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WASHINGTON STATE

DEPARTMENT OF

E COL 0 GY

Biological Testing Methods 80-12

For the Designation of Dangerous Waste

Part A: Static Acute Fish Toxicity Test

Part B: Acute Oral Rat Toxicity Test

Publication #80-12, Revised June 1999

Hazardous Waste and Toxics Reduction Program

Washington State Department of Ecology

printedon recycled paper
For a copy of this document, please contact:

Department of Ecology

Publications

PO Box 47600

Olympia, WA 98504-7600

The Department of Ecology is an equal opportunity agency and does not discriminate

on the basis of race, creed, color, disability, age, religion, national origin, sex, marital

status, disabled veteran's status, Vietnam Era veteran's status or sexual orientation.

If you have special accommodation needs, or require this document in alternative

format, please contact the Hazardous Waste and Toxics Reduction Program at (360) 407-

6700 (Voice) or (360) 407-6006 (TDD).

Regional TDD numbers are:

+-~._-~- ~----~ _._--~_.__.. - ----~- ---

Central Regional Office

Eastern Regional Office

Northwest Regional Office

Southwest Regional Office

(509) 454-7673

(509) 458-2055

(425) 649-4259

(360) 407-6306 L
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WASHINGTON STATE

DEPARTMENT OF

ECOL 0 GY

Biological Testing Methods 80-12

For the Designation of Dangerous Waste.

Part A: Static Acute Fish Toxicity Test

Part B: Acute Oral Rat Toxicity Test

Publication #80-12, Revised June 1999

Hazardous Waste and Toxics Reduction Program

Washington State Department of Ecology

printedon recycled paper
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Table of Contents

Glossary v

Terminology vii

Part A: Static Acute Fish Toxicity Test

Introduction A-1

Toxicity Identification Evaluation (TIE) A-2

Health and Safety A-2

Methods and Materials A·4

Holding Facilities A-4

Test Chambers A-5

Cleaning and Disinfection A-6

Dilution Water A-7

Test Organisms A-8

Care and Handling A-9

Holding A-9

Stress Indicators A-IO

Sample Preparation A-IO

Sample Water Content Determination & Reporting A-IO

Particle Size Reduction A-II

Rotary Agitation Method A-II

Test Procedure A·12

Replicates and Control. A-12

Test Temperature A-12

pH A-13

·Dissolved Oxygen A-13

Loading : A-14

Feeding A-14

Duration and Acceptability A-14

Test Observations and Measurements A-14
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Criteria for Death A-IS

Termination A-IS

Data Analysis A-IS

Quality Assurance I Quality Control A·15

Procedures A-IS

Reference Toxicants A-16

Calculation of Results A·17

Approach A-17

Hypothesis A-17

Calculation A-18

Sample size A-18

Mortalities A-18

F Test A-18

Equal variance t test A-19

Modified t test A-19

Critical F and t values A-19

Data Reporting A·22

References A·25

Appendices

A. Neutralized Fish Toxicity Test Procedure A-27

B. Good Laboratory Procedure Standards A-29

C. Water Quality A-3S

D. Dissolved Oxygen Table A-36

E. Recommended Fish Loading A-37

F. Sample Data Sheet. A-39

G. Recommended Test Conditions and Procedures for Fish Bioassay A-43

Part B: Acute Oral Rat Toxicity Test

Introduction B-1

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Methods B·1

Sample Collection and Preparation ; B-1

Test Animals '" B-2

Pretest Conditioning B-2

Procedure B-2

Calculation ofResults B·3

Approach : B-3

Hypothesis B-4

Calculation (N < 30) ; B-4

Calculation (N > 30) B-7

Data Reporting 8·8

References 8·9

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°c

CaC03

CaS04

CaS04

e2H20

cm

CO2

COD

d

DO

DW

EHW

g

gal

gpm

h

HCL

H20

in

KCL

L

m

mg

mg/L

MgS04

min

mL

mg/kg

NaHC03

NaOH

ng/L

NH3

ppb

Glossary

degree(s) Celsius

calcium carbonate

calcium sulfate

calcium sulfate dihydrate (gypsum)

centimeter

carbon dioxide

chemical oxygen demand

day

dissolved oxygen

dangerous waste

extremely hazardous waste

degree(s) Fahrenheit

grams

gallons

gallons per minute

hour

hydrochloric acid

water

inch(es)

potassium chloride

liter

meter

milligram

milligram per liter

magnesium sulfate

................................................................................................ minute(s)

milliliter

milligram per kilogram

sodium bicarbonate

sodium hydroxide

nanograms per liter

ammonia

..................................................................................... parts per billion

v
ppm

pptr

QA/QC

qt

rpm

TOC

WAC

>

<

>

5

parts per million

parts per trillion

quality assurance/quality control

quart

rotations per minute

total organic carbon

Washington Administrative Code

greater than

less than

greater than or equal to

less than or equal to

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• Terminology

Grammatical Terms

Can...means "is/are able to"

May...means "is/are allowed to"

Might...means "could possible;" never used as a synonym for "may" or "can"

Must... expresses an absolute requirement

Should...means the specified condition is recommended and ought to be met if

possible

Technical Terms

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Acclimation...means to become physiologically adapted to a particular level of

one or more environmental variables, such as temperature. The term usually

refers to controlled laboratory conditions.

• Acute toxicity... a discernible, adverse effect (lethal or sublethal) induced in the

test organisms within a short period of exposure to a test material, usually 4

days for fish.

Alevin...a recently hatched, non-feeding fish with an evident yolk sac for

nutritive requirements. Sometimes referred to as yolk-sac fry.

Conductivity...is a numerical expression of the ability of an aqueous solution to

carry an electrical current. This ability depends on the concentrations of ions in

solution, their valence and mobility, and on the temperature of the solution.

Conductivity is normally reported in the Systeme International dunites (51) unit

of millisiemens/meter, or as micromhos/centimeter (1 mS/m = 10 umhos/cm)..

Control...a treatment in an investigation or study duplicating all conditions and

factors possibly affecting the investigation results, except the specific condition

being studied. In aquatic toxicity tests, the control must duplicate all conditions

of the exposure treatment(s), but must contain no test material.

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The control is used to determine the absence of measurable toxicity due to basic test

conditions (e.g., quality of the control/dilution water, health or handling of test

organisms).

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Dangerous waste ...those solid wastes designated in WAC 173-303-070through

173-303-100 as dangerous, extremely hazardous, or mixed waste.

Deionizeduiater...water that has been passed through resin columns to remove

ions from solution and thereby purify it.

Distilled water...water that has been passed through a distillation apparatus of

borosilicate glass or other material, to remove impurities.

Extremely hazardous waste...those dangerous and mixed wastes designated in

WAC 173-303-100 as extremely hazardous. Also, those dangerous wastes

showing statistically significant mortality in test concentrations of 10 mg/L.

Fingerling...a young (underyearling), actively feeding fish.

Flow-through ...describes scenario in which holding vessels are renewed

continuously by the constant inflow of fresh solution or holding water, or by a

frequent intermittent flow.

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Hardness...the concentration of cations in water that will react with a sodium 0

soap to precipitate an insoluble residue. In general, hardness is a measure of the

concentration of calcium and magnesium ions in water, and is expressed as

mg/L calcium carbonate (CaC03) or equivalent.

Hatch date...the date the alevins emerge from the egg. At this point, the alevins

live off the yolk sac, and in nature, bury into the water body gravel.

LC50...the median lethal concentration (i.e., the concentration of material in

water that is estimated to be lethal to 50 percent of the test organisms). The LC50

and its 95 percent confidence limits are usually derived by statistical analysis of

mortalities in several test concentrations, after a fixed period of exposure. "The

duration of exposure must be specified (e.g., 96 hour LC50).

Leachate... any liquid, including any components suspended in the liquid, that

has percolated through or drained from dangerous waste.

Lethal... means causing death by direct action. Death of fish is

defined as the cessation of all visible signs of movement or other

activity; no response from gentle prodding.

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Loading...the weight of organisms per liter of test solution; this is limited to

minimize the depletion of DO, the accumulation of injurious concentrations of

metabolic waste products, and/or stress induced by crowding.

Lux ... a unit of illumination based on units per square meter. One lux =0.0929

foot-candles and one foot-candle =10.76 lux.

Monitoring...the routine (e.g., daily, weekly, monthly, quarterly) checking of

quality or collection and reporting of information. In the context of this method,

it means either the periodic (routine) checking and measurement of certain

biological or water-quality variables, or the collection and testing of samples of

effluent, elutriate, leachate, or receiving water for toxicity.

Percentage (%)...is a concentration expressed in parts per hundred parts. One

percent represents one unit or part of material (e.g., effluent, elutriate, leachate,

or receiving water) diluted with water to a total of 100 parts. Concentrations can

be prepared on a volume-to-volume or weight-to-weight basis, and are

expressed as the percentage of test material in the final solution.

pH...the negative logarithm of the activity of hydrogen ions in gram equivalents

per liter. The pH value expresses the degree of intensity of both acidic and

alkaline reactions on a scale from 0 to 14, with 7 representing neutrality.

Numbers less than 7 signify increasingly greater acidic reactions, and numbers

greater than 7 indicate increasingly basic, or alkaline, reactions.

Photoperiod...the duration of illumination and darkness within a 24 hour day.

Pretreatment...in this method, is treatment of a sample or dilution thereof, prior

to exposure of fish.

Reconstituted water...deionized or glass-distilled water to which reagent-grade

chemicals have been added. The resultant synthetic fresh water is free from

contaminants and has the desired pH and hardness characteristics.

Reference toxicant...a standard chemical used to measure the sensitivity of the

test fish in order to establish confidence in the toxicity data obtained for a test

material. In most instances a toxicity test with a reference toxicant is performed

to assess the sensitivity of the organisms at the time the test material is

evaluated, and the precision of results obtained by the laboratory.

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Replicate...One of several identical experiments, procedures, or samples.

Duplicate is a special case of replicates consisting of two samples or

measurements.

Static...describes toxicity tests in which test solutions are not renewed during

the test.

Static replacement...describes toxicity tests in which test solutions are renewed

(replaced) periodically during the test, usually every 24 hours. Synonymous

terms are "renewal", "batch replacement", and "semi-static".

Swim-up...post-alevin emergence from the gravel and commencing of active

feeding. Point at which to begin marking the age of the fish.

