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

Revised June 2009 

Publication no. 80-12 
Publication and Contact Information 

This guidance is available on the Department of Ecology’s website at 

www.ecy.wa.gov/biblio/8012.html 

For more information contact: 

Hazardous Waste and Toxics Reduction Program 

P.O. Box 47600 

Olympia, WA 98504-7600 

Phone: 360-407-6700 

Washington State Department of Ecology - www.ecy.wa.gov 

o Headquarters, Olympia 360-407-6000 

o Northwest Regional Office, Bellevue 425-649-7000 

o Southwest Regional Office, Olympia 360-407-6300 

o Central Regional Office, Yakima 509-575-2490 

o Eastern Regional Office, Spokane 509-329-3400 

To ask about the availability of this document in a format for the visually impaired, call the Hazardous 

Waste and Toxics Reduction Program at 360-407-6700. Persons with hearing loss can call 711 for 

Washington Relay Service. Persons with a speech disability can call 877-833-6341.
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

Hazardous Waste and Toxics Reduction Program 

Washington State Department of Ecology 

Olympia, Washington 
This page is purposely left blank
 Table of Contents 

Part A: Static Acute Fish Toxicity Test 

Introduction ................................................................................................ 1 

 Health and Safety ...................................................................................................... 1 

Methods and Materials .............................................................................. 3 

 Holding Facilities ........................................................................................................ 3 

 Test Chambers .......................................................................................................... 4 

 Cleaning and Disinfection ..................................................................................... 4 

 Dilution Water ....................................................................................................... 5 

 Test Organisms .......................................................................................................... 6 

 Care and Handling ................................................................................................ 7 

 Holding ................................................................................................................. 7 

 Stress Indicators ................................................................................................... 7 

 Sample Preparation ................................................................................................... 8 

 Sample Requirements .......................................................................................... 8 

 Particle Size Reduction ........................................................................................ 8 

 Rotary Agitation Method ....................................................................................... 8 

Test Procedure ........................................................................................... 9 

 Replicates and Control ............................................................................................... 9 

 Test Temperature ...................................................................................................... 9 

 pH ............................................................................................................................. 9 

 Dissolved Oxygen ...................................................................................................... 9 

 Loading .................................................................................................................... 10 

 Feeding .................................................................................................................... 10 

 Duration and Acceptability ....................................................................................... 10 

 Test Observations and Measurements .................................................................... 10 

 Criteria for Death ...................................................................................................... 11 

 Termination......................................................................................................... 11 

 Data Analysis ...................................................................................................... 11 

Quality Assurance / Quality Control ....................................................... 11 

 Procedures .............................................................................................................. 11 

 Reference Toxicants ................................................................................................ 12 

Calculation of Results ............................................................................. 12 

 Approach ................................................................................................................. 13 

 Hypothesis ............................................................................................................... 13 

 Calculation ............................................................................................................... 13

 Sample size ........................................................................................................ 13 
 Mortalities ........................................................................................................... 13 

 F Test ................................................................................................................. 14 

 Equal variance t test ........................................................................................... 14 

 Modified t test ..................................................................................................... 14 

 Critical F and t values ......................................................................................... 15 

Data Reporting ......................................................................................... 17 

Part B: Acute Oral Rat Toxicity Test 

Introduction .............................................................................................. 19 

Methods .................................................................................................... 19 

 Sample Collection and Preparation .......................................................................... 19 

 Test Animals ............................................................................................................ 19 

 Pretest Conditioning ................................................................................................. 20 

 Procedure ................................................................................................................ 20 

Calculation of Results ............................................................................. 20 

 Approach ................................................................................................................. 21 

 Hypothesis ............................................................................................................... 21 

 Calculation (N < 30) ................................................................................................. 21 

 Calculation (N = 30) ................................................................................................. 23 

Data Reporting ......................................................................................... 24 

References ................................................................................................ 24 

Appendices 

A. Neutralized Fish Toxicity Test Procedure ........................................................... 27 

B. Good Laboratory Procedure Standards .............................................................. 29 

C. Water Quality ...................................................................................................... 33 

