# Biological Testing Methods 80-12

For the Designation of Dangerous Waste

Part A: Static Acut e Fish Toxicity Test Part B: Acute Oral Rat Toxicity Test

Revised June 2009 Publication no. 80-12

## Publication and Contact Information

This guidance is available on the De partment of Ecology’s website at www.ecy.wa.gov/biblio/8012.html

For more information contact:

Hazardous Waste and Toxics Reduction Program P.O. Box 47600 Olympia, WA 98504-7600

Phone: 360-407-6700

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

o Headquarters, Olympia 360-407-6000

o Northwest Regional Office, Bellevue 425-649-7000

o Southwest Regional Office, Olympia 360-407-6300

o Central Regional Office, Yakima 509-575-2490

o Eastern Regional Office, Spokane 509-329-3400

To ask about the availability of this document in a format for the visually impaired, call the Hazardous Waste and Toxics Reduction Program at 360-407-6700. Persons with hearing loss can call 711 for Washington Relay Service. Persons with a speech disability can call 877-833-6341.

# Biological Testing Methods 80-12

For the Designation of Dangerous Waste

Part A: Static Acut e Fish Toxicity Test

Part B: Acute Oral Rat Toxicity Test

Hazardous Waste and Toxics Reduction Program Washington State Department of Ecology Olympia, WashingtonThis page is purposely left blank

# Table of Contents

Part A: Static Acute Fish Toxicity Test

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



### Methods and Materials .............................................................................. 3 Holding Fa cilitie s ........................................................................................................ 3 Test C hambers .......................................................................................................... 4 Cleaning and Disinfect ion ..................................................................................... 4 Dilution Water ....................................................................................................... 5 Test Organisms .......................................................................................................... 6 Care and Handling ................................................................................................ 7 Hold ing ................................................................................................................. 7 Stress Indi cators ................................................................................................... 7 Sample Pr eparation ................................................................................................... 8 Sample Re quirement s .......................................................................................... 8 Particle Size Reducti on ........................................................................................ 8 Rotary Agit ation Me thod ....................................................................................... 8



### Test Procedure ........................................................................................... 9 Replicates and Control ............................................................................................... 9 Test Tem peratur e ...................................................................................................... 9 pH .......................................................................................................................... ... 9 Dissolved Ox ygen ...................................................................................................... 9 Loading .................................................................................................................... 1 0 Feeding .................................................................................................................... 1 0 Duration and Acc eptabilit y ....................................................................................... 10 Test Observations and Measurem ents .................................................................... 10 Criteria for Death ...................................................................................................... 11 Terminat ion......................................................................................................... 11 Data Analysis ...................................................................................................... 11


### Quality Assurance / Quality Control ....................................................... 11 Procedures .............................................................................................................. 11 Reference To xicants ................................................................................................ 12



### Calculation of Results ............................................................................. 12 Approac h ................................................................................................................. 13 Hypothesis ............................................................................................................... 13 Calculat ion ............................................................................................................... 13 Sample size ........................................................................................................ 13Mortalit ies ........................................................................................................... 13 F Test ................................................................................................................. 14 Equal variance t test ........................................................................................... 14 Modified t test ..................................................................................................... 14 Critical F a nd t values ......................................................................................... 15


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



## Part B: Acute Oral Rat Toxicity Test

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

Methods .................................................................................................... 19 Sample Collectio n and Preparation .......................................................................... 19 Test Anim als ............................................................................................................ 19 Pretest Conditioning ................................................................................................. 20 Procedur e ................................................................................................................ 20



### Calculation of Results ............................................................................. 20 Approac h ................................................................................................................. 21 Hypothesis ............................................................................................................... 21 Calculation (N < 30) ................................................................................................. 21 Calculation (N = 30) ................................................................................................. 23


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

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

Appendices A. Neutralized Fish Toxici ty Test Pr ocedure ........................................................... 27 B. Good Laboratory Pr ocedure Standar ds .............................................................. 29 C. Water Q uality ...................................................................................................... 33 D. Dissolved Oxy gen Table ..................................................................................... 34 E. Recommended Fi sh Loading .............................................................................. 35 F. Sample Data Sheet ............................................................................................ 36 G. Recommended Test Conditions and Pr ocedures for Fish Bioassay ................... 39



### Glossary .................................................................................................... 41

Terminology.............................................................................................. 43

# List of Figures and Tables

Figures

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



## Tables

Table 1. Preparation of Dilution Wate r Using Reagent Grade Chemicals ......................................5

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

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

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

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

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

Table 7. Summary for Modified T Test .........................................................................................1 7

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

Table 9. Approximate Saturation and Concentrati on Values of Dissolved Oxygen in Freshwater at 12 ° C ............................................................................................................................. 34

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

Table 11. Discrete and Cumulative Binomial Probabilities for P o =0.5 .........................................221 

# Part A: Method 80-12 Static Acute Fish Toxicity Test

Introduction

The Washington State Department of Ecology (Ecology) developed the acute fish toxicity test (Method 80-12) to determine if a waste meets the definition of dangerous waste in the Dangerous Waste Regulations , Chapter 173-303 WAC. Appendix A cont ains a modified test procedure for conducting neutralized fish toxicity tests.

Method 80-12 provides a simple, low cost method for testing the toxicity of a waste against the requirements of WAC 173-303. This met hod determines if the sample waste LC 50 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 LC 50 , for this method’s purposes, is the median lethal concentration of waste that kills 50 percent of the test fish within 96 hours.

If the toxicity of a waste is unk nown, the waste must be tested fo r dangerous waste designation using Method 80-12. The waste concentr ations of 100 mg/L and 10 mg/L were selected to correspond with the definitions of dangerous waste and extr emely hazardous waste, re spectively. State-only special waste uses a regulatory threshold of 10 mg/L.

Waste designated by Method 80-12 must be regulated and managed as specified in WAC 173-303, and must be sampled according to procedures in WAC 173-303-110, Sampling and Testing Methods.

Any person whose waste fails the acute fish toxic ity test, and who believes that mortality resulted from effects other than acute t oxicity (e.g., disease or physical stress), may petition Ecology to exempt the waste from designation. They may also petition Ecology to modify the test procedure. Any person who believes that test failure resulted only because of pH may choose to demonstrate this by performing a neutralized fish bioassay pe r Appendix A. The purpose of this demonstration would be to revise waste designation as prov ided in WAC 173-303-910( 5). Any pH wastes containing hazardous constituents with inherent toxicity (e.g., ammoni a, cyanide) will not qualify for designation changes. If additional toxicity informa tion is needed to further characterize a waste, a toxicity identification evaluation (TIE) procedure may be useful (EPA, 1991a ). Petitions must be submitted per WAC 173-303-910.



### Health and Safety

Development and maintenance of an effective health and safety program in the laboratory requires an ongoing commitment by laborator y management, and includes:

• Appointment of a laboratory health and safety of ficer with the responsibility and authority to develop and maintain a safety program.2 • Preparation of a formal, written, health and safe ty plan, which is provide d to each laboratory staff member.

• Ongoing training program on laboratory safety.

• Regularly scheduled, documen ted, safety inspections.

Toxicity testing may involve sign ificant risks to personal safety and health. Personnel conducting toxicity tests should protect themselves by taking all safety precautions necessary to prevent bodily injury. Observe procedures to prevent inhalation or skin absorption of wastes. Each laboratory should be equipped with all necessary safety e quipment prior to conducti ng toxicity tests.

Personal safety gear, such as rubber aprons, lab coats, respirators, gl oves, safety glasses, and safety shoes, should be used by personnel. Each laborat ory should have safety equipment such as first aid kits, fire extinguishers, fire alarms, and fire blankets.

Because the chemical composition of waste is usually poorly known, consider them potential health hazards. Minimize exposure to waste sa mples. Fume and canopy hoods over test areas must be used whenever necessa ry. Strong acids and volatile organic solvents employed in glassware cleaning must be used in a fume hood or with an exhaust canopy over the work area.

The following recommended laboratory procedures will promote a safe working environment and minimize health risks. This list is by no means exhaustive. Ecology encourages testing laboratories to implement these, and other, recommendations promo ting the health and safety of laboratory personnel.

• Testing industrial wastes s hould be performed in complian ce with accepted rules pertaining to the handling of hazardous materials. Ecology recommends that personnel conducting toxicity tests not work alone.

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

• Immediately wash any body part that may have come in contact with wastes. Although a water rinse often suffices, note that some wastes may be activated with water.

• Adequately label all containers to indicate contents.

• Good housekeeping habits contribute to safety and reliable results.

• Use electrical equipment approved by Underwri ters Laboratories. Ground-fault interrupters should be installed in all “wet” labs where electrical equipment is used.

• Staff training in basic first aid and cardio -pulmonary resuscitation (CPR) is strongly recommended.

• All personnel should receive tetanus immuni zations. The following are optional and dependent on the type of waste anticipated: hepatitis B (if suspected contact with human bodily fluids, including blood); hepatitis A, t yphoid fever, and polio (if suspected contact with human feces or urine). Polio may be transmitted from infant immunizations through fecal material. Please err on the side of caution. If your laboratory has any questions, contact the Washington Department of Labor and Industries or si milar agency in your state.3 • Wastes generated during toxicity testing must be handled properly and safely disposed. Each testing facility will have its own waste dispos al requirements based on local, state, and federal rules and regulations. All persons re sponsible for, or otherwise involved in, performing testing activities must know, unde rstand, and comply with these rules and regulations. Local fire offici als should be notified of any potentially hazardous conditions.

