in.) tempered plate glass held together with

clear silicone adhesive. Silicone adhesive absorbs some

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3.

organochlorine and organophosphorus pesticides. -which are difficult

to remove. Therefore. as little of the adhesive, as possible should

be in contact with water; extra-,beads of adhesive inside the

containers should be remOved. Stainless steel (No. 304 or No,. 316)

. can be used in the construction of test chambers. but should be of

welded, not soldered. construction. Commercially ,availabl~ test

chambers may be used if they meet the above requirements.

The tank configuration should. ata minimum. satisfy the following

criteria: The test solution tank should be at least 15 cm deep for

fish over 0.5g each. and at least five em deep for smaller fish.

Chambers filled to within 15 em of the top need to be covered to

prevent the test fish from jumping out.

Cleaning and Disinfection

Test chambers should be cleaned before use. New test chambers

should be washed with detergent and rinsed first with water. then

with reagent grade acetone. followed by copious rinsing with water.

then with acid (such as 5'percent concentrated nitric acid). and

finally copious rinsing with tap or other clean water. At the end

of every test; the chambers should be (1) emptied. (2) immediately

rinsed with water. (3), cleaned bya procedure appropriate for

removing' the toxicant tested (e. g.. acid to remove metals and bases;

detergent. organic solvent. or activated carbon to remove organic

compounds), (4) disinfected to remove fish pathogens. and (5),

copious rinsing with water. Acid is useful for removing mineral

deposits. and 200 mg hypochlorite/liter for one hour is useful for

removing organic matter and for disinfection.' (A solution

containing 200 mg hypochlorite/liter is conveniently made by adding:

6 ml of liquid household chlorine bleach to1 liter of water.)

However. acid and hypochlorite should not be used together because

hazardous chlorine fumes may be produced. Tanks may also be'

sterilized with an iodophor. Commercially prepared iodofors using

polyYinylpyrrolidone-iodine (PVP-I) as the active ingredient. are

available as Vescodine. Betadine. Argentyne. or other product names.

For disinfection of equipment such as fish tanks. prepare a 50 ppm

'solution of titrateable iodine by dilutinga commercially availabie

product. The equipment should be immersed in or washed with this

solution for at least 10 minutes followed by copious rinsing with

clean water. Iodophors and hypochlorites are very acutely toxic to

,most aquatic organisms. Hypochlorite can be removed by flushing

with water or by reacting it with an equivalent concentration of

sodium thiosulfate or sodium sulfite. Alternatively~ aftera tank

is washed with hypochlorite and rinsed. the equipment may be air

dried overnight to ,allow dissipation of residual chlorine.

When fish are first brought intoa facility. they should be

quarantined for at least 14 days. To maintain test fish in good

condition during holding and acclimation. crowding should be

avoided. The dissolved oxygen concentration must be maintained

between 60 and 100 percent saturation; gentle aeration may be used

if necessary.

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4. Dilution yater

An adequate supply of a 4i1ution water that is acceptable to the

test fish and the purpose of the test must be available. The

minimal criterion for an acceptable dilution water for acute

toxicity tests is that healthy test fish will survive in it for the

duration of acclimation and testing without showing signs of stress,

such as discoloration or unusual behavior. A better criterion for

an acceptable freshwater dilution water is that test fish will

survive, grow, and reproduce satisfactorily in it. Freshwater

dilution water is considered to be of constant quality if the

monthly ranges of the hardness, alkalinity, and specific conductance

are less than 10 percent of their respective averages and if the

monthly range of pH is less than 0.8 unit.

Chlorinated water should never be used for dilution water because

residual chlorine is toxic to aquatic organisms. Dechlorinated

water should only be used as a last resort, because removal of residual chlorine is often incomplete. In addition, municipal water

supplies ·often contain unacceptably high concentrations of copper,

lead, zinc, and fluoride. Toxicity tests can be adversely affected

by the use of dilution water containing these materials. When

necessary, .water containing the above materials can be treated to

make such water suitable for bioassay testing. Metals can usually

be removed with che1ating agents. . Chlorine may be removed by

filtering through activated carbon then treating with a sodium

thiosulfate or sodium sulfite drip. Sodium thiosulfate is better

for dechlorinating water than sodium sulfite. Both are more

reliable than a carbon filter, especially for removing chloramines.

The dilution water should be intensively aerated by such means as

air stones, surface aerators, or screen tubes before introduction of

the toxicant. Adequate aeration wi11.bring the concentration of

dissolved oxygen and other gases into equilibrium with air and

minimize oxygen demand and the concentration of volatiles. The

concentration of dissolved oxygen in the dilution water should be

between 90 and 100 percent saturation. Test chambers should be

rinsed with dilution water just before. use.

The hardness of the dilution water can affect the outcome of the

fish toxicity test. The hardness of the dilution water used in

fresh water toxicity tests must not exceed 100 mgll (expressed as

CaCo,) and should not be less than 40 mg/l. (Irregular toxicity

test results may occur when the hardness falls below this level.)

Use reagent-grade chemicals to adjust hardness and alkalinity. The

lab report should include information which confirms the final water

quality parameters are within the ranges found in Table 1 for ·soft·

to -moderately hard· water.

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-Adapted from Methods for Measuring the Acute Toxicity of Effluents

.to Freshwater and Marine Organisms, 1985, by EPA.

bAdd reagent grade chemicals to distilled or deionized water.

cApproximate equilibrium pH after aerating 24 h.

dExpressed in mg/L as CaCO,~

Water OUality

Water quality parameters in addition to pH, hardness, and alkalinity

can adversely affect the outcome of acute fish toxicity tests. Even

sub-lethal concentrations of certain materials in the dilution water

may contribute to fish mortality because of the additive effect of

similar materials in the waste being tested, or because of synergism'

resulting from dissimilar materials. Some' of the more common

materials and parameters of concern are presented in Table 2 along

with recommended ranges or recommended maximum concentrations. If

erratic results are experienced in the toxicity test" the dilution

water should be checked for the materials included in Table 2, or

for other materials which may be present in· the dilution water

source .