D. Dissolved Oxygen Table ..................................................................................... 34 

E. Recommended Fish Loading .............................................................................. 35 

F. Sample Data Sheet ............................................................................................ 36 

G. Recommended Test Conditions and Procedures for Fish Bioassay ................... 39 

Glossary .................................................................................................... 41 

Terminology.............................................................................................. 43 
List of Figures and Tables 

 

Figures 

Figure 1. Test Fish Standards...........................................................................................................6 

Tables 

Table 1. Preparation of Dilution Water Using Reagent Grade Chemicals ......................................5 

Table 2. Critical F Values With Two-Tailed a=0.05 .....................................................................15 

Table 3. Critical T Values With One-Tailed a=0.10 ......................................................................15 

Table 4. Test Results ......................................................................................................................15 

Table 5. Summary for Equal Variance T Test ...............................................................................16 

Table 6. Test Results ......................................................................................................................16 

Table 7. Summary for Modified T Test .........................................................................................17 

Table 8. Water Quality Parameters ................................................................................................33 

Table 9. Approximate Saturation and Concentration Values of Dissolved Oxygen in Freshwater 

at 12°C .............................................................................................................................34 

Table 10. Recommended Fish Loadings ........................................................................................35 

Table 11. Discrete and Cumulative Binomial Probabilities for Po=0.5 .........................................22 

1 

Part A: Method 80-12 

Static 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. 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. If additional toxicity information is needed to further characterize a waste, a 

toxicity identification evaluation (TIE) procedure may be useful (EPA, 1991a). Petitions must be 

submitted per WAC 173-303-910. 

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: 

• Appointment of a laboratory health and safety officer with the responsibility and authority to 

develop and maintain a safety program. 
2 

• Preparation of a formal, written, health and safety plan, which is provided to each laboratory 

staff member. 

• Ongoing training program on laboratory safety. 

• 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 poorly known, consider them 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 all 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 all “wet” labs where electrical equipment is used. 

• Staff training in basic first aid and cardio-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 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. 
3 

• 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 (2002), 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 7 days 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 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 16 hours of light/8 hours of darkness 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/m2/s 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. 
4 

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 surface-area-to-volume ratio). 

Test chambers may be constructed of 3-mm (0.125 in), double strength, or 6-mm (0.25 in) tempered 

plate glass held together with clear silicone adhesive. Silicone adhesive absorbs some 

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 adhesive inside the containers 

should be removed. Stainless steel (No. 304 or No. 316) can be used in the construction of test 

chambers, but should be of welded, not soldered, construction. 

Commercially 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 ammonia-based 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. 

• 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. 

• 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 mL of 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. 

 

For disinfection of equipment such as fish tanks, prepare a 50 mg/L solution of titrateable iodine by 

diluting a commercially available product. The equipment should be immersed in or washed with 
5 

this solution for at least 10 minutes followed by copious rinsing with clean water. Iodophors and 

hypo-chlorites are acutely toxic to most aquatic organisms. Hypochlorite can be removed by 

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

sodium sulfite. After a tank is washed with hypochlorite and rinsed, air-dry the equipment overnight 

to allow dissipation of residual chlorine. 

Dilution Water 

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 units to produce consistent quality dilution water. 

Ideally, non-chlorinated 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 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 CaCO3. 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 Chemicalsa

Water Type 

NaHCO3

mg/Lb

CaSO4•2H2O 

mg/Lb

MgSO4

mg/Lb

KCl 

mg/Lb

pHc Total 

Resulting 

Hardnessd

Total 

Resulting 

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 

a

 Adapted from EPA (2002) 

b

 Add reagent grade chemicals to distilled or deionized water. 

c

 Approximate equilibrium pH after aerating 24 hours. 

d

 Expressed as mg/L CaCO3. 
6 

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 mykiss (formerly Salmo gairdneri); 

and brook trout, Salvelinus fontinalis. The test temperature for all species is 12 + 1 oC. 

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. 

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

All test fish should be obtained from a hatchery that has been certified disease-free 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 or on the observations and 

judgment of a knowledgeable individual. 

• Treating all fish that arrive on-site using treatments commonly used by sources (hatcheries) 

which produce certified disease-free fish. 

FIGURE 1. 
7 

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. 

