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• WASHINGTON STATE DEPARTMENT OF 

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# Biological Testing Methods 80-12 Forthe Designation ofDangerous Waste

Part A: Static Acute Fish Toxicity Test

PartB: Acute Oral Rat Toxicity Test

Publication #80-12, Revised June 1999 Hazardous Waste and Toxics Reduction Program Washington State Department of Ecology printedon recycledpaperFor a copy ofthis document, please contact: Department ofEcology Publications PO Box 47600 Olympia, WA 98504-7600

The Department of Ecology is an equal opportunity agency and does not discriminate on the basis of race, creed, color, disability, age, religion, national origin, sex, marital status, disabled veteran's status, Vietnam Era veteran's status or sexual orientation. Ifyou have special accommodation needs, or require this document in alternative format, please contact the Hazardous Waste and Toxics Reduction Program at (360) 407- 6700 (Voice) or (360) 407-6006 (TDD). Regional TDD numbers are:**+-~._-~- ~----~** **_._--~_.** __..-**----~-** --- Central Regional Office Eastern Regional Office Northwest Regional Office Southwest Regional Office (509) 454-7673 (509) 458-2055 (425) 649-4259 (360) 407-6306 

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• WASHINGTON STATE DEPARTMENT OF 

# ECOL 0 GY Biological Testing Methods 80-12 Forthe Designation ofDangerous Waste.

Part A: Static Acute Fish Toxicity Test

PartB: Acute OralRat Toxicity Test

Publication#80-12, Revised June 1999 Hazardous Waste and Toxics Reduction Program Washington State Department of Ecology printedon recycledpaper

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• Table of Contents
Glossary v Terminology vii 

## PartA: Static Acute Fish ToxicityTest Introduction A-1 Toxicity Identification Evaluation (TIE) A-2 Health and Safety A-2 

### Methods and Materials 

### A·4 Holding Facilities A-4 Test Chambers A-5 Cleaning and Disinfection A-6 DilutionWater A-7 Test Organisms A-8 Care and Handling A-9 Holding A-9 Stress Indicators A-IO Sample Preparation A-IO Sample Water Content Determination & Reporting A-IO Particle Size Reduction A-II Rotary Agitation Method A-II 

### Test Procedure A·12 Replicates and Control. A-12 Test Temperature A-12 pH A-13 ·Dissolved Oxygen A-13 Loading : A-14 Feeding A-14 Duration and Acceptability A-14 Test Observations and Measurements A-14

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### I Criteria for Death A-IS Termination A-IS Data Analysis A-IS 

### Quality Assurance 

### IQuality Control A·15 Procedures A-IS Reference Toxicants A-16 

### Calculation of Results A·17 Approach A-17 Hypothesis A-17 Calculation A-18 Sample size A-18 Mortalities A-18 F Test A-18 Equal variance t test A-19 Modified t test A-19 Critical F and t values A-19 

### Data Reporting A·22 References A·25 Appendices A. Neutralized FishToxicity Test Procedure A-27 B. Good Laboratory Procedure Standards A-29 C. Water Quality A-3S D. Dissolved OxygenTable A-36 E. Recommended Fish Loading A-37 F. Sample Data Sheet. A-39 G. Recommended Test Conditions and Procedures for Fish Bioassay A-43 

## Part B: Acute Oral RatToxicityTest Introduction B-1 ii 

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• Methods B·1 Sample Collection and Preparation ; B-1 TestAnimals '" B-2 PretestConditioning B-2 Procedure B-2 

### Calculation ofResults B·3 Approach : B-3 Hypothesis B-4 Calculation (N < 30) ; B-4 Calculation (N > 30) B-7 

### Data Reporting 8·8 References 8·9

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• °c CaC03 CaS04 CaS04e2H20 cm CO2 COD d DO DW EHW g gal 

### gpm h HCL H 20 in KCL L m mg mg/L MgS04 min mL mg/kg NaHC03 NaOH ng/L NH3 ppb 

# Glossary
degree(s) Celsius calcium carbonate calcium sulfate calcium sulfate dihydrate (gypsum) centimeter carbon dioxide chemical oxygen demand day dissolved oxygen dangerous waste extremely hazardous waste degree(s) Fahrenheit grams gallons gallons per minute hour hydrochloric acid water inch(es) potassium chloride liter meter milligram milligramper liter magnesium sulfate

................................................................................................ minute(s) milliliter milligramperkilogram sodiumbicarbonate sodiumhydroxide nanograms per liter ammonia

..................................................................................... parts perbillion

vppm pptr QA/QC qt 

### rpm TOC WAC > < > 5 parts per million parts per trillion quality assurance/quality control quart rotations per minute total organic carbon Washington Administrative Code greater than less than greater than or equal to less than or equal to

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# • Terminology Grammatical Terms Can...

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

### Might
...

### means "could possible;" never used as a synonymfor "may" or "can" 

### Must
...

### expresses an absolute requirement 

### Should
...

### means the specified condition is recommended and ought to be met if possible 

## Technical Terms

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### Acclimation
.. .means to become physiologically adapted to a particular level of one or more environmental variables, such as temperature. The term usually refers to controlled laboratory conditions.



### • Acute toxicity... a discernible, adverse effect (lethal or sublethal) induced in the test organisms within a shortperiod of exposure to a testmaterial, usually 4 days for fish. 

### Alevin...arecently hatched, non-feeding fish with an evident yolk sac for nutritive requirements. Sometimes referred to as yolk-sac fry. 

### Conductivity...is a numerical expression ofthe ability of an aqueous solution to carry an electrical current. This ability depends on the concentrations ofions in solution, their valence and mobility, and on the temperature of the solution. Conductivity is normally reported in the Systeme International dunites (51) unit ofmillisiemens/meter, or as micromhos/centimeter (1 mS/m = 10 umhos/cm)
.. 

### Control...a treatmentin an investigation or study duplicating all conditions and factors possibly affecting the investigationresults, exceptthe specific condition being studied. In aquatic toxicity tests, the control must duplicate all conditions ofthe exposure treatment(s), butmust contain no testmaterial. 

# • The control is used to determine the absence ofmeasurable toxicity due to basic test conditions (e.g., quality ofthe control/dilution water, health or handling oftest organisms).

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### Dangerous waste ...those solid wastes designated inWAC 173-303-070through 173-303-100 as dangerous, extremely hazardous, or mixed waste. 

### Deionizeduiater
...water that has been passed through resin columns to remove ions from solution and thereby purify it. 

### Distilledwater...waterthathas beenpassed through a distillation apparatus of borosilicate glass or other material, to remove impurities. 

### Extremely hazardous waste.. .those dangerous and mixed wastes designated in WAC 173-303-100 as extremely hazardous. Also, those dangerous wastes showing statistically significant mortality in test concentrations of 10 mg/L. 

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

### Flow-through
...describes scenario in which holding vessels are renewed continuously by the constant inflow offresh solution or holding water, or by a frequentintermittent flow.

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### Hardness
...the concentration ofcations inwater that will reactwith a sodium 

# 0 soap to precipitate an insoluble residue. In general, hardness is a measure ofthe concentration of calcium and magnesiumions in water, and is expressed as mg/L calcium carbonate (CaC03) or equivalent. 

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

## off the yolk sac, and in nature, bury into the waterbody gravel. LC50
...the median lethal concentration (i.e., the concentration ofmaterial in water thatis estimated to be lethal to 50percent ofthe 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 ofexposure. "The duration ofexposure mustbe specified (e.g., 96 hour LC50). 

### Leachate
...

### any liquid, including any components suspended in the liquid, that has percolated through or drained from dangerous waste. 

### Lethal
... means causing deathby direct action. Death offish is defined as the cessation of all visible signs ofmovement or other activity; no response from gentle prodding.
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• Loading
...the weight of organisms per liter oftest solution; this is limited to minimize the depletion of DO, the accumulation ofinjurious concentrations of metabolic waste products, and/or stress induced by crowding. 

### Lux ... aunit ofilluminationbased on units per square meter. One lux 

## =0.0929 foot-candles and one foot-candle 

## =10.76 lux. 

### Monitoring...the routine (e.g., daily, weekly, monthly, quarterly) checking of quality or collection and reporting ofinformation. In the context of this method, it means either the periodic (routine) checking and measurement of certain biological or water-quality variables, or the collection and testing of samples of effluent, elutriate, leachate, or receiving water for toxicity. 

### Percentage (%)
...is a concentration expressed in parts per hundred parts. One percentrepresents one unit orpart ofmaterial (e.g., effluent, elutriate, leachate, or receiving water) diluted with water to a total of 100parts. Concentrations can be prepared on a volume-to-volume orweight-to-weightbasis, and are expressed as the percentage oftest material in the final solution
. pH
...the negative logarithm ofthe activity ofhydrogen ions in gram equivalents per liter. The pH value expresses the degree of intensity ofboth acidic and alkaline reactions on a scale from 0 to 14, with 7 representing neutrality. Numbers less than 7 signify increasingly greater acidic reactions, and numbers greater than 7indicate increasinglybasic, or alkaline, reactions. 

### Photoperiod
...the duration ofillumination and darkness within a 24 hour day. 

### Pretreatment
...

### in this method, is treatment of a sample or dilution thereof, prior to exposure of fish. 

### Reconstitutedwater ...deionized or glass-distilled water to which reagent-grade chemicals have been added. The resultant synthetic fresh water is free from contaminants and has the desired pH and hardness characteristics. 

### Reference toxicant...a standard chemical used to measure the sensitivity ofthe testfish in order to establish confidence in the toxicity data obtained for a test material. In most instances a toxicity test with a reference toxicantis performed to assess the sensitivity of the organisms at the time the testmaterial is evaluated, and the precision ofresults obtained by the laboratory.
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### Replicate...One of several identical experiments, procedures, or samples. Duplicate is a special case ofreplicates consisting oftwo samples or measurements. Static
...describes toxicity tests in which test solutions are not renewed during the test. 

### Static replacement
...describes toxicity tests inwhich test solutions are renewed (replaced) periodically during the test, usually every 24hours. Synonymous terms are "renewal", "batchreplacement", and "semi-static". Swim-up
...post-alevin emergence from the gravel and commencing of active feeding. Point at which to begin marking the age ofthe fish. 