Toxicity...the inherent potential or capacity of a material to cause adverse effects

in living organisms.

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Toxicity test...a determination of the effect of a material on a group of selected

organisms under defined conditions. An aquatic toxicity test usually measures ()

the proportions of organismsaffected by their exposure to specific

concentrations of chemical, effluent, elutriate, leachate, or receiving water.

()

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Part A: Method 80-12

State Acute Fish Toxicity Test

Introduction

The Washington State Department of Ecology (Ecology) developed the

acute fish toxicity test (Method 80-12) to determine if a waste meets the

definition of dangerous waste in the Dangerous Waste Regulations,

Chapter 173-303 WAC. Appendix A contains a modified test procedure

for conducting neutralized fish toxicity tests.

Method 80-12 provides a simple, low cost method for testing the toxicity

of a waste against the requirements of WAC 173-303. This method

determines if the sample waste LC50 is significantly less than or equal to

the regulatory threshold of 100 mg/L dangerous waste (DW), 10 mg/L

extremely hazardous waste (EHW), or 10 mg/L special waste. The LC50,

for this method's purposes, is the median lethal concentration of waste

that kills 50 percent of the test fish within 96 hours.

If the toxicity of a waste is unknown, the waste must be tested for

dangerous waste designation using Method 80-12. The waste

concentrations of 100 mg/L and 10 mg/L were selected to correspond

with the definitions of dangerous waste and extremely hazardous waste,

respectively. State-only special waste uses a regulatory threshold of 10

mg/L.

Waste designated by Method 80-12 must be regulated and managed as

specified in WAC 173-303, and must be sampled according to procedures

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 petition Ecology to exempt the waste from

designation under WAC 173-303-101. They may also petition Ecology to

modify the test procedure. Any person who believes that test failure

resulted only because of pH may choose to demonstrate this by

performing a neutralized fish bioassay per Appendix A. The purpose of

this demonstration would be to revise waste designation as provided in

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

with inherent toxicity (e.g., ammonia, cyanide) will not qualify for

designation changes. Petitions must be submitted per WAC 173-303-910.

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Toxicity Identification Evaluation (TIE)

The Toxicity Identification Evaluation (TIE) procedure (EPA, 1988) is used

to identify toxicants or classes of toxicants in a dangerous waste that

designates by only failing the state fish bioassay. Depending on the

outcome of the TIE, Ecology may allow an alternative management

standard, sufficiently protective of human health and the environment,

for a state-only toxic waste.

EPA developed the TIE procedure to aid wastewater dischargers in

identifying toxicants within a complex effluent as part of a Toxicity

Reduction Evaluation (TRE). A TRE is an evaluation intended to

determine remedial actions necessary to comply with NPDES water

quality-based limits (EPA, 1989). Although TIE procedures have

primarily been applied to NPDES-permitted effluents, the methods are

applicable to other types of aqueous samples, including ambient waters,

sediment pore waters, sediment elutriates, and hazardous waste

leachates.

EPA published the TIE procedure in three phases, with provisions for

evaluating samples exhibiting both acute and chronic toxicity. Follow the

acute toxicity methodology in the TIE to be consistent with the acute fish C)

bioassay. Phase I characterizes the chemical and physical nature of the

waste without specifically identifying the toxicants (EPA, 1991; EPA,

1992). Phase II describes methods to identify toxicants (e.g., nonpolar

organics, ammonia, cationic metals) but is incomplete and does not

provide methods for many constituents (EPA, 1993a). Phase III describes

methods to confirm that the suspected toxicants, rather than

methodological artifacts, are the true cause of toxicity (EPA, 1993b).

Please contact the Hazardous Waste and Toxics Reduction Program at

360/407-6700 for further information.

Health and Safety .

Development and maintenance of an effective health and safety program

in the laboratory requires an ongoing commitment by laboratory

management, and includes: .

• the appointment of a laboratory health and safety officer with the

responsibility and authority to develop and maintain a safety

program;

• the preparation of a formal, written, health and safety plan, which is

provided to each laboratory staff member;

• an ongoing training program on laboratory safety; and ()

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• regularly scheduled, documented, safety inspections.

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. Observe procedures to prevent

inhalation or skin absorption of wastes. Each laboratory should be equipped

with all necessary safety equipment prior to conducting toxicity tests.

Personal safety gear, such as rubber aprons, lab coats, respirators, gloves,

safety glasses, and safety shoes, should be used by personnel. Each

laboratory should have safety equipment such as first aid kits, fire

extinguishers, fire alarms, and fire blankets.

Because the chemical composition of waste is usually only poorly known,

consider them as potential health hazards. Minimize exposure to waste

samples. Fume and canopy hoods over test areas must be used whenever

necessary. Strong acids and volatile organic solvents employed in

glassware cleaning must be used in a fume hood or with an exhaust

canopy over the work area.

The following recommended laboratory procedures will promote a safe

working environment and minimize health risks. This list is by no means

exhaustive. Ecology encourages testing laboratories to implement these,

and other, recommendations promoting the health and safety of

laboratory personnel.

• Testing industrial wastes should be performed in compliance with

accepted rules pertaining to the handling of hazardous materials.

Ecology recommends that personnel conducting toxicity tests not work

alone.

• Wastes should be considered potential health hazards and exposure to

them should be minimized.

• Immediately wash any body part that may have come in contact with

wastes. Although a water rinse often suffices, note that some wastes

may be activated with water.

• Adequately label ali containers to indicate contents.

• Good housekeeping habits contribute to safety and reliable results.

• Use electrical equipment approved by Underwriters Laboratories.

Ground-fault interrupters should be installed in ali "wet" labs where

electrical equipment is used.

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• Staff training in basic first aid and cardia-pulmonary resuscitation

(CPR) is strongly recommended.

• All personnel should receive tetanus immunizations. The following

are optional and dependent on the type of waste anticipated: hepatitis

B (if suspected contact with human bodily fluids, including blood);

hepatitis A, typhoid fever, and polio (if suspected contact with human

feces and/or urine). Polio may be transmitted from infant

immunizations through fecal material. Please err on the side of

caution. If your laboratory has any questions, contact the Washington

Department of Labor and Industries or similar agency in your state.

• Wastes generated during toxicity testing must be handled properly

and safely disposed. Each testing facility will have its own waste

disposal requirements based on local, state, and Federal rules and

regulations. All persons responsible for, or otherwise involved in,

performing testing activities must know, understand and comply with

these rules and regulations. Local fire officials should be notified of

any potentially hazardous conditions.

For further guidance on safe laboratory practices, consult EPA (1986),

EPA (1993c), and Walters and Jameson (1984). Also see Appendix B for

Good Laboratory Procedure Standards.

Methods and Materials

Holding Facilities

Facilities should include tanks for holding/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; this

may include either once-through or recirculating systems with at least 50

percent renewal. Holding, acclimation, and dilution-water tanks and

headboxes should be equipped for temperature control and aeration. Air

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

water may be used. Test fish should be shielded from disturbances during

holding, acclimation, and testing. The test facility should be well ventilated and free of fumes.

Fish should be quarantined at least 7days when first brought into a

facility. Avoid crowding to maintain test fish in good condition during

holding and acclimation. The dissolved oxygen (DO) concentration must

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be maintained between 60 percent and 100 percent saturation; gentle

aeration may be used if necessary. Aeration drives off the ammonia from

generated biological waste.

A photoperiod of-B-16 hours of light per 24 hours should be provided

during the test. Preferably, though not required, light intensity should be

raised gradually over a 15 minute period at the beginning of the

photoperiod. Gradually lower the light intensity over the same period of

time at the end of the photoperiod. Use a dimmer switch or other suitable

device to make this easier. The light intensity should be in the range of

natural outdoor light or ambient laboratory illumination (about 10-20

uE/m2Is or 50-100 ft-c). Avoid high intensity and extreme lighting conditions.

Whether fabricated in the lab or purchased commercially, tanks should

not be constructed of materials, or have surface coatings, which will leach

or dissolve into aqueous solutions. Tanks and other equipment that

contact stock solutions or test solutions should be chosen to minimize

sorption of toxicants from water. Use glass, stainless steel, and

fluoroplastics whenever possible to minimize leaching, dissolution, and

sorption. Concrete and rigid (unplasticized) 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.

Test Chambers

Test chambers should have a minimum solution depth of 15 cm.

Chambers filled to within the top 15 cm should be covered to prevent test

fish from jumping out. Test chambers commonly used in static tests for

fish are wide-mouth glass jars or aquaria, 3.8 L (1 gal) or 19.0 L (5 gal).

Special glass or stainless steel test chambers can be constructed to accommodate test fish requiring particular physical conditions (large surfacearea-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 organochlorine and

organophosphorus pesticides, which are difficult to remove. As little of

the adhesive as possible should be in contact with water; extra beads of

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adhesive inside the containers should be removed. Stainless steel (No.

304 or No. 316) can be used inthe construction of test chambers, but

should be of welded, not soldered, construction.

Commercially available test chambers meeting the above requirements may

be used. Disposable, non-toxic plastic (e.g., polypropylene) liners may be

used. Ecology highly recommends laboratories using disposable liners

perform several rounds of in-house testing to determine that the liners are

truly non-toxic before Using in dangerous waste designation testing.

Cleaning and Disinfection

Clean test chambers before use. Wash new test chambers with ammoniabased detergent suitable for biological laboratories, then rinse in the

following sequence with:

• clean water,

• acid, such as 5 percent concentrated nitric acid,

• copious clean water,

• reagent grade acetone, and

• copious clean water.

At every test termination, chambers should be cleaned in this sequence:

• emptied;

• immediately rinsed with clean water;

• cleaned using appropriate procedures for removing the toxicant (e.g.,

acid to remove metals and bases; detergent, organic solvent, or

activated carbon to remove organic compounds); and

• rinsed with copious clean water.

Acid is useful for removing mineral deposits and 200 mg hypochlorite/L for one

hour is useful for removing organic matter and for disinfection. A solution

containing 200 mg hypochlorite/L is conveniently made by adding 6 m1of

liquid household chlorine bleach to 1 L of water. 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

iodophors using polyvinylpyrrolidone-iodine (PVP-I) as the active ingredient,

are available as Wescodine, Betadine, Argentyne, or other product names.

o

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

titrateable iodine by diluting a commercially available 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 hypo-chlorites are C.)·

acutely toxic to most aquatic organisms. Hypochlorite can be removed by

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flushing with water or by reacting it with an equivalent concentration of sodium

thiosulfate or sodium sulfite. After a tank is washed with hypochlorite and

rinsed, air-dry the equipment overnight to allow dissipation of residual chlorine.