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 flow-through system with a minimum flow rate of five volume 

exchanges per day (ASTM, 2006a). When possible, hold fish in dilution water and as close to test 

temperature as possible. During long holding periods, however, it is generally 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 reacclimatize for 7 days. The following 

physical characteristics indicate stress: 

• Darkened color (normal is silver/gray) • Flashing 

• Emaciation • Flipping 

• Erratic swimming • Fungus/Fin Rot 

• Excessive mucus production • Gasping at surface 

• External parasites • Hemorrhaging 

• Not eating • Hyperventilation 
8 

Sample Preparation 

Sample Reporting Requirements 

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. 

Sample analysis must occur with 45 days of sample collection. 

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 + 2o

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 cm2

, 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 is it recommended. For 

materials that do not obviously meet the criteria, sample-specific methods would need to be 

developed and employed to measure the surface area. Such methodology is currently not 

available. 

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 + 2o

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 lying 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 with deionized water, at a minimum, are 

acceptable methods. 
9 

Test Procedure 

Replicates and Control 

Use a minimum of ten fish in each of three replicate test tanks exposed to each treatment. 

Additional fish and replicates may be used (e.g., 15 fish in each of four replicates). Replicates 

must be true replicates with no water connections between the replicate test chambers. Randomize 

the replicate test chambers and impartially distribute 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. 

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°C. 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. 

The 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 
10 

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 D 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 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 

Loading 

Loading in test chambers should not exceed 0.8 g/L. The g/L 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. 

• Concentrations of metabolic products does not increase above acceptable levels. 

• 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. 

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. 

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. 
11 

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 are 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. 

Quality Assurance / Quality Control 

Procedures 

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. 
12 

• Laboratory services. 

• Use and care of instruments, glassware, and chemicals. 

• Data management. 

• 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. 

• 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. Reference toxicant testing is required for each new batch of fish 

regardless of whether a test has been run that month on another batch of fish. More frequent testing with a reference toxicant is appropriate when situations occur which may affect test results. 

For example, a change in dilution or holding water conditions, new equipment or piping in the 

laboratory, or a change of analysts, all indicate the need for additional use of a reference toxicant. 

Various compounds are commonly used as reference toxicants and include phenol, sodium 

chloride (NaCl), potassium chloride (KCl), zinc chloride (ZnCl2), and zinc sulfate (ZnSO4). This 

is not an all inclusive list by any means. 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. 
13 

Approach 

The statistical analysis is based on methodology presented by Zar (1984), EPA (2002), 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 if sample 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, 2002). 

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

test samples (Zar, 1984; EPA, 2002). 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. 

Thus, the appropriate t test is used to compare the difference between test and control mortality 

proportions against a constant, po = 0.5, corresponding to the LC50. Up to ten percent mortality in 

the control sample is allowed. A one-tailed t test is used, since the analysis determines only if the 

sample LC50 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). 

Hypothesis 

In terms of concentration, the one-tailed null hypothesis (Ho) 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 - pCm) ≥ po, where pTm and pCm 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 Ho 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 

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 ten (n ≥10). In addition, the number of fish per 

replicate tank should not exceed a biomass loading density of 0.8 g/L. 

2. Mortalities: Mortalities are expressed as proportions:

pC = proportion of mortalities in control replicate. 

pT = proportion of mortalities in test replicate. 
14 

The bioassay is invalid if overall control mortalities exceed 10 percent (EPA, 2002). 

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, 2002): 

F = sC

2

/sT

2

 or F = sT

2

/sC

2

, whichever is larger. 

where: F = F statistic 

 sC

2

 = variance for the control group. 

 sT

2

 = 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 is zero, the 

variances are considered 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) / (2sp

2

 /N)0.5 (general equation) or 

t = (pTm - pCm - 0.5) / [(sT

2

 + sC

2

)/N]0.5 

where: t = t statistic 

pTm = mean pT

pCm = mean pC

po = 0.5 = proportion of mortalities at LC50

sp

2

 = pooled variance = (sT

2

 + sC

2

)/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, Ho is rejected, and it is concluded that the waste does not 

designate as a regulated waste. Note that in the case where (sT

2

 + sC

2

) = 0, Ho is rejected when 

(pTm - pCm) < 0.5 and Ho 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): 

df = (N - 1)(sC

2

 + sT

2

)

2

 / [(sC

2

)

2

 + (sT

2

)

2

] 

If the computed degrees of freedom are non-integer, use the next smaller integer. Note that if 

either sC

2

 = 0 or sT

2

 = 0, then df = N - 1. The calculated t statistic is compared to a critical t value 
15 

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 section numbers 3-5 under 'Calculation'. 