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PARAMETER

pH

Alkalinity

Ammonia (NH3)

Cadmium

Chloride

Copper

'TABLE 2. VATER QUALITY PARAMETERSRECOMMENDED ENVIRONMENTAL LIMIT

7.2-7.8

30-70 (as CaCO,)

0.02 ppm (un-ionized form)

0.4 ppb in soft water

2-3 ppm is 80ft water { 100 ppm)

less than 5 ppb

C)

HarClness

Lead

Mercury (organic or

inorganic)

Nickel

Nitrogen

Nitrite

Oxygen

Ozone Residual

PCB (Polychlorinated

Biphenyls as

Arochlor 1254)

Total Suspended and

settleable solids

Zinc

40-100 mg/l (as CaCO,)

0.03 ppm

0.2 ppb

88 ppb

Maximum total gas pressure 110' of saturation

100 ppb

At least 8 ppm at l2-C

3 ppb

0.002 ppm·

80 ppm or less

65 ppb

C)

-Adapted from Required Hatchery Environment for Smoltification by

G. Wedemeyer, et ale (1981)

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•• III. TEST ORGANISMS

The following table lists the .test organisms that can be used in the

static fish toxicity test.

TABLE 3. SPECIES AND TEST TEMPERATURES

Species· Test Temperature (OC>

Freshwater

Vertebrates

Coho salmon, Oncorhynchus kisutcb

Rainbow trout, .Oncorhynchus

(formerly gairdneri)

Brook trout, Salyelinus fontinalis

·The species must be verified.

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12 * 1

12 * 1

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All fish used in a test should be of the same age, and from the same

source since organisms of the same spec~es from different sources may produce different results in an acute toxicity test.. Usual sources of freshwater fish are private, state, and federal hatcheries.

All fish used in a test should be as uniform as possible in size. Fish

that are between 30 and 120 days old should be used. The age of a fish is

reported from the time the fish is at the sWim-up, or button-up stage, and

not from the time the egg hatches. Unless there is a specific reason for

doing so, very young (not yet actively feeding) fish should not be used.

In any single test, all fish should be from the same year class, and the

standard length.: (tip of snout to end of cauda:l peduncle) of the longest

fish should be no more than twice that of the shortest fish (Figure 1) .

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Figure 1

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All efforts should be made to ensure that test fish are disease-free prior

to using them in a toxicity test. To this end, one may:

a) Select fish from a source (hatchery) which will certify that all

fish are disease-free.

b) Treat all fish which arrive on-site using treatments commonly used

by sources (hatcheries) which produce'certified disease- free fish.

c) Discrimlnately select test fish from a reputable source, and/or on

the observations and judgment of knowledgeable individual.

All test fish should be obtained from a hatchery that has been certified

disease-free for the following diseases; bacterial kidney disease

(RenibacteriUJD salmoninarum); Costia (Ichythobodo): bacterial gill disease

(Myxobacteria species); and FUrunculosis (Aeromouas salmonicida).

As an alternative to obtaining certified disease-free fish, one may treat

fish on-site to control external parasites and latent bacterial infection.

The treatment requires two .steps: (1) Immerse the fish in an aerated

solution of 200 ppm Formalin for 60 minutes, and (2) 24 hours after the

Formalin treatment, begin and continue a commercial feed medicated diet

(e.g., Terramycin) for 10 days. If the fish are severely diseased, it is

better to destroy the entire lot immediately.

Between use with different groups of test fish, holding and acclimation

tanks should be sterilized according to the procedures in Chapter 11.3,

Cleaning and Disinfection.

1. Care and Handlin&

Test fish must be cared for and handled properly so they are not

wmecessarily stressed. To avoid unnecessary stress, test fish

should not be subjected to rapid changes in temperature or water

quality. In general, aquatic organisms should not be subjected to

mor~ than a 3-C. change in water temperature in any l2-hour period.

The test fish must be maintained in the dilution water at the test

temperature for at least the last 48 hours before they are placed in

the test chambers.

Test fish should be handled as little as possible. When handling is

necessary, it should be done as gently, carefully, and quickly as

possible, so that the fish are not unnecessarily stressed. Fish

that .touch dry surfaces or are dropped or injured during handling

should be discarded. Dip nets are best for handling fish. Such

nets are commercially available, or can be made from small-mesh

nylon netting, nylon or silk bolting cloth, plankton netting, or

similar material. Equipment used to handle aquatic organisms should

be sterilized between uses by autoclaving or by treatment with an

iodophor, 200 mg hypochlorite per liter, or 30 percent formalin plus

one percent benzalkonium chloride for at least one hour; Hands

should be washed or sterilized before handling test fish.

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3.

Boldine

After collection or transportation. fish should be acclimated to

laboratory conditions for 14 days before they are used in tests.

However. fish which have been acclimated for only 7 days JBIX be used

in tests. Fish should be held in uncontaminated. aerated water 'of

constant temperature and quality in a flow-through system with a

flow rate of at least one volUllle exchange per hour. llhen possible.

the fish should be ,held in dilution water and as close to the

temperature at which they are to be tested (Table 3) as possible.

During long holding periods. however. it is generally easier and

safer to hold fish at temperatures lower than 12·C because the metabolic rate and the number and severity of disease outbreaks are

reduced. The recommended fish loading for holding tanks. is

prOVided for various water temperatures. fish size. and species. in

Table'Dl in Appendix D.

If stock fish have 5' mortality between 24 hours after arrival onsite and the end of the 14 day acclimation period. the entire lot

should not be used for testing. If at ariy time during the holding

period there is an abnormal number of mortalities. the fish should

not be used for testing.

Duality ,

All fish in a test must be of acceptable quality. Test fish should:

be carefully observed daily during holding and acclimation for signs'_

of disease. stress. physical damage. abnormal beha~ior and

mortality. Dead. injured. and abnormal individuals must be

discarded. Visual examination of the behavior and external

appearance of the fish should cover the following:, Are they eating?

Is flipping.' flashing. or erratic swi.amling evident? Are they

gasping at the surface? Is their color normal? Is hyperventilation. hemorrhaging. or excessive mucus production evident?

Are "fin rot. fungus. or external parasites present? Are test

oqanisms emaciated? If the organisms show any signs of disease or.

stress. appropriate action should be taken.

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IV. SAMPLE PREPARATION

General

A sample of the waste to be tested shall be obtained using an

appropriate sampling method approved by the Department. All samples

must be extracted using the method and equipment described below.

In addition, solid samples must be reduced in size prior to

extraction unless -the sample meets the particle size criteria

described in IV.2. below.

Samples should be mixed prior-to opening the sample container. A

- wrist-action shaker, a three-dimensional shaker-mixer such as

Iurbula, -or other appropriate - device may be used for miXing.

Viscous samples should be stirred after opening to make the sample

as homogeneous as possible.

Two methods of sample extraction are presently allowed for samples

being tested using the fish toxicity test. Samples may be extracted

using a wrist-action shaker or a rotary agitation apparatus. In

each method, the sample is allowed to extract over a six-hour mixing

period. The extraction water (solution) will be th~ stock dilution

water used in the fish toxicity test.

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2.