For further guidance on safe laboratory practi ces, consult EPA (1986), EPA (2002), and Walters and Jameson (1984). Also see Appendix B for Good Laboratory Procedure Standards.



## Methods and Materials

Holding Facilities

Facilities should include tanks for holding/acclimating test fish and a constant-temperature room or recirculating water bath for the test chambers. Holding tanks must be operated on a flow-through basis; this may include either once-through or recirculating sy stems with at least 50 percent renewal. Holding, acclimation, and dilution-wa ter tanks and headboxes should be equipped for temperature control and aeration. Air used for aeration must be free of oil and fumes; filters to remove oil and water may be used. Test fish shou ld be shielded from disturbances during holding, acclimation, and testing. The test facility s hould be well ventilated and free of fumes.

Fish should be quarantined at l east 7 days when first brought into a facility. Avoid crowding to maintain test fish in good condition during hol ding and acclimation. The dissolved oxygen (DO) concentration must be maintained between 60 percent a nd 100 percent saturation; gentle aeration may be used if necessary. Aeration drives o ff the ammonia from gene rated biological waste.

A photoperiod of 16 hours of light/8 hours of da rkness should be provided during the test. Preferably, though not required, light intensity should be raised gradually over a 15 minute period at the beginning of the photoperi od. Gradually lower the light inte nsity over the same period of time at the end of the photoperiod. Use a dimmer switch or other suitable device to make this easier. The light intensity shoul d be in the range of natural ou tdoor light or ambient laboratory illumination (about 10-20 uE/m 2 /s or 50-100 ft-c). Avoid high intensity and extreme lighting conditions.

Whether fabricated in the lab or purchased co mmercially, tanks should no t be constructed of materials, or have surface coati ngs, which will leach or dissolve into aqueous solutions. Tanks and other equipment that contact stoc k solutions or test solutions should be chosen to minimize sorption of toxicants from water. Use glass, stai nless steel, and fluoroplasti cs whenever possible to minimize leaching, dissolution, and sorption. Conc rete and rigid (unplastic ized) plastics may be used for holding and acclimation tanks and in the wa ter supply system. Cast iron pipe may be used in freshwater supply systems, but colloidal iron w ill be added to the dilution water and strainers will be needed to remove rust particles. Natura l rubber, copper, brass, galvanized metal, solder, and lead should not come in contact with dilution water, stock soluti ons, or test solutions before or during exposure of test fish.4 

### Test Chambers

Test chambers should have a mini mum solution depth of 15 cm. Ch ambers filled to within the top 15 cm should be covered to prevent test fish from jumping out. Test chambers commonly used in static tests for fish are wi de-mouth glass jars or aquaria, 3.8 L (1 gal) or 19.0 L (5 gal). Special glass or stainless steel test chambers can be constructe d to accommodate test fish requiring particular physical conditions (large surface-area-to-volume ratio).

Test chambers may be constructed of 3-mm (0.125 in), double strength, or 6-mm (0.25 in) tempered plate glass held together with clear silicone adhesive. Silicone a dhesive absorbs some organochlorine and organophosphorus pesticides, whic h are difficult to remove. As little of the adhesive as possible should be in contact with wate r; extra beads of adhesive inside the containers should be removed. Stainless steel (No. 304 or No. 316) can be us ed in the construction of test chambers, but should be of welded, not soldered , construction.

Commercially available test chambers meeting th e above requirements may be used. Disposable, non-toxic plastic (e.g., polypropylen e) liners may be used. Ecology highly recommends laboratories using disposable liners perform several rounds of in-house testing to determine that the liners are truly non-toxic before using in dangerous waste designation testing.

Cleaning and Disinfection Clean test chambers before use. Wash new test chambers with ammonia-based detergent suitable for biological laboratories, then ri nse in the following sequence with:

• Clean water.

• Acid, such as 5 percent c oncentrated nitric acid.

• Copious clean water.

• Reagent grade acetone.

• Copious clean water.

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

• Emptied.

• Immediately rinsed with clean water.

• Cleaned using appropriate procedures for rem oving the toxicant (e.g., acid to remove metals and bases; detergent, organic solvent, or activated carbon to rem ove organic compounds.

• Rinsed with copious clean water.

Acid is useful for removing mineral deposits and 200 mg hypochlorite/L for one hour is useful for removing organic matter and for disinfection. A solution containing 200 mg hypochlorite/L is conveniently made by adding 6 mL of liquid household chlorine bleach to 1 L of water. Acid and hypochlorite should not be us ed together because hazardous ch lorine fumes may be produced. Tanks may also be sterilized with an iodop hor. Commercially prep ared iodophors using polyvinylpyrrolidone-iodine (PVP-I) as the active ingr edient, are available as Wescodine, Betadine, Argentyne, or other product names.

For disinfection of equipment such as fish tanks, prepare a 50 mg/L solution of titrateable iodine by diluting a commercially available pr oduct. The equipment should be immersed in or washed with5 this solution for at least 10 mi nutes followed by copious rinsing with clean water. Iodophors and hypo-chlorites are acutely toxic to most aquatic organisms. Hypochlorite can be removed by flushing with water or by reacting it with an eq uivalent concentration of sodium thiosulfate or sodium sulfite. After a tank is washed with hypoch lorite and rinsed, air-dry the equipment overnight to allow dissipation of residual chlorine.

Dilution Water An adequate supply of quality dilution water must be available. Acceptable test dilution water allows healthy fish to survive for the duration of acclimation and testing wit hout showing stress, such as discoloration or unusual behavi or. A better criterion for acceptab le dilution water is test fish surviving and growing satisfactorily in it. Monthly hardness, al kalinity, and specific conductance ranges must be within less than 10 percent of their respective averag es. The monthly pH range must be less than 0.8 units to produce c onsistent quality dilution water.

Ideally, non-chlorinated dilution water is preferred, but may not be realistic. Dechlorination using activated carbon filters or other methods may be used to dechlorinate dilution water as thoroughly as possible. Chlorine may also be removed using an activated carbon f ilter then treating with a sodium thiosulfate or sodium sulf ite drip. Sodium thiosulfate is bette r for dechlorinating water than sodium sulfite and may be more reliable than activ ated carbon for removing chloramines.

Metals can usually be removed with chelating agents. Municipal water supplies often contain unacceptably high concentrations of copper, lead, zinc, and fluoride; buildings may contain copper pipes and lead solder. Toxicity tests can be adversely affected by the use of dilution water containing these materials.

The dilution water should be intensively aerated usi ng air stones, surface aerators, or screen tubes before introducing the toxicant. Adequate aeratio n will bring the concentration of dissolved oxygen and other gases into equilibrium with air and mi nimize oxygen demand and volatiles concentration. The dilution water dissolved oxygen should be be tween 90 - 100 percent saturation. Rinse test chambers with dilution wa ter just before use.

Dilution water hardness can affect th e outcome of the test. Moderate ly hard dilution water must be used in this method and should not be less than 40 mg/L CaCO 3 . Low hardness/alkalinity may render test organisms more sensitiv e to some toxicants. Use reag ent-grade chemicals to adjust hardness and alkalinity. Tabl e 1 is for guidance purposes.

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

Water Type NaHCO 3 mg/L b CaSO 4 • 2H 2 O mg/L b MgSO 4 mg/L b KCl mg/L b pH c Total Resulting Hardness d Total Resulting Alkalinity d
Soft 48.0 30.0 30.0 2.0 7.2-7.6 40-48 30-35 Moderately Hard 96.0 60.0 60.0 4.0 7.4-7.8 80-100 60-70

a Adapted from EPA (2002)
b Add reagent grade chemicals to distilled or deionized water.

c Approximate equilibrium pH after aerating 24 hours.
d Expressed as mg/L CaCO 3 .6 

### Test Organisms
Use one of the following species in the static fish toxicity test (all species must be verified): Coho salmon, Oncorhynchus kisutch ; rainbow trout, Oncorhynchus mykiss (formerly Salmo gairdneri ); and brook trout, Salvelinus fontinalis . The test temperature for all species is 12 + 1 o C.

All fish used in a test should be of the same ag e and from the same source. Organisms of the same species from different sources may produce different test results. Usual sour ces of freshwater fish are private, state, and federal hatcheries.

Test fish should be between 30 and 90 days from swim-up and as uniform in size as possible. Circumstances may arise where this range cannot be met; some variation in age is acceptable, provided the variation is no more than four or five days. In any si ngle test, all fish should be from the same batch. The standard length (tip of s nout to end of caudal pedunc le) of the longest fish should be no more than twice that of the shortest fish (Figure 1). All test fish should be obtained from a hatchery that has been certified disease-free for the following diseases; bact erial kidney disease ( Renibacterium salmoninarum ); Costia ( Ichythobodo ); bacterial gill disease ( Myxobactriea sp. ); and Furunculosis ( Aeromonas salmonicida ). If fish are diseased, destroy the entire lot immediately. Make all efforts to ensure test fish are disease-free prior to initiation of toxic ity test. Opti ons include:

• Selecting fish from a source (hatchery) which will certify that all fi sh are disease-free.

• Discriminately selecting test fish from a reputable source or on the observations and judgment of a knowledgeable individual.