Dilution Wate:r;

An adequate supply of quality dilution water must be available. Acceptable test

dilution water allows healthy fish to survive for the duration of acclimation and

testing without showing stress, such as discoloration or unusual behavior. A

better criterion for acceptable dilution water is test fish surviving and growing

satisfactorily in it. Monthly hardness, alkalinity, and specific conductance

ranges must be within less than 10 percent of their respective averages. The

monthly pH range must be less than 0.8 unit to produce consistent quality

dilution water.

Ideally, non-ehlorinated dilution water is preferred, but may not be realistic.

Dechlorination using activated carbon filters or other methods may be used to

dechlorinate dilution water as thoroughly as possible. Chlorine may also be

removed using an activated carbon filter then treating with a sodium thiosulfate or

sodium sulfite drip. Sodium thiosulfate is better for dechlorinating water than

sodium sulfite and may be more reliable than activated carbon for removing

chloramines. .

Metals can usually be removed with chelating agents. Municipal water supplies

often contain unacceptably high concentrations of copper, lead, zinc, and

fluoride; buildings may contain copper pipes and lead solder: Toxicity tests can

be adversely affected by the use of dilution water containing these materials.

The dilution water should be intensively aerated using either air stones, surface

aerators, or screen tubes before introducing the toxicant. Adequate aeration will

bring the concentration of dissolved oxygen and other gases into equilibrium

with air and minimize oxygen demand and volatiles concentration. The dilution·

water dissolved oxygen should be between 90 - 100 percent saturation. Rinse

test chambers with dilution water just before use.

Dilution water hardness can affect the outcome of the test. Moderately hard

dilution water must be used in this method and should not be less than 40 mg/L

CaC03• Low hardness/alkalinity may render test organisms more sensitive to

some toxicants. Use reagent-grade chemicals to adjust hardness and alkalinity.

Table 1 is for guidance purposes.

Table 1. Preparation of Dilution Water Using Reagent Grade Cherncals'

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NaHCOa CaS04-2HP MgS04 KCI pHc Total Resulting Total Resulting ('

Water Type mg/Lb mg/Lb mg/Lb mg/Lb Hardness" Alkalinityd

Soft 48.0 30.0 30.0 2.0 7.2-7.6 40-48 30-35

Moderately Hard 96.0 60.0 60.0 4.0 7.4-7.8 80-100 60-70

• Adapted from EPA (1993c)

b Add reagent grade chemicals to distilled ordeionized water.

e Approximate equilibrium pH after aerating 24hours.

• Expressed asmg/L CaCa,.

Test Organisms

Use one of the following species in the static fish toxicity test (all species must be

verified): coho salmon, Oncorhynchus kisutch; rainbow trout, Oncorhynchus mykis

(formerly Salmo gairdneri); and brook trout, Salvelinus fontinalis. The test

temperature for all species is 12 ± 1 "C.

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

Organisms of the same species from different sources may produce different test

results. Usual sources of freshwater fish are private, state, and federal

hatcheries.

Maxillary

Preopercle

Bran hiostegals (gills)

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Standard Length

FIGURE 1.

Test fish should between 30 and 90 days from swim-up and as uniform in size as

possible. Circumstances may arise where this range cannot be met; some

variation in age is acceptable, provided the variation is no more than four or five

days. In any single test, all fish should be from the same batch. The standard

length (tip of snout to end of caudal peduncle) of the longest fish should be no (J"

more than twice that of the shortest fish (Figure 1).

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• All test fish should be obtained from a hatchery that has been certified diseasefree for the following diseases; bacterial kidney disease (Renibacterium

salmoninarum); Costia (Ichythobodo); bacterial gill disease (Myxobactriea sp.); and

Furunculosis (Aeromonas salmonicida). If fish are diseased, destroy the entire lot

immediately. Make all efforts to ensure test fish are disease-free prior to

initiation of toxicity test. Options include:

• selecting fish from a source (hatchery) which will certify that all fish are

disease-free;

• discriminately selecting test fish from a reputable source, and/or on the

observations and judgment of knowledgeable individual; or

• treating all fish that arrive on-site using treatments commonly used by

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

Between use of different test fish groups, holding and acclimation

tanks should be sterilized according to the procedures in Cleaning

and Disinfection.

Care and Handling

Minimize unnecessary stress to test fish. To avoid unnecessary stress, do not

subject fish to rapid changes in temperature or water quality. -Generally, aquatic

organisms should not be subjected to more than a 3°C change in water

temperature in any 12 hour period. Maintain test fish in dilution water at test

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

Handle test-fish as little as possible. If necessary, handle them as gently, carefully, and quickly as possible. Discard fish that touch dry surfaces, are dropped,

or injured during handling. Dip nets are best for handling fish and are

commercially available or 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 treating

with an iodophor, 200 mg hypochlorite/L, or 30 percent formalin plus 1 percent

benzalkonium chloride for at least 1 hour. Sterilize or wash hands before and

after handling fish.

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Holding

After collection or transportation, quarantine and acclimate fish to laboratory

conditions for a minimum of 7 days before used in tests. Hold fish in

uncontaminated, aerated water of constant temperature and quality in a flowthrough system with a minimum flow rate of five volume exchanges per day

(ASTM,1993). When possible, hold fish in dilution water and as close to test

temperature as possible. During long holding periods, however, it is generally

9
easier and safer to hold fish at temperatures lower than 12°C as the metabolic

rate and the number and severity of disease outbreaks are reduced.

- Recommended fish loading for holding tanks is provided for various water

temperatures, fish size, and species, in Appendix- D.

If stock fish have 5 percent or greater mortality between 24 hours after arrival

on-site and the end of the 7 day acclimation period, the entire lot should not be

used for testing. If at any time during the holding period there is an abnormal

number of mortalities, do not use the fish for testing.

Stress Indicators

Test fish should be carefully observed daily during holding and acclimation for

signs of disease, stress, physical damage, abnormal behavior and mortality.

Dead, injured, and abnormal individuals must be discarded. Visually examine

the behavior and external appearance of the fish. If the stressor can be

determined, remove stressor and reacclimate for 7 days. The following physical

characteristics indicate stress:

c

Darkened color (normal is silver/gray)

Emaciation

Erratic swimming

Excessive mucus production

External parasites

Not eating

Sample Preparation

Flashing

Flipping

Fungus/Fin Rot

Gasping at surface

Hemorrhaging

Hyperventilation

o·

Sample Water Content Determination

and Reporting Requirements

Ecology requires reporting the percent water of materials to be tested for

dangerous waste designation using Method 80-12. The following procedure is

used to estimate the water content of samples that will undergo toxicity testing

using either 80-12 A or Bmethods.

• Obtain the tare weight to the nearest O.Ol-g of a clean dry ceramic or metal

crucible or equivalent container.

• Weigh out a representative sub-sample of the material to be tested (100-g

minimum) into the above container. Since samples must be representative,

stirring of samples with liquid content> 0.5% during sub-sampling is C·.-)

required.

------------------------~-=-~,..,.,......=====-------- - 10
• • Dry the material at 60 to 80aC until two successive weighings to the nearest

O.01-g yield the same value.

• Calculate the percent non-volatile solids using the following equation:

• Include the calculated test-material percent solids in the final report showing

the raw data obtained.

• Retain procedural laboratory bench records in archive for at least five years.

I.

Weight of dried material in the container - container tare weight

Initial (wet) weight of material in container - container tare weight X 100 = % solids

Caution: Test materials having ignitability, corrosivity, or reactivity

characteristics must be handled with appropriate caution when water content

and toxicity tests are conducted.

See SW846 Chapter 7 in EPA, 1986 for ignitability, corrosivity, or reactivity

definitions.

All samples must be extracted using the method described below. Solid samples

must be reduced in size prior to extraction unless the sample meets the particle

size criteria described below.

Ensure all phases of the sample are homogenous. Stirring may be required if the

sample is not homogenous. Samples may be extracted using a rotary agitation

apparatus. The rotary agitation apparatus must be capable of rotating the

extraction bottles at 30 ± 2 rpm. The sample is allowed to extract for 18 ± 2 hours

at 23 ±2°C. The extraction water is the stock dilution water used in the toxicity test.

Particle Size Reduction

Prior to conducting the extraction, determine the need for reducing the sample

particle size. Particle reduction is required unless the solid has a surface area

per gram of material equal to or greater than 3.1 em', or is smaller than 1 cm in

its narrowest dimension (i.e., is capable of passing through a 9.5 mm (0.375 inch)

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.

Note: Surface area criteria are meant for filamentous (e.g., paper, cloth, and

similar) waste material. Actual measurement of surface area is not required, nor

11
is it recommended. For materials that do not obviously meet the criteria, C~~

sample-specific methods would need to be developed and employed to measure /

the surface area. Such methodology is currently not available.

Rotary Agitation Method

• Weigh out the correct amount of a well-mixed sample and transfer into 500

or 1000 ml extractor bottle.

• Add 200 ml of test dilution water to the extractor bottle.

• Place a Teflon cap liner over the mouth of the extractor bottle and screw on

the cap.

• Mix the sample on the rotary agitation apparatus for 18 ± 2 hours at 23 ± 2°C,

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 of water and laying on its side on the tank

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

• Begin the test as described below.

• Extreme care should be exercised throughout the course of the extraction so

as not to contaminate the exterior of the extraction flask. For example,

sealing sterile flasks in sterile aluminum foil prior to use or copiously rinsing 0

with deionized water, at a minimum, are acceptable methods.

Test Procedure

Replicates and Control

Use a minimum of 10 fish in each of 3 replicate test tanks exposed to each

treatment. Additional fish and replicates may be used (e.g., 15 fish in each of 4

replicates). Replicates must be true replicates with no water connections

between the replicate test chambers. Randomize the replicate test chambers and

impartially distributed a representative sample of test fish to the chambers.

One randomization method adds to each test chamber no more than 20 percent

of the number of test fish to be placed in each test chamber. Repeat this process

until each test chamber contains the desired number of test fish. Alternatively,

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.