The rotary agitation apparatus must be capable of rotating the

extraction bottles in an end-over-end fashion at 30 t 2 rpm. A list

of known suitable rotary extraction devices is included in IV, 5.

Particle Size Reduction

Prior t~ conducting the extraction, a determination must be made

regarding the need to reduce the particle size of the sample.

Particle reduction is required, unless the solid has a surface area

per gram of material equal to or greater than 3.1 cm2 , or is smaller

than -1 em in its narrowest- dimension (1.e., is -capable of passing

thx.ough a 9.5 mm (0.375 incb) standard sieve).

If the surface area is smaller or the particle size larger than

described above, prepare the solid portion of the waste for

extraction by crushing, cutting, or grinding the waste to a surface

area or particle-size as described above.

~: Surface area criteria are meant for filamentous (e.g., paper,

- cloth, and similar) waste material. Actual measurement of surface

area is not required, nor is it recommended. For materials that do

not obviously meet the criteria, sample-specific methods would need

to be developed and employed to measure the -surface area. Such

methodology is currently not available.

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3 . Sample Extraction:· Wrist Action Shaker Method' '

a. Weigh out the corr~ct amount of a well-mixed sample and

, transfer into a 500 ml screw-top Erlenmeyer flask.

b. Add 200 ml of extraction water (solution) to the Erlenmeyer

flask.

c. Place a teflon cap liner over the mouth of the Erlenmeyer

flask and screw the cap on.

d. Mix the sample on the wrist-action shaker for six hours,

remove the cap and pour the extraction water into the dilution

water in the fish test tank. Rinse all loose material from

the flask into fish test tank with a 200 ml aliquot of stock

dilution water Place the flask into the fish tank so that

the flask is full of water and laid on its side on the tank

bottom. Drop the cap liner into the fish test tank.

e. Begin the test as described in V. Test Procedure.

, f. Care should be exercised throughout the course of the

extraction so as not to contaminate the exterior of the

extraction flask.

4. Sample Extraction: Rotary Aa;1tation Method

'. a.

b.

C c.

d.

•

e.

Weigh out the correct amount of a well-mixed sample and

transfer into a 1000 ml extractor bottle'.

Add 200 ml of extraction water (solution) to the extractor

bottle.

Place a teflon cap liner over the mouth of the extractor

- bottle and screw the cap on.

Kix the sample on the rotary agitation apparatus for six

hours, remove the cap and pour the extraction water into the

dilution. water in the fish test tank. Rinse all loose

material from the flask into the fish test tank with a 200 ml

. aliquot of stock dilution water. Place the bottle into the

fish tank so that the bottle is full o'f water and laid on its

side on the tank bottom. Drop the cap liner into the fish

test tank.

Begin the test as described in V. Test Procedure.

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f. Care should be exercised throughout the course of the·

extraction so as not to contaminate the exterior of the

extraction flask:

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5. Botary A&itatiQD Apparatus

The following rotary agitation devices are suitable for extracting

samples prior to con~ucting a fish toxicity text. This is not an

exhaustive list of rotary agitation devices suitable for performing

sample extractions. Any device that rotates the extraction vessel

in an end-over-end fashion at 30 • 2 rpm is acceptable.

luiS,'I. "5'" "'tHip Apptnbl'

()

Mo4tl 10,

...- ---

1, Azaal7tloal t.niDa "a=~, PA 4-1at~1. Eat~ac~o~ (DC205)

aIIIll e-.ul~iDa (215) '''-4490 '-Ioul. Eat~ac~o~ (DC20)

Se~, IDa. 12-~U. Eat~~o~ (DC201)

2, .u.oo1&~ed De.laD 61--=1&. VA 2-V••••l (3740-2)

aIIIll Mfa" Co, (70S) 549-5999 4-V••••l (S740-4)

6-V....l (S740-6)

S, Blwl_~ ~,VA 4-V....l (04-00-00)

Mach1JM .. ('04) 845-6424 .-V••••l (08-00-00)

De.laD, IDa,

4. IRA Hach1De SUp &.D~, PR 8-V••••l (OliOO1)

aIIIll 1.ebo~.UZ7 (80') 752-4004

5, L&r. LeDda, Mfa, 1lh1~ Lab, 111 5-V....l (5VIlE)

(31S) 449-4116 10-V••••l (lOVRE)

6. M11110n Cozp, ladfozd, teA 4-1o~~la Eat~ac~or (YTSOOJWN)

(aGO) 225-SS84

1Federal Register, Vol. 55, No. 61, p. 11867 (Mar. 29, 1990).

-14-

o

o
•

v. TEST PROCEDURE

1. Conductins the Fish Toxicity Test

Proper conduct of the toxicity test requires that the test include

all of the steps listed below. It is important that steps (a)

through (e) be done in the sequence shown.

a. Fill the test and control tanks with dilution water, allowing

for 400 ml of water from the sample extraction step. The

dilution water must have hardness. alkalinity, and pH wi thin

the range for -soft- to -moderately hard- water as described

in Table 1.

b. Introduce the waste into the test tank. The waste includes

any undissolved sample, the extract, the rinse, and the

extraction flask or bottle .

.c. Collect a sample for measuring hardness, alkalinity, and

conductivity.

d. Measure and record D.O.• pH. and temperature ..

h. Terminate the test after 96 hours.

f. Observe and record data as indicated on the data sheet.

e. Introduce the fish into the test tank.

• g. Adjust the temperature and D.O. as necessary.

i. Complete the data reporting sheet.

2. Adjustins the pH .

Th~ pH of the test solution KAY NOT be adjusted. Any adjusting of

the pH will render the test null and void. there are provisions for

demonstrating that the waste kills fish only because of the pH.

Appendix A includes a procedure for conducting such toxicity test.

I t is not uncommon for the pH to drift during. the 96 -hour test

period. This drift is credited, in part, to fish respiration which

increases the CO2 content of the water. which in turn tends to

buffer the solution. Do not adjust the pH when this' drift is

observed. Record the pH at the time intervals indicated on the data

sheet.

•

3.. Replicates and Control

At least 10 fish in each of three replicate test tanks must be

exposed to each treatment. Replicates must be true replicates with

no water connections between the replicate test chambers.

Treatments should be randomized. Random assignment of one test

chamber for each treatment in a row, followed by random assignment

-15-
of a second test chamber for each treatment· 'in another or an

extension of the same row, is more desirable than total 0

randomization. A represet\tative sample of the test fish should be

impartially distributed to the test chambers by adding to each test

chamber no more than 20 percent of the number of test fish to be

placed in each test chamber and repeating the process until each

test chamber .contains the desired number of test fish. Alternatively, the fish can be assigned either by random assignment of one

fish to each test chamber, random assignment of a second fish to

each chamber, etc., or by total randomization. It is often convenient to assign fish to other containers and then add them to the

test chambers all at once.