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

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Toxicity test...a determination ofthe effect of a material on a group of selected organisms under defined conditions. An aquatic toxicity testusually measures () the proportions of organismsaffectedby their exposure to specific concentrations ofchemical, effluent, elutriate, leachate, or receiving water.



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• Part A: Method 80-12 State Acute Fish Toxicity Test Introduction The Washington State Department of Ecology (Ecology) developed the acute fish toxicity test (Method 80-12) to determine if a waste meets the definition ofdangerous waste in the Dangerous Waste Regulations, Chapter 173-303 WAC. Appendix A contains a modified test procedure for conducting neutralized fish toxicity tests. Method 80-12provides a simple, low costmethod for testing the toxicity of a waste againstthe requirements ofWAC 173-303. This method determines ifthe 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 ofwaste thatkills 50percentof the testfish within 96 hours. Ifthe toxicity of a waste is unknown, the waste mustbe tested for dangerous waste designation using Method 80-12. The waste concentrations of 100mg/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 mustbe regulated and managed as specified inWAC 173-303, and mustbe sampled according to procedures inWAC 173-303-110,Sampling and Testing Methods. Any personwhose waste fails the acute fish toxicity test, and who believes thatmortality resulted from effects other than acute toxicity (e.g., disease or physical stress), may petition Ecology to exemptthe waste from designation underWAC 173-303-101. They may also petitionEcology to modify the test procedure. Any person who believes thattestfailure resulted 

### only because ofpH 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 inherenttoxicity (e.g., ammonia, cyanide) will not qualify for designation changes. Petitions mustbe submitted perWAC 173-303-910.
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## Toxicity Identification Evaluation (TIE) The Toxicity Identification Evaluation (TIE) procedure (EPA, 1988) is used to identify toxicants or classes oftoxicants in a dangerous waste that designates by only failing the state fish bioassay. Depending on the outcome ofthe TIE, Ecology may allow an alternative management standard, sufficiently protective ofhumanhealth and the environment, for a state-only toxic waste. EPA developed the TIE procedure to aid wastewater dischargers in identifying toxicants within a complex effluent as part of a Toxicity Reduction Evaluation (TRE). A TRE is an evaluationintended to determine remedial actions necessary to comply with NPDES water quality-based limits (EPA, 1989). AlthoughTIE procedures have primarilybeenapplied to NPDES-permitted effluents, the methods are applicable to other types of aqueous samples, including ambient waters, sedimentpore waters, sedimentelutriates, and hazardous waste leachates. EPA published the TIE procedure in three phases, with provisions for evaluating samples exhibitingboth acute and chronic toxicity. Follow the acute toxicity methodology in the TIE to be consistentwiththe acute fish 

# C) bioassay. Phase I characterizes the chemical and physical nature ofthe waste without specifically identifying the toxicants (EPA, 1991; EPA, 1992). Phase II describes methods to identify toxicants (e.g., nonpolar organics, ammonia, cationic metals) but is incomplete and does not provide methods for many constituents (EPA, 1993a). Phase III describes methods to confirmthat the suspected toxicants, rather than methodological artifacts, are the true cause oftoxicity (EPA, 1993b). Please contact the Hazardous Waste and Toxics Reduction Program at 360/407-6700 for further information. 

## Health and Safety
. Development and maintenance of an effective health and safety program in the laboratory requires an ongoing commitmentby laboratory management, and includes:
. • the appointment of a laboratory health and safety officer with the responsibility and authority to develop and maintain a safety program;
• the preparation of a formal, written, health and safety plan, which is provided to each laboratory staffmember;

• an ongoing training program on laboratory safety; and ()
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• • regularly scheduled, documented, safety inspections
. Toxicity testing may involve significantrisks to personal safety and health. Personnel conducting toxicity tests should protect themselves by taking all safety precautions necessary to preventbodily injury. Observe procedures to prevent inhalation or skin absorption ofwastes. Each laboratory should be equipped with all necessary safety equipmentprior 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 ofwaste is usually only poorly known, consider them as potential health hazards. Minimize exposure to waste samples. Fume and canopy hoods over test areas mustbe used whenever necessary. Strong acids and volatile organic solvents employed in glassware cleaning mustbe 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 ofhazardous materials. Ecology recommends thatpersonnel conducting toxicity tests not work alone.

• Wastes should be considered potential health hazards and exposure to them should be minimized.

• Immediately wash anybody part that may have come in contactwith wastes. Although a water rinse often suffices, note that some wastes maybe activated with water.

• Adequately label ali containers to indicate contents.

• Good housekeeping habits contribute to safety and reliable results. • Use electrical equipment approved by Underwriters Laboratories. Ground-faultinterrupters should be installed in ali "wet" labs where electrical equipmentis used.

3• Stafftraining inbasic first aid and cardia-pulmonary resuscitation (CPR) is strongly recommended. • All personnel should receive tetanus immunizations. The following are optional and dependent on the type of waste anticipated: hepatitis B (ifsuspected contactwith humanbodily fluids, including blood); hepatitis A, typhoid fever, and polio (ifsuspected contactwithhuman feces and/or urine). Polio may be transmitted from infant immunizations through fecal material. Please err on the side of caution. If your laboratory has any questions, contact the Washington Department ofLabor and Industries or similar agency inyour state.

• Wastes generated during toxicity testing mustbe 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 shouldbe notified of any potentially hazardous conditions. For further guidance on safe laboratory practices, consult EPA (1986), EPA (1993c), and Walters and Jameson (1984). Also see Appendix B for Good Laboratory Procedure Standards. 

## Methods and Materials Holding Facilities Facilities should include tanks for holding/acclimating test fish and a constant-temperature room or recirculating waterbathfor the test chambers. Holding tanks mustbe operated on a flow-throughbasis; this may include either once-through or recirculating systems with at least50 percentrenewal. Holding, acclimation, and dilution-water tanks and headboxes shouldbe equipped for temperature control and aeration. Air used for aeration mustbe free of oil and fumes; filters to remove oil and water maybe used. Testfish shouldbe shielded from disturbances during holding, acclimation, and testing. The test facility should be well ventilated and free of fumes. Fish shouldbe quarantined at least 7days when first broughtinto a facility. Avoid crowding to maintain testfish in good condition during holding and acclimation. The dissolved oxygen (DO) concentration must
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• be maintained between 60 percent and 100percent saturation; gentle aeration maybe used ifnecessary. Aeration drives off the ammonia from generated biological waste. A photoperiod of-B-16hours oflightper 24 hours should be provided during the test. Preferably, thoughnotrequired, light intensity should be raised gradually over a 15 minute period at the beginning ofthe photoperiod. Gradually lower the light intensity over the same period of time at the end ofthe photoperiod. Use a dimmer switch or other suitable device to make this easier. The lightintensity should be in the range of natural outdoor light or ambientlaboratory illumination (about 10-20 

### uE/m 2Is or 50-100 ft-c). Avoid high intensity and extreme lighting conditions. Whether fabricated in the lab or purchased commercially, tanks should notbe constructed ofmaterials, or have surface coatings, which will leach or dissolve into aqueous solutions. Tanks and other equipmentthat contact stock solutions or test solutions should be chosen to minimize sorption oftoxicants 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. Castiron pipe maybe used in freshwater supply systems, but colloidal iron will be addedto the dilutionwater and strainers will be needed to remove rust particles. Natural rubber, copper, brass, galvanized metal, solder, and lead should not come in contactwith dilution water, stock solutions, or test solutions before or during exposure oftestfish. 

## 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 fromjumping out. Test chambers commonly used in static tests for fish are wide-mouth glassjars or aquaria, 3.8 L (1 gal) or 19.0 L (5 gal). Special glass or stainless steel test chambers canbe constructed to accommodate testfish requiring particularphysical conditions (large surfacearea-to-volume ratio). Testchambers 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 5adhesive inside the containers shouldbe removed. Stainless steel (No. 304 or No. 316) canbe used inthe construction oftest chambers, but should be ofwelded, not soldered, construction. Commercially available testchambers meeting the above requirements may be used. Disposable, non-toxicplastic (e.g.,polypropylene) liners maybe used. Ecologyhighly recommends laboratories using disposable liners perform several rounds ofin-house testing to determinethatthe liners are trulynon-toxicbefore Usingindangerous waste designationtesting. 

### Cleaning and Disinfection Clean test chambers before use. Wash new test chambers with ammoniabased detergent suitable for biological laboratories, then rinse in the following sequence with:

• clean water,
• acid, such as 5 percent concentrated nitric acid,

• copious clean water,
• reagent grade acetone, and

• copious clean water. At every testtermination, chambers shouldbe cleaned inthis sequence:
• emptied;

• immediately rinsed with clean water;
• cleaned using appropriate procedures for removing the toxicant (e.g., acid to remove metals and bases; detergent, organic solvent, or activated carbon to remove organic compounds); and

• rinsed with copious cleanwater. 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 madeby adding 6 m1 of liquid household chlorine bleach to 1 L ofwater. Acid and hypochlorite should not be used togetherbecause 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 productnames. 

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For disinfection ofequipment such as fish tanks, prepare a 50ppm solution of titrateable iodine by diluting a commercially available product. The equipment shouldbe immersed in or washed with this solution for at least 10 minutes followed by copious rinsing with cleanwater. Iodophors and hypo-chlorites are 

# C.)· acutely toxic to most aquatic organisms. Hypochlorite canbe removed by
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• flushing withwater orby 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 overnightto allow dissipation ofresidual chlorine. 