Table 2. Critical F Values With Two-Tailed α = 0.05 

Denominator df Numerator 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 9.36 

5 8.43 7.76 7.39 7.15 

Table 3. Critical T Values With One-Tailed α = 0.10 

df t

1 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 Critical t values are negative, since the interest lies only in whether (pT - 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 ten 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. 

Table 4. Test Results 

Replicate Dilution Water Control 

(n=10) 

10 mg/L Sample (n=10) 100 mg/L Sample 

(n=10) 

 (N=3) Number 

Dead 

Proportion 

Dead (pC) 

Number 

Dead 

Proportion 

Dead (pT) 

Number 

Dead 

Proportion 

Dead (pT) 

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 (pm) 0.067 0.133 0.500 

Variance (s2)

1 0.0033 0.0033 0.0100 1 Variance equation is s2

=Σ(p-pm)

2

/N-1 where p = pC or pT
16 

After 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 & 

denominator each = (N - 1) 

2, 2 2, 2 

Critical F (see Table 12) 39.0 39.0 

Calculated t1 statistic -9.25 -1.01 

Critical t df2 = [2(N-1)] 4 4 

Critical t (see Table 23) -1.53 -1.53 

Does waste designate? NO YES 

Waste Code not applicable WT02 

1 10 mg/L calculated t = (0.133-0.067-0.5)/[(0.0033+0.0033)/3]0.5 & 100 mg/L calculated t = (0.5-0.067-0.5)/[(0.0033+0.0100)/3]0.5

2

 2(N-1) is only used in the equal 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), Ho 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), Ho 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. 

Table 6. Test Results 

Replicate Dilution Water Control 

(n=15) 

10 mg/L Sample (n=15) 100 mg/L Sample 

(n=15) 

(N=4) Number 

Dead 

Proportion 

Dead (pC) 

Number 

Dead 

Proportion 

Dead (pT) 

Number 

Dead 

Proportion 

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

)

1

 0 0.0278 0.0130 

1 Variance equation is s2

=Σ(p-pm)

2

/N-1 where p = pC or pT
17 

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 2 and 3, 

respectively. 

Table 7. Summary for Modified T Test 

10 mg/L 100 mg/L 

Calculated F statistic The variances are considered statistically unequal because the control 

variance is zero. Therefore, the modified t test is used. 

Calculated t1 statistic -3.39 0.88 

Critical t df2 = (N-1) 3 3 

Critical t (see Table 2 3) -1.64 -1.64 

Does waste designate? NO YES 

Waste code not applicable WT02 

1

 10 mg/L calculated t = (0.217-0-0.5)/[(0+0.0278)/4]0.5 & 100 mg/L calculated t = (0.550 00.5)/[(0+0.0130)/4]0.5

2

 (N - 1) is used in the modified t test when either sC

2

 = 0 or sT

2

 = 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), Ho 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), Ho 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. 

• Detailed description of waste tested, its source, lot number, date of sample collection, known 

composition, known physical and chemical properties. 

• Dilution water source, chemical characteristics, description of pretreatment, any additives. 

• 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 LC50. 
18 

• 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. 

• Anything unusual about the test, any deviations from procedures, and any other relevant 

information. 
19 

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 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., corn 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 weight, except when 

an aqueous solution is used, where 2 mL/100 g is acceptable (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, 1991b). 
20 

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 seven 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 mg/kg 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/100 g body weight, except when an 

aqueous solution is used, where 2 mL/100 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. 

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 14 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 sign 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). 
21 

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 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). 