Every test requires a control that consists of, the same dilution

water, conditions, number of replicates, procedures, and test

species used in the test tanks, except that none of the toxicants

being tested is added.·

5. Dissolved OXygen

The dissolved oxygen in each test chamber should be 80 percent (8.5

mgll at l2·C) of saturation at the begirming of the test. The D.O.

in each test chamber must not fall below 60 percent (6.4 mgll at

l2·C) of'saturation during the first 48 hours of the test, and must

not fall below 5.0 mgll during the remainder of the test. If the

D.O. falls below 5.0 mgll during the test and the mortality exceeds

the pass/fail threshold, the test must be voided. Test solutions

may be gently aerated if the D.O. does not meet the above criteria. 0

Once aeration is initiated, it must be continued throughout the . .

duration of the test. Turbulence should be avoided because it might.!

stress the fish, resuspend fecal matter, and greatly increase

volatilization.. Because aeration readily occurs at the surface,

efficient aeration can be achieved with minimum turbulence by using

an air lift to tranSfer solution from the bottom to the surface.

Aera&ion should be the same in all test chambers, including the

controls. If aeration is used, it is desirable to conduct a

stmultaneous test without aeration to determine if aeration affects

the results of the test.

A test is not a valid toxicity test if more than 10 percent of the

organisms die in the control test chambers.

5. Test Temperature

The test temperature for the test organism must be l2·C (see Table

3). The actual test temperature must not deviate from lrC by more

than * 1.0·C at an~ time during the ~est.

6. Loaeline

The glliter of test fish (wet weight, blotted dry) to. solution in

the test chambers must not be ao high that it affects the results of

the test. Therefore, the loading must be limited to ensure that (1)

the concentrations' of dissolved oxygen and toxicant are not

-16-

o

--~------------,
• , 7.

decreased below acceptable levels, (2)' the' 'concentration of

metabolic products does not increase above acceptable levels, and

(3) the fish are not stressed because of crowding. The loading in

test chambers must not exceed 0.8 glliter. Lower loadings may be

used if the concentration of dissolved oxygen does not remain above

60 percent saturation for the first 48, hours of the test and above

40 percent saturation after 48 hours.

Pissolvin& Toxicant

The toxicant should be added to the dilution water, at 100 mg/liter

or 1000 mglliter, without the use of any solvents or other carriers,

'except water. A gentle swirl with a glass rod is usually sufficient

to disperse the t:0xicant.,

8. Feeding,

Test fish should not be fed during acute toxicity tests or for a

minimum of 48 hours before the test. Fecal matter and uneaten food

will decrease the dissolved oxygen concentration which may affect

the biological activity of some toxicants.

9. Duration

•

•

•

10.

The test begins when the test fish are first exposed to the toxicant.

and ends 96 hours later.

Bio1odca1 Data

Death is the adverse effect most often 'used in acute toxicity tests:

with aquatic organisms. The criteria for death is lack of movement,.

especially the absence of respiratory movements in fish and lack of,'

reaction to gentle prodding. The loss of equilibrium, defined as

the inability' to make coordinated movement and maintain a normal

upright position, is not to be 'considered' a mortality. The latter

responses can be useful in interpretation of test results and should

be noted in the comments section of the data reporting sheet.

It is extremely important that procedures used to determine whether

test fish are dead" immobilized, or otherwise affected, and to

remove dead fish do not stress the live test fish. Movement of test

chambers and prodding must be done very gently.

The number of dead or affected fish in each test chamber must be

counted ,every 24 hours after the beginning of the test. More

observations are desirable, especially near the beginning of the

test. A suggested schedule is to count the number of dead or

affected fish in each chamber 3, 6, 12, and 24 hours after the

beginning of the test and twice a day thereafter to the end of the

test.

Dead fish must be removed as soon as they are observed, or once

every 24' hours. Weights and lengths of test fish may be determined

by weighing and measuring and discarding either; (1) a

-17-
representative group ·of fish before the test;· or (b) the control

fish that are alive at the end of the test. The length should be 0

reported as standard length and the weights should be wet weight. '

blotted dry. .

Fish used in a test, including those used in a control treatment.

must not be used in a later test.

11. Chemical and Physical Data for Test Tanks

The dissolved oxygen concentration and the pH must be measured in

all test tanks at the beginning, every 24 bours (as long as live

fish are present), and at the end of the test or at the time the

last fish dies in the test tank. Hardness. alkalinity. pH, and

conductivity must be measured in the test and control tanks at the

start of the test. It is desirable to measure the same parameters

at the end of the test. Measurement of calcium. magnesium, sodium,

potassium, ch~oride, sulfate, particulate matter. and total organic

carbon (TOC) or chemical oxygen demand (COD) is also desirable. For

all tests, continuous temperature monitoring in at least one test

tank is desirable. As an alternative, the maximum and minimum temperature must be recorded every 24 hours. As a second alternative.

temperatures in at least one test tank must be recorded at least

every six hours.

o

-18-

()
•

•

VI~ QUALITY ASSURANCE/QUALITY CONTROL

1. Procedures for Quality ~surancetouality Control

Because reliable laboratory data is important to making good

environmental decisions, a good quality assurance/quality control

(QA/QC) program is essential to ensure the reliability of the data.

Quality assurance (QA) is the total integrated program for assuring

the reliability of monitoring and measurement data. A comprehensive

quality assurance program would address the entire system affecting

data quality, from collecting a representative sample in the field

"to preparing the final report. The QA program should address:

(a)"sample collection and sample preservation; (b) laboratory

services; (c) use and care of instruments, glassware, and chemicals;

(3) data management; and (e) laboratory personnel. Quality control

(QC) is the routine application of statistically based procedures to

evaluate and control the accuracy of results from analytical measurements. A comprehensive quality control program" includes procedures for estimating and controlling precision and bias (e.g.,

accuracy of results) and for determining lower reporting limits

(e.g., detection limits). A comprehensive quality control program

would address specific topics related to sample analysis, including;

(a) defining data quality objectives; (b) choosing analytical

methods "which will meet those objectives; (c) estimating within-lab

precision and bias; (d) setting up control charts; (e) participating

in inter-lab tests; and (f) analyzing samples.

For specific recommendations regarding the conduct of a fish

bioassay, refer to Appendix A, Good Laboratory Procedure Standards.