### Dilution Wate:r; An adequate supply of quality dilution watermustbe 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 criterionfor acceptable dilution water is testfish surviving and growing satisfactorily in it. Monthly hardness, alkalinity, and specific conductance ranges mustbe within less than 10 percent oftheir respective averages. The monthly pH range mustbe less than 0.8 unit to produce consistent quality dilution water. Ideally, non-ehlorinated dilutionwateris preferred, butmaynotbe realistic. Dechlorinationusing activated carbonfilters or othermethods maybe used to dechlorinate dilution water as thoroughly as possible. Chlorinemay also be removed using an activated carbonfilter thentreating with a sodiumthiosulfate or sodiumsulfite drip. Sodiumthiosulfate is betterfor dechlorinating waterthan sodiumsulfite and maybe more reliablethan activated carbonfor removing chloramines.
. Metals can usually be removed with chelating agents. Municipal water supplies often containunacceptablyhigh concentrations ofcopper, lead, zinc, and fluoride; buildings may contain copper pipes and lead solder: Toxicity tests can be adversely affected by the use of dilution water containing these materials. The dilution water should be intensively aerated using either air stones, surface aerators, or screen tubes before introducing the toxicant. Adequate aeration will bring the concentration ofdissolved 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 waterjustbefore use. Dilutionwater hardness can affect the outcome ofthe test. Moderately hard dilution water mustbe used in this method and should notbe less than 40 mg/L CaC03• Low hardness/alkalinity may render test organisms more sensitive to some toxicants. Use reagent-grade chemicals to adjusthardness and alkalinity. Table 1 is for guidance purposes. 

## Table 1. Preparation of Dilution Water Using Reagent Grade 

## Cherncals'

7NaHCOa CaS0 4-2HP MgS04 KCI pHc Total Resulting Total Resulting (' WaterType mg/Lb mg/Lb mg/Lb mg/Lb Hardness" Alkalinityd Soft 48.0 30.0 30.0 2.0 7.2-7.6 40-48 30-35 Moderately Hard 96.0 60.0 60.0 4.0 7.4-7.8 80-100 60-70

• Adapted from EPA (1993c)
b Add reagent grade chemicals todistilled ordeionized water.
e Approximate equilibrium pH after aerating 24hours.
• Expressed asmg/L CaCa,. 

## Test Organisms Use one ofthe following species in the static fish toxicity test (all species mustbe verified): coho salmon, Oncorhynchus kisutch; rainbow trout, Oncorhynchus mykis (formerly Salmo gairdneri); and brook trout, Salvelinusfontinalis. The test temperature for all species is 12 

### ± 1 "C. All fish used in a test shouldbe ofthe same age and from the same source. Organisms of the same species from different sources may produce differenttest results. Usual sources offreshwater fish are private, state, and federal hatcheries.

Maxillary PreopercleBran hiostegals (gills) 

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Standard Length FIGURE 1. Test fish should between 30 and 90 days from swim-up and as uniform in size as possible. Circumstances may arise where this range cannotbe 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 snoutto end ofcaudal peduncle) ofthe longestfish shouldbe no 

# (J" more than twice that ofthe shortest fish (Figure 1). 8• All testfish should be obtained from a hatchery thathas been certified diseasefree for the following diseases; bacterial kidney disease 

### (Renibacterium salmoninarum); Costia 

### (Ichythobodo); bacterial gill disease 

### (Myxobactriea sp.); and Furunculosis 

### (Aeromonas salmonicida). If fish are diseased, destroy the entire lot immediately. Make all efforts to ensure testfish are disease-free prior to initiation oftoxicity test. Options include:
• selecting fish from a source (hatchery) which will certify that all fish are disease-free;

• discriminately selecting test fish from a reputable source, and/or on the observations andjudgment ofknowledgeable individual; or
• treating all fish that arrive on-site using treatments commonly used by sources (hatcheries) which produce certified disease-free fish. Between use ofdifferenttest fish groups, holding and acclimation tanks should be sterilized according to the procedures in Cleaning and Disinfection. 

### Care and Handling Minimize unnecessary stress to testfish. To avoid unnecessary stress, do not subject fish to rapid changes in temperature or water quality. -Generally, aquatic organisms should notbe subjected to more than a 3°C change in water temperature in any 12hour period. Maintain testfish 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. Ifnecessary, handle them as gently, carefully, and quickly as possible. Discard fish thattouch dry surfaces, are dropped, or injured during handling. Dip nets are bestfor handling fish and are commercially available or made from small-meshnylonnetting, 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, 200mg hypochlorite/L, or 30 percent formalin plus 1 percent benzalkonium chloride for atleast 1 hour. Sterilize or wash hands before and afterhandling 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 constanttemperature and quality in a flowthrough systemwith a minimum flow rate of five volume exchanges per day (ASTM,1993). When possible, hold fish in dilution water and as close to test temperature as possible. During long holding periods, however, itis generally
9easier 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 mortalitybetween 24 hours after arrival on-site and the end ofthe 7 day acclimation period, the entire lot should notbe used for testing. If at any time during the holding period there is an abnormal number ofmortalities, 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 mustbe discarded. Visually examine the behavior and external appearance ofthe fish. Ifthe stressor canbe determined, remove stressor and reacclimate for 7 days. The following physical characteristics indicate stress: 

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Darkened color (normal is silver/gray) Emaciation Erratic swimming Excessive mucus production External parasites Noteating 

## Sample Preparation Flashing Flipping Fungus/Fin Rot Gasping at surface Hemorrhaging Hyperventilation 

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Sample Water ContentDetermination and Reporting Requirements Ecology requires reporting the percentwater ofmaterials to be tested for dangerous waste designation using Method 80-12. The following procedure is used to estimate the water content of samples that will undergo toxicity testing using either 80-12 A or Bmethods.

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

• Weigh out a representative sub-sample ofthe material to be tested (100-g minimum) into the above container. Since samples mustbe representative, stirring of samples with liquid content> 0.5% during sub-sampling is 

# C·.-) required.**------------------------~-=-~,..,.,......=====--------** 

## - 10

# • • Dry the material at 60 to 80aC until two successive weighings to the nearest O.01-g yield the same value.

• Calculate the percentnon-volatile solids using the following equation:

• Include the calculated test-material percent solids in the final report showing the raw data obtained.
• Retainprocedural laboratorybench records in archive for atleast five years. 

# I. Weight ofdried material in the container

- container tare weight Initial (wet) weightofmaterialincontainer

- containertare weight X 100 

## = % solids

Caution: Test materials having ignitability, corrosivity, or reactivity characteristics mustbe handled with appropriate caution when water content and toxicity tests are conducted. See SW846Chapter 7 inEPA, 1986for ignitability, corrosivity, orreactivity definitions. All samples mustbe extracted using the method described below. Solid samples mustbe reduced in size prior to extraction unless the sample meets the particle size criteria described below. Ensure all phases ofthe sample are homogenous. Stirring may be required ifthe sample is nothomogenous. Samples may be extracted using a rotary agitation apparatus. The rotary agitation apparatus mustbe capable ofrotating the extractionbottles at 30 ± 2 rpm. The sample is allowed to extractfor 18

### ± 2hours at 23

## ±2°C. The extractionwateris the stockdilutionwaterused 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 ofmaterial equal to or greater than 3.1 em', or is smaller than 1 cmin its narrowest dimension (i.e., is capable ofpassing through a 9.5 mm (0.375 inch) standard sieve). Ifthe surface area is smaller or the particle size larger than described above, prepare the solid portion ofthe waste for extractionby crushing, cutting, or grinding the waste to a surface area or particle-size. Note: Surface area criteria are meantfor filamentous (e.g., paper, cloth, and similar) waste material. Actual measurement of surface area is notrequired, nor
11is itrecommended. For materials that do not obviously meetthe criteria,**C~~** sample-specific methods would need to be developed and employed to measure / the surface area. Such methodology is currently not available. 

### Rotary Agitation Method

• Weigh outthe correct amount of a well-mixed sample and transfer into 500 or 1000 ml extractorbottle.

• Add 200 ml oftest dilution water to the extractorbottle.

• Place a Teflon cap liner over the mouth ofthe extractorbottle and screw on the cap. • Mix the sample on the rotary agitation apparatus for 18 

### ± 2hours at 23

### ± 2°C, remove the cap and pour the extraction water into the dilution waterin the fish test tank. Rinse all loose material from the flask into the fish testtank with a 200 ml aliquot of stock dilution water. Place the bottle into the fish tank so that the bottle is full ofwater and laying onits side on the tank bottom. Drop the cap liner into the fish test tank.

• Beginthe test as described below.

• Extreme care should be exercised throughoutthe course ofthe extraction so as notto contaminate the exterior ofthe extraction flask. For example, sealing sterile flasks in sterile aluminum foil prior to use or copiously rinsing 

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

## Test Procedure Replicates and Control Use a minimum of 10 fish in each of 3 replicate test tanks exposed to each treatment. Additional fish and replicates maybe used (e.g., 15 fish in each of 4 replicates). Replicates mustbe true replicates withno water connections between the replicate test chambers. Randomize the replicate test chambers and impartially distributed a representative sample oftestfish to the chambers. One randomization method adds to each test chamber no more than 20percent ofthe number oftestfish to be placed in each test chamber. Repeatthis process until each test chamber contains the desired number oftest fish. Alternatively, fish canbe assigned eitherby random assignment of one fish to each test chamber, random assignment of a second fish to each chamber, etc., or by total randomization. It is often convenient to assign fish to other containers and then add them to the test chambers. 

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•

• Every test requires a control consisting ofthe same dilution water, conditions, number ofreplicates, procedures, and test species used in the test tanks. None ofthe toxicants being tested is added to the control. 

## Test

## Temperature The testtemperature for all test species mustbe 12°e. The actual test temperature mustnot deviate from 12°C by more than ± 1.0°C at any time during the test. Continuous temperature monitoring in at least one testtank is desirable. Alternatively, the maximum and minimum temperature mustbe recorded every 24hours. As a second alternative, temperatures in at least one test tank mustbe recorded at least every 6 hours. 