• Treating all fish that arrive on-site using treatments commonly used by sources (hatcheries) which produce certified disease-free fish. FIGURE 1.7 Between use of different test fish groups, hol ding and acclimation tanks should be sterilized according to the procedures in Cleaning and Disinfection

.
Care and Handling Minimize unnecessary stress to test fish. To avoi d unnecessary stress, do not subject fish to rapid changes in temperature or water quality. Generally, a quatic organisms should not be subjected to more than a 3°C change in water temperature in any 12 hour period. Maintain test fish in dilution water at test temperature for at least the last 48 hours before they are placed in test chambers.

Handle test-fish as little as possi ble. If necessary, handle them as gently, carefully, and quickly as possible. Discard fish that touch dry surfaces, are dropped, or injured during handling. Dip nets are best for handling fish and ar e commercially available or made from small-mesh nylon netting, nylon or silk bolting cloth, plankton netting, or similar material. E quipment used to handle aquatic organisms should be sterilized between uses by autoclaving or treating with an iodophor, 200 mg hypochlorite/L, or 30 percent formalin plus 1 percen t benzalkonium chloride for at least 1 hour. Sterilize or wash hands before and after handling fish.

Holding After collection or transportati on, quarantine and acclimate fish to laboratory conditions for a minimum of 7 days before used in tests. Hold fish in uncontaminated, aerated water of constant temperature and quality in a flow-through system with a minimum flow rate of five volume exchanges per day (ASTM, 2006a). When possible, hol d fish in dilution water and as close to test temperature as possible. During long holding periods, however, it is generally easier and safer to hold fish at temperatures lower than 12°C as the metabolic rate and the nu mber and severity of disease outbreaks are reduced. Recommended fish loading for holding tanks is provided for various water temperatures, fish size, and species, in Appendix D.

If stock fish have 5 percent or greater mortality between 24 hours after arrival on-s ite and the end of the 7 day acclimation period, the entire lot should not be used for testing. If at any time during the holding period there is an abnor mal number of mortal ities, do not use the fish for testing.

Stress Indicators Test fish should be carefully observed daily du ring holding and acclimation for signs of disease, stress, physical damage, abnormal behavior, and mortality. Dead, injured, and abnormal individuals must be discarded. Vi sually examine the behavior and ex ternal appearance of the fish. If the stressor can be determin ed, remove stressor and reacclimatize for 7 days. The following physical characteristics indicate stress:

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

• Emaciation • Flipping

• Erratic swimming • Fungus/Fin Rot

• Excessive mucus production • Gasping at surface

• External parasites • Hemorrhaging

• Not eating • Hyperventilation8 

### Sample Preparation
Sample Reporting Requirements All samples must be extracted using the method de scribed below. Solid samples must be reduced in size prior to extraction unless the sample meet s the particle size criteria described below. Sample analysis must occur with 45 days of sample collection.

Ensure all phases of the sample are homogenous. Stirring may be required if the sample is not homogenous. Samples may be extracted using a rota ry agitation apparatus. The rotary agitation apparatus must be capable of rota ting the extraction bottles at 30 ± 2 rpm. The sample is allowed to extract for 18 + 2 hours at 23 + 2 o C. The extraction water is the stock dilution water used in the toxicity test.

Particle Size Reduction Prior to conducting the extraction, determine the need for reducing the sample particle size. Particle reduction is required unless the solid has a surface area per gram of material equal to or greater than 3.1 cm 2 , or is smaller than 1 cm in its narrowest dimension (i.e., is capable of passing through a 9.5 mm (0.375 in ch) standard sieve).

If the surface area is sm aller or the particle size larger than described above, prepare the solid portion of the waste for extraction by crushing, cutti ng, or grinding the waste to a surface area or particle-size.

Note: Surface area criteria are meant for fila mentous (e.g., paper, cloth, and similar) waste material. Actual measurement of surface area is not required, nor is it recommended. For materials that do not obviously meet the criter ia, sample-specific methods would need to be developed and employed to measure the surf ace area. Such methodology is currently not available.

Rotary Agitation Method

• Weigh out the correct amount of a well-mixe d sample and transfer into 500 or 1000 mL extractor bottle.

• Add 200 mL of test dilution wa ter to the extractor bottle.

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

• Mix the sample on the rotary agitation apparatus for 18 + 2 hours at 23 + 2 o C, remove the cap and pour the extraction water in to the dilution water in the fi sh test tank. Rinse all loose material from the flask into th e fish test tank with a 200 mL al iquot of stock dilution water. Place the bottle into the fish tank so that the bottle is full of wa ter and lying on its side on the tank bottom. Drop the cap liner into the fish test tank.

• Begin the test as described below.

• Extreme care should be exercised throughout the course of the extract ion so as not to contaminate the exterior of the extraction flask. For example, sealing sterile flasks in sterile aluminum foil prior to use or copiously rinsi ng with deionized water, at a minimum, are acceptable methods.9 

## Test Procedure

Replicates and Control
Use a minimum of ten fish in each of three rep licate test tanks exposed to each treatment. Additional fish and replicates may be used (e.g., 15 fi sh in each of four re plicates). Replicates must be true replicates with no water connections between the repl icate test chambers. Randomize the replicate test chambers and impartially distribute a representative sample of test fish to the chambers.

One randomization method adds to each test cham ber no more than 20 percent of the number of test fish to be placed in each test chamber. Rep eat this process until each test chamber contains the desired number of test fish. Alternatively, fish can be assigned either by random assignment of one fish to each test chamber, random assignment of a second fish to each chamber, etc., or by total randomization. It is often c onvenient to assign fish to other c ontainers and then add them to the test chambers.

Every test requires a control consisting of the same dilution water, conditions, number of replicates, procedures, and test speci es used in the test tanks. None of the toxicants being tested is added to the control.



### Test Temperature
The test temperature for all test species must be 12°C. The actual test temperature must not deviate from 12°C by more than ± 1.0 °C at any time during the test. Continuous temperature monitoring in at least on e test tank is desirabl e. Alternatively, the maximum and minimum temperature must be record ed every 24 hours. As a second alternative, temperatures in at least one test tank mu st be recorded at least every 6 hours.



### pH

Do not adjust the pH of the test solution. Any pH adjusting will render the test void. There are provisions for demonstrating that the waste kills fish only because of pH. Appendix A includes a procedure for conducting this type of toxicity test.

The pH may tend to drift during the 96 hour test period. This drift is du e, in part, to fish respiration, increasing the CO 2 content of the water, which tends to buffer the solution. Do not adjust the pH when this drift is observed. Record the pH at the time intervals indicated on the data sheet.


### Dissolved Oxygen
The saturation value, not concentration, determin es the ease of oxygen transport across the gill surface diffusion gradient. The dissolved oxygen in each test chamber should be at least 80 percent (e.g., 8.5 mg/L at 12°C at 250 feet above sea level) of sa turation at the beginning of the test. The dissolved oxygen should not fall belo w 60 percent (e.g., 6.4 mg/L at 12°C at 250 feet above sea level) of satura tion during the first 48 hour s of the test. It must not fall below 50 percent10 saturation during the remainder of the test. The test must be voided if the dissolved oxygen falls below 50 percent saturation during the test and mortality exceeds the pass/fail threshold. Please refer to Appendix D for information on dissolved oxygen saturation and concentration levels at different altitudes.

Test solutions may be gently ae rated if dissolved oxygen does not meet the above criteria. Once aeration is initiated, continue throughout test duration. Avoid turbulence; it stresses fish, resuspends fecal matter, and increases volatiliza tion. Because evaporation readily occurs at the surface, efficient aeration can be achieved with minimum turbulence by using an air lift to transfer solution from the bottom to the surface. Aeratio n should be identical in all test chambers


### Loading
Loading in test chambers should not exceed 0.8 g/L. The g/L of test fish (w et weight, blotted dry) to solution in test chambers should not be so high as to affect test results. To determine loading, use same number of fish as per replicate but do not use these fish in the actual test. Lower loadings are recommended if dissolved oxygen does not remain above 60 per cent saturation for the first 48 hours of the test and above 50 percent satu ration after 48 hours. Limit loading to ensure:

• Concentrations of dissolved oxygen and toxicant are not decreased below acceptable levels.

• Concentrations of metabolic products doe s not increase above acceptable levels.

• Fish are not stressed due to crowding.


### Feeding
Do not feed test fish during acute toxicity tests or for a minimum of 48 hours before the test. If the test fish are fed less than 48 hours before te st initiation, fecal matte r and uneaten food may decrease the dissolved oxygen concentration and ot herwise affect the biol ogical activity of some toxicants.


### Duration and Acceptability
Begin the test by placing the flask of waste and le achate in the test chambe r on its side. The cap should also be placed in the test chamber.

The flask exterior should be sterile or as clean as possibl e to avoid intr oducing additional contamination. “Blank” flasks used in the ex traction process should be used for the control chambers.

The test begins when test fish ar e first exposed to the toxicant and ends 96 hours later. Control test chambers must show 90 percent survival or greater for test ac ceptance or validity.



### Test Observations and Measurements
Measure dissolved oxygen and the pH in all test tanks at the be ginning. Repeat these measurements every 24 hours (as long as live fish are present). Also record the dissolved oxygen and pH at the end of the test or at the time the last fish dies in the test ta nk. Hardness, alkalinity, pH, and conductivity must be measured in the test and control tanks at th e start of the test.11 Measure the same parameters at the end of the test. Measurement of calcium, magnesium, sodium, potassium, chloride, sulfate, particulate matter, and total organic car bon (TOC) or chemical oxygen demand (COD) is also desirable.