•

2. Reference Toxicants

A reference toxicant is used to establish the validity of toxicity

data generated. The reference toxicant measures every aspect of

toxie-ity testing, including: The condition of the test fish; the

pusence and effects of stressors (other than the compound being

tested) in holding and testing; and, the analytical techniques being

used. If a reference toxicant LC50 differs significantly from that

previously established in a laboratory, a full review of test

conditions and procedures is indicated.

Test fish must be evaluated with a reference toxicant within the

fourteen days immediately preceding a toxicity test, or preferably,

concurrently"with the test. More frequent testing with a reference

toxicant is appropriate when situations occur which may affect test

results. For example, a change in dilution or holding water conditions, new equipment or piping in the laboratory, or a change of

analysts, all indicate the need for additional use of a reference

toxicant.

A variety of compounds are commonly used as reference toxicants.

Three reference toxicants are available from EPA; including sodium

dodecylsulfate, sodium pentachlorophenate, and cadmium chloride

-19-
(Environmental Monitoring and Support Laboratory" (EKSL). Cincinnati.

Ohio 45268).

VII. CALCULATION OF RESULT

As mentioned in the introduction. this test procedure does not determine

the LCso• but rather provides information -about- the LCso of a test

toxicant, specifically. whether the LCso is above or below a preestablished

concentration. Therefore. this method is classified as a range finding

test.

Data collected during the test will be analyzed according to the binomial

distribution. Data from the three replicates will be pooled-for several

reasons: (1) ease of interpretation. (2) to increase the number of

organisms (n) from 10 to 30. and (3) variation between the three

replicates will provide information on sample representativeness and on

laboratory methodology.

1. Examples

For a waste material to be designated an extremely hazardous waste

(EHW). greater than ten cumulative deaths out of 30 test organisms

-must occur within 96 hours at a concentration 100 mgll. This waste

is said - to have an LCso less than 100 mgll at the 95 percent

confidence level (example 1).

o

Example 1. Number of Cumulative Deaths

0 24 48 72 96 (hours) 0

Tank A 2 3 5 6 6

Tank B 3 3 4 4 4

Tank C 1 2 3 3 .:.,1-

13 EHW @100 mgll

Similarly. a waste material would be designated a dangerous waste if

greater the 11 cumulative deaths out of 30teBt organisms occurred

- within 96 hours at a concentration of 1000 mgll. This material is

said to have an LCso of less than 1000 mgll at the 90 percent

confidence level (example 2).

Example 2. Number of Cumulative Deaths

0 24 48 72 96 (hours)

Tank D 3 4 5 5 "6

Tank E 2 4 4 5 5

Tank F 3 3 "3 5 • 16 DY @1000 mg/l

-20-

o
•

•

•

Normally, any control deaths should be cause for concern with the

test conditions. For those borderline tests, corrections for up to

10 'percent mortality in cOJ\trol can be achieved by Abbott's Formula

(Equation 1). .

(Equation 1)

P=.... _-~

p.-C

l-C

P - corrected proportion for control mortality

p* - observed test fish mortality as a proportion

(i.e., .3, .4, etc.) .

C - observed control fish mortality as a proportion

Z is a test statistic that enables us to determine whether or not

the test statistic exceeds any preselected confidence interval

(Equation 2).

(Equation 2)

Z - test statistic

Po - 0.50 (i.e., LC50 )

N - 30 (pooled organisms)

If the test statistic Z is less than the break point value for a

c}wsen confidence interval (Table 4), then the hypothesis that the

LC50 is less than 100 or 1000 mg/l is rejected. An application using

Abbott's formula appears in Example 3. .

Table 4. Confidence Interval

'..

Confidence Interval

90' for 1000 mg/l

95' for 100 mg/l

Example 3.

Break Point

-1.28

-1.64

•

A waste material has been tested at 100 mg/l and killed 11 fish

within 96 hours. It appears the material is an EHW. However, one

control death was observed (less than or equal to 10 percent), does

this change the test conclusions? .

-21-
Step 1. Input variables into Equation 1:

Where p* - 11/30 - 0.31

C - 1/10 - 0.10

P= 0.37-0.10

1-0.10

P=0.30

Step 2. Input P into Equation 2:

Z= . P-Po

Po(~Po)

Where Po - 0.50

.N - 30

P - 0.30

Z 0.30-0.50

= I .50(1-.50)

30

Z= .30-.50

.09128

Z=-2.19

Since Z is less than the break point of -1.64 the material is not an

EHW at the 95 percent confidence interval.

2. Precision

The results of acute toxicity tests conducted in different

laboratories using the same species, dilution water, toxicant, and

pr~cedures will usually differ by no more than a factor of two.

3. Reportinl

A report of the results of a test must include the following:

a. Name of test, iuvestigator, and laboratory, and date test was.

begun;

-22-

o

o

o
•

b • A detailed description of the toxicant, including its source,

lot number, composition (identity and concentration of major

ingredients and maJor impurities), and known physical and

chemical propertie·s;

c. The source of the dilution water, its chemical characteristics, and a description of any pretreatment, including any

additives to the dilution water;

d. Detailed information about the test organisms, including

scientific name and how verified (and strain for salmonids

when appropriate), weight (wet, blotted dry), length (standard

length for fish), age, life stage, source, history, observed

diseases, treatments, acclimation procedure, and food used;

e. A description of experimental desim and the test chambers,

the depth volume of solution in the chambers, the way the test

was begun, the number of organisms per -treatment, the-loading,

and the lighting.

f. A description of any aeration perfOrmed on test solutions

before or during the test;

g. Definition of the criterion used to determine the effect and

a summary of general observations on other effects or

symptoms;

• h. Number and percentage of fish that died in the test and the

control tanks;

i. Identification and concentration of reference toxicant used

and the resulting mortality and LCso•

j. Methods used for, and the results (with standard deviation)

- of, all chemical analyses of water quality and toxicant

concentration~ including validation studies and reagent

blanks; -

k , The average and range of the followin& parameters: acclimation temperature, test temperature, - pH, hardness,

alkalinity, dissolved oxygen, and conductivity;

1. Anything unusual about the test, any deviation from these

procedures, and any other relevant information.

.,

II. The volume and concentration of neutralizing solution used to

adjust the pH of the test solution.

•

A sample reporting form is attached. Any alternative forms should

provide, at a minimum, the information required on the attached

sample form•

-23-
• (h)

(1)

Observe and'record data as indicated on the data sheet.

Adjust the temperature an4 D.O. ·as necessary.

•

•

(j) Terminate the test after 96 bourse

(k) Complete the data reporting sheet.