Hypothesis 

In terms of concentration, the one-tailed null hypothesis (Ho) 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: pT

a ≥po, 

where pT

a

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

consistent with statistical methodology outlined by EPA (1986). Rejecting Ho 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, 2002). Corrections for up to 10 

percent mortality in the control are applied with Abbott's formula (Finney, 1971; EPA, 2002): 

 pT

a = (pT - pC) / (1 - pC) 

 where: pT

a = control-adjusted proportion of mortalities in test group 

 pT = observed proportion of mortalities in test group 

 pC = observed proportion of mortalities in control group 

Note that pT

a = pT when pC = 0. If pC ≤ 0.10 and pC > pT, then pTa = 0 as a result of data smoothing (EPA, 

2002). That is, pT and pC are each set equal to their average, (pT + pC) / 2, so that pT

a = 0. 

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 NpT

a

, 

where NpT

a

 = control-adjusted number of mortalities. NpT

a

 may be non-integer, in which case, the next 
22 

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)!]po

X(1 - po)(N - X) (general equation) 

 or 

 P(X) = [N! / X!(N - X)!]0.5N

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 LD50 

If the resultant cumulative binomial probability is less than or equal to alpha (≤ 0.10), then Ho 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 11 below. 

Table 11. Discrete and Cumulative Binomial Probabilities for po = 0.5 

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

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

D C D C D C D 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 to determine if the waste sample designates as a 
23 

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 = pT

a

 and NpT

a

 = (10)(0.300) = 3. For po = 0.5 and N = 

10, discrete binomial probabilities for 0, 1, 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 ≤

3 deaths is greater than the established alpha level of 0.10 (i.e., 0.1719 > 0.10). Therefore, Ho 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 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, pT

a

 = 0.278 and NpT

a

 = (20)(0.278) = 5.56. NpT

a

 is 

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

for 0, 1, 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 ≤ 6 deaths is less than 

the established alpha level of 0.10 (i.e., 0.0577 < 0.10). Therefore, Ho 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 pT

a

 differs from po = 0.5 in terms of a normal distribution: 

 

 Z = (pT

a

 - po) / [pT

a

(1 - pT

a

)/N]0.5 (general equation) 

 or 

 Z= (pT

a

 - 0.5) / [pT

a

(1 - pT

a

)/N]0.5

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 ≤ -1.28, Ho 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, pT

a

 = 0.348 and Z = -2.26. Because the 

calculated Z statistic (-2.26) is less than the critical Z value (-1.28), Ho is rejected and the waste does not 

designate as dangerous waste. 
24 

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. 

• 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. 

• Statement of whether the waste sample passes or fails the bioassay and designates as a dangerous 

waste, special waste, or extremely hazardous waste, according to the Dangerous Waste Regulations, 

Chapter 173-303 WAC. 

References

ASTM. 2006a . Standard guide for conducting early life-stage toxicity tests with fishes: E1241-05. In: 

2006 ASTM Standards on Aquatic Toxicology and Hazard Evaluation, ASTM, Philadelphia, 

Pennsylvania. 

ASTM. 2006b. Standard test method for estimating acute oral toxicity in rats. Method E 1163-98 

(Reapproved 2002). 2006 Annual Book of ASTM Standards, Volume 11.045, American Society for 

Testing and Materials (ASTM), Philadelphia. 

ASTM. 2006c. Standard test method for conducting a 90-day oral toxicity study in rats. Method E 

1372-95 (Reapproved 2003). 2006 Annual Book of ASTM Standards, Volume 11.045, 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. 
25 

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

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

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. 1991a. Methods for aquatic toxicity identification evaluation. Phase I toxicity characterizations, 

2nd ed. EPA/600/6-91/003. Environmental Research Laboratory, Duluth, MN. 

EPA. 1991b [[[SEE TABLE 8 footnote]]] 

EPA. 1991b. 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 2002. Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and 

marine organisms. 5th ed. EPA-821-R-02-012 Environmental Monitoring Systems Laboratory, Office of 

Research and Development, EPA, Cincinnati, OH. 

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

studies of toxicity. Environ. Toxicol. Chem. 14(7):1247-1256. 

Federal Register Vol. 48 No. 230, November 29, 1983, Good Laboratory Practice Standards, pp. 53946 ff. 

Finney, DJ. 1971. Probit Analysis. 3rd ed., Cambridge University Press, London. 