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Every test requires a control consisting of the same dilution water, conditions,

number of replicates, procedures, and test species used in the test tanks. None

of the toxicants being tested is added to the control.

Test Temperature

The test temperature for all test species must be 12°e. The actual test

temperature must not deviate from 12°C by more than ± 1.0°C at any time

during the test.

Continuous temperature monitoring in at least one test tank is desirable.

Alternatively, 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 6 hours.

pH

Do not adjust the pH of the test solution. Any pH adjusting will render the test

void. There are provisions for demonstrating that the waste kills fish only

because of pH. Appendix A includes a procedure for conducting this type of

toxicity test.

pH may tend to drift during the 96 hour test period. This drift is due, in part, to

fish respiration, increasing the CO2 content of the water which 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.

Dissolved Oxygen

The saturation value, not concentration, determines the ease of oxygen transport

across the gill surface diffusion gradient. The dissolved oxygen in each test

chamber should be at least 80 percent (e.g., 8.5 mg/L at 12°C at 250 feet above

sea level) of saturation at the beginning of the test. The dissolved oxygen should .

not fall below 60 percent (e.g., 6.4 mg/L at 12°C at 250 feet above sea level) of

saturation during the first 48 hours of the test. It must not fall below 50 percent

saturation during the remainder of the test. The test must be voided if the

dissolved oxygen falls below 50 percent saturation during the test and mortality

exceeds the pass/fail threshold. Please refer to Appendix E for information on

dissolved oxygen saturation and concentration levels at different altitudes.

Test solutions may be gently aerated if dissolved oxygen does not meet the

above criteria. Once aeration is initiated, continue throughout test duration.

Avoid turbulence; it stresses fish, resuspends fecal matter, and increases

volatilization. Because evaporation readily occurs at the surface, efficient

13
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aeration can be achieved with minimum turbulence by using an air lift to

transfer solution from the bottom to the surface. Aeration should be identical in

all test chambers with the exception of the control chambers. Aerate at least onehalf of the control tanks to verify that mortality is caused by the toxicity of the waste

sample and not by the physical act of aeration (e.g., too turbulent, aeration

accidentally stops at night, etc.). Do not aerate the water in the remaining control

tanks.

Loading

Loading in test chambers should not exceed 0.8 giL. The giL of test fish (wet

weight, blotted dry) to solution in test chambers should not be so high as to

affect test results. To determine loading, use same number of fish as per replicate

but do not use these fish in the actual test. Lower loadings are recommended if

dissolved oxygen does not remain above 60 percent saturation for the first 48

hours of the test and above 50 percent saturation after 48 hours. Limit loading to

ensure:

• concentrations of dissolved oxygen and toxicant are not decreased below

acceptable levels;

• concentration of metabolic products does not increase above acceptable

levels; and

• fish are not stressed due to crowding.

Feeding . Do not feed test fish during acute toxicity tests or for a minimum of 48 hours

before the test. If the test fish are fed less than 48 hours before test initiation,

fecal matter and uneaten food may decrease the dissolved oxygen concentration

and otherwise affect the biological activity of some toxicants.

Duration and Acceptability

Begin the test by placing the flask of waste and leachate in the test chamber on

its side. The cap should also be placed in the test chamber.

The flask exterior should be sterile or as clean as possible to avoid introducing

additional contamination. "Blank" flasks used in the extraction process should

be used for the control chambers.

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The test begins when test fish are first exposed to the toxicant and ends 96 hours

later. Control test chambers must show 90 percent survival or greater for test ()

acceptance or validity.

14
• Test Observations and Measurements

Measure dissolved oxygen and the pH in all test tanks at the beginning. Repeat

these measurements every 24 hours (as long as live fish are present). Also record

the dissolved oxygen and pH 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. Measure the same parameters at

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

chloride, sulfate, particulate matter, and total organic 'carbon (TOC) or chemical

oxygen demand (COD) is also desirable.

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

24 hours after starting the test. More observations are desirable, especially near

test initiation. Ecology suggests counting the number of dead or affected fish in

each chamber 4,8, and 24 hours (or similar time increments) after the beginning

of the test. Repeat the count twice daily thereafter to the end of the test.

Remove dead fish as soon as they are observed, or once every 24 hours. Do not

stress live test fish when determining whether test fish are dead, immobilized, or

otherwise affected, and removing dead fish, Movement of test chambers and

prodding must be very gentle.

• Criteria for Death

Death is the adverse effect most often used in acute toxicity tests. 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 considered mortality. The latter responses can be useful in interpretation of test·

results and should be noted in the comment section of the data-reporting sheet.

Termination

Weights and lengths of test fish may be determined by weighing and measuring

and discarding either; (a) a 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 reported as

standard length and the weights should be wet weight, blotted dry. Destroy all fish,

both control and treatment, at termination of test, after data collection is complete.

•

Data Analysis

Refer to Calculation of Results. The number of mortalities needed to designate

a waste is no longer a single number for dangerous waste or extremely

hazardous waste. This "threshold" number of mortalities is now a function of

the mean and variance in the control group and the mean and variance in the

test group and does not utilize a data transformation.

15
Quality Assurance I Quality Control

Procedures for Quality Assurance/Quality Control

Reliable laboratory data is critical for making sound environmental decisions. A

good quality assurance/quality control (QA/QC) program is essential to ensure

data reliability. Quality assurance (QA) is the total integrated program for

assuring the reliability of monitoring data. A comprehensive QA program

addresses everything affecting data and should address:

• sample collection and preservation;

• laboratory services;

• use and care of instruments, glassware, and chemicals;

• data management; and,

• 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 QC program includes procedures for estimating and controlling

precision and bias, as well as:

• defines data quality objectives,

• chooses analytical methods which will meet those objectives,

• estimates within-lab precision and bias,

• sets up control charts, and

• participates in inter-lab tests.

Reference Toxicants

Reference toxicants establish the validity of toxicity data. The reference toxicant

measures all aspects of toxicity testing including organism quality, presence and

. effects of stressors other than test compounds, and the quality of analytical

techniques. If reference toxicant results (LC50s) differ significantly from previously established results (LC50 control charts), a full review of laboratory test

conditions and procedures is indicated. Results that are not within two standard

deviations of the mean LC50, should be considered "red flags."

Evaluate procedures and test fish with a reference toxicant once a month 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

16

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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 and include

phenol, NaCl, KCl, ZnC12

, and ZnS04• This is not an all inclusive list by any

means. Reference toxicants available from EPA include CuS04 and CdC12

(Environmental Monitoring and Support Laboratory (EMSL), Cincinnati, Ohio

45268). To ensure nominal concentrations of reference toxicants, concentrations

should be measured periodically.

Calculation of Results

The bioassay results are calculated using the statistical method described below;

two examples are given. Within a 90 percent confidence limit, the test sample

fails the bioassay by exhibiting a median lethal concentration (LC50) less than or

equal to (i.e., toxicity greater than or equal to) the regulatory threshold (i.e., 100

or 10 mg/L) when the calculated t statistic is greater than a critical t value.

Approach

The statistical analysis is based on methodology presented by Zar (1984), EPA

(1993c), and Erickson and McDonald (1995). Because observations on individual

fish in the same tank cannot necessarily be considered statistically independent,

the experimental unit is the tank and not individual fish. This avoids a statistical

error in experimental design referred to as pseudoreplication (Hurlbert, 1984).

The two-sample t test is used. This test assumes both control and test groups are

randomly selected from normal populations with equal variances. However, the

frequency of samples displaying varying proportions of dead and live fish, as

determined in acute lethality bioassays, is described by the binomial

distribution. Use of the two-sample t test is robust enough to stand considerable

departures from its theoretical assumptions, especially ifsample sizes are equal

(Zar, 1984). Untransformed sample data have been shown to be normally

distributed, in most cases, as assessed by Shapiro-Wilk's test for normality (EPA,

1993c).

The type of t test used depends on the homogeneity of variance assumption

between control and test samples (Zar, 1984; EPA, 1993c). This assumption is

evaluated with the variance ratio F test. If variances are equal, the equal

variance t test is used. If variances are not equal, a modified t test is used.

17
Thus, the appropriate t test is used to compare the difference between test and C~

control mortality proportions against a constant, Po =0.5, corresponding to the

LC50. Up to 10 percent mortality in the control sample is allowed. A one-tailed t

test is used, sincethe analysis determines only ifthe sample LC50 issignificantly greater

than the regulatory threshold. This objective is consistent with the procedure for

waste designation from bioassay data outlined in WAC 173-303-100(5)(c).

Hypothesis

In terms of concentration, the one-tailed null hypothesis (H') to be tested is LC50

100 mg/L (dangerous waste threshold) or LC50 10 mg/L (extremely

hazardous waste threshold or special waste threshold). In terms of mortality,

each of these hypotheses corresponds to a one-tailed Ho: (PTm - PcJ 3 Po' where

PTm and Pem are the mean proportion of mortalities in the test and control

samples, respectively, and Po = 0.5 is the proportion of mortalities at the LC50.

An alpha level of 0.10 is used in the t test (i.e., one-tailed 90 percent confidence

level), consistent with statistical methodology outlined by EPA (1986). Rejecting

H, demonstrates the sample does not designate as a regulated waste.

Calculation

1) Sample size

The number of replicates in the control and test groups should be equal and 0

should each consist of at least three (N 3). The number of fish per replicate

tank should be equal across all replicates and should consist of at least 10

(n 10). In the rare event that a test is initiated with unequal replicates, please

contact the Department of Ecology for statistical guidance. In addition, the

number of fish per replicate tank should not exceed a biomass loading density of

0.8 giL.

2) Mortalities

Mortalities are expressed as proportions:

Pe = proportion of mortalities in control replicate

PT = proportion of mortalities in test replicate.

The bioassay is invalid if overall control mortalities exceed 10 percent (EPA,

1993c).

3) F test

The variance ratio F test is used to identify the appropriate t test. A two-tailed F

test is conducted at an alpha level of 0.05 with N -1 degrees of freedom (df) in (.)\

both numerator and denominator (Zar, 1984; EPA, 1993c):

18
•

•

F = SC2/s/ or F = s/lsC

2, whichever is larger, where:

F = F statistic

Sc2 = variance for the control group '2 ST = variance for the test group.

If the calculated F value is less than or equal to the critical F value, the variances

are equal and the equal variance t test is used. If the calculated F value is greater

than the critical F value, the variances are not equal and the modified t test

should be used. Note that in the case where both variances are zero, the variances are

equal. However, when only one variance iszero, the variances areconsidered statistically

unequal.