The number of dead or affected fish in each te st chamber must be counted every 24 hours after starting the test. More observati ons are desirable, especially near test initiation. Ecology suggests counting the number of dead or affected fish in each chamber 4, 8, and 24 hours (or similar time increments) after the beginning of the test. Repeat the count twice daily thereafter to the end of the
test.

Remove dead fish as soon as they are observed, or once every 24 hours. Do not stress live test fish when determining whether test fish are dead, immobilized, or otherwis e affected, and removing dead fish. Movement of test chambe rs and prodding must be very gentle.



### Criteria for Death

Death is the adverse effect most ofte n used in acute toxicity tests. The criteria for death are lack of movement, especially the absence of respiratory m ovements in fish and lack of reaction to gentle prodding. The loss of equilibrium, defined as the inability to make coordinated movement and maintain a normal upright position, is not considered mortality. The latter re sponses can be useful in interpretation of test results and should be not ed in the comment section of the data-reporting sheet.

Termination Weights and lengths of test fish may be dete rmined by weighing and measuring and discarding either; (a) a representative group of fish before the test; or (b) the control fish that are alive at the end of the test. The length should be reported as standard length and th e weights should be wet weight, blotted dry. Destroy all fi sh, both control and treatment, at termination of test, after data collection is complete.

Data Analysis Refer to Calculation of Results . The number of mort alities needed to designate a waste is no longer a single number for dangerous waste or extremely hazardous waste. This “threshold” number of mortalities is now a function of the mean and variance in the control group and the mean and variance in the test group a nd does not utilize a data transformation.



## Quality Assurance / Quality Control

Procedures
Reliable laboratory data is critical for maki ng sound environmental decisions. A good quality assurance/quality control (QA/QC) program is e ssential to ensure data reliability. Quality assurance (QA) is the total integrated program fo r assuring the reliability of monitoring data. A comprehensive QA program addresses everything affecting data and should address:

• Sample collection and preservation.12 • Laboratory services.

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

• Data management.

• Laboratory personnel.

Quality control (QC) is the routine application of statistically based procedures to evaluate and control the accuracy of results from analytic al measurements. A comprehensive QC program includes procedures for estimating and c ontrolling precision a nd bias, as well as:

• Defines data quality objectives.

• Chooses analytical methods which will meet those objectives.

• Estimates within-lab precision and bias.

• Sets up control charts.

• Participates in inter-lab tests.



### Reference Toxicants
Reference toxicants establish the validity of toxi city data. The reference toxicant measures all aspects of toxicity testing includ ing organism quality, presence and effects of stressors other than test compounds, and the quality of analytical techniques. If reference toxicant results (LC 50 s) differ significantly from previ ously established results (LC 50 control charts), a full review of laboratory test conditions and pro cedures is indicated. Results th at are not within two standard deviations of the mean LC 50 , should be considered “red flags.”

Evaluate procedures and test fish with a reference toxicant once a month or, preferably, concurrently with the test. Reference toxicant testing is required for ea ch new batch of fish regardless of whether a test has been run that mont h on another batch of fish. More frequent testing with a reference toxicant is appropriate when s ituations occur which may affect test results. For example, a change in dilution or holding wa ter conditions, new equipment or piping in the laboratory, or a change of analysts, all indicate the need for additional use of a reference toxicant.

Various compounds are commonly used as refe rence toxicants and include phenol, sodium chloride (NaCl), potassium chlori de (KCl), zinc chloride (ZnCl 2 ), and zinc sulfate (ZnSO 4 ). This is not an all inclusive list by any means. To ensu re nominal concentrations of reference toxicants, concentrations should be measured periodically.



## Calculation of Results

The bioassay results are calculated using the statis tical method described below; two examples are given. Within a 90 percent confid ence limit, the test sample fails th e bioassay by exhibiting a median lethal concentration (LC 50 ) less than or equal to (i.e., toxicity greater than or equa l to) the regulatory threshold (i.e., 1 00 or 10 mg/L) when the calculated t sta tistic is greater than a critical t value.13 

### Approach
The statistical analysis is based on met hodology presented by Zar (1984), EPA (2002), and Erickson and McDonald (1995). Because observati ons on individual fish in the same tank cannot necessarily be considered statistically indepe ndent, the experimental unit is the tank and not individual fish. This avoids a statistical error in experimental design referred to as pseudoreplication (Hurlbert, 1984).

The two-sample t test is used. This test a ssumes both control and test groups are randomly selected from normal populations with equal va riances. However, the frequency of samples displaying varying proportions of dead and live fis h, as determined in acute lethality bioassays, is described by the binomial distribu tion. Use of the two-sample t test is robust enough to stand considerable departures from its theoretical assump tions, especially if sample sizes are equal (Zar, 1984). Untransformed sample data have been show n to be normally distributed, in most cases, as assessed by Shapiro-Wilk’s test for normality (EPA, 2002).

The type of t test used depends on the homogene ity of variance assumpti on between control and test samples (Zar, 1984; EPA, 2002). This assumpti on is evaluated with the variance ratio F test. If variances are equal, the equal va riance t test is used. If variances are not equal, a modified t test is used.

Thus, the appropriate t test is used to compare the difference be tween test and control mortality proportions against a constant, p o = 0.5, corresponding to the LC 50 . Up to ten percent mortality in the control sample is allowed. A one-tailed t test is used, since the analysis determines only if the sample LC 50 is significantly greater than the regu latory threshold. This objective is consistent with the procedure for waste designation from bioassay data outlined in WAC 173-303-100(5)(c).


### Hypothesis
In terms of concentration, the one-tailed null hypothesis (H o ) to be tested is LC 50 ≤ 100 mg/L (dangerous waste threshold) or LC 50 ≤ 10 mg/L (extremely hazardous waste threshold or special waste threshold). In terms of mortality, each of these hypothese s corresponds to a one-tailed H o : (p Tm - p Cm ) ≥ p o , where p Tm and p Cm are the mean proportion of morta lities in the test and control samples, respectively, and p o = 0.5 is the proportion of mortalities at the LC 50 . An alpha level of 0.10 is used in the t test (i.e., one -tailed 90 percent confidence leve l), consistent with statistical methodology outlined by EPA (1986). Rejecting H o demonstrates the sample does not designate as a regulated waste.



### Calculation
1. Sample size: The number of replicates in the cont rol and test groups should be equal and should each consist of at least three (N

 ≥ 3). The number of fish pe r replicate tank should be equal across all replicates and shoul d consist of at least ten (n

 ≥

 10). In addition, the number of fish per replicate tank should not exceed a biomass loading density of 0.8 g/L.

2. Mortalities: Mortalities are expressed as proportions:
p C = proportion of mortalitie s in control replicate.
p T = proportion of mortali ties in test replicate.14 The bioassay is invalid if overall control mortalities exceed 10 percent (EPA, 2002).

3. F test: The variance ratio F test is used to identify the appropriate t test. A two-tailed F test is conducted at an alpha level of 0.05 with N - 1 degrees of freedom (df) in both numerator and denominator (Zar, 1984; EPA, 2002):

F = s C 2 /s T 2 or F = s T 2 /s C 2 , whichever is larger.

where: F = F statistic

s C 2 = variance for the control group.

s T 2 = variance for the test group.

If the calculated F value is less than or equal to the critical F value, the variances are equal and the equal variance t test is used. If the calculated F value is greater than the critical F value, the variances are not equal and the modi fied t test should be used. Note that in the case where both variances are zero, the variances are equal. However, when only one variance is zero, the variances are considered statistically unequal.

4. Equal variance t test: With an equal number of replic ates in the control and test groups, the equal variance t test ha s the following form (Zar, 1984): t = (p Tm - p Cm - p o ) / (2s p 2 /N) 0.5 (general equation) or t = (p Tm - p Cm - 0.5) / [(s T 2 + s C 2 )/N] 0.5

where: t = t statistic
p Tm = mean p T p Cm = mean p C p o = 0.5 = proportion of mortalities at LC 50
s p 2 = pooled variance = (s T 2 + s C 2 )/2 N = number of replicates in control or test group.

The number of degrees of freedom in an equal varian ce t test with an equal number of replicates in control and test groups is 2(N - 1) . The calculated t sta tistic is compared to a one-tailed critical t value at an alpha level of 0.10 with 2(N - 1) degree s of freedom (df). When the calculated t value is less than or equal to the critical t value, H o is rejected, and it is concluded that the waste does not designate as a regulated waste. Note that in the case where (s T 2 + s C 2 ) = 0, H o is rejected when (p Tm - p Cm ) < 0.5 and H o is accepted when (p Tm - p Cm ) ≥ 0.5.

5. Modified t test: With an equal number of replicates in the control and test groups, the modified t test for unequal variances has the same form as the equal variance t test above (Zar, 1984). However, the number of de grees of freedom differs and is equal to the following (Zar, 1984): df = (N - 1)(s C 2 + s T 2 ) 2 / [(s C 2 ) 2 + (s T 2 ) 2 ]

If the computed degrees of freedom are non- integer, use the next smaller integer. Note that if either s C 2 = 0 or s T 2 = 0, then df = N - 1. The calculated t stat istic is compared to a critical t value15 with the modified degrees of freedom. The interp retation then follows that described above for the equal variance t test.