## pH Do not adjustthe pH ofthe test solution. Any pH adjusting will render the test void. There are provisions for demonstrating that the waste kills fish only because ofpH. Appendix A includes a procedure for conducting this type of toxicity test. pH may tend to driftduring the 96 hour test period. This drift is due, in part, to fish respiration, increasing the CO2 content ofthe water which tends to buffer the solution. Do not adjustthe pH when this driftis 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 80percent (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
. notfall 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 ofthe test. Itmustnotfall below 50percent saturation during the remainder of the test. The test mustbe voided ifthe dissolved oxygen falls below 50percent saturation during the test and mortality exceeds the pass/fail threshold. Please refer to Appendix Efor information on dissolved oxygen saturation and concentrationlevels at different altitudes. Test solutions may be gently aerated ifdissolved oxygen does notmeetthe above criteria. Once aeration is initiated, continue throughouttest duration. Avoid turbulence; it stresses fish, resuspends fecal matter, and increases volatilization. Because evaporationreadily occurs at the surface, efficient
13I

! • aeration canbe achieved with minimumturbulence by using an air liftto transfer solution from thebottomto the surface. Aeration should be identical in all test chambers with the exception of the control chambers. Aerate at least onehalfofthe control tanks to verify thatmortalityis causedbythe toxicity of the waste sample and notbythe physical act ofaeration (e.g., too turbulent, aeration accidentally stops at night, etc.). Do notaerate the waterin the remaining control tanks. 

## Loading Loading in test chambers should not exceed 0.8 giL. The giL oftest fish (wet weight, blotted dry) to solution in test chambers should notbe so high as to affect test results. To determineloading, 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 60percent saturation for the first 48 hours of the test and above 50 percent saturation after 48 hours. Limitloading to ensure:
• concentrations of dissolved oxygen and toxicant are not decreased below acceptable levels;

• concentration of metabolic products does not increase above acceptable levels; and

• fish are not stressed due to crowding. 

## Feeding
. Do notfeed test fish during acute toxicity tests or for a minimum of 48 hours before the test. If the testfish 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 testby 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 shouldbe sterile or as clean as possible to avoid introducing additional contamination. "Blank" flasks used in the extractionprocess should be used for the control chambers. 

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The testbegins when testfish are first exposed to the toxicant and ends 96hours later. Control test chambers must show 90 percent survival or greater for test () acceptance or validity.
14

## • Test Observations and Measurements Measure dissolved oxygen and the pH in all testtanks at the beginning. Repeat these measurements every 24hours (as long as live fish are present). Also record the dissolved oxygen and pH at the end ofthe test or at the time the lastfish dies in the test tank. Hardness, alkalinity, pH, and conductivity mustbe measured in the test and control tanks at the start ofthe test. Measure the same parameters at the end of the test. Measurement ofcalcium, magnesium, sodium, potassium, chloride, sulfate, particulate matter, and total organic'carbon (TOC) or chemical oxygen demand (COD) is also desirable. The number ofdead or affected fish in each test chamber mustbe counted every 24 hours after starting the test. More observations are desirable, especially near testinitiation. Ecology suggests counting the number ofdead or affected fish in each chamber 4,8, and 24 hours (or similar time increments) after the beginning ofthe 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 oftest chambers and prodding mustbe very gentle. 

## • Criteria for Death Deathis the adverse effectmostoftenused in acutetoxicity tests. The criteriafor death is lack ofmovement, especiallythe absence ofrespiratorymovements in fish and lack ofreactionto gentleprodding. The loss ofequilibrium, defined as the inabilityto make coordinatedmovementand maintain anormal uprightposition, is notconsidered mortality. The latterresponses canbe useful in interpretationoftest· results and shouldbe noted in the comment sectionof the data-reporting sheet. 

### Termination Weights and lengths of test fish maybe determinedbyweighing and measuring and discarding either; (a) arepresentative group of fish before the test; or (b) the control fish thatare alive at the end ofthe test. The length shouldbe reported as standardlength and the weights shouldbe wetweight, blotted dry. Destroy all fish, bothcontrol and treatment, at terminationof test, after data collectionis complete. 

# • Data Analysis Refer to Calculation ofResults. The number ofmortalities needed to designate a waste is no longer a single numberfor dangerous waste or extremely hazardous waste. This "threshold" number of mortalities is now a function of the mean and variance in the control group and the mean and variance in the test group and does not utilize a data transformation.
15

## Quality Assurance I Quality Control Procedures for Quality Assurance/Quality Control Reliable laboratory data is critical for making sound environmental decisions. A good quality assurance/quality control (QA/QC) programis essential to ensure data reliability. Quality assurance (QA) is the total integrated programfor assuring the reliability ofmonitoring data. A comprehensive QA program addresses everything affecting data and should address:

• sample collection and preservation;

• laboratory services;

• use and care ofinstruments, glassware, and chemicals;

• data management; and,

• laboratory personnel. Quality control (QC) is the routine application of statisticallybased procedures to evaluate and control the accuracy ofresults 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 meetthose objectives,

• estimates within-lab precision and bias,

• sets up control charts, and

• participates in inter-lab tests. 

### Reference Toxicants Reference toxicants establish the validity of toxicity data. The reference toxicant measures all aspects oftoxicity testing including organism quality, presence and

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

techniques. Ifreference toxicant results (LC50s) differ significantly from previously established results (LC50 control charts), a full review oflaboratory test conditions and procedures is indicated. Results that are not within two standard deviations ofthe mean LC50, shouldbe considered "red flags." Evaluate procedures and testfish with a reference toxicant once a month or, preferably, concurrently with the test. More frequent testing with a reference toxicantis appropriate when situations occur which may affect test results. For example, a change in dilution or holding water conditions, new equipment or

16 

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• piping in the laboratory, or a change of analysts, all indicate the need for additional use of a reference toxicant. A variety ofcompounds are commonly used as reference toxicants and include phenol, NaCl, KCl, ZnC12, and ZnS04• This is not an all inclusive listby any means. Reference toxicants available from EPA include CuS04 and CdC12 (Environmental Monitoring and Support Laboratory (EMSL), Cincinnati, Ohio 45268). To ensure nominal concentrations ofreference 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 10mg/L) when the calculated t statistic is greater than a critical t value. 

## Approach The statistical analysis is based on methodology presented by Zar (1984), EPA (1993c), and Erickson and McDonald (1995). Because observations on individual fish in the same tank cannotnecessarilybe considered statistically independent, the experimental unitis 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 ofsamples displaying varying proportions of dead and live fish, as determined in acute lethalitybioassays, is described by the binomial distribution. Use ofthe 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 testfor normality (EPA, 1993c). The type of t test used depends on the homogeneity ofvariance assumption between control and test samples (Zar, 1984; EPA, 1993c). This assumption is evaluated with the variance ratio F test. Ifvariances are equal, the equal variance t testis used. Ifvariances are notequal, a modified t testis used.

17Thus, the appropriate t testis used to compare the difference betweentest and**C~** control mortality proportions against a constant, 

### Po 

## =0.5, corresponding to the LC50. Up to 10 percent mortality in the control sample is allowed. 

### A one-tailed t test is used, sincetheanalysis determines only

## ifthesample LC50 issignificantlygreater than theregulatory threshold. This objective is consistentwith the procedure for waste designation frombioassay data outlined inWAC 173-303-100(5)(c). 

## Hypothesis In terms ofconcentration, the one-tailed null hypothesis (H') to be tested is LC50 100 mg/L (dangerous waste threshold) or LC50 10 mg/L (extremely hazardous waste threshold or special waste threshold). In terms ofmortality, each ofthese hypotheses corresponds to a one-tailed H

### o : (PTm

-PcJ 3 

### Po' where 

### PTm and 

### Pem are the mean proportion ofmortalities inthe test and control samples, respectively, and 

### Po = 0.5 is the proportion ofmortalities at the LC50. An alpha level of 0.10 is used in the t test (i.e., one-tailed 90 percent confidence level), consistentwith statistical methodology outlinedby EPA (1986). Rejecting H, demonstrates the sample does not designate as a regulated waste. 

## Calculation 1) Sample size The number ofreplicates in the control and test groups shouldbe equal and 

# 0 should each consist of at leastthree (N 3). The number of fish per replicate tank should be equal across all replicates and should consist of at least 10 (n 10). In the rare event that a testis initiated withunequal replicates, please contactthe Department ofEcology for statistical guidance. In addition, the number offish per replicate tank should not exceed a biomass loading density of 0.8 giL. 

### 2) Mortalities Mortalities are expressed as proportions: 

### Pe = proportion ofmortalities in control replicate 

### PT = proportion of mortalities in test replicate. The bioassay is invalid if overall control mortalities exceed 10percent (EPA, 1993c). 

### 3) F test The variance ratio Ftestis 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 offreedom (df) in (.)\ both numerator and denominator (Zar, 1984; EPA, 1993c):
18

# •

• F = 

### SC2/s/or F = 

### s/lsC 2, whichever is larger, where: F = F statistic Sc2 = variance for the control group

'2 ST = variance for the test group. Ifthe calculated F value is less than or equal to the critical F value, the variances are equal and the equal variance t testis 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 thecase where bothvariances are zero, thevariances are equal. However, when only onevariance iszero, thevariances areconsideredstatistically unequal. 

### 4) Equal variance t test With an equal number ofreplicates in the control and test groups, the equal variance t test has the following form (Zar, 1984): t = (PTm PCm

- PO> 

### 1 (2s 2

### INt 5 (general equation); or, t = (PTm

- PCm - 0.5) 

### 1 [(;/ + 

### sc 2

### )/ Nt 5 where: t = t statistic PTm = meanpT PCm = mean Pc Po = 0.5 = proportion ofmortalities at LC50 Sp2 = pooled variance =(s/ 

### + 

### sc 2)/ 2 N = number ofreplicates in control or test group. The number ofdegrees offreedom in an equal variance t test with an equal number ofreplicates 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 offreedom (df). When the calculated t value is less than or equal to the critical t value, H, is rejected, and it is concluded thatthe waste does not designate as a regulated waste. Note thatin the case where (s/ + sc 2) = 0, H, is rejected when (PTm

- Pc,J < 0.5 and H, is accepted when (PTm

- PCm) 0.5. 

### 5) Modified t test ' With an equal number ofreplicates in the control and test groups, the modified t . test 

### forunequal variances has the same form as the equal variance t test above (Zar,1984). However, the number of degrees offreedom differs and is equal to the following (Zar, 1984):

19Ifthe computed degrees offreedom are noninteger, use the next smallerinteger. Note that ifeither sc 2 

## =0 or 

### 5.r2 

## =0, then df

## =N - 1. The calculated t statistic is compared to a critical t value with the modified degrees offreedom. 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 offreedom canbe found in most statisticsbooks (e.g., Zar,1984). Fornumber of degrees offreedom, see above text.