3. Recording the Data

When conducting a neutralized toxicity test. record the following

information on ·the data reporting sheet:

(a) The neutralizing material (i.e .• NaOH or HC1).

(b) The normality of the neutralizing solution.

(c) The volume of the neutralizing solution.

(d) Indicate whether' readjustment of pH was necessary 30 minutes after

initial pH adjustment.

-25-
APPENDIX·A

NEUTRALIZED FISH TOXICITY TEST PROCEDURE

1. . General

The neutralized fish toxicity test procedure is similar in all respects to

the test procedure described in the main body of this manual except as

noted in this appendix. The only difference is that aquarium water is

neutralized the waste is introduced into the aquarium, and before

the fish are placed into the aquarium.

The aquarium water may be neutralized using only aqueous solutions of

hydrochloric acid or sodium hydroxide. Add the neutralizing solution

dropwise while stirring the contents of the test tank, and measuring .the

pH continuously. The· pH of the aquarium water should be adjusted to 7.5

and allowed to stand 30 minutes to detect any pH drift. If the pH drifts

outside the range 7.2 - 7.8, the aquarium water should be readjusted to pH

.7 .5, and the fish introduced immediately after. Further pH adjustment is

not allowed.

o

2.

The neutralizing· solutions should be as concentrated as practicable to

minimize diluting the aquarium water. Under no circumstances should more

than 50 m1 be Used to adjust the pH. The actual volume and normality of

neutralizing solution used should be reported on the. data reporting

sheet.

Conducting the Neutralized Fish Toxicity Test

Proper conduct of the Neutralized Fish Toxicity Test requires that the

test include all of the steps listed below. It is important that. steps

(a) through (e) be done in the sequence ,shown.

(a) Fill the test tank and the control tank with dilution water allowing

for 490 m1 of water from the sample extraction step. The dilution

w"er must have hardness, alkalinity, and pH within the range for

·soft-to -moderately hard· water as described in Table 1.

(b) Introduce the waste into the test .tank. The waste includes any

wtdisso1ved sample, the extract, the. rinse, and the extraction flask

or bottle.

(c) Measure and record the pH.

(d) Adjust the pH to 7.5 and wait 30 minutes. If the pH drifts outside

the range 7.2-7.8 re-adjust the pH and proceed immediately to (e),

(f), and (g). .

(e) Collect a sample for measuring hardness, alkalinity, and

conductivity.

(f) Measure and record D.O., pH, and temperature.

o

(g) Introduce the fish into'the test tank.

-24-
•

•

•

1.

2.

APPENDIX B

EFFLUENT TOXICITY TEST PROCEDURE

General

The effluent toxicity test procedure is similar in all respects to the

test procedure described in the main body of this manual except as noted

in this appendix.

This procedure shall be used for the salmonid toxicity limit required in

NPDES permits. The purpose of the static effluent toxicity test required

by the Department of Ecology is to evaluate the effect of an effluent on

the resident and migratory salm~nid fishes in state waters. The effluent

"tOXicity standards for Washington State require that 80 percent of the

salmonid fish tested survive in at least 65 percent concentration of

effluent for 96 hours.

The sample shall not be dechlorinated prior to conducting .the test.

Samples shall be maintained at a 4·C in the dark during shipping and

storage. The sample" should be collected with minimum head space. The

recommended holding time is 72 hours.

Conducting the Effluent Toxicity Test

Proper conduct of the Effluent Toxicity Test requires that the test

include all of the steps listed below.

(a) Fill the test tank with a solution of effluent and dilution water.

The solution strength must be as specified in the NPDES permit. The:

dilution water must have hardness. alkalinity. and pH within the

"ranges specified in Table 1.

(b) Collect a sample for measuring hardness, alkalinity • and

conductivity. "

(c) Measure and record D.O., pH, and temperature.

(d) Introduce the fish into the test tank.

(e) Observe and record data as indicated on the data sheet.

(f) Adjust the temperature and D.O. as necessary.

(g) Terminate the test after 96 hours.

(h) Complete the data reporting sheet.

-26-
•

.'

e,

•

APPENDIX C

GOOD LABORATORY FRQCEDUU 0 STANDARDS1

The information included below was adapted from EPA's Good Laboratory Practice

(GLP) Standards. These standards are intended to be recommended standards rather

than requirements. Laboratories are encouraged to develop QA/QC programs which

address these recommended standards•

A. ORGANIZATION AND PERSONNEL

1. Personnel

Each individual engaged in the conduct of or responsible for the

supervision of a study shall have education, training, and experience, or combination thereof, to enable that individual to perform

the assigned functions.

Each testing facility shall maintain a current summary of training

and experience and job description for each individual engaged in or

supervising the conduct of a study.

There shall be a sufficient number of personnel for the timely and

proper conduct of the study according to the protocol.

Personnel shall take necessary personal sanitation and health

precautions designed to avoid contamination of test and control

substances and test systems.

Personnel engaged in a study shall wear clothing appropriate for the

duties they perform. Such clothing shall be changed as often as

necessary to prevent microbiological, radiological, or chemical

contamination of test systems and test and control substances.

Any individual found at any time to have an illness that may

adyersely affect the quality and integrity of the study shall be

excluded from direct contact with test systems, test and control

, substances and any other operation or function that may adversely

affect the study until the condition is corrected. All personnel

shall be instructed to report to their immediate supervisors any

health or medical conditions that may reasonably be considered to

have an adver~e affect on a study.

0., 2. Biolo&istin Char&e

•

For each study, a biologist, or a scientist, or other professional

of appropriate education, training, and experience, or combination

thereof, shall be identified as the biologist in charge. The

biologist in charge has overall responsibility for the technical

conduct of the study, as well, as for the interpretation, analysis,

lFederal Register, Vol. 48, No. 230, p. 53922ff (Nov. 29, 1983).

-27-
documentation, and reporting of results, and represents the single

point of study control. The biologist in charge shall assure that:

(a) The protocol, including any change, is approved as provided by

Ecology and ~s followed.

(b) All experimental data, including observations of unanticipated

responses of the test system are accurately recorded and

verified.

o

(c) Unforeseen circumstances that may affect the quality and

integrity of the study are noted when· they occur, and

corrective action is taken and documented.

" (d) Test systems are as specified in the protoc91.

(e) All applicable good laboratory practice regulations are

followed.

(f) All raw data, documentation, protocols, specimens, and final

reports are transferred to the archives during or at the close

of the study.

3. Protocol'

Each study shall have an approved written protocol that clearly

indicates the objectives and all methods for the conduct of the

study. The protocol shall contain but not be necessarily be limited

to the following information: 0

(a) A descriptive title and statement of the purpose of the study.