6. Critical F and t values: Tables 2 and 3 list critical F and t values, respectively. Appropriate critical values for other degrees of freedom can be found in most statistics books (e.g., Zar, 1984). For number of degrees of freedom, see section numbers 3-5 under 'Calculation'.

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

Denominator df Numerator df 2 3 4 5 2 39.0 39.2 39.2 39.3 3 16.0 15.4 15.1 14.9 4 10.6 9.98 9.60 9.36 5 8.43 7.76 7.39 7.15

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

df t 1 df t 1 -3.078 6 -1.440 2 -1.886 7 -1.415 3 -1.638 8 -1.397 4 -1.533 9 -1.383 5 -1.476 10 -1.372

1 Critical t values are negative, since the interest lies only in whether (p T - p C ) is significantly smaller than 0.5.

Example 1: Equal Variance t Test This test consists of two treatments and one control, each in triplicate. Two sets of three replicates of ten fish each are exposed to 10 and 100 mg/L, respectively, of waste using the 80-12 bioassay procedure to determine if the waste sample desi gnates as dangerous waste or extremely hazardous waste. The control group is exposed to dilu tion water only.

Table 4. Test Results

Replicate Dilution Water Control (n=10) 10 mg/L Sample (n=10) 100 mg/L Sample (n=10) (N=3) Number Dead Proportion Dead (p C ) Number Dead Proportion Dead (p T ) Number Dead Proportion Dead (p T ) 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 (p m ) 0.067 0.133 0.500 Variance (s 2 ) 1 0.0033 0.0033 0.0100

1 Variance equation is s 2
= Σ (p-p
m ) 2 /N-1 where p = p C or p T16 After 96 hours, control and test tank mortalities are ta bulated. Proportions are based on number of fish per replicate (e.g., 1/10 = 0.100). The critical F and t values are found in Tables 2 and 3, respectively.

Table 5. Summary for Equal Variance T Test

10 mg/L 100 mg/L Calculated F statistic 0.0033/0.00 33 = 1.00 0.0100/0.0033 = 3.03 Critical F df; numerator & denominator each = (N - 1) 2, 2 2, 2

Critical F (see Table 1 2 ) 39.0 39.0 Calculated t 1 statistic -9.25 -1.01 Critical t df 2 = [2(N-1)] 4 4 Critical t (see Table 2 3 ) -1.53 -1.53 Does waste designate? NO YES Waste Code not applicable WT02

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

2 2(N-1) is only used in the equal variance t test

With the appropriate equations, calculate F = 1.00 (10 mg/L) and F = 3.03 (100 mg/L). Because both of the calculated F statistics are less than the critical F value (39.0) , the variances are equal and the equal variance t test is performed. The ca lculated t statistics are -9.25 (10 mg/L) and -1.01 (100 mg/L). Because the 10 mg/L calculated t statis tic (-9.25) is less than the critical t value (- 1.53), H o is rejected and the waste does not designate as extremely hazardous waste. However, because the 100 mg/L calculated t st atistic (-1.01) is grea ter than the critical t value (-1.53), H o is
not rejected and the waste does designate as dangerous waste.


### Example 2: Modified t Test
This test consists of two treatments and one cont rol, each in quadruplicat e. Two sets of four replicates of 15 fish each are exposed to 10 a nd 100 mg/L, respectively, of waste using the 80-12 bioassay procedure to determine if the waste samp le designates as dangerous waste or extremely hazardous waste. The control group is exposed to dilution water only.

Table 6. Test Results

Replicate Dilution Water Control (n=15) 10 mg/L Sample (n=15) 100 mg/L Sample (n=15) (N=4) Number Dead Proportion Dead (p C ) Number Dead Proportion Dead (p T ) Number Dead Proportion Dead (p T ) 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 (p m ) 0 0.217 0.550 Variance (s 2 ) 1 0 0.0278 0.0130

1 Variance equation is s 2
= Σ (p-p
m ) 2 /N-1 where p = p C or p T17 After 96 hours, control and test tank mortalities are ta bulated. Proportions are based on number of fish per replicate (e.g., 1/15 = 0.067). The critical F and t values are found in Tables 2 and 3, respectively.

Table 7. Summary for Modified T Test

10 mg/L 100 mg/L Calculated F statistic The variances are cons idered statistically unequal because the control variance is zero. Therefore, the modified t test is used. Calculated t 1 statistic -3.39 0.88 Critical t df 2 = (N-1) 3 3 Critical t (see Table 2 3 ) -1.64 -1.64 Does waste designate? NO YES Waste code not applicable WT02

1 10 mg/L calculated t = (0.217-0-0.5)/[(0+0.0278)/4] 0.5 & 100 mg/L calculated t = (0.550 00.5)/[(0+0.0130)/4] 0.5

2 (N - 1) is used in the m odified t test when either s C 2 = 0 or s T 2 = 0

Zero variance in the control group dictates that the variances be considered unequal by the F test rule stated above. Therefore, the modified t test is employed and the calcul ated t for 10 mg/L is - 3.39 and for 100 mg/L is 0.88. Because the 10 mg/L cal culated t statis tic (-3.39) is less than the critical t value (-1.64), H o is rejected and the waste does not designate as extremely hazardous waste. However, because the 100 mg/L calculated t statistic (0.88) is greate r than the critical t value (-1.64), H o is not rejected and the waste does designate as dangerous waste.


## Data Reporting

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

• Name of test, investigator, labor atory, and date test was begun.

• Detailed description of waste tested, its source, lot number, date of sample collection, known composition, known physical and chemical properties.

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

• Detailed information about test organisms to include scientific name and how verified, wet weight, blotted dry, standard length for fis h, age, life stage, source, history, observed diseases, treatments, acclima tion procedure, food used.

• Description of experimental design and test chambers, test solution volume depth in chambers, number fish per replicate and tr eatment, loading, and lighting description.

• Description of any aeration perf ormed before or during test.

• Definition of criterion used to determine endpoi nts/effects, summary of general observations.

• Number and percentage of fish deaths, per re plicate and per treatment , in control and test chambers on a daily basis.

• Date run, identification and concentration of re ference toxicant used and resulting mortality and LC 50
.18 • Methods used for and results with standard de viation of all chemical analyses of water quality and toxicant concentr ation, including validation st udies and reagent blanks.

• Value of following: acclimation temperature, test temperature, pH, hardness, alkalinity, dissolved oxygen, and conductivity.

• Anything unusual about the test, any deviations from procedures, and any other relevant information.19 

# Part B: Method 80-12 Acute Oral Rat Toxicity Test

Introduction

The acute oral rat bioassay is ge nerally used to determine the rela tive acute toxicity of an unknown substance. This acute oral rat bioassay was developed by the Washington Department of Ecology (Ecology) for the purpose of determining if a wast e meets the definition of dangerous waste in the Dangerous Waste Regulations , Chapter 173-303 WAC.

This method determines lethality an d signs of acute toxicity from a wa ste sample administ ered in a single dose by gavage to a limited number of rats. The bioassay determines if the test sample exhibits a median lethal dose (LD 50 ) either greater than or less than or equa l 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 ar e doses of 5000, 500, and 50 mg/kg (weight of test substanc e per unit body weight), respectiv ely. The waste sample fails the bioassay by exhibiting an LD 50 statistically less than or equal to (i.e., toxicity greater than or equal to) the pertinent regulatory threshold.



## Methods

Methodology has been adapted from acute oral toxicity testing, outlined by the EPA in the Code of Federal Regulations (CFR, 1993) and by Committee E-35 on Pesticides with th e American Society for Testing and Materials (ASTM, 1994a; ASTM, 1994b).


### Sample Collection and Preparation
A representative sample of waste should be obtai ned, using methods approve d by Ecology. The waste sample should be homogenized and eith er dissolved or suspended in an appropriate vehicle, so that the material is in a homogeneous liquid form amenable to gavage. In decreasing orde r of preference, water, saline, or oil (e.g., corn oil) are recommended for the vehicle. The test solution concentration (i.e., weight of waste sample per volume solution) shoul d not exceed 1 mL/100 g body weight, except when an aqueous solution is used, wher e 2 mL/100 g is acceptable (CFR, 1993).

The vehicle and test solution should be at the same temperature as the test room. Record the type of vehicle, test solution concen tration, and temperature and phot operiod of the testing room.



### Test Animals
Use common laboratory strains of albino rats (e.g., Sprague-Dawley) weighing 190 to 300 g, prefasted. At each dose level tested (5000, 500, or 50 mg/kg), use a minimum of five males and five females. Larger samples are allowed; maintain an equal numb er of male and female rats. Include the same number of animals, and equal numbers of males and fe males, in the vehicle control group as in the test group. Female rats should be nulliparous and nonpre gnant. Use both males and females to incorporate possible gender differences in acute toxicity (Dixon, 1965) a nd renal metabolism (EPA, 1991b).20 

### Pretest Conditioning

Animals should not have be en used for any other tests and should be maintained acco rding to accepted laboratory practices for the care and handling of test animals. Identify and cag e individually each animal. Rat chow (or equivalent) and water must be available ad libitum until 18 to 20 hours before test initiation, at which time depriv e animals of food. Examine each animal for overt signs of dise ase. Acclimate to the test environment for a minimum of seven da ys. During acclimation, observe animals for adve rse health effects. Any animals demonstrating si gns of spontaneous disease should be eliminated prior to test initiation. Use only animals judged to be healthy.