Hurlbert, SH. 1984. Pseudoreplication and the design of ecological field experiments. Ecol. 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. U.S. 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., Bio-Engineering in Fisheries. Special Publication by American Fisheries Society, Washington, D.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. 

Zar, JH. 1984. Biostatistical Analysis. 2nd ed., Prentice Ha 

27 

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 hydrochloric acid (HCl) or sodium 

hydroxide (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 tanks and control tanks 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 tanks. 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 tanks and control tanks. 

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

9. Maintain temperature and dissolved oxygen as described in the section on “Test Procedure.” 

10. Terminate the test after 96 hours. 

11. Complete the data reporting sheet. 
28 

Recording the Data 

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

• Neutralizer (i.e., NaOH or HCl). 

• Normality of neutralizer. 

• Volume of neutralizer. 

• Indicate if pH readjustment was necessary 30 minutes after initial pH adjustment. 
29 

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 job 

description for each individual engaged in or supervising the conduct of a study. 

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

according to the protocol. 

Personnel shall take necessary personal sanitation and health precautions designed to avoid 

contamination of test and control substances and test systems. 

Personnel engaged in a study shall wear clothing appropriate for the duties they perform. Such 

clothing shall be changed as often as necessary to prevent microbiological, radiological, or chemical contamination of test systems and test and control substances. 

Any individual found at any time to have an illness that may 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 effect 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 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. 
30 

Unforeseen circumstances that may affect the quality and integrity of the study are noted when 

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 title and statement of the purpose of the study. 

• 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. 

• The route of administration and the reason for its choice. 

• Each dosage level, expressed in milligrams per kilogram of body weight or other appropriate 

units, of the test or control substance to be administered and the method and frequency of 

administration. 

• 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. 
31 

• 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 therefore 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. 

Maintenance and Calibration of Equipment

Equipment shall be adequately inspected, cleaned, and maintained. Equipment used for the 

generation, measurement, or assessment of data shall be adequately tested, calibrated, or 

standardized. 

Written standard operating procedures (SOPs) shall set forth in sufficient detail the methods, 

materials, and schedules to be used in the routine inspection, cleaning, maintenance, testing, 

calibration, or standardization of equipment, and shall specify remedial action to be taken in the 

event of failure or malfunction of equipment. The written SOPs 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, 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. 
32 

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. 

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. 

• Handling of animals found moribund or dead during study. 

• Necrospy 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 SOPs 

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. 

• Historical file of SOPs, and all revisions thereof, including the dates of such revisions, shall 

be maintained. 

Reagents and Solutions 

All reagents in the laboratory areas shall be labeled to indicate identity, titer or concentration, 

storage requirements, and expiration date. Solutions prepared in the laboratory shall be labeled to 

indicate identify, concentration, date of preparation, and preparer’s name or initials. Deteriorated 

or outdated reagents and solutions shall not be 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-821-R-02-012 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 Parametersa 

PARAMETER RECOMMENDED 

pH 7.2 - 7.8 

Alkalinity 30 - 70 mg/L as CaCO3

Ammonia (NH3) 0.02 mg/L unionized 

Cadmium (Cd) 100 ng/L 

Chloride (Cl) 2 - 3 mg/L 

Copper (Cu) less than 5 μg/L 

Hardness 40-100 mg/L as CaCO3

Lead (Pb) 0.03 mg/L or less 

Mercury (Hg) organic or inorganic 100 ng/L 

Nickel (Ni) 88 μg/L or less 

Nitrogen (N) maximum total gas pressure 110 percent of saturation 

Nitrite (NO2) 100 μg/L 

Oxygen (O2) at least 8 mg/L at 12oC 

Ozone (O3) Residual 3 μg/L 

PCB as Aroclor 1254 50 ng/L or less 

Total Suspended and Settleable Solids 80 mg/L or less 

Zinc (Zn) less than 65 μg/L 

Total organochlorine pesticides 50 ng/L or less 

 aAdapted from Wedemeyer et al. (1981) and EPA (2002). 
34 

Appendix D 

Table 9. Approximate Saturation1 and Concentration Values of Dissolved Oxygen 

in Freshwater at 12°C 

Altitude (ft) DO (mg/L) a

100% 

Saturation 

DO (mg/L) at

80% 

Saturation

DO (mg/L) a

60% 

Saturation

Altitude 

(ft) 

DO (mg/L) at

100% 

Saturation

DO (mg/L) a

80% 

Saturation 

DO (mg/L) 

at 60% 

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 1 Based on Pillard (1996). 
35 

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 salmonids used in toxicity tests. 