TABLE2. CRITICAL F VALUES WITH TWD-TAILED a

## = p.OS Denominator Numeratordf df 2 3 4 5 2 39.0 39.2 39.2 39.3 3 16.0 15.4 15.1 14.9 4 10.6 9.98 9.60 c 9.36 5 8.43 7.76 7.39 7.15
TABLE3. CRITICAL TVALUES WITHONE-TAILEDa=O.10 df t' df t 1 -3.078 6 -1.440 2 -1.886 7 -1.415 3 -1.638 8 -1.397 4 -1.533 9 -1.383 5 -1.476 10 -1.372

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

is significantly smaller than 0.5. Example 1: Equal Variance t Test This test consists oftwo treatments and one control, each in triplicate. Two sets ofthree replicates of 10 fish each are exposed to 10 and 100 mg/L, respectively, ofwaste using the 80-12bioassay procedure to determine ifthe waste sample designates as dangerous waste or extremely hazardous waste. The control group is exposed to dilution water only. 

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Replicate

(N=3) 

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### I

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## '~

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• A 1 0.100 1 0.100 4 0.400 B 1 0.100 2 0.200 6 0.600 C 0 0 1 0.100 5 0.500 Mean (pJ 0.067 0.133 0.500 Variance (S2)1 0.0033 0.0033 0.0100

Mer 96hours, control and testtank mortalities are tabulated. Proportions are based on number offish perreplicate (e.g., 

### 1/10 = 0.100). The critical F and t values are found in Tables 2 and 3, respectively.

TABLE5. SUMMARYFOR EQUAL VARIANCET TEST 10 mg/L 100 mg/L Calculated F statistic 0.0033/0.0033

### =1.00 0.0100/0.0033

### =3.03 Critical F df; numerator & 2,2 2,2' denominator each 

### =(N - 1)

Critical F (see Table 1) 39.0 39.0

Calculated t statistic -9.25 -1.01 Critical t df" 

### =r2(N-1)1 4 4 Critical t (see Table 2) -1.53 -1.53 Does waste desiqnate? NO YES Waste Code not applicable WT02 '10 mglL calculated t= (0.133-0.067-0.5)/[(0.0033+0.0033)/3]'5 & 100 mgIL calculated t=(0.5-0.067-0.5)/[(0.0033+0.0100)/3]'5

, 2(N-1) isonly used intheequal variance t test
With the appropriate equations, calculate F

## =1.00 (10mg/L) and F

## =3.03 (100 mg/L). Becauseboth ofthe calculated F statistics are less than the critical F value (39.0), the variances are equal and the equal variance t testis performed. The calculated t statistics are -9.25 (10mg/L) and -1.01 (100mg/L). Because the 10 mg/L calculated t statistic (-9.25) is less than the critical t value (-1.53), H, is rejected and the waste does not designate as extremely hazardous waste. However, because the 100 mg/L calculated t statistic (-1.01) is greater than the critical t value (-1.53),H, is not rejected and the waste does designate as dangerous waste. Example 2: Modified t Test This test consists oftwo treatments and one control, each in quadruplicate. Two sets offour replicates of 15 fish each are exposed to 10 and 100mg/L,· respectively, ofwaste using the 80-12bioassay procedure to determine if the waste sample designates as dangerous waste or extremely hazardous waste. The control group is exposed to dilution water only
.

21TABLE6. TESTRESULTS Replicate DilutionWater Control (n:15) 10mgILSample (n:15) 100 mgILSample (n:15)

(N:4) Number Proportion Number Proportion Number Proportion Dead
. Dead**(p~)** Dead Dead (PT) Dead Dead (PT) A 0 0 4 0.267 9 0.600 B 0 0 6 0.400 8 0.533 C 0 0 3 0.200 10 0.667 D 0 0 0 0 6 0.400 Mean (PM) 0 0.217 0.550 Variance (S2)' 0 0.0278 0.0130

1Vanance equation ISs'=1:{p-p.J'/N-l where p = peor p, After 96 hours, control and testtank mortalities are tabulated. Proportions are based on number offish per replicate (e.g., 1/15 

### =0.067). The critical F and t values are found in Tables 1 and 2, respectively. TABLE7. SUMMARYFOR MODIFIED TTEST 10mg/L 100 mg/L Calculated Fstatistic The variances are considered statistically unequal because the control variance iszero. Therefore, the modified t test isused. Calculated t' statistic -3.39 0.88 Critical tdf2 =(N-1 ) 3 3 Critical t (see Table2) -1.64 -1.64 Does waste designate? NO YES Waste code not applicable WT02

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

a(N- 1)isused inthemodified t testwhen either se' = 0 orsr' = 0 Zero variance in the control group dictates that the variancesbe 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 100mg/L is 0.88. Because the 10 mg/L calculated t statistic (-3.39) is less than the critical t value (- 1.64), H, is rejected and the waste does not designate as extremely hazardous waste. However, because the 100 mg/L calculated t statistic (0.88) is greater than the critical t value (-1.64), H, is not rejected and the waste does designate as . dangerous waste. . . 

## Data Reporting The report must contain the following information (a sample reporting form is found in Appendix E):

• name oftest, investigator, laboratory, and date testwas begun;

22 ( <, I 

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• • detailed description ofwaste tested, its source, lotnumber, known composition, known physical and chemical properties;

• dilution water source, chemical characteristics, description ofpretreatment, any additives;

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

• detailed informationabouttest organisms to include scientific name andhow verified, wetweight, blotted

### dry, standard lengthfor fish, age, life stage, source, history, observed diseases, treatments, acclimationprocedure, food used;

• description ofexperimental 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 ofcriterionused to determine endpoints/effects, summary of general observations;

• number and percentage offish deaths, perreplicate and per treatment, in control and test chambers on a dailybasis;

• date run, identification and concentration ofreference toxicant used and resulting mortality and LCSO;

• methods used for and results with standard deviation of all chemical analyses ofwater quality and toxicant concentration, including validation studies and reagentblanks;

• value offollowing: acclimation temperature, test temperature, pH, hardness, alkalinity, dissolved oxygen, and conductivity; and

• anything unusual aboutthe test, any deviations from procedures, and any otherrelevantinformation
.

23

# • References ASTM. 1993. Standard guide for conducting early life-stage toxicity tests with fishes: E1241-92. pp. 180-207. In: 1993 ASTM Standards on Aquatic Toxicology and Hazard Evaluation, ASTM, Philadelphia, Pennsylvania. EnvironmentCanada. 1990. Biological testmethod: acute lethality test using rainbow trout. Conservation and Protection. Report EPS 1/RM/9. Ottawa, Canada. EPA. 1985.Methods for measuring the acute toxicity ofeffluents to freshwater and marine organisms. EPA/600/4-85/013. EPA. 1986. Testmethods for evaluating solid waste. 3rd ed., SW-846,Volume II,Part III,Chapter9. Office of Solid Waste and EmergencyResponse, Washington, DC. 

# •

• EPA. 1989. Generalized methodology for conducting industrial toxicity reduction evaluations (TREs). EPA/600/2-88/070. Water Engineering Research Laboratory, Cincinnati, OH. EPA. 1991. Methods for aquatic toxicity identification evaluations. Phase I toxicity characterizations, 2nd ed. EPA/600/6-91/003. Environmental Research Laboratory, Duluth, MN. EPA. 1992. Toxicity identification evaluation. Characterization of chronically toxic effluents, phase I. EPA/600/6-91/005F. EPA. 1993a. Methods for aquatic toxicity identification evaluations. Phase II toxicity identificationprocedures for samples exhibiting acute and chronic toxicity. EPA/600/R-92/080. EPA. 1993b. Methods for aquatic toxicity identification evaluations. Phase III toxicity confirmationprocedures for samples exhibiting acute and chronic toxicity. EPA/600/R-92/081.

- EPA. 1993c.Methods for measuring the acute toxicity ofeffluents and receiving waters to freshwater and marine organisms. 4th Ed. by CI Weber, Environmental Monitoring Systems Laboratory, Office ofResearch and Development, EPA, Cincinnati, OH. EPA/600/4-90/027F. Erickson, WP and LLMcDonald. 1995. Tests for bioequivalence of control media and testmedia in studies oftoxicity. Environ. Toxicol. Chern. 14(7):1247-1256. Federal Register Vol. 48 No. 230, November 29, 1983, Good Laboratory Practice Standards, pp. 53946ff. 25Hurlbert, SH. 1984. Pseudoreplication and the design of ecological field experiments. Eco!. Monogr. 54(2):187-211. Pillard, D. 1996. ENSR Corporation. Personal communication. 100percent saturation concentrations calculated from regression models. Original data obtained from "Instruction Manual YSIModels 54ARC and 54ABPDissolved Oxygen Meters" pp. 16-17. YSI Inc., Yellow Springs, OH. Walters, DB, and CW Jameson. 1984. Health and safety for toxicity testing. Butterworth Publ., Woburn, MA. Wedemeyer, GA, and

## JW Wood, 1977. Stress as a predisposing factor in fish disease. 

## us. Fish and Wildlife Service. FDL-38. Washington, D.C. Wedemeyer, GA, RL Saunders, and WC Clarke, 1981. The hatchery environmentrequired to optimize smoltificationin the artificial propagation of anadromous salmonids. pp. 6-20. In: E. Kiney and L. Allen, eds., BioEngineering in Fisheries. Special Publicationby American Fisheries Society, Washington, D.C. 

# c

Wedemeyer, GA, DJ McLeay, C Phillip Goodyear. 1984. Assessing the tolerance offish and fish populations to environmental stress: the problems and methods ofmonitoring. 

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



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• Appendix A Neutralized Fish Toxicity Test Procedure The neutralized fish toxicity test procedure is identical to the PartA 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 aquariumwatermaybe neutralized using only aqueous HCl or NaOH. Add the neutralizing solution dropwise while stirringthe contents ofthe test tank, and measuring the pH continuously. The pH of the aquariumwater shouldbe adjusted to 7.5and allowed to stand 30minutes to detectany pH drift. Ifthe pH drifts outsidethe range 7.2 - 7.8, the aquariumwater shouldbe readjusted to pH 7.5, and the fishintroducedimmediatelythereafter. FurtherpH adjustmentis notallowed. 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 ofneutralizing solution used on the data reporting sheet
. 