4) Equal variance t test

With an equal number of replicates in the control and test groups, the equal

variance t test has the following form (Zar, 1984):

t = (PTm PCm - PO> 1 (2s 2INt5

(general equation); or,

t = (PTm - PCm - 0.5) 1 [(;/ + sc2

)/ Nt 5

where: t = t statistic

PTm = meanpT

PCm = mean Pc

Po = 0.5 = proportion of mortalities at LC50

Sp2 = pooled variance =(s/ + sc2)/ 2

N = number of replicates in control or test group.

The number of degrees of freedom in an equal variance t test with an equal

number of replicates in control and test groups is 2(N -1). The calculated t

statistic is compared to a one-tailed critical t value at an alpha level of 0.10 with

2(N -1) degrees of freedom (df). When the calculated t value is less than or

equal to the critical t value, H, is rejected, and it is concluded that the waste does

not designate as a regulated waste. Note that in the case where (s/ + sc2) = 0, H,

is rejected when (PTm - Pc,J < 0.5 and H, is accepted when (PTm - PCm) 0.5.

5) Modified t test '

With an equal number of replicates in the control and test groups, the modified t .

test for unequal variances has the same form as the equal variance t test above

(Zar,1984). However, the number of degrees of freedom differs and is equal to

the following (Zar, 1984):

19
If the computed degrees of freedom are noninteger, use the next smaller integer.

Note that if either sc2=0 or 5.r2=0, then df =N - 1. The calculated t statistic is

compared to a critical t value with the modified degrees of freedom. The

interpretation then follows that described above for the equal variance t test.

6) Critical F and t values

Tables 2 and 3 list critical F and t values, respectively. Appropriate critical

values for other degrees of freedom can be found in most statistics books (e.g.,

Zar,1984). For number of degrees of freedom, see above text.

TABLE 2. CRITICAL F VALUES WITH TWD-TAILED a =p.OS

Denominator Numerator df

df 2 3 4 5

2 39.0 39.2 39.2 39.3

3 16.0 15.4 15.1 14.9

4 10.6 9.98 9.60 c 9.36

5 8.43 7.76 7.39 7.15

TABLE 3. CRITICAL TVALUES WITHONE-TAILEDa=O.10

df t' df t

1 -3.078 6 -1.440

2 -1.886 7 -1.415

3 -1.638 8 -1.397

4 -1.533 9 -1.383

5 -1.476 10 -1.372

1 Criticai t values are negative, since the interest lies only in whether (Pr - Pc)

is significantly smaller than 0.5.

Example 1: Equal Variance t Test

This test consists of two treatments and one control, each in triplicate. Two sets

of three replicates of 10 fish each are exposed to 10 and 100 mg/L, respectively,

of waste using the 80-12 bioassay procedure to determine if the waste sample

designates as dangerous waste or extremely hazardous waste. The control

group is exposed to dilution water only.

o

Replicate

(N=3) ()

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[

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•

A 1 0.100 1 0.100 4 0.400

B 1 0.100 2 0.200 6 0.600

C 0 0 1 0.100 5 0.500

Mean (pJ 0.067 0.133 0.500

Variance (S2)1 0.0033 0.0033 0.0100

Mer 96 hours, control and test tank mortalities are tabulated. Proportions are

based on number of fish per replicate (e.g., 1/10 = 0.100). The critical F and t

values are found in Tables 2 and 3, respectively.

TABLE 5. SUMMARY FOR EQUAL VARIANCE T TEST

10 mg/L 100 mg/L

Calculated F statistic 0.0033/0.0033 =1.00 0.0100/0.0033 =3.03

Critical F df; numerator & 2,2 2,2'

denominator each =(N - 1)

Critical F (see Table 1) 39.0 39.0

Calculated t statistic -9.25 -1.01

Critical t df" =r2(N-1)1 4 4

Critical t (see Table 2) -1.53 -1.53

Does waste desiqnate? NO YES

Waste Code not applicable WT02

'10 mglLcalculated t =(0.133-0.067-0.5)/[(0.0033+0.0033)/3]'5 & 100 mgIL calculated t =(0.5-0.067-0.5)/[(0.0033+0.0100)/3]'5

, 2(N-1) is only used intheequal variance t test

With the appropriate equations, calculate F =1.00 (10 mg/L) and F =3.03 (100

mg/L). Because both of the calculated F statistics are less than the critical F value

(39.0), the variances are equal and the equal variance t test is performed. The

calculated t statistics are -9.25 (10 mg/L) and -1.01 (100 mg/L). Because the 10

mg/L calculated t statistic (-9.25) is less than the critical t value (-1.53), H, is

rejected and the waste does not designate as extremely hazardous waste.

However, because the 100 mg/L calculated t statistic (-1.01) is greater than the

critical t value (-1.53), H, is not rejected and the waste does designate as

dangerous waste.

Example 2: Modified t Test

This test consists of two treatments and one control, each in quadruplicate. Two

sets of four replicates of 15 fish each are exposed to 10 and 100 mg/L,·

respectively, of waste using the 80-12 bioassay procedure to determine if the

waste sample designates as dangerous waste or extremely hazardous waste. The

control group is exposed to dilution water only.

21
TABLE 6. TEST RESULTS

Replicate DilutionWater Control (n:15) 10mgILSample (n:15) 100 mgILSample (n:15)

(N:4) Number Proportion Number Proportion Number Proportion

Dead . Dead (p~) Dead Dead (PT) Dead Dead (PT)

A 0 0 4 0.267 9 0.600

B 0 0 6 0.400 8 0.533

C 0 0 3 0.200 10 0.667

D 0 0 0 0 6 0.400

Mean (PM) 0 0.217 0.550

Variance (S2)' 0 0.0278 0.0130

1 Vanance equation IS s'=1:{p-p.J'/N-l where p = peor p,

After 96 hours, control and test tank mortalities are tabulated. Proportions are

based on number of fish per replicate (e.g., 1/15 =0.067). The critical F and t

values are found in Tables 1 and 2, respectively.

TABLE 7. SUMMARY FOR MODIFIED TTEST

10mg/L 100 mg/L

Calculated Fstatistic The variances are considered statistically unequal because the

control variance is zero. Therefore, the modified t test isused.

Calculated t' statistic -3.39 0.88

Critical t df2 =(N-1 ) 3 3

Critical t (see Table 2) -1.64 -1.64

Does waste designate? NO YES

Waste code not applicable WT02

1 110 mglLcalculated t = (0.217-0-0.5)1[(0+0.0278)/4)'5 & 100 mglLcalculated t = (0.55000.5)1[(0+0.0130)/4]'5

a (N- 1)is used inthemodified t testwhen either se' = 0 or sr' = 0

Zero variance in the control group dictates that the variances be considered

unequal by the F test rule stated above. Therefore, the modified t test is

employed and the calculated t for 10 mg/L is -3.39 and for 100 mg/L is 0.88.

Because the 10 mg/L calculated t statistic (-3.39) is less than the critical t value (-

1.64), H, is rejected and the waste does not designate as extremely hazardous

waste. However, because the 100 mg/L calculated t statistic (0.88) is greater

than the critical t value (-1.64), H, is not rejected and the waste does designate as .

dangerous waste. . .

Data Reporting

The report must contain the following information (a sample reporting form is

found in Appendix E):

• name of test, investigator, laboratory, and date test was begun;

22

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• detailed description of waste tested, its source, lot number, known

composition, known physical and chemical properties;

• dilution water source, chemical characteristics, description of pretreatment,

any additives;

• calculated test-material percent solids (% solids) showing raw data obtained;

• detailed information about test organisms to include scientific name and how

verified, wet weight, blotted dry, standard length for fish, age, life stage, source,

history, observed diseases, treatments, acclimation procedure, food used;

• description of experimental design and test chambers, test solution volume

depth in chambers, number fish per replicate and treatment, loading, and

lighting description;

• description of any aeration performed before or during test;

• definition of criterion used to determine endpoints/effects, summary of

general observations;

• number and percentage of fish deaths, per replicate and per treatment, in

control and test chambers on a daily basis;

• date run, identification and concentration of reference toxicant used and

resulting mortality and LCSO;

• methods used for and results with standard deviation of all chemical

analyses of water quality and toxicant concentration, including validation

studies and reagent blanks;

• value of following: acclimation temperature, test temperature, pH, hardness,

alkalinity, dissolved oxygen, and conductivity; and

• anything unusual about the test, any deviations from procedures, and any

other relevant information.

23
• References

ASTM. 1993. Standard guide for conducting early life-stage toxicity tests with

fishes: E1241-92. pp. 180-207. In: 1993 ASTM Standards on Aquatic Toxicology

and Hazard Evaluation, ASTM, Philadelphia, Pennsylvania.

Environment Canada. 1990. Biological test method: acute lethality test using

rainbow trout. Conservation and Protection. Report EPS 1/RM/9. Ottawa,

Canada.

EPA. 1985. Methods for measuring the acute toxicity of effluents to freshwater

and marine organisms. EPA/600/4-85/013.

EPA. 1986. Test methods for evaluating solid waste. 3rd ed., SW-846,Volume II, Part

III,Chapter 9. Office of Solid Waste and Emergency Response, Washington, DC.

•

•

EPA. 1989. Generalized methodology for conducting industrial toxicity

reduction evaluations (TREs). EPA/600/2-88/070. Water Engineering Research

Laboratory, Cincinnati, OH.

EPA. 1991. Methods for aquatic toxicity identification evaluations. Phase I

toxicity characterizations, 2nd ed. EPA/600/6-91/003. Environmental Research

Laboratory, Duluth, MN.

EPA. 1992. Toxicity identification evaluation. Characterization of chronically

toxic effluents, phase I. EPA/600/6-91/005F.

EPA. 1993a. Methods for aquatic toxicity identification evaluations. Phase II

toxicity identification procedures for samples exhibiting acute and chronic

toxicity. EPA/600/R-92/080.

EPA. 1993b. Methods for aquatic toxicity identification evaluations. Phase III

toxicity confirmation procedures for samples exhibiting acute and chronic

toxicity. EPA/600/R-92/081. -

EPA. 1993c. Methods for measuring the acute toxicity of effluents and receiving

waters to freshwater and marine organisms. 4th Ed. by CI Weber,

Environmental Monitoring Systems Laboratory, Office of Research and

Development, EPA, Cincinnati, OH. EPA/600/4-90/027F.