### Procedure
Administer doses of 5000, 500, or 50 mg/kg to evaluate whether the waste sample designates as dangerous waste, special waste, or extr emely hazardous waste, respectively. This dose is expressed as weight of the test substance per unit body weight. Determine the fasted bo dy weight of each rat at the start of the test to calculate the volume of test solution (i.e., waste sample in vehicl e) needed to delive r the desired dose.

Administer the test solution in a sing le dose by gavage, using a stomach tube or suitab le intubation cannula. As stated above, th e volume of test solu tion should not exceed 1 mL/100 g body we ight, except when an aqueous solution is used, where 2 mL/100 g is accep table (CFR, 1993). Deliver a uniform dose to rats with varying body weights by ad justing the volume of the pr epared test solution of known waste concentration.

A concurrent vehicle control group should be treated id entically to the test group with the notable exception that only vehicle (i.e., no waste sample) should be ad ministered. Control group animals should receive a volume of vehicle per unit body weight (mL/kg) equal to the volume of test solution pe r unit body weight (mL/kg) administered to the test group animals. Record the volumes of vehicl e and test solution administered to control and test group animals, respectively.

After dosing, immediatel y return all animals to individual cages with ad libitum food and water. Observe animals for mortality and signs of acute toxicity immedia tely after dosing, at one and four hou rs after dosing, and daily thereafter for 14 consecutiv e days. Cage-side observ ations should include evaluation of skin and fur, eyes and mucous membranes, respiratory, circulator y, autonomic and central nervous systems, somatomotor activity, and behavior patt erns. Particular attention should be directed toward noting tremors, convulsions, lethargy, sa livation, and diarrhea. Record any mortalities an d sign of acute toxicity.

Temperature of the testing r oom should be recorded daily. Body we ights should be determ ined and recorded at the start of the test, on day 7, and at death.

Sacrifice surviving animals at the end of the test on day 14. Perform gross necropsies on all animals in both control and test groups. Record all gross pathology changes.


## Calculation of Results

The bioassay result is calculated using the statistical method described below. Three examples are also presented. Within a 90 percent conf idence limit, the test sample fails the bioassay by exhibiting a median lethal dose (LD 50 ) less than or equal to (i.e., toxicity greater than or equal to) the regulatory threshold (i.e., 5000, 500, or 50 mg/kg) when the cumulative binomial probability exceeds the established alpha level for small samples (N < 30), or when the calculated Z st atistic is greater than -1.28 for larger samples (N ≥ 30).21 

### Approach
The frequency of samples di splaying varying proportions of dead and live rats, as determined in acute lethality bioassays, is described by the binomial dist ribution. Due to the stat istical independence among observations on individually caged rats, the experimental unit is the individual rat, and results from the dangerous waste oral rat bioassay can be analyzed with a binomial goodness of fit test. The observed proportion of mortalities in the test group is co mpared to a constant, p o = 0.5, which corr esponds to the LD 50
. A one-tailed test is employed , since we wish only to determine if the sample LD 50 is significantly greater than the regulatory threshold. This objective is c onsistent with the procedure for waste designation from bioassay data outlined in WAC 173-303-100(5)(c).

Two binomial methods are applied, depending on sample size (N) of the test group. Both methods incorporate a correction for up to 10 percent mortality in the control gr oup. For N < 30 , the cumulative binomial probability of mortalities is determined directly from the binom ial distribution. For N ≥ 30, the normal approximation to the binomial test is employ ed (Zar, 1984).


### Hypothesis
In terms of concentration, th e one-tailed null hypothesis (H o ) to be tested is LD 50 ≤ 5000 mg/kg (dangerous waste threshold), LD 50 ≤ 500 mg/kg (special wa ste threshold), or LD 50 ≤ 50 mg/kg (extre mely hazardous waste threshold). In terms of mortality, each of these hypothes es corresponds to a one-tailed H o : p T a ≥ p o , where p T a is the proportion of cont rol-adjusted mortalities in the test group and p o = 0.5 is the proportion of mortalities at the LD 50 . An alpha level of 0.10 is used (i.e., on e-tailed 90 percent confidence level), consistent with statistical methodology outlined by EPA (1986). Rejecting H o demonstrates that the sample does not designate as a regulated waste.


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

2) Control correction The bioassay is invalid if control mortalities exceed 10 percent (E PA, 2002). Correc tions for up to 10 percent mortality in the control are applied with Abbott's fo rmula (Finney, 1971; EPA, 2002):

p T a = (p T - p C ) / (1 - p C ) where: p T a = control-adjusted proportion of mortalities in test group
p T = observed proportion of mortalities in test group
p C = observed proportion of mo rtalities in control group

Note that p T a = p T when p C = 0. If p C ≤ 0.10 and p C > p T , then p T a = 0 as a result of data smoothing (EPA, 2002). That is, p T and p C are each set equal to their average, (p T + p C ) / 2, so that p T a = 0.

3) Binomial probability The cumulative binomial probability for the control-adju sted number of mortali ties in the test group is determined by summing the discrete probabilities a ssociated with all mortalities from zero to Np T a

, where Np T a = control-adjusted numbe r of mortalities. Np T a may be non-integer, in which case, the next22 highest integer should be used. Disc rete binomial probabilities can be ob tained from statistical tables or from the following equation (Zar, 1984):

P(X) = [N! / X!(N - X)!]p o X (1 - p o ) (N - X) (general equation)

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

where: P(X) = discrete probabilit y of X mortalities in test group N = number of rats in test group X = number of morta lities in test group
p o = 0.5 = proportion of mortalities at the LD 50
If the resultant cumulative binomial probabi lity is less than or equal to alpha ( ≤ 0.10), then H o is rejected and it is concluded that the waste does not designate as a regulated waste. Discrete and cumulative binomial probabilities for p o = 0.5 and several sample sizes are presented in Table 11 below.



### Table 11. Discrete and Cumulati ve Binomial Probabilities for p o 

### = 0.5
Discrete (D) and Cumulative (C) Binomial Probabilities for p o = 0.5 and N = 10, 15, 20, and 25.
X N = 10 N = 15 N = 20 N = 25 D C D C D C D C 0 0.0010 0.0010 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 1 0.0098 0.0108 0.0005 0.0005 0.0000 0.0000 0.0000 0.0000 2 0.0439 0.0547 0.0032 0.0037 0.0002 0.0002 0.0000 0.0000 3 0.1172 0.1719 0.0139 0.0176 0.0011 0.0013 0.0001 0.0001 4 0.2051 0.3770 0.0417 0.0593 0.0046 0.0059 0.0004 0.0005 5 0.2461 0.6231 0.0916 0.1509 0.0148 0.0207 0.0016 0.0021 6 0.2051 0.8282 0.1527 0.3036 0.0370 0.0577 0.0053 0.0074 7 0.1172 0.9454 0.1964 0.5000 0.0739 0.1316 0.0143 0.0217 8 0.0439 0.9893 0.1964 0.6964 0.1201 0.2517 0.0322 0.0539 9 0.0098 0.9991 0.1527 0.8491 0.1602 0.4119 0.0609 0.1148 10 0.0010 1.0000 0.0916 0.9407 0.1762 0.5881 0.0974 0.2122 11 0.0417 0.9824 0.1602 0.7483 0.1328 0.3450 12 0.0139 0.9963 0.1201 0.8684 0.1550 0.5000 13 0.0032 0.9995 0.0739 0.9423 0.1550 0.6550 14 0.0005 1.0000 0.0370 0.9793 0.1328 0.7878 15 0.0000 1.0000 0.0148 0.9941 0.0974 0.8852 16 0.0046 0.9987 0.0609 0.9461 17 0.0011 0.9998 0.0322 0.9783 18 0.0002 1.0000 0.0143 0.9926 19 0.0000 1.0000 0.0053 0.9979 20 0.0000 1.0000 0.0016 0.9995 21 0.0004 0.9999 22 0.0001 1.0000 23 0.0000 1.0000 24 0.0000 1.0000 25 0.0000 1.0000



### Example 1 Ten rats each in the control and test group (N = 10) are orally dos ed at 0 and 5000 mg/kg of waste, respectively, according to the bioassay procedure to determine if the waste sample designates as a23 dangerous waste. After 14 days, 3 ra ts have died in the test group (p T = 0.300) and zero control mortalities (p C = 0) are observed. Therefore, p T = p T a and Np T a = (10)(0.300) = 3. For p o = 0.5 and N = 10, discrete binomial probabilities for 0, 1, 2, and 3 deaths are 0.0010, 0.0098, 0.0439, and 0.1172, respectively (Table 1). Summing th ese probabilities, it is apparent that the cumulative probability for ≤ 3 deaths is greater than the established al pha level of 0.10 (i.e., 0.1719 > 0.10). Therefore, H o is not rejected and the waste designa tes as a dangerous waste.