(b) Identification of the test and control substance by chemical

name, Chemical Abstract Service Registry Number (CAS), or

- other established identification system. '

(c) The name and address of the sp::,nsor and the name and address

of the testing facility at which thestuciy is being conducted.

(d) The proposed,starting and completion dates.

(e) Justification for selection of the test system.

(f) Where applicable, the number, body weight range, sex, s~urce

of supply, species, strain, substrain, and age of the test

system.

(g) The procedure for identification of the test system.

(h) A description of the experimental design, including the

methods for the control of bias.

(i) A description' and/or identification of the diet used in the

study as well as solvents, emulsifiers and/or other materials

-28- o
•

..

(j)

(k)

used to solubilize or sUspend the test or ·control substances·

before mixing with the carrier. 2 The description shall

include specificati~ns for acceptable levels of contaminants

that are reasonably expected to be present in the dietary

materials and are known to be capable of interfering with the

purpose or conduct of the study if present at levels greater

than established by the specifications.

The route of administration and the reason for its choice.

Each dosage level, expressed in milligrams per kilogram of

body weight or other appropriate units, of the test or control

substance to be administered and the method and frequency of

administration.

(1) Method by which the degree of absorption of the test and

control substances by the test system will be determined if

necessary to achieve the objectives of the study.

(m) The type and frequency of tests, analyses, and measurements to

be made •.

(n) The records to be maintained.

(0) The date of approval of the protocol by the sponsor and the

signature of the biologist in charge .

• (p) A statement of the proposed statistical methods to be used.

...

•

(q) All changes in or revisions of an approved protocol and the

reasons therefor shall be documented, signed by the biologist

in charge, dated, and maintained with the protocol.

4. Conduct of a Study

The study shall be conducted in accordance with the protocol.

The test systems shall be monitored in conformity with the protocol.

Specimens shall be identified by test system, study, nature, and

date of collection. This information·shall be located on the

specimen container or shall accompany the specimen in a manner that

precludes error in the recording and storage of data .

Records of gross findings for a specimen from postmortem observations. shall be available to a pathologist wllen examining that

specimen histopathologically.

2NOTE: In state's test procedure, per IV.7, the only allowed .toxicant

carrier is water.

-29-
All data generated during the conduct of a study, except those that·

are generated as direct computer input. shall be recorded directly. 0

promptly. and legibly in .ink. All data entries shall be dated on

the day of entry and signed or initialed by the person entering the

data. Any change in entries shall be made so as not to obscure the

original entry. shall indicate the reason for such change. and shall

be dated and signed or identified at the time of the change. In

computer driven data collection systems. the individual responsible

for direct data input shall be identified at the time of data input.

Any change in computer entries shall be made so as not to obscure

the original entry. shall indicate the reason for change, and shall

be dated and the responsible individual shall be identified.

5. Maintenance and Calibration of Equipment

Equipment shall be adequately inspected. cleaned, and maintained.

Equipment used for the generation, measurement, or assessment of

data shall be adequately tested. calibrated, and/or standardized.

A written standard operating procedures shall set forth. in sufficient detail the methods. materials, and schedules to be used in the

routine inspection, cleaning, maintenance, testing, calibration,

and/or standardization of equipment, and shall specify remedial

action .to be taken in the event of failure .or malfunction of

equipment. The written standard operating procedures shall

.designate the person responsible .for the performance of each

operation, and copies of the standard operating procedures shall be

made available to laboratory personnel.

Written records shall be maintained of all inspection, maintensnce,

testing. calibrating, and/or standardiZing operations. These

records, containing the date of the operation, shall describe

whether the maintenance operations were routine and followed the

written standard operating procedures. Written records shall be

kep~of non-routine repairs performed on equipment as a result of

failure and malfunction. Such records shall document the nature of

the defect, how and when the defect was discovered, and any remedial

action taken in response to the defect.

•

o

B. TESTING FACILITIES OPERATION

1. Standard Operating Procedures

A testing facility shall have standard operating procedures in

writing setting forth study methods that management is satisfied are

adequate to ensure the quality and integrity of the data generated

in the course of a study. All deviations in a study from standard

operating procedures shall be authorized by the study director and ~,

shall be documented in the raw data.

Significant changes in established standard operating procedures

shall be properly authorized in writing by management.

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•• Standard operating procedures shall be established for. but not

limited.to. the following:

(a) Animal room preparation.

(b) Animal care.

(c) Receipt. identification. storage. handling. mixing. and method

of sampling of the test and control substances.

(d) Test system observations•

• (e) Laboratory tests.

(f) Handling of animals found moribund or dead during study.

(g) Necropsy of animals or postmortem examination of animals.

(h) Collection and identification of specimens.

(i) Histopathology.

(j) Data handling. storage, and retrieval.

(k) Maintenance and calibration of equipment.

•

(1)

(m)

Transfer, proper placement, and identification of animals.

Each laboratory area shall have immediately available

laboratory manuals and standard operating procedures pertinent

to the laboratory' procedures being performed, e. g."

toxicology, histology, clinical chemistry, hematology.,

teratology, necropsy. Published literature may be used as a

supplement to standard operating procedures.

f'

•

2.

(n) A historical file of standard operating procedures, and all

revisions thereof, including the dates of such revisions,

shall be maintained.

ReSlents and Solutions

All reagents and solutions in the laboratory areas shall be labeled

to indicate identity, titer or concentration, storage requirements,

and expiration date. Deteriorated or outdated reagents and

solutions shall not be used.

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•

•

APPENDIX D

RECOMMEND~ FISH LOADING

Prior to use in a toxicity test, the fish are Usually maintained at the test

facility in a circulated holding tank. It has been determined through experience

that a minimum level of circulation is required to maintain the viability of the

test fish. The recommended level of circulation varies depending on the· species

of fish, the size of fish, and the temperature. For small fish, the size of the

fish is usually reported in terms of number of fish per pound. The table below

lists the recommended fish minute loadings (pounds of fish per gallon per minute

of inflow), to promote the health and quality of salmonid fish used in toxicity

tests.

•

Species

Coho Salmon

Fall and Spring

Chinook Salmon

TABLE Dl. RECOMMENDED FISH LOADINGS·

Water Temperature Pounds of Fish per GPMb

·C ·F loooe sooe looe

3 38 3.5 5.0 8.0

9 48 2.7 4.0 6.0

.14 58 2.2 3.0 4.5

-17 63 2.0 3.5

3 38 3.0 4.0 6.0

9 48 2.5 3.0 5.0

14 58 2.0 2.2 3.5

17 63 1.2 3.0

f'

•

• Adapted from Fish Diseases Leaflet 38 (see bibliography).

b GPM means gallons per minute.

e Fish size (number of fish per pound).