Erickson, WP and LL McDonald. 1995. Tests for bioequivalence of control media

and test media in studies of toxicity. Environ. Toxicol. Chern. 14(7):1247-1256.

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

Standards, pp. 53946 ff.

25
Hurlbert, SH. 1984. Pseudoreplication and the design of ecological field

experiments. Eco!. Monogr. 54(2):187-211.

Pillard, D. 1996. ENSR Corporation. Personal communication. 100 percent

saturation concentrations calculated from regression models. Original data

obtained from "Instruction Manual YSI Models 54ARC and 54ABP Dissolved

Oxygen Meters" pp. 16-17. YSI Inc., Yellow Springs, OH.

Walters, DB, and CW Jameson. 1984. Health and safety for toxicity testing.

Butterworth Publ., Woburn, MA.

Wedemeyer, GA, and JW Wood, 1977. Stress as a predisposing factor in fish

disease. us. Fish and Wildlife Service. FDL-38. Washington, D.C.

Wedemeyer, GA, RL Saunders, and WC Clarke, 1981. The hatchery

environment required to optimize smoltification in the artificial propagation of

anadromous salmonids. pp. 6-20. In: E. Kiney and L. Allen, eds., BioEngineering in Fisheries. Special Publication by American Fisheries Society,

Washington, D.C.

c

Wedemeyer, GA, DJ McLeay, C Phillip Goodyear. 1984. Assessing the tolerance

of fish and fish populations to environmental stress: the problems and methods

of monitoring. C

Zar, JH. 1984. Biostatistical analysis. 2nd ed., Prentice Hall, Englewood Cliffs,

NJ.

o

26
•

•

•

Appendix A

Neutralized Fish Toxicity Test Procedure

The neutralized fish toxicity test procedure is identical to the Part A method

except as noted below. The only difference is that aquarium water is neutralized

after 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 HCl or NaOH. 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 thereafter. Further pH adjustment is not allowed.

The neutralizing solutions should be as concentrated as practicable to minimize

diluting the aquarium water. Under no circumstances should> 50 ml be used to

adjust pH. Report the actual volume and normality of neutralizing solution

used on the data reporting sheet.

Conducting the Neutralized Fish Toxicity Test

The neutralized fish toxicity test requires all of the proceeding steps for validity.

Conduct steps 1 "- 5 in the sequence shown.

1. Fill the test tank and control tank with dilution water allowing for 400 ml of

water from the sample extraction step. The dilution water must show

hardness, alkalinity, and pH within the range for "soft" to "moderately hard"

water as described in Table 1.

2. Introduce the waste into the test tank. The waste includes any undissolved

sample, the extract, the rinse, and the extraction flask or bottle.

3. Measure and record the pH.

4. 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 steps 5, 6, and 7.

5. Collect a sample for measuring hardness, alkalinity, and conductivity.

6. Measure and record dissolved oxygen, pH, and temperature.

7. Introduce the fish into the test tank.

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

9. Adjust the temperature and dissolved oxygen as necessary.

10. Terminate the test after 96 hours.

11. Complete the data reporting sheet.

27
Recording the Data

For neutralized tests, record the following information on the data reporting

sheet:

• neutralizer (i.e., NaOH or Hel),

• normality of neutralizer,

• volume of neutralizer, and

• indicate if pH readjustment was necessary 30 minutes after initial pH

adjustment.

28

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Appendix B

Good Laboratory Standards

The following was adapted from EPA's Good Laboratory Practice (GLP)

Standards. These standards are recommended, not required. Laboratories are

encouraged to develop QA/QC programs which address these recommended

standards.

Organization and Personnel: 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 fob 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 adversely 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 adverse affect on a study.

Biologist in Charge

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

29
the technical conduct of the study, as well as for the interpretation, analysis, (-

documentation, and reporting of results, and represents the single point of study /

control. The biologist in charge shall assure that:

• The protocol, including any change, is approved as provided by Ecology and

is followed.

• All experimental data, including observations of unanticipated responses of '

the test system are accurately recorded and verified.

Unforeseen circumstances that may affect the quality and integrity of the study

are notedwhen they occur, and corrective action is taken and documented.

• Test systems are as specified in the protocol.

• All applicable good laboratory practice regulations are followed.

• All raw data, documentation, protocols, specimens, and final reports are

transferred to the archives during or at the close of the study.

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:

'. A descriptive titleand statement of the purpose of the study. C • Identification of the test and control substance by chemical name, Chemical

Abstract Service Registry Number (CAS), or other established identification

system.

• The name and address of the sponsor and the name and address of the

testing facility at which the study is being conducted.

• The proposed starting and completion dates.

• Justification for selection of the test system.

• Where applicable, the number, body weight range, sex, source of supply,

species, strain, substrain, and age of the test system.

• The procedure for identification of the test system.

• A description of the experimental design, including the methods for the

control of bias.

• A description and/or identification of the diet used in the study as well as

solvents, emulsifiers and/or other materials used to solubilize or suspend the

test or control substances before mixing with the carrier. The description

shall include specifications for acceptable levels of contaminants that are

reasonably expected to be present in the dietary materials and are known to

be capable of interfering with the purpose or conduct of the study if present

at levels greater than established by the specifications. C' • The route of administration and the reason for its choice.

30
•

•

•

• 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.

• 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.

• . The type and frequency of tests, analyses, and measurements to be made.

• The records to be maintained.

• .The date of approval of the protocol by the sponsor and the signature of the

biologist in charge.

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

• 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.

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 when examining that specimen histopathologically.

All data generated during the conduct of a study, except those that are generated

as direct computer input, shall be recorded directly, promptly, and legibly in

ink. All data entries shall be dated on the day of entry and signed or initialed by

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

obscure the original entry, shall indicate the reason for such change, and shall be

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

31
Maintenance and Calibration of Equipment C

Equipment shall be adequately inspected, cleaned, and maintained. Equipment j

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, maintenance, testing,

calibrating, and/or standardizing operations. These records, containing the date

of the operation, shall describe whether the maintenance operations were routine

and followed the written standard operating procedures. Written records shall

be kept of 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.

Testing Facilities Operation:

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.

o

Standard operating procedures (SOPs) shall be established for, but not limited

to, the following:

• animal room preparation;

• animal care;

• receipt, identification, storage, handling, mixing, and method of sampling of

the test and control substances;

• test system observations;

• laboratory tests; 0

32
•

•

• handling of animals found moribund or dead during study;

• necropsy of animals or postmortem examination of animals;

• collection and identification of specimens;

• histopathology;

• data handling, storage, and retrieval;

• maintenance and calibration of equipment;

• 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; and,

• a historical file of standard operating procedures, and all revisions thereof,

including the dates of such revisions, shall be maintained.

Reagents 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 ~e used.

33
•

•

•

Appendix C

Water Quality

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

adversely affect the outcome of acute fish toxicity tests. Sub-lethal

concentrations of certain materials in 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. The more

common materials and parameters of concern are presented below with

recommended ranges or maximum concentrations. If erratic results are experienced in the toxicity test, the dilution water should be checked for the materials

included in Table 8, or for other materials which may be present in the dilution

water source. Also refer to EPA/600/4-90/027F Methods for Measuring the

Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine

Organisms for additional guidance on water quality.

TABLE 8. WATER QUALITY PARAMETER~

PARAMETER RECOMMENDED

pH 7.2 -7.8

Alkalinity 30- 70mgll asCaCO,

Ammonia (NH ) 0.02 ppm unionized

Cadmium (Cd) 100 noll

Chloride 2 - 3 ppm

Copper (Cu) less than 5 ppb

Hardness 40-100 mQ/l,as CaCO,

lead (Pb) 0.03 ppm orless

Mercury (Hg) organic orinorganic 100 ngll

Nickel (Ni) 88ppb orless

Nitrogen (N) maximum total gas pressure 110 percent of

saturation

Nitrite 100 ppb

Oxygen atleast 8 ppm at 12"C

Ozone Residual 3 ppb

PCB asArochlor 1254 50ngll orless

Total Suspended and Settlable Solids 80ppm orless

Zinc (Zn) less than 65ppb

Total organochlorine pesticides 50ngll orless

'Adapted from Wedemeyer et al,(1981) and EPA (1991).

35
36

- - - -~--

Appendix D C_~

TABLE 9.APPROXIMATE SATURATlONlAND CONCENTRATION VALUES OFDISSOLVED OXYGEN IN

FRESHWATER AT 1~C

Altitude (ft) DO (mglL) at DO (mglL) at DO (mglL) at Altitude DO (mgll)at DO (mglL) at DO (mgll) at

100% 80% 60% (ft) 100% 80% 60%

Saturation Saturation Saturation Saturation Saturation Saturation

0 10.71 8.57 6.43 4000 9.35 7.48 5.61

100 10.68 8.54 6.41 4250 9.26 7.41 5.56

250 10.62 8.50 6.37 4500 9.18 7.34 5.51

500 10.54 8.43 6.32 4750 9.09 7.27 5.45

750 10.45 8.36 6.27 5000 9.01 7.21 5.41

1000 10.37 8.30 6.22 5250 8.92 7.14 5.35

1250 10.28 8.22 6.17 5500 8.84 7.07 5.30

1500 10.20 8.16 6.12 5750 8.75 7.00 5.25

1750 10.11 8.09 6.07 6000 8.67 6.94 5.20

2000 10.03 8.02 6.02 6250 8.58 6.86 5.15

2250 9.94 7.95 5.96 6500 8.50 6.80 5.10

2500 9.86 7.89 5.92 6750 . 8.41 6.73 5.05

2750 9.77 7.82 5.86 7000 8.33 6.66 5.00

3000 9.69 7.75 5.81 7250 8.24 6.59 4.94

3250 9.60 7.68 5.76 7500 8.16 6.53 4.90

3500 9.52 7.62 5.71 7750 8.07 6.46 4.84

3750 9.43 7.54 5.66 8000 7.99 6.39 4.79 \ 1Based onPillard (1996).

o

t------

, ~=------------------------~...,.....,--"=""""""":":""""- __---
•

•

•

Appendix E

Recommended Fish Loading

Prior to use in toxicity tests, fish are usually maintained at the test facility in a

circulated holding tank. A minimum level of circulation is required to maintain

the viability of test fish. The recommended level of circulation varies depending

on the species of fish, size of fish, and 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.