## Conducting the Neutralized Fish Toxicity Test The neutralized fish toxicity test requires all ofthe proceeding steps for validity. Conduct steps 1 "- 5 in the sequence shown. 1. Fill the testtank and control tank with dilution water allowing for 400 ml of water from the sample extraction step. The dilution water must show hardness, alkalinity, and pH within the range for "soft" to "moderately hard" water as described in Table 1. 2. Introduce the waste into the test tank. The waste includes any undissolved sample, the extract, the rinse, and the extraction flask orbottle. 3. Measure and record the pH. 4. Adjustthe pH to 7.5 and wait 30 minutes. Ifthe pH drifts outside the range 7.2-7.8,re-adjustthe pH and proceed immediately to steps 5, 6, and 7. 5. Collect a samplefor measuring hardness, alkalinity, and conductivity. 6. Measure and record dissolved oxygen, pH, and temperature. 7. Introduce the fish into the test tank. 8. Observe and record data as indicated on the data sheet. 9. Adjustthe temperature and dissolved oxygen as necessary. 10. Terminate the test after 96 hours
. 11. Complete the data reporting sheet.
27

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

• neutralizer (i.e., NaOH or Hel),

• normality ofneutralizer,

• volume ofneutralizer, and

• indicate ifpH readjustment was necessary 30 minutes afterinitial pH adjustment.

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• Appendix B Good Laboratory Standards The following was adapted from EPA's Good Laboratory Practice (GLP) Standards. These standards are recommended, notrequired. Laboratories are encouraged to develop QA/QC programs which address these recommended standards. 

## Organization and Personnel: Personnel Eachindividual engaged in the conduct of or responsible for the supervision of a study shall have education, training, and experience, or combinationthereof, to enable thatindividual to performthe assigned functions. Each testing facility shall maintain a current summary oftraining and experience and fob description for eachindividual engaged in or supervising the conduct of a study. There shall be a sufficientnumber-ofpersonnel for the timely and proper conduct ofthe study according to the protocol. Personnel shall take necessary personal sanitation and health precautions designed to avoid contamination oftest 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 oftest systems and test and control substances. Any individual found at any time to have an illness that may adversely affect the quality and integrity ofthe study shall be excluded from direct contactwith test systems, test and control substances and any other operation or function that may adversely affectthe study until the conditionis corrected. All personnel shall be instructedto reportto theirimmediate supervisors anyhealth or medical conditions thatmay reasonablybe consideredto have an adverse affect on a study. 

### Biologist in Charge For each study, a biologist, or a scientist, or other professional of appropriate education, training, and experience, or combination thereof, shall be identified as the biologist in charge. The biologistin charge has overall responsibility for 29the technical conduct of the study, as well as for the interpretation, analysis, (- documentation, and reporting ofresults, and represents the single point ofstudy / control. The biologist in charge shall assure that:

• The protocol, including any change, is approved as provided by Ecology and is followed. • All experimental data, including observations of unanticipated responses of ' the test system are accurately recorded and verified. Unforeseen circumstances that may affectthe quality and integrity ofthe study are notedwhen they occur, and corrective action is taken and documented.

• Test systems are as specified inthe 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 ofthe study. 

## Protocol Each study shall have an approved written protocol that clearly indicates the objectives and all methods for the conduct ofthe study. The protocol shall containbutnotbe necessarily be limited to the following information: '. A descriptive titleand statement ofthe purpose ofthe study. 

# C

• Identification ofthe test and control substance by chemical name, Chemical Abstract Service Registry Number (CAS), or other established identification system.

• The name and address ofthe 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 ofthe test system.
• Where applicable, the number, body weightrange, sex, source of supply, species, strain, substrain, and age ofthe test system.
• The procedure for identification ofthe test system. • A description ofthe experimental design, including the methods for the control ofbias. • A description and/or identification ofthe 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 withthe carrier. The description shall include specifications for acceptable levels ofcontaminants that are reasonably expected to be presentin the dietary materials and are known to be capable ofinterfering with the purpose or conduct ofthe study ifpresent atlevels greater than established by the specifications. 

# C'

• The route of administration and the reason for its choice.
30

# •

•

• • Each dosage level, expressed in milligrams perkilogram ofbody weight or other appropriate units, ofthe 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 systemwill be determined ifnecessary to achieve the objectives of the study. • . The type and frequency oftests, analyses, and measurements to be made.

• The records to be maintained. • .The date ofapproval ofthe protocol by the sponsor and the signature ofthe biologist in charge. • A statement of the proposed statistical methods to be used. • All changes in orrevisions of an approved protocol and the reasons therefor shallbe documented, signed by the biologist in charge, dated, and maintained with the protocol. 

## Conduct ofa 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 ofcollection. This information shall be located on the specimen container or shall accompany the specimenin a manner thatprecludes error in the recording and storage of data. Records ofgross findings for a specimenfrom postmortem observations shall be available to a pathologistwhen examining that specimenhistopathologically. All data generated during the conduct of a study, exceptthose 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 ofthe change. In computer driven data collection systems, the individual responsible for direct data input shall be identified atthe time of data input. Any change in computer entries shall be made so as notto obscure the original entry, shall indicate the reason for change, and shall be dated and the responsible individual shall be identified
.

31

## Maintenance and Calibration ofEquipment 

# C Equipment shall be adequately inspected, cleaned, and maintained. Equipment j used for the generation, measurement, or assessment of data shall be adequately tested, calibrated, and/or standardized. A written standard operating procedures shall set forth in sufficient detail the methods, materials, and schedules to be used in the routine inspection, cleaning, maintenance, testing, calibration, and/or standardization ofequipment, and shall specify remedial action to be taken in the event offailure or malfunction of equipment. The written standard operating procedures shall designate the person responsible for the performance of each operation, and copies of the standard operating procedures shall be made available to laboratory personnel. Written records shall be maintained of all inspection, maintenance, testing, calibrating, and/or standardizing operations. These records, containing the date ofthe operation, shalldescribe whether the maintenance operations were routine and followed the written standard operating procedures. Written records shall be kept ofnon-routine repairs performed on equipment as a result offailure and malfunction. Such records shall documentthe nature ofthe defect, how and when the defect was discovered, and any remedial action taken in response to the defect. 

## TestingFacilities 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 ofthe data generated inthe 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. Significantchanges in established standard operating procedures shall be properly authorized in writing by management. 

# o

Standard operating procedures (SOPs) shall be established for, butnotlimited to, the following:

• animal roompreparation;

• animal care;
• receipt, identification, storage, handling, mixing, and method of sampling of the test and control substances;

• test system observations;

• laboratory tests; 

# 0

32

# •

• • handling of animals found moribund or dead during study;

• necropsy of animals or postmortem examination of animals;

• collection and identification of specimens;

• histopathology;

• data handling, storage, and retrieval;

• maintenance and calibration of equipment;

• transfer, properplacement, and identification of animals;

• each laboratory area shall have immediately available laboratory manuals and standard operating procedures pertinentto the laboratory procedures being performed (e.g., toxicology, histology, clinical chemistry, hematology, teratology, necropsy). Published literature maybe used as a supplement to standard operating procedures; and, • a historical file of standard operating procedures, and all revisions thereof, including the dates of such revisions, shall be maintained. 

## Reagents and Solutions All reagents and solutions in the laboratory areas shall be labeled to indicate identity, titer or concentration, storage requirements, and expiration date. Deteriorated or outdated reagents and solutions shall not**~e** used
.

33

# •

•

• Appendix C Water Quality Water quality parameters, in addition to pH, hardness, and alkalinity, can adversely affect the outcome of acute fish toxicity tests. Sub-lethal concentrations ofcertain materials in dilution water may contribute to fish mortalitybecause ofthe additive effect of similar materials in the waste being tested, or because of synergismresulting from dissimilar materials. The more common materials and parameters ofconcern are presented below with recommended ranges or maximum concentrations. Iferratic results are experienced in the toxicity test, the dilution water should be checked for the materials included inTable 8, or for other materials which maybe present in the dilution water source. Also refer to EPA/600/4-90/027F Methods for Measuring the Acute Toxicity ofEffluents and Receiving Waters to Freshwater and Marine Organisms for additional guidance on water quality.
TABLE8. WATER QUALITY**PARAMETER~** PARAMETER RECOMMENDED pH 7.2 -7.8 Alkalinity 30- 70mgll asCaCO, Ammonia (NH ) 0.02 ppm unionized Cadmium (Cd) 100 noll Chloride 2 - 3ppm Copper (Cu) lessthan 5 ppb Hardness 40-100 mQ/l,as CaCO, lead (Pb) 0.03 ppm orless Mercury (Hg) organic orinorganic 100 ngll Nickel (Ni) 88ppb orless Nitrogen (N) maximum total gas pressure 110 percent of saturation Nitrite 100 ppb Oxygen atleast 8ppm at 12"C Ozone Residual 3ppb PCB asArochlor 1254 50ngll orless Total Suspended and Settlable Solids 80ppm orless Zinc (Zn) less than 65ppb Total organochlorine pesticides 50ngll orless

'Adapted from Wedemeyer etal,(1981) and EPA (1991)

.