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• • •

11I11 11 111 .. 1 11111

'"1''1''' " [ COL 0 GY DATA SHEET FOR STATIC BASIC ACUTE FISH TOXICITY TEST

laboratory _

. Analyst --:- _

IndustrylToxlcant Beginning Date T1me _

Address Ending Date Time

Collector Test Organlsm --------------- ~---~

Date Sample Collected Required Test Temperature Range, _

Test , To1III To1III

laboratoly Reference Cone. tMnber ofCllntil1lve Dea'" Dissolved Oxygen (mgll) pH 25C TlWllper81Ur1 (C) Hanlness AIkaIInItr ConcludIvItr

Number ner (mgll) I (mgn. (mgnes uMHOSIan

No. CaCOsl caco,l

0 24 48 72 ge 0 24 48 72 ge 0 24' 48 72 98 0 24 48 72 98 0 Ie 0 18 0 Ie

Sample Description _

Average Weight Mean length longest __---'- Shortest Ratio (Ionglshort) _

Number of organisms per chamber Ratio of flesh to water _ 'Comments _

__________ EWH> 10/30

_______ __ OW> 11/30

Data Verified By ~=~ Date _

ECY 030-1-40

Mllhod onFile wtth \he DIpattmInt of EClIlIDgy

GENERAL PROCEDURE FOR STATIC BASIC ACUTE FISH TOXICITY TEST
•

v

•

•

•

u.S. Environmental Protection Agency, Karch 1985, Methods for Measuring

the Acute Toxicity of Effluents to Freshwater and Karine Organisms.

(EPA/600/4-85/0l3)

2. Federal Register Vol. 48 No. 230, November 29, 1983, Good Laboratory

Practice Standards, p. 53946 ff.

3; Wedem~yer, G.A., and J.W. Wood, 1977. Stress as a Predisposing Factor in

Fish Disease. U.S. Fish and Wildlife Service, Fish Disease Leaflet

38 (FDL-38), Washington, D.C.

4. Wedemeyer, G.A., D.J. McLeay, C. Phillip Goodyea~. 1984. Assessing the

Tolerence of Fish and Fish Populations to Environmental Stress: The

Problems and Methods of Monitoring.

5. Wedemeyer, G.A., R.L. Saunders, and W.C. Clarke, 1981. The Hatchery

Environment Required to Optimize Smoltification in the Artificial

Propagation of Anadromous Salmonids. Pages 6-20 in E. Kineyand L.

Allen, eds., Bio-Engineering in Fisheries. Special Publication by

American Fisheries Society, Washington, D.C.

-34':'
•

•

•

•

,

•

PART B

ACUTE ORAL RAT TOXICITY TEST

1980
•

•

PART B

ACUTE ORAL R4T TOXICITY TEST

I. .INTRODUCTION

The purpose of oral rat testing is to determine the relative biological

risk of potentially hazardous waste to man and wildlife. The results will

, determine whether a waste is classes as extremely hazardous waste or 'as a

dangerous waste under VAC 173-302 -The Hazardous Vaste Regulation. - Test

analyses are not based upon leachate extraction procedures and as such, do

not attempt to simulate changes that ,the waste might undergo after

disposal. The toxicity test is to be construed' as an index of acute

single exposure of biological risk to the generator, transporter, treater

and disposal site operator. The test is not intended to determine what

changes in toxicity might occur, after disposal due to environmental

reactions. These conditions are difficult to predict and quantify

especially for the sanitary landfill scenario.

The Department uses oral rat tests whenever a generator has not or cannot

adequately characterize the toxicity of a waste because of:

a. A lack of knowledge of the chemical composition and presence of

toxic constituents.

b. A lack of knowledge of the biological hazards of toxic constituents

or the waste as a whole .

• c. The inability to book designate a waste's biological toxicity.

•

•

11. STANDARDS

To determine whether a waste is extremely hazardous, a representative

sample ,of the waste shall be tested at 500 milligrams of waste per

kilogram of rat body weight. Tests showing significant toxicity as

defined herein shall be deemed extremely hazardous wastes (1.e., LDso S 500

mg/kg) . To determine whether a waste is a' dangerous waste, a

representative sample shall be tested at 5,000 milligrams of waste per

kilogram of rat body weight (1.e., LDso S 5,000 mg/lcg). The death of two

or less test animals per dose will be statistically evident that the LD50

is greater than the ,standard at the 95 percent confidence level. The

death of any control animal will invalidate the entire test.

111. REPRESENTATIVE SAMPLE

A representative sample of the waste to be tested shall be obtained using

methods established by the Department of Ecology. The need to obtain a

truly representative waste sample is as important as the biological

testing itself, and its inadequacy may be grounds for rejecting an

otherwise acceptable result. '

,IV. SAMPLE PREPARATION

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I
The waste shall be tested in the form it is to be' disposed with the'

following exceptions:

a. Waste may be ground and coarsely filtered to allow its oral

administration by stomach tube.

b. Liquid and solid pllases should be homogenized before administration.

o

c. Upon review and approval on a case-by-case basis by the Department

of Ecology.'

V. TEST ORGANISMS

Male albino rats weighing between 200 and 300 grams shall be used for test

and control animals.

Ten rats shall be used at each dose level, and ten rats for each positive

control test. (See lbelow)

. VI. DOSING

All animals shall be dosed by gavage. All animals shall receive the same

concentration and approximate volume of dosing solution, not ·to exceed 4-5

mls per animal'.

VII. TEST DURATION

The animals shall be observed for 14 days.

VIII .. TEST PROCEDURE

Food shall be withheld from animals the night prior to dosing.

Observations for mortality and clinical pharmacological or toxicological

signs will ~e recorded immediately after dosing at one and four hours, and

daily thereafter for 14 consecutive days. All surviving animals will be

sacrificed at termination on the 14th day. Gross necropsies will be

performed on animals that die and those ·sacrificed at termination.

IX. DATA REPORTING

The test report shall include the following information:

a. Tabulation of response data by dose level (Le.·, number of animals

dying per "number of animals exposed).

lNOTE: Ecology may require a positive control group for particular wastes

or toxicants when the sensitivity of the test animal is in doubt. When used, the

positive control group should serve as an internal quality control to ascertain

whether the test animals are sensitive or resistant to the test material by

testing their response to a known toxic agent•.

B-2

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o·
•

",'

•

•

•

•

b •

c.

d.

Tille of death after do~ing.

Clinical pharmacological•. and/or toxicological signs.

Gross necropsy findings.

B-3