Table 10. Recommended Fish Loadingsa

SPECIES WATER 

TEMPERATURE 

POUNDS FISH PER GPM 

o

C oF 1000b 500b 100b

Coho Salmon 3 

 9 

14 

17 

38 

48 

58 

63 

3.5 

2.7 

2.2 

---- 

5.0 

4.0 

3.0 

2.0 

8.0 

6.0 

4.5 

3.5 

Fall/Spring 

Chinook Salmon 

 3 

 9 

14 

17 

38 

48 

58 

63 

3.0 

2.5 

2.0 

---- 

4.0 

3.0 

2.2 

1.2 

6.0 

5.0 

3.5 

3.0 a Adapted from Wedemeyer & Wood (1977) 

 b Fish size (number of fish per pound)
36 

Appendix F

Sample Data Sheet 

The Data Sheet for Static Basic Acute Fish Toxicity Test is printed on page 37. Please 

copy this form to report test data. 
37 

  Industry/Toxicant Beginning Date Address Ending Date Collector Test Organism Date Sample Collected 

 Required Test Temperature 

Range 

Laboratory 

Reference 

Number 

Test 

Contai

ner 

No. 

Conc

. 

(mg/l) 

Number of Cumulative 

Deaths 

Dissolved Oxygen (mg/l) pH 25 C Temperature (C) 

 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 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 Department of Ecology 

DATA SHEET FOR STATIC BASIC ACUTE FISH TOXICITY TEST 
38 

GENERAL PROCEDURE FOR STATIC BASIC ACUTE FISH TOXICITY TEST Data Verified By ________________Date _________________ 
39 

Appendix G 

Recommended Test Conditions and Procedures for Fish Bioassay 

Test type • Static non-renewal 

Duration • 96 hours 

Light quality • Ambient laboratory illumination 

Light intensity • 10-20 uE/m2

/s (50-100 ft-c) 

Photoperiod • 16 hours light, 8 hours darkness 

Test chamber size • 3.8 L or 1 gal minimum 

Minimum depth • 15 cm test solution 

Test solution renewal • none 

Source of fish • hatchery stock, free of known diseases 

Control water • moderately hard water; see Table 1 

Fish age • 30 - 90 days 

Temperature • 12 oC + 1o

C 

Oxygen/aeration 

• None unless DO<80 percent saturation at altitude; 

ideal DO is 80 to 100 percent saturation 

pH • no pH adjustment 

Feeding • not required 

Cleaning • not required 

Number of fish per test chamber • minimum 10 

Number of replicate chamber per concentration • minimum 3 

Number of fish per concentration • minimum 30 

Observations 

• fish mortality, appearance, and behavior at 24, 48, 

72, and 96 hours minimum 

Measurements 

• temperature, DO, pH, in each tank daily; conductivity 

test 

Endpoints • mortality 

Test acceptability • 90 percent survival or greater in controls 

Reference toxicant 

• CuSo4, CdC12, KCl, NaCl, or ZnSO4 (not inclusive); 

evaluate at least once/month and with each new 

batch of fish 

Solvents 

• Extraction water is the control water used in test 

controls 
40 

Concentration 

• 100 mg/L to determine dangerous waste; 10 mg/L to 

determine extremely hazardous waste 

Control water • Moderately hard water 

Sampling date 

• Date of sample collection; tests done on sample 

collected more than 45 days prior to extraction must 

be noted in the report 

Transport and storage 

• Extract within seven days of sample receipt; store in 

dark at 4°C; test within 8 days of sample collection 

Extraction 

• Extract using rotary agitation device (30 + 2 rpm) for 

18 hours + 2 hours at 23°C + 2°C 
41 

Glossary

o

C degree(s) Celsius 

CaCO3 calcium carbonate 

CaSO4 calcium sulfate 

CaSO4•2H2O calcium sulfate dihydrate (gypsum) 

cm centimeter 

CO2 carbon dioxide 

COD chemical oxygen demand 

d day 

DO dissolved oxygen 

DW dangerous waste 

EHW extremely hazardous waste 

o

F degree(s) Fahrenheit 

g grams 

gal gallons 

gpm gallons per minute 

h hour 

HCL hydrochloric acid 

H2O water 

in inch(es) 