### Example 2 Twenty rats each in the control a nd test group (N = 20) are orally dos ed at 0 and 5000 mg/kg of waste, respectively, according to the bioassay procedure in or der to determine if the waste sample designates as a dangerous waste. After 14 days, 7 rats have died in the test group (p T = 0.350) and 2 control mortalities (p C = 0.100) are observed. Therefore, p T a = 0.278 and Np T a = (20)(0.278) = 5.56. Np T a is then rounded to 6 deaths, as described above. For p o = 0.5 and N = 20, discrete binomial probabilities for 0, 1, 2, 3, 4, 5, and 6 deaths are 0, 0, 0.0002, 0.0011, 0.0046, 0.0148, and 0.0370, respectively (Table 1). Summing these probabilities, it is a pparent that the cumulative probability for ≤ 6 deaths is less than the established alpha level of 0.10 (i.e., 0.0577 < 0.10). Therefore, H o 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 pe rcent (EPA, 993). The control correction is performed with Abbott's formula as described above.

2) Normal approximation Based on the mean and standard deviation for a bi nomial distribution, the following formula has been adapted from Zar (1984) to evaluate how p T a differs from p o = 0.5 in terms of a normal distribution:

Z = (p T a - p o ) / [p T a (1 - p T a )/N] 0.5 (general equation)

or Z= (p T a - 0.5) / [p T a (1 - p T a )/N] 0.5

where: Z = normal deviate
p o = 0.5 = proportion of mortalities at the LD 50 N = number of rats in test group

The calculated Z test statistic is co mpared to a one-tailed critical Z va lue at an alpha level of 0.10 (i.e., Z = -1.28). When the calculated Z value is

 ≤ -1.28, H o is rejected, and it is c oncluded that the waste does not designate as a regulated waste.



### Example 3 Fifty rats per control and test groups (N = 50) are orally dosed at 0 and 5000 mg/kg of waste, respectively, according to the bioassay procedure in or der to determine if the waste sample designates as a dangerous waste. After 14 days, 20 rats have died in the test group (p T = 0.400) and 4 control mortalities (p C = 0.080) are observed. From these data, p T a = 0.348 and Z = -2.26. Because the calculated Z statistic (-2.26) is less than the critical Z value (-1.28), H o is rejected and the waste does not designate as dangerous waste.24 

## Data Reporting

The report must contain the following information:

• Study initiation and termination dates.

• Description of testing facility.

• Source, genetic strain, and sex of control and test animals.

• Description of sample preparati on, including type of vehicle used and test solution concentration.

• Dose of test solution and volumes of vehicle and test solution admini stered to respective control and test groups.

• Animal body weights at test in itiation, day 7, and at death in both control and test group.

• Mortalities and signs of acute to xicity immediately after dosing, at one and four hours after dosing, and daily thereafter for 14 consecutive days in both control and test groups.

• Photoperiod and daily temp erature of testing room.

• Gross necropsy results.

• Statistical analysis to evaluate whether the waste sample LD 50 is either greater than or less than or equal to the administered dose which correspon ds to an established regulatory threshold.

• Statement of whether the waste sample passes or fails the bioassay and designates as a dangerous waste, special waste, or extremel y hazardous waste, according to the Dangerous Waste Regulations

, Chapter 173-303 WAC.



## References

ASTM. 2006a . Standard guide for conducting early life-stage toxicity tests with fishes: E1241-05. In: 2006 ASTM Standards on Aquatic Toxicology and Hazard Evaluation, ASTM, Philadelphia, Pennsylvania.

ASTM. 2006b. Standard test method for estimating acute oral toxicity in rats. Method E 1163- 98 (Reapproved 2002). 2006 Annual Book of ASTM Standards, Volume 11.04 5, American Society for Testing and Materials (ASTM), Philadelphia.

ASTM. 2006c. Standard test method for conducting a 90-day oral toxicity st udy in rats. Method E 1372-95 (Reapproved 2003). 2006 Annual Book of ASTM Standards, Volume 11.04 5, American Society for Testing and Mate rials (ASTM), Philadelphia.

CFR. 1993. Health effects testing guidelines: Acute oral toxicity. Environmental Protection Agency (EPA), Title 40, Part 798.1175. Code of Fe deral Regulations (CFR), Washington, DC.

Dixon, WJ. 1965. The up-and-down method for small sa mples. J. Amer. Stat. Assoc. 60:967-978.25 Environment Canada. 1990. Biolog ical test method: acute lethal ity test using rainbow trout. Conservation and Protection. Repor t EPS 1/RM/9. Ottawa, Canada.

EPA. 1986. Test methods for evaluating solid waste. 3rd ed., SW-846, Volume II, Part III, Chapter 9. Office of Solid Waste and Emergency Response, Washington, DC.

EPA. 1991a. Methods for aquatic toxicity identifica tion evaluation. Phase I toxi city characterizations, 2 nd ed. EPA/600/6-91/003. Environmental Research Laboratory, Duluth, MN.

EPA. 1991b [[[SEE TA BLE 8 footnote]]]

EPA. 1991b. Alpha 2u-globulin: Associat ion with chemically induced rena l toxicity and neoplasia in the male rat. EPA/625/3-91/019F. Risk Assessment Fo rum, Environmental Prot ection Agency (EPA), Washington, DC.

EPA 2002. Methods for measuring the acute toxicity of effluents and rece iving waters to freshwater and marine organisms. 5th ed. EPA-821-R-02-012 Environm ental Monitoring Systems Laboratory, Office of Research and Development, EPA, Cincinnati, OH.

Erickson, WP and LL McDonald. 1995. Tests for bioequi valence of control media and test media in studies of toxicity. Environ. Toxicol. Chem. 14(7):1247-1256.

Federal Register Vol. 48 No. 230, November 29, 1983, Good Laboratory Practic e Standards, pp. 53946 ff. Finney, DJ. 1971. Probit Analysis. 3rd ed., Cambridge University Press, London.

Hurlbert, SH. 1984. Pseudoreplication and the design of ecological field experi ments. Ecol. Monogr. 54(2):187-211.

Pillard, D. 1996. ENSR Corporation. Personal comm unication. 100 percent saturation concentrations calculated from regression models. Original data obtained from “I nstruction Manual YSI Models 54ARC and 54ABP Dissolved Oxygen Meters” pp. 16-17. YSI Inc., Yellow Springs, OH.

Walters, DB, and CW Jameson. 1984. Health and safe ty for toxicity testing. Butterworth Publ., Woburn, MA.

Wedemeyer, GA, and JW Wood, 1977. Stress as a predis posing factor in fish di sease. U.S. Fish and Wildlife Service. FDL-38. Washington, D.C.

Wedemeyer, GA, RL Saunders, and WC Clarke, 1981. The hatchery e nvironment required to optimize smoltification in the artificial propagation of anadro mous salmonids. pp. 6-20. In: E. Kiney and L. Allen, eds., Bio-Engineering in Fisheries. Special Publication by American Fisheries Society, Washington, D.C.

Wedemeyer, GA, DJ McLeay, C Phillip Goodyear. 1984. Assessing the toleran ce of fish and fish populations to environmental stress: th e problems and methods of monitoring. Zar, JH. 1984. Biostatistical Analysis. 2nd ed., Prentice Ha27 

# Appendix A

Neutralized Fish Toxicity Test Procedure

The neutralized fish toxicity test procedure is identical to the Part A method except as noted below. The only difference is that aquarium water is ne utralized after the waste is introduced into the aquarium, and before the fish are placed into the aquarium.

The aquarium water may be neutralized using on ly aqueous hydrochloric acid (HCl) or sodium hydroxide (NaOH). Add the neutrali zing solution dropwise while stirri ng the contents of the test tank, and measuring the pH continuo usly. The pH of the aquarium water should be adjusted to 7.5 and allowed to stand 30 minutes to detect any pH dr ift. If the pH drifts outside the range 7.2 - 7.8, the aquarium water should be readjusted to pH 7.5, and the fish introduced im mediately thereafter. Further pH adjustment is not allowed.

The neutralizing solutions should be as concentrated as practicable to minimize diluting the aquarium water. Under no circumstances should > 50 mL be used to adjust pH. Report the actual volume and normality of neutralizing solution used on the data reporting sheet.


### Conducting the Neutralized Fish Toxicity Test
The neutralized fish toxicity test requires all of the proceeding st eps for validity. Conduct steps 1 - 5 in the sequence shown.

1. Fill the test tanks and control tanks with dilution water allo wing for 400 mL of water from the sample extraction step. The dilution water mu st show hardness, alkalinity, and pH within the range for “soft” to “moderately ha rd” water as described in Table 1.

2. Introduce the waste into the test tanks. The waste incl udes any undissolved sample, the extract, the rinse, and the extraction flask or bottle.

3. Measure and record the pH.

4. Adjust the pH to 7.5 and wait 30 minutes. If th e pH drifts outside th e range 7.2-7.8, re-adjust the pH and proceed immediately to steps 5, 6, and 7.

5. Collect a sample for measuring ha rdness, alkalinity, and conductivity.

6. Measure and record dissolve d oxygen, pH, and temperature.

7. Introduce the fish into the te st tanks and control tanks.

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

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

10. Terminate the test after 96 hours.

11. Complete the data reporting sheet.28 

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

• Neutralizer (i.e., NaOH or HCl).

• Normality of neutralizer.

• Volume of neutralizer.

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

# Appendix B

Good Laboratory Standards

The following was adapted from EPA’s Good Laboratory Practice (GLP) Standards. These standards are recommended, not required. Labo ratories are encouraged to develop QA/QC programs, which address these recommended standards.


### Organization and Personnel: Personnel
Each individual engaged in the conduct of or re sponsible for the supervision of a study shall have education, training, and experience, or combination thereof, to enable that individual to perform the assigned functions.