3536 - - -**-~--** 

# Appendix D**C_~** TABLE9.APPROXIMATESATURATlON l AND CONCENTRATION VALUES OFDISSOLVED OXYGENIN FRESHWATERAT**1~C** Altitude (ft) DO (mglL) at DO (mglL) at DO (mglL) at Altitude DO (mgll)at DO (mglL) at DO (mgll) at 100% 80% 60% (ft) 100% 80% 60% Saturation Saturation Saturation Saturation Saturation Saturation 0 10.71 8.57 6.43 4000 9.35 7.48 5.61 100 10.68 8.54 6.41 4250 9.26 7.41 5.56 250 10.62 8.50 6.37 4500 9.18 7.34 5.51 500 10.54 8.43 6.32 4750 9.09 7.27 5.45 750 10.45 8.36 6.27 5000 9.01 7.21 5.41 1000 10.37 8.30 6.22 5250 8.92 7.14 5.35 1250 10.28 8.22 6.17 5500 8.84 7.07 5.30 1500 10.20 8.16 6.12 5750 8.75 7.00 5.25 1750 10.11 8.09 6.07 6000 8.67 6.94 5.20 2000 10.03 8.02 6.02 6250 8.58 6.86 5.15 2250 9.94 7.95 5.96 6500 8.50 6.80 5.10 2500 9.86 7.89 5.92 6750 . 8.41 6.73 5.05 2750 9.77 7.82 5.86 7000 8.33 6.66 5.00 3000 9.69 7.75 5.81 7250 8.24 6.59 4.94 3250 9.60 7.68 5.76 7500 8.16 6.53 4.90 3500 9.52 7.62 5.71 7750 8.07 6.46 4.84 3750 9.43 7.54 5.66 8000 7.99 6.39 4.79 \ 1 Based onPillard (1996).



# o t------**~=------------------------~...,.....,--"=""""""":":""""-** 

### __---

,

# •

•

• Appendix E Recommended Fish Loading Prior to use in toxicity tests, fish are usually maintained at the testfacility in a circulated holding tank. A minimumlevel ofcirculation is required to maintain the viability oftest fish. The recommended level of circulation varies depending on the species of fish, size offish, and temperature. For small fish, the size ofthe fish is usually reported in terms ofnumber of fish per pound. The table below lists the recommended fish minute loadings (pounds offish per gallon per minute ofinflow), to promote the health and quality of salmonid fish used in toxicity tests. TABLE 10. RECOMMENDED FISHLOADINGS" SPECIES WATER POUNDS FISH PER GPM TEMPERATURE °C of 1000b 500 b 100 b Coho Salmon 3 38 3.5 5.0 8.0 9 48 2.7 4.0 6.0 14 58 2.2 3.0 4.5 17 63 ---- 2.0 3.5 Fall/Spring 3 38 3.0 4.0 6.0 ChinookSalmon 9 48 2.5 3.0 5.0 14 58 2.0 2.2 3.5 17 63 ---- 1.2 3.0

•Adapted from Wedemeyer &Wood (19n) b Fish size (number offish perpound)

37

# •

•

•

' 

# Appendix F Sample Data Sheet , The 

### Data Sheetfor StaticBasicAcute Fish Toxicity Testis printed onpage 4l. Please copy this form to reporttest data
.

39

# - 1111"'''' '"" 11,11.I'f' II [tOLOGY IndustryfToxicant Address Collector Date Sample Collected 

# • DATA SHEET FOR STATIC BASIC ACUTE FISH TOXICITY TEST Beginning Date Ending Date Test Organism Required Test Temperature Range 

# •
Laboratory

_ Analyst Time Time

Laboratory Test Total Total Conductivity Reference Contai Cone. Number of Cumulative Dissolved Oxygen (mgll) pH25C Temperature (C) Hardness Alkalinity uMHOStcm Number ner No. (mgtl) Deaths (mgtl as (mgtl as CaC03) CaC03) 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 96 0 96 0 96

Sample Description ----:-:--------- Average Weight Mean Length Longest Shortest Ratio(long/short)

_ Number oforganisms perchamber Ratio offlesh to water

_ Comments,

_
Method on File with the Depertment of Ecology GENERAL PROCEDURE FOR STATICBASIC ACUTE FISH TOXICITY TEST Date

_

A-41 DataVerified By

# • Appendix G Recommended Test Conditions and Procedures for Fish Bioassay



# •

• Testtype Duration Light quality Light intensity Photoperiod Test chambersize Minimum**de~th** Test solution renewall Source offish Control water Fish age Temperature Oxygen/aeration
pH Feeding Cleaning Numberoffish pertest chamber • static non-renewal .96 hours

• ambient laboratory illumination • 10-20 

### uE/m2/s (50-100 ft-c) • 16 hours light, 8 hours darkness 

### • 3.8 L or 1 gal minimum • 15 em test solution

• none

• hatchery stockfree ofknown diseases

• moderately hard water; see Table 1 • 20 - 90 days

• none unless DO < 80 percent saturation at altitude; ideal DO is 80 to 100percent saturation

• no pH adjustment

• not required

• not required

• minimum 10 A-43Number ofreplicate chamber per concentration Number offish per concentration Observations

Measurements
Endpoints Test acceptability
Reference toxicant

Solvents
Concentration

Control water Transport and Storage

Extraction

New Parameter calculate • minimum3

• minimum30

• fish mortality, appearance, and behavior at 24, 48, 72,and 96hours minimum

• temperature, DO, pH, in each tank daily; conductivity at beginning/end of test

• mortality • 90 percent survival or greater in controls
.

• CuSo4 , CdC12 , KCl, NaCl, or 

### znS04 (notinclusive); evaluate at least once/month

• extraction wateris the control water used in test controls
.

• 100.ppmto determine dangerous waste; 10 ppm to determine extremely hazardous waste .

• moderately hard water

• extractwithin seven days of sample receipt..store in dark at 4°C;test within 8 days of sample receipt

• extract using rotary agitation device (30

### ± 2 rpm) for 18 hours 

### ± 2 hours at 23°C

### ± 2°C

• sample water content determination; test-material percent solids (% solids)

A-44 

# c



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• Part B: Method 80·12 Acute Oral Rat Toxicity Test Introduction The acute oral ratbioassay is generally used to determine the relative acute toxicity of an unknown substance. This acute oral ratbioassay was developed by the Washington Department ofEcology (Ecology) for the purpose of determining if a waste meets the definition ofdangerous 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 ofrats. The bioassay determines ifthe 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 oftest substance per unitbody 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 pertinentregulatory 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 ofwaste.shouldbe obtained, using methods approved by the Department ofEcology. The waste sample should be homogenized and either dissolved or suspended in an appropriate vehicle, so thatthe material is in a homogeneous liquid form: amenable to gavage. In decreasing order ofpreference, water, saline, or oil (e.g., com oil) are recommended for the vehicle. The test solution concentration (i.e., weight ofwaste sample per volume solution) should notexceed 1 ml/100 g body
B-1weight, exceptwhen an aqueous solution is used, where 2 ml/l00 g is acceptable 

# C (CFR, 1993).
.. The vehicle and test solution should be at the same temperature as the testroom. Record the type ofvehicle, test solution concentration, and temperature and photoperiod ofthe 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 ofmale and female rats. Include the same number of animals, and equal numbers ofmales and females, in the vehicle control group as in the test group. Female rats should be nulliparous and nonpregnant. Use both males and females to incorporate possible gender differences in acute toxicity (Dixon, 1965) and renal metabolism (EPA, 1991). - I i



## Pretest Conditioning Animals should nothave been used for any other tests and shouldbe 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 mustbe available ad libitum until 18 to 20hours before testinitiation, at which time deprive animals offood. Examine each animal for overt signs of disease. Acclimate to the test environment for a minimum of 7 days. During acclimation, observe animals for adverse health effects. Any animals demonstrating signs of spontaneous disease shouldbe eliminated prior to testinitiation. Use only animalsjudged to be healthy. 

## Procedure Administer doses of 5000, 500, or50 mglkg to evaluate whetherthe waste sample designates as dangerous waste, special waste, or extremely hazardous waste, respectively. This dose is expressed as weight ofthe test substance per unitbody weight. Determine the fasted body weight ofeach 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 oftest solution should not exceed 1 ml/l00 g body weight, exceptwhen an aqueous solution is used, where 2 ml/l00 g is acceptable (CFR, 1993). Deliver a uniform dose to rats with varying body weights by adjusting the volume ofthe prepared test solution of known waste concentration. B-2 

# o

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# •

•

• A concurrentvehicle 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 ofvehicle per unitbody weight (ml/kg) equal to the volume oftest solution per unitbody weight (ml/kg) administered to the test group animals. Record the volumes of vehicle and test solution administered to control and test group animals, respectively. After dosing, immediately return all animals to individual cages with ad libitum food and water. Observe animals for mortality and signs of acute toxicity immediately after dosing, at one and four hours after dosing, and daily thereafter for**1~** consecutive days. Cage-side observations should include evaluation of skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity, and behaviorpatterns. Particular attention shouldbe directed toward noting tremors, convulsions, lethargy, salivation, and diarrhea. Record any mortalities and signs of acute toxicity
.. Temperature ofthe testing room should be recorded daily. Body weights should be determined and recorded atthe start ofthe test, on day 7, and at death
. Sacrifice surviving animals at the end ofthe test on day 14. Perform gross necropsies on all animals in both control and test groups. Record all gross pathology changes. 

## Calculation ofResults The bioassay resultis calculated using the statistical method described below. Three examples are also presented. Within a 90 percent confidence limit, the test sample fails thebioassayby 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 cumulativebinomial probability exceeds the established alpha level for small samples (N < 30), or when the calculated Z statistic is greater than -1.28 for larger samples (N 30). 

## Approach The frequency of samples displaying varying proportions of dead and live rats, as determined in acute lethalitybioassays, is described by thebinomial distribution. Due to the statistical independence among observations on individually caged rats, the experimental unitis the individual rat, and results B-3from the dangerous waste oral ratbioassay canbe analyzed with a binomial goodness of fit test. The observed proportion ofmortalities in the test group is compared to a constant, Po= 0.5, which corresponds to the LD50. A one-tailed testis employed, since we wish only to determine if the sample LD50 is significantlygreater than the regulatory threshold. This objective is consistent with the procedure for waste designation from bioassay data outlined inWAC 173-303-100(5)(c). Twobinomial methods are applied, depending on sample size (N) ofthe test group. Both methods incorporate a correction for up to 10 percent mortality in the control group. ForN < 30, the cumulativebinomial probability ofmortalities is determined directly from the binomial distribution. For N 30, the normal approximation to the binomial testis employed (Zar, 1984). 