TABLE 10. RECOMMENDED FISH LOADINGS"

SPECIES WATER POUNDS FISH PER GPM

TEMPERATURE

°C of 1000b 500b 100b

Coho Salmon 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

Fall/Spring 3 38 3.0 4.0 6.0

Chinook Salmon 9 48 2.5 3.0 5.0

14 58 2.0 2.2 3.5

17 63 ---- 1.2 3.0

• Adapted from Wedemeyer &Wood (19n)

b Fish size (number offish perpound)

37
•

•

•'

Appendix F

Sample Data Sheet , The Data Sheet for Static Basic Acute Fish Toxicity Test is printed on page 4l.

Please copy this form to report test data.

39
-

11,11.I'f' 1111"'''' '""II

[tOLOGY

IndustryfToxicant

Address

Collector

Date Sample

Collected

DATA SHEET FOR STATIC BASIC ACUTE FISH TOXICITY TEST

•

Beginning Date

Ending Date

Test Organism

Required Test Temperature

Range

•

Laboratory _

Analyst

Time

Time

Laboratory Test Total Total Conductivity

Reference Contai Cone. Number of Cumulative Dissolved Oxygen (mgll) pH25C Temperature (C) Hardness Alkalinity uMHOStcm

Number ner No. (mgtl) Deaths (mgtl as (mgtl as

CaC03) CaC03)

0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 96 0 96 0 96

Sample Description ----:-:---------

Average Weight Mean Length Longest Shortest Ratio (long/short) _

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

Method on File with the Depertment of Ecology

GENERAL PROCEDURE FOR STATIC BASIC ACUTE FISH TOXICITY TEST

Date _

A-41

DataVerified By
• Appendix G

Recommended Test Conditions and Procedures

for Fish Bioassay

•

•

Test type

Duration

Light quality

Light intensity

Photoperiod

Test chamber size

Minimum de~th

Test solution renewall

Source of fish

Control water

Fish age

Temperature

Oxygen/aeration

pH

Feeding

Cleaning

Number offish per test chamber

• static non-renewal

.96 hours

• ambient laboratory illumination

• 10-20 uE/m2/s (50-100 ft-c)

• 16 hours light, 8 hours darkness

• 3.8 L or 1 gal minimum

• 15 em test solution

• none

• hatchery stock free of known diseases

• moderately hard water; see Table 1

• 20 - 90 days

• none unless DO < 80 percent

saturation at altitude; ideal DO is 80 to

100 percent saturation

• no pH adjustment

• not required

• not required

• minimum 10

A-43
Number ofreplicate chamber per

concentration

Number offish per concentration

Observations

Measurements

Endpoints

Test acceptability

Reference toxicant

Solvents

Concentration

Control water

Transport and Storage

Extraction

New Parameter

calculate

• minimum3

• minimum30

• fish mortality, appearance, and

behavior at 24, 48, 72,and 96 hours

minimum

• temperature, DO, pH, in each tank

daily; conductivity at beginning/end of

test

• mortality

• 90 percent survival or greater in

controls .

• CuSo4 , CdC12 , KCl, NaCl, or znS04

(not inclusive); evaluate at least

once/month

• extraction water is the control water

used in test controls .

• 100.ppm to determine dangerous

waste; 10 ppm to determine extremely

hazardous waste .

• moderately hard water

• extract within seven days of sample

receipt..store in dark at 4°C; test within 8

days of sample receipt

• extract using rotary agitation device

(30 ± 2 rpm) for 18 hours ± 2 hours at

23°C± 2°C

• sample water content determination;

test-material percent solids (% solids)

A-44

c

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Part B: Method 80·12

Acute Oral Rat Toxicity Test

Introduction

The acute oral rat bioassay is generally used to determine the relative acute

toxicity of an unknown substance. This acute oral rat bioassay was developed by

the Washington Department of Ecology (Ecology) for the purpose of

determining if a waste meets the definition of dangerous waste in the Dangerous

Waste Regulations, Chapter 173-303 WAC.

This method determines lethality and signs of acute toxicity from a waste sample

administered in a single dose by gavage to a limited number of rats. The

bioassay determines if the test sample exhibits a median lethal dose (LD50)

either greater than or less than or equal to a regulatory threshold corresponding

to a waste designation, dangerous waste, special waste, or extremely hazardous

waste. Applicable regulatory thresholds for dangerous waste, special waste, and

extremely hazardous waste are doses of 5000,500, and 50 mg/kg (weight of test

substance per unit body weight), respectively. The waste sample fails the

.bioassay by exhibiting an LD50 statistically less than or equal to (i.e., toxicity

greater than or equal to) the pertinent regulatory threshold.

Methods

Methodology has been adapted from acute oral toxicity testing, outlined by the

EPA in the Code of Federal Regulations (CFR, 1993) and by Committee E-35 on

Pesticides with the American Society for Testing and Materials (ASTM, 1994a;

ASTM, 1994b).

Sample Collection and Preparation

A representative sample of waste .should be obtained, using methods approved

by the Department of Ecology.

The waste sample should be homogenized and either dissolved or suspended in

an appropriate vehicle, so that the material is in a homogeneous liquid form:

amenable to gavage. In decreasing order of preference, water, saline, or oil (e.g.,

com oil) are recommended for the vehicle. The test solution concentration (i.e.,

weight of waste sample per volume solution) should not exceed 1 ml/100 g body

B-1
weight, except when an aqueous solution is used, where 2 ml/l00 g is acceptable C

(CFR, 1993). ..

The vehicle and test solution should be at the same temperature as the test room.

Record the type of vehicle, test solution concentration, and temperature and

photoperiod of the testing room.

Test Animals

Use common laboratory strains of albino rats (e.g., Sprague-Dawley) weighing

190 to 300 g, prefasted. At each dose level tested (5000,500, or 50 mg/kg), use a

minimum of five males and five females. Larger samples are allowed; maintain

an equal number of male and female rats. Include the same number of animals,

and equal numbers of males and females, in the vehicle control group as in the

test group. Female rats should be nulliparous and nonpregnant. Use both males

and females to incorporate possible gender differences in acute toxicity (Dixon,

1965) and renal metabolism (EPA, 1991).

- I

i

Pretest Conditioning

Animals should not have been used for any other tests and should be

maintained according to accepted laboratory practices for the care and handling

of test animals. Identify and cage individually each animal. Rat chow (or

equivalent) and water must be available ad libitum until 18 to 20 hours before

test initiation, at which time deprive animals of food. Examine each animal for

overt signs of disease. Acclimate to the test environment for a minimum of 7

days. During acclimation, observe animals for adverse health effects. Any

animals demonstrating signs of spontaneous disease should be eliminated prior

to test initiation. Use only animals judged to be healthy.

Procedure

Administer doses of 5000, 500, or 50 mglkg to evaluate whether the waste

sample designates as dangerous waste, special waste, or extremely hazardous

waste, respectively. This dose is expressed as weight of the test substance per

unit body weight. Determine the fasted body weight of each rat at the start of .

the test to calculate the volume of test solution (i.e., waste sample in vehicle)

needed to deliver the desired dose.

Administer the test solution in a single dose by gavage, using a stomach tube or

suitable intubation cannula. As stated above, the volume of test solution should

not exceed 1 ml/l00 g body weight, except when an aqueous solution is used,

where 2 ml/l00 g is acceptable (CFR, 1993). Deliver a uniform dose to rats with

varying body weights by adjusting the volume of the prepared test solution of

known waste concentration.

B-2

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•

•

•

A concurrent vehicle control group should be treated identically to the test

group with the notable exception that only vehicle (i.e., no waste sample) should

be administered. Control group animals should receive a volume of vehicle per

unit body weight (ml/kg) equal to the volume of test solution per unit body

weight (ml/kg) administered to the test group animals. Record the volumes of

vehicle and test solution administered to control and test group animals,

respectively.

After dosing, immediately return all animals to individual cages with ad libitum

food and water. Observe animals for mortality and signs of acute toxicity

immediately after dosing, at one and four hours after dosing, and daily

thereafter for 1~ consecutive days. Cage-side observations should include

evaluation of skin and fur, eyes and mucous membranes, respiratory,

circulatory, autonomic and central nervous systems, somatomotor activity, and

behavior patterns. Particular attention should be directed toward noting

tremors, convulsions, lethargy, salivation, and diarrhea. Record any mortalities

and signs of acute toxicity..

Temperature of the testing room should be recorded daily. Body weights should

be determined and recorded at the start of the test, on day 7, and at death.

Sacrifice surviving animals at the end of the test on day 14. Perform gross

necropsies on all animals in both control and test groups. Record all gross

pathology changes.

Calculation of Results

The bioassay result is calculated using the statistical method described below.

Three examples are also presented. Within a 90 percent confidence limit, the

test sample fails the bioassay by exhibiting a median lethal dose (LD50) less than

or equal to (i.e., toxicity greater than or equal to) the regulatory threshold (i.e.,

5000, 500, or 50 mg/kg) when the cumulative binomial probability exceeds the

established alpha level for small samples (N < 30), or when the calculated Z

statistic is greater than -1.28 for larger samples (N 30).

Approach

The frequency of samples displaying varying proportions of dead and live rats,

as determined in acute lethality bioassays, is described by the binomial

distribution. Due to the statistical independence among observations on

individually caged rats, the experimental unit is the individual rat, and results

B-3
from the dangerous waste oral rat bioassay can be analyzed with a binomial

goodness of fit test. The observed proportion of mortalities in the test group is

compared to a constant, Po= 0.5, which corresponds to the LD50. A one-tailed

test is employed, since we wish only to determine if the sample LD50 is

significantly greater than the regulatory threshold. This objective is consistent

with the procedure for waste designation from bioassay data outlined in WAC

173-303-100(5)(c).