KCL potassium chloride 

L liter 

m meter 

mg milligram 

mg/L milligram per liter 

MgSO4 magnesium sulfate 

min minute(s) 

mL milliliter 

mg/kg milligram per kilogram 

NaHCO3 sodium bicarbonate 

NaOH sodium hydroxide 

ng/L nanograms per liter 

NH3 ammonia 

QA/QC quality assurance/quality control 

qt quart 

rpm rotations per minute 

TOC total organic carbon 
42 

WAC Washington Administrative Code 

> greater than 

< less than 

> greater than or equal to 

< less than or equal to 
43 

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

Acclimatize...to become physiologically adapted to a particular level of one or more environmental 

variables, such as temperature or water chemistry. 

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

short period of exposure to a test material, usually constituting a non-substantial portion of their life span. 

Conductivity...is a numerical expression of an aqueous solution’s ability to carry an electrical current. 

This ability depends on the concentrations of ions in solution, their valence and mobility, and on the 

solution temperature. Conductivity is normally reported in the Système International d’unités (SI) unit 

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

Control...a treatment that is essentially free of contaminants and is used routinely to assess the 

acceptability of a test. In Salmonid tests, the control must duplicate all conditions of the exposure 

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

Dangerous waste ...those solid wastes designated per WAC 173-303-070 through 173-303-100 as 

dangerous, extremely hazardous, or mixed waste. 

Deionized water...water passed through resin columns to remove ions. 

Distilled water...water passed through a distillation apparatus of borosilicate glass or other material, to 

remove impurities. 

Extremely hazardous waste...those dangerous and mixed wastes designated per WAC 173-303-100 as 

extremely hazardous. Also, those dangerous wastes showing statistically significant mortality in test 

concentrations of 10 mg/L. 

Flow-through...continuous renewal of holding water or test solution. 

Hardness...a measure of the concentration of calcium and magnesium ions in water, expressed as mg/L 

calcium carbonate (CaCO3) or equivalent. 
44 

LC50...the median lethal concentration (i.e., the concentration of material in water 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 which 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, such as no response from gentle prodding. 

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

dissolved oxygen (DO) or toxicant below acceptable levels, the accumulation of injurious concentrations 

of metabolic waste products, or stress induced by crowding. 

Lux...a unit of illumination equal to 1 lumen per square meter. One lux = equals 0.0929 foot-candles 

and one foot-candle = equals 10.76 lux. 

Observations...routine checks of biological and water quality variables, which may include fish 

survival and behavior, water temperature, dissolved oxygen, pH, hardness and conductivity. 

pH...the negative logarithm of the hydrogen ions activity in gram equivalents per liter. The pH value 

expresses the degree of intensity of both acidic and alkaline solutions on a scale from 0 to 14, with 7 

representing neutrality. Numbers less than 7 signify increasingly greater acidic solutions, and numbers 

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

Photoperiod...duration of illumination and of darkness over a 24 hour day. 

Reconstituted water...standard, synthetic water prepared with de-ionized water and reagent grade 

chemicals or mineral water; to be used for culturing of organisms, as control water, and as dilution water 

for test solutions. 

Reference toxicant...a known toxicant used to measure all aspects of toxicity testing, including 

organism quality, presence and effects of stressors other than test compounds, and the quality of 

analytical techniques. Data from regular reference toxicant testing is used to establish the precision of 

bioassay results generated by the laboratory. 

Replicate...Each of several experimental units that are tested simultaneously using the sample 

experimental conditions. 

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

Swim-up...stage at which Salmonids emerge from the gravel and begin to actively feed. It is the point at 

which to begin marking the age of the fish. 

Toxicity...inherent potential or capacity of a material to cause adverse effects in living organisms.