# c

Hypothesis In terms ofconcentration, the one-tailed null hypothesis (HJ to be tested is LD50 :::; 5000 mg/kg (dangerous waste threshold), LD50:::; 500 mg/kg (special waste threshold), or LD50:::; 50 mg/kg (extremely hazardous waste threshold). In terms ofmortality, each ofthese hypotheses corresponds to a one-tailed Ho : PTa Po'where PTais the proportion ofcontrol-adjusted mortalities in the test group and Po= 0.5 is the proportion ofmortalities atthe LD50. Analpha level of 0.10 is 

# 0 used (i.e., one-tailed 90 percentconfidence level), consistentwith statistical methodology outlined by EPA (1986). Rejecting H, demonstrates that the sample does not designate as a regulated waste. 

## Calculation (N <30) 1) Minimum sample size A minimum of 10 rats (N = 10) is to be used each in the control and test group with an equal number ofmales and females. 

### 2) Control correction The bioassay is invalid ifcontrol mortalities exceed 10 percent (EPA, 1993). Corrections for up to 10 percentmortality in the control are applied with Abbott's formula (Finney, 1971; EPA, 1993): 

## p/= (PT- Pc) / (1 - Pc) where: p/== control-adjusted proportion ofmortalities in test group PT=observed proportion of mortalities in test group Pc= observed proportion ofmortalities in control group

B-4 () I

# •

•

•• Note that p/= PT when Pc=

## o. If**Pc~** 0.10 and Pc? PT' then PTa= 0 as a result of data smoothing (EPA, 1993). Thatis, PT and Pc are each set equal to their average, (PT+Pc) / 2, so that PTa= O. 

### 3) Binomial probability The cumulative binomial probability for the control-adjusted number of mortalities in the test group is determined by summing the discrete probabilities associated with all mortalities from zero to 

### Np,', where Np/ = control-adjusted number ofmortalities. Np,' may be noninteger, in which case, the next highest integer shouldbe used. Discrete binomial probabilities canbe obtained from statistical tables or from the following equation (Zar, 1984): P(X) = [N! / X!(N - X)!]PoX (l - pJ(N-X) (general equation)
or P(X) = [N! / X!(N - X)!]O.SN where: P(X) = discrete probability of Xmortalities in test group N = number of rats in test group X = number of mortalities in test group Po= 0.5 = proportion of mortalities at the LDSO Ifthe resultant cumulative binomial probability is less than or equal to alpha ( 0.10), then H, is rejected and it is concluded that the waste does not designate as a regulated waste. Discrete and cumulative binomial probabilities for Po= 0.5 and several sample sizes are presented in Table 1 on page B-6.

B-5

# c Table 1. Discrete (D) and Cumulative (C Binomial ProbabilitiesforPn= 0.5 and N=10, 15,20, and 25. X N = 10 N = 15 N=20 N=25 0 C 0 C 0 C 0 C 0 0.0010 0.0010 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 1 0.0098 0.0108 0.0005 0.0005 0.0000 0.0000 0.0000 0.0000 2 0.0439 0.0547 0.0032 0.0037 0.0002 0.0002 0.0000 0.0000 3 0.1172 0.1719 0.0139 0.0176 0.0011 0.0013 0.0001 0.0001 4 0.2051 0.3770 0.0417 0.0593 0.0046 0.0059 0.0004 0.0005 5 0.2461 0.6231 0.0916 0.1509 0.0148 0.0207 0.0016 0.0021 6 0.2051 0.8282 0.1527 0.3036 0.0370 0.0577 0.0053 0.0074 7 0.1172 0.9454 0.1964 0.5000 0.0739 0.1316 0.0143 0.0217 8 0.0439 0.9893 0.1964 0.6964 0.1201 0.2517 0.0322 0.0539 9 0.0098 0.9991 0.1527 0.8491 0.1602 0.4119 0.0609 0.1148 10 0.0010 1.0000 0.0916 0.9407 0.1762 0.5881 0.0974 0.2122 11 0.0417 0.9824 0.1602 0.7483 0.1328 0.3450 12 0.0139 .0.9963 0.1201 0.8684 0.1550 0.5000 13 0.0032 0.9995 0.0739 0.9423 0.1550 0.6550 14 0.0005 1.0000 0.0370 0.9793 0.1328 0.7878 15 0.0000 1.0000 0.0148 0.9941 0.0974 0.8852 16 0.0046 0.9987 0.0609 0.9461 17 0.0011 0.9998 0.0322 0.9783 18 0.0002 1.0000 0.0143 0.9926 19 0.0000 1.0000 0.0053 0.9979



## - 20 0.0000 1.0000 0.0016 0.9995/ 21 0.0004 0.9999 22 0.0001 1.0000 23 0.0000 1.0000 24 0.0000 1.0000 25 0.0000 1.0000 Example 1 Ten rats each in the control and test group (N 

## =10) are orally dosed at 0 and 5000 mg/kg of waste, respectively, according to the bioassay procedure in order to determine if the waste sample designates as a dangerous waste. After 14 days, 3 rats have died inthe test group 

### (PT = 0.300) and zero control mortalities 

### (Pc = 0) are observed. Therefore, 

### PT= PTa and Np,' 

### = (10)(0.300) 

### = 3. For 

### Po =0.5 and N 

## =10, discrete binomial probabilities for 0, I, 2, and 3 deaths are 0.0010, 0.0098,0.0439, and 0.1172, respectively (Table 1). Summing these probabilities, it is apparentthatthe cumulative probability for deaths is greater than the established alpha level of 0.10 (i.e., 0.1719 > 0.10). Therefore, H, is not rejected and the waste designates as a dangerous waste. Example 2 Twenty rats each in the control and test group (N 

## =20) are orally dosed at 0 and 5000 mg/kg of waste, respectively, according to the bioassay procedure in order 

# ().

-- ....

## _-----_._--------------------------------- 8-6

# ·'

• to determine ifthe waste sample designates as a dangerous waste. After 14 days, 7 rats have died in the test group (PT

## =0.350) and 2 control mortalities (Pc

## = 0.100) are observed. Therefore, PTa 

## =0.278 and Np," 

## =(20)(0.278)

## =5.56. Np,' is then rounded to 6 deaths, as described above. For

### Po

## =0.5 and N 

## =20, discrete binomial probabilities for 0, I, 2, 3, 4, 5, and 6 deaths are 0, 0, 0.0002, 0.0011, 0.0046,0.0148, and 0.0370, respectively (Table 1). Summing these probabilities, it is apparentthatthe cumulative probability for :s; 6 deaths is less than the established alpha level of 0.10 (i.e., 0.0577 < 0.10). Therefore, H, is rejected and the waste does not designate as a dangerous waste. 

## Calculation (N • 30) 1) Control correction The bioassay is invalid if control mortalities exceed 10 percent (EPA, 993). The control correctionis performed with Abbott's formula as described above. 

### 2) Normal approximation Based on the mean and standard deviation for a binomial distribution, the following formula has been adapted from Zar (1984) to evaluate how p/ differs from 

### Po 

## =0.5 in terms of a normal distribution: 

## z =(PTa

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

## z= (p/ - 0.5) / [PTa (l - p/)

### /Nt.5 where: Z 

### =normal deviate 

### Po = 0.5 = proportion ofmortalities atthe LD50 N = number ofrats in test group The calculated Z test statistic is compared to a one-tailed critical Z value at an' alpha level of 0.10 (i.e., Z 

## =-1.28). When the calculated Z value is :s; -1.28, H, is rejected, and itis 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 ofwaste, respectively, according to the bioassay procedure in order to determine if the waste sample designates as a dangerous waste. After 14 days, 20 rats have died in the test group (PT

## =0.400) and 4 control mortalities (Pc

## = 0.080) are observed. From these data, PTa

## =0.348 and Z 

## =-2.26. Because the

B-7calculated Z statistic (-2.26) is less than the critical Z value (-1.28), Ho is rejected 

# C and the waste does not designate as dangerous waste. 

## Data Reporting The report must contain the following information:
• study initiation and termination dates;

• description of testing facility;

• source, genetic strain, and sex ofcontrol and test animals;

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

• dose oftest 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 inboth control and test group;
• mortalities andsigns of acute toxicity immediately after dosing, at one and four hours after dosing, and daily thereafter for 14 consecutive days inboth control and test groups;
• photoperiod and daily temperature oftesting room;

• gross necropsy results; 

# 0

• statistical analysis to evaluate whether the waste sample LD50 is either greater than or less than or equal to the administered dose which corresponds to an established regulatory threshold; and,

• statement of whether the waste sample passes or fails the bioassay for designating as a dangerous waste, special waste, or extremely hazardous waste, according to the Dangerous Waste Regulations, Chapter 173-303 WAC.



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•

• References

ASTM. 1994a. Standard test method for estimating acute oral toxicity in rats. Method E 1163-90. 1994Annual Book of ASTM Standards, Volume 11.04, American Society for Testing and Materials (ASTM), Philadelphia. ASTM. 1994b. Standard test method for conducting a 90-day oral toxicity study in rats. Method E 1372-90. 1994 Annual Book of ASTM Standards, Volume 11.04,American Society for Testing and Materials (ASTM), Philadelphia. CFR. 1993. Health effects testing guidelines: Acute oral toxicity. Environmental Protection Agency (EPA), Title 40, Part 798.1175. Code ofFederal Regulations (CFR),Washington, DC Dixon, 

## WJ. 1965.The up-and-down method for small samples. J. Amer. Stat. Assoc. 60:967-978. EPA. 1986. Test methods for evaluating solid waste. 3rd ed., SW-846, Volume II, Part III, Chapter 9. Office ofSolid Waste and Emergency Response, Washington, DC EPA. 1991. Alpha 2u-globulin: Association with chemically induced renal toxicity and neoplasia in the male rat. EPA/625/3-91/019F. Risk Assessment Forum, Environmental Protection Agency (EPA), Washington, DC EPA. 1993.Methods for measuring the acute toxicity ofeffluents and receiving waters to freshwater and marine organisms. 4th ed., EPA/600/4-90/027F. Ed. by CIWeber, Environmental Monitoring Systems Laboratory, Office of Research and Development, EPA, Cincinnati, OH. Finney, DJ. 1971. ProbitAnalysis. 3rd ed., Cambridge University Press, London. Zar, JH. 1984. Biostatistical Analysis. 2nd ed., Prentice Hall, Englewood Cliffs, 

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