Two binomial methods are applied, depending on sample size (N) of the test

group. Both methods incorporate a correction for up to 10 percent mortality in

the control group. For N < 30, the cumulative binomial probability of mortalities

is determined directly from the binomial distribution. For N 30, the normal

approximation to the binomial test is employed (Zar, 1984).

c

Hypothesis

In terms of concentration, the one-tailed null hypothesis (HJ to be tested is LD50

:::; 5000 mg/kg (dangerous waste threshold), LD50:::; 500 mg/kg (special waste

threshold), or LD50:::; 50 mg/kg (extremely hazardous waste threshold). In

terms of mortality, each of these hypotheses corresponds to a one-tailed Ho: PTa

Po' where PTa is the proportion of control-adjusted mortalities in the test group

and Po = 0.5 is the proportion of mortalities at the LD50. An alpha level of 0.10 is 0

used (i.e., one-tailed 90 percent confidence level), consistent with statistical

methodology outlined by EPA (1986). Rejecting H, demonstrates that the sample

does not designate as a regulated waste.

Calculation (N <30)

1) Minimum sample size

A minimum of 10 rats (N = 10) is to be used each in the control and test group

with an equal number of males and females.

2) Control correction

The bioassay is invalid if control mortalities exceed 10 percent (EPA, 1993).

Corrections for up to 10 percent mortality in the control are applied with

Abbott's formula (Finney, 1971; EPA, 1993):

p/= (PT- Pc) / (1 - Pc)

where: p/ == control-adjusted proportion of mortalities in test group

PT=observed proportion of mortalities in test group

Pc= observed proportion of mortalities in control group

B-4

() I
•

•

••

Note that p/= PT when Pc= o. If Pc~ 0.10 and Pc? PT' then PTa = 0 as a result of

data smoothing (EPA, 1993). That is, PT and Pc are each set equal to their

average, (PT+Pc) / 2, so that PTa = O.

3) Binomial probability

The cumulative binomial probability for the control-adjusted number of

mortalities in the test group is determined by summing the discrete probabilities

associated with all mortalities from zero to Np,', where Np/ = control-adjusted

number of mortalities. Np,' may be noninteger, in which case, the next highest

integer should be used. Discrete binomial probabilities can be obtained from

statistical tables or from the following equation (Zar, 1984):

P(X) = [N! / X!(N - X)!]PoX(l - pJ(N-X) (general equation)

or

P(X) = [N! / X!(N - X)!]O.SN

where: P(X) = discrete probability of X mortalities in test group

N = number of rats in test group

X = number of mortalities in test group

Po= 0.5 = proportion of mortalities at the LDSO

If the resultant cumulative binomial probability is less than or equal to alpha (

0.10), then H, is rejected and it is concluded that the waste does not designate as

a regulated waste. Discrete and cumulative binomial probabilities for Po= 0.5

and several sample sizes are presented in Table 1 on page B-6.

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c Table 1.

Discrete (D) and Cumulative (C Binomial Probabilities for Pn= 0.5 and N=10, 15,20, and 25.

X N = 10 N = 15 N=20 N=25

0 C 0 C 0 C 0 C

0 0.0010 0.0010 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000

1 0.0098 0.0108 0.0005 0.0005 0.0000 0.0000 0.0000 0.0000

2 0.0439 0.0547 0.0032 0.0037 0.0002 0.0002 0.0000 0.0000

3 0.1172 0.1719 0.0139 0.0176 0.0011 0.0013 0.0001 0.0001

4 0.2051 0.3770 0.0417 0.0593 0.0046 0.0059 0.0004 0.0005

5 0.2461 0.6231 0.0916 0.1509 0.0148 0.0207 0.0016 0.0021

6 0.2051 0.8282 0.1527 0.3036 0.0370 0.0577 0.0053 0.0074

7 0.1172 0.9454 0.1964 0.5000 0.0739 0.1316 0.0143 0.0217

8 0.0439 0.9893 0.1964 0.6964 0.1201 0.2517 0.0322 0.0539

9 0.0098 0.9991 0.1527 0.8491 0.1602 0.4119 0.0609 0.1148

10 0.0010 1.0000 0.0916 0.9407 0.1762 0.5881 0.0974 0.2122

11 0.0417 0.9824 0.1602 0.7483 0.1328 0.3450

12 0.0139 .0.9963 0.1201 0.8684 0.1550 0.5000

13 0.0032 0.9995 0.0739 0.9423 0.1550 0.6550

14 0.0005 1.0000 0.0370 0.9793 0.1328 0.7878

15 0.0000 1.0000 0.0148 0.9941 0.0974 0.8852

16 0.0046 0.9987 0.0609 0.9461

17 0.0011 0.9998 0.0322 0.9783

18 0.0002 1.0000 0.0143 0.9926

19 0.0000 1.0000 0.0053 0.9979 - 20 0.0000 1.0000 0.0016 0.9995/

21 0.0004 0.9999

22 0.0001 1.0000

23 0.0000 1.0000

24 0.0000 1.0000

25 0.0000 1.0000

Example 1

Ten rats each in the control and test group (N =10) are orally dosed at 0 and

5000 mg/kg of waste, respectively, according to the bioassay procedure in order

to determine if the waste sample designates as a dangerous waste. After 14

days, 3 rats have died in the test group (PT = 0.300) and zero control mortalities

(Pc = 0) are observed. Therefore, PT= PTa and Np,' = (10)(0.300) = 3. For Po = 0.5

and N =10, discrete binomial probabilities for 0, I, 2, and 3 deaths are 0.0010,

0.0098,0.0439, and 0.1172, respectively (Table 1). Summing these probabilities, it

is apparent that the cumulative probability for deaths is greater than the

established alpha level of 0.10 (i.e., 0.1719 > 0.10). Therefore, H, is not rejected

and the waste designates as a dangerous waste.

Example 2

Twenty rats each in the control and test group (N =20) are orally dosed at 0 and

5000 mg/kg of waste, respectively, according to the bioassay procedure in order (). -- ...._-----_._---------------------------------

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to determine if the waste sample designates as a dangerous waste. After 14

days, 7 rats have died in the test group (PT =0.350) and 2 control mortalities (Pc = 0.100) are observed. Therefore, PTa =0.278 and Np," =(20)(0.278) =5.56. Np,' is

then rounded to 6 deaths, as described above. For Po =0.5 and N =20, discrete

binomial probabilities for 0, I, 2, 3, 4, 5, and 6 deaths are 0, 0, 0.0002, 0.0011,

0.0046,0.0148, and 0.0370, respectively (Table 1). Summing these probabilities, it

is apparent that the cumulative probability for :s; 6 deaths is less than the

established alpha level of 0.10 (i.e., 0.0577 < 0.10). Therefore, H, is rejected and

the waste does not designate as a dangerous waste.

Calculation (N • 30)

1) Control correction

The bioassay is invalid if control mortalities exceed 10 percent (EPA, 993). The

control correction is performed with Abbott's formula as described above.

2) Normal approximation

Based on the mean and standard deviation for a binomial distribution, the

following formula has been adapted from Zar (1984) to evaluate how p/ differs

from Po =0.5 in terms of a normal distribution:

z =(PTa - pJ / [PTa(l - p/)/N]O,S (general equation)

or

z= (p/ - 0.5) / [PTa(l - p/)/Nt.5

where: Z =normal deviate

Po = 0.5 = proportion of mortalities at the LD50

N = number of rats in test group

The calculated Z test statistic is compared to a one-tailed critical Z value at an'

alpha level of 0.10 (i.e., Z =-1.28). When the calculated Z value is :s; -1.28, H, is

rejected, and it is concluded that the waste does not designate as a regulated

waste.

Example 3

Fifty rats per control and test groups (N = 50) are orally dosed at 0 and 5000

mg/kg of waste, respectively, according to the bioassay procedure in order to

determine if the waste sample designates as a dangerous waste. After 14 days,

20 rats have died in the test group (PT =0.400) and 4 control mortalities (Pc = 0.080) are observed. From these data, PTa =0.348 and Z =-2.26. Because the

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calculated Z statistic (-2.26) is less than the critical Z value (-1.28), Ho is rejected C

and the waste does not designate as dangerous waste.

Data Reporting

The report must contain the following information:

• study initiation and termination dates;

• description of testing facility;

• source, genetic strain, and sex of control and test animals;

• description of sample preparation, including type of vehicle used and test

solution concentration;

• dose of test solution and volumes of vehicle and test solution administered to

respective control and test groups;

• animal body weights at test initiation, day 7, and at death in both control and

test group;

• mortalities and signs of acute toxicity immediately after dosing, at one and

four hours after dosing, and daily thereafter for 14 consecutive days in both

control and test groups;

• photoperiod and daily temperature of testing room;

• gross necropsy results; 0 • statistical analysis to evaluate whether the waste sample LD50 is either greater

than or less than or equal to the administered dose which corresponds to an

established regulatory threshold; and,

• statement of whether the waste sample passes or fails the bioassay for

designating as a dangerous waste, special waste, or extremely hazardous

waste, according to the Dangerous Waste Regulations, Chapter 173-303 WAC.

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References

ASTM. 1994a. Standard test method for estimating acute oral toxicity in rats.

Method E 1163-90. 1994 Annual Book of ASTM Standards, Volume 11.04,

American Society for Testing and Materials (ASTM), Philadelphia.

ASTM. 1994b. Standard test method for conducting a 90-day oral toxicity study

in rats. Method E 1372-90. 1994 Annual Book of ASTM Standards, Volume

11.04, American Society for Testing and Materials (ASTM), Philadelphia.

CFR. 1993. Health effects testing guidelines: Acute oral toxicity. Environmental

Protection Agency (EPA), Title 40, Part 798.1175. Code of Federal Regulations

(CFR),Washington, DC

Dixon, WJ. 1965. The up-and-down method for small samples. J. Amer. Stat.

Assoc. 60:967-978.

EPA. 1986. Test methods for evaluating solid waste. 3rd ed., SW-846, Volume II,

Part III, Chapter 9. Office of Solid Waste and Emergency Response, Washington,

DC

EPA. 1991. Alpha 2u-globulin: Association with chemically induced renal

toxicity and neoplasia in the male rat. EPA/625/3-91/019F. Risk Assessment

Forum, Environmental Protection Agency (EPA), Washington, DC

EPA. 1993. Methods for measuring the acute toxicity of effluents and receiving

waters to freshwater and marine organisms. 4th ed., EPA/600/4-90/027F. Ed. by

CI Weber, Environmental Monitoring Systems Laboratory, Office of Research

and Development, EPA, Cincinnati, OH.

Finney, DJ. 1971. Probit Analysis. 3rd ed., Cambridge University Press, London.

Zar, JH. 1984. Biostatistical Analysis. 2nd ed., Prentice Hall, Englewood Cliffs,

NJ·

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