Each testing facility shall ma intain a current summary of training and experience and job description for each individual engaged in or supervising the conduct of a study.

There shall be a sufficient number of personne l for the timely and proper conduct of the study according to the protocol.

Personnel shall take necessary personal sanita tion and health precautions designed to avoid contamination of test and contro l substances and test systems.

Personnel engaged in a study shall wear clothing a ppropriate for the duties they perform. Such clothing shall be changed as ofte n as necessary to prevent microbi ological, radiological, or chemical contamination of test systems and test and control substances.

Any individual found at any time to have an illn ess that may adversely affect the quality and integrity of the study shall be excluded from direct contact with test systems, test and control substances and any other operation or function th at may adversely affect the study until the condition is corrected. All personn el shall be instructed to repor t to their immediate supervisors any health or medical conditions that may reas onably be considered to have an adverse effect on a study.


### Biologist in Charge
For each study, a biologist, or a scientist, or ot her professional of appropr iate education, training, and experience, or combination thereof, shall be iden tified as the biologist in charge. The biologist in charge has overall responsibility for the techni cal conduct of the study, as well as for the interpretation, analysis, documentation, a nd reporting of results, and repres ents the single point of study control. The biologist in charge shall assure that:

• The protocol, including any change, is appr oved as provided by Ec ology and is followed.

• All experimental data, including observations of unanticipated responses of the test system are accurately recorded and verified.30 Unforeseen circumstances that may affect the qu ality and integrity of the study are noted when they occur, and corrective act ion is taken and documented.

• Test systems are as spec ified in the protocol.

• All applicable good laboratory pr actice regulations are followed.

• All raw data, documentation, protocols, specimen s, and final reports are transferred to the archives during or at the close of the study.



### Protocol
Each study shall have an approved written protocol that clearly indicates th e objectives and all methods for the conduct of the study. The protocol shall contain but not be necessarily be limited to the following information:

• A descriptive title and statemen t of the purpose of the study.

• Identification of the test and c ontrol substance by chemical name, Chemical Abstract Service Registry Number (CAS), or other es tablished identification system.

• The name and address of the sponsor and the na me and address of the testing facility at which the study is being conducted.

• The proposed starting and completion dates.

• Justification for selection of the test system.

• Where applicable, the number, body weight rang e, sex, source of supply, species, strain, substrain, and age of the test system.

• The procedure for identification of the test system.

• A description of the experimental design, in cluding the methods for the control of bias.

• A description and/ or identification of the diet used in the study as well as solvents, emulsifiers and/ or other materials used to solubilize or suspend the test or control substances before mixing with the carrier. The descripti on shall include specifications for acceptable levels of contaminants that are reasonably expe cted to be present in the dietary materials and are known to be capable of interfering with the purpose or conduct of th e study if present at levels greater than established by the specifications.

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

• Each dosage level, expressed in milligrams per kilogram of body weight or other appropriate units, of the test or control substance to be administered and the method and frequency of administration.

• Method by which the degree of ab sorption of the test and cont rol substances by the test system will be determined if necessary to achieve the objectives of the study.

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

• The records to be maintained.31 • The date of approval of the protocol by the s ponsor and the signature of the biologist in charge.

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

• All changes in or revisions of an approved protocol and the reasons therefore shall be documented, signed by the biologist in charge, dated, and maintained with the protocol.


### Conduct of a Study
The study shall be conducted in accordance with the protocol.

The test systems shall be monitored in conformity with the protocol. Specimens shall be identified by test system, study, nature, and date of collect ion. This information shall be located on the specimen container or shall accompany the specim en in a manner that precludes error in the recording and storage of data.

Records of gross findings for a sp ecimen from postmortem observa tions shall be available to a pathologist when examining that specimen histopathologically.

All data generated during the conduc t of a study, except those that are generated as direct computer input, shall be recorded directly, pr omptly, and legibly in ink. All data entries shall be dated on the day of entry and signed or initiale d by the person entering the data. Any change in entries shall be made so as not to obscure the or iginal entry, shall indicate the r eason for such change, and shall be dated and signed or identified at the time of th e change. In computer driven data collection systems, the individual responsible for direct data i nput shall be identified at the time of data input. Any change in computer entries shall be made so as not to obscure the origin al entry, shall indicate the reason for change, and shall be dated and th e responsible individual shall be identified.


### Maintenance and Calibration of Equipment
Equipment shall be adequately inspected, clean ed, and maintained. Equipment used for the generation, measurement, or assessment of data shall be adequately tested, calibrated, or standardized.

Written standard operating procedures (SOPs) sha ll set forth in sufficient detail the methods, materials, and schedules to be used in the routine inspection, cleani ng, maintenance, testing, calibration, or standardiza tion of equipment, and shall specify remedial action to be taken in the event of failure or malfunction of equipment. The written SOPs shall designate the person responsible for the performance of each operation, and copies of th e standard operating procedures shall be made available to laboratory personnel.

Written records shall be maintained of all in spection, maintenance, testing, calibrating, or standardizing operations. These records, containing the date of the operation, shall describe whether the maintenance operations were routin e and followed the written standard operating procedures. Written records shal l be kept of non-routine repairs performed on equipment as a result of failure and malfunction. Such records shall document the nature of the defect, how and when the defect was discovered, and any remedi al action taken in response to the defect.32 

### Testing Facilities Operation: Standard Operating Procedures
A testing facility shall have st andard operating procedures in wr iting setting forth study methods that management is satisfied are adequate to ensu re the quality and integrity of the data generated in the course of a study. All de viations in a study from standa rd operating procedures shall be authorized by the study director and shall be documented in the ra w data. Significant changes in established standard operating pr ocedures shall be properly author ized in writing by management.

SOPs shall be established for, but not limited to, the following:

• Animal room preparation.

• Animal care.

• Receipt, identification, storage, handling, mixi ng, and method of sampling of the test and control substances.

• Test system observations.

• Laboratory tests.

• Handling of animals found mo ribund or dead during study.

• Necrospy of animals or postmortem examination of animals.

• Collection and identification of specimens.

• Histopathology.

• Data handling, storage, and retrieval.

• Maintenance and calibration of equipment.

• Transfer, proper placement, a nd identification of animals.

• Each laboratory area shall have immediat ely available laboratory manuals and SOPs pertinent to the laboratory procedures being performed (e.g., toxicol ogy, histology, clinical chemistry, hematology, teratology, necropsy). Published literature may be used as a supplement to standard operating procedures.

• Historical file of SOPs, and all revisions thereo f, including the dates of such revisions, shall be maintained.


### Reagents and Solutions
All reagents in the laboratory ar eas shall be labeled to indicate identity, titer or concentration, storage requirements, and expiration date. Solutions prepared in the laboratory shall be labeled to indicate identify, concentration, date of preparation, and preparer’s na me or initials. Deteriorated or outdated reagents and so lutions shall not be used.33 

# Appendix C

Water Quality

Water quality parameters, in addition to pH, har dness, and alkalinity, ca n adversely affect the outcome of acute fish toxicity test s. Sub-lethal concentrations of certain materials in dilution water may contribute to fish mortality because of the additive effect of similar materials in the waste being tested, or because of synergism resulti ng from dissimilar materials. The more common materials and parameters of concern are presen ted below with recommended ranges or maximum concentrations. If erratic result s are experienced in the toxicity test, the dilution water should be checked for the materials included in Table 8, or for other materials, which may be present in the dilution water source. Al so refer to EPA-821-R-02-012 Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms for additional guidance on water quality.

Table 8. Water Quality Parameters a

PARAMETER RECOMMENDED pH 7.2 - 7.8 Alkalinity 30 - 70 mg/L as CaCO 3 Ammonia (NH 3 ) 0.02 mg/L unionized Cadmium (Cd) 100 ng/L Chloride (Cl) 2 - 3 mg/L Copper (Cu) less than 5 μg/L Hardness 40-100 mg/L as CaCO 3 Lead (Pb) 0.03 mg/L or less Mercury (Hg) organic or inorganic 100 ng/L Nickel (Ni) 88 μg/L or less Nitrogen (N) maximum total gas pres sure 110 percent of saturation Nitrite (NO 2 ) 100 μg/L Oxygen (O 2 ) at least 8 mg/L at 12 o C Ozone (O 3 ) Residual 3 μg/L PCB as Aroclor 1254 50 ng/L or less Total Suspended and Settleable Solids 80 mg/L or less Zinc (Zn) less than 65 μg/L Total organochlorine pesticides 50 ng/L or less

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

# Appendix D

Table 9. Approximate Saturation 1 and Concentration Valu es of Dissolved Oxygen in Freshwater at 12 ° C Altitude (ft ) DO (mg/L) a 100% Saturation DO (mg/L) a t 80% Saturation DO (mg/L) a 60% Saturation A ltitude (ft) DO (mg/L) a t 100% Saturation DO (mg/L) a 80% Saturation DO (mg/L) at 60% Saturation
0 10.71 8.57 6.43 4000 9.35 7.48 5.61 100 10.68 8.54 6.41 4250 9.26 7.41 5.56 250 10.62 8.50 6.37 4500 9.18 7.34 5.51 500 10.54 8.43 6.32 4750 9.09 7.27 5.45 750 10.45 8.36 6.27 5000 9.01 7.21 5.41 1000 10.37 8.30 6.22 5250 8.92 7.14 5.35 1250 10.28 8.22 6.17 5500 8.84 7.07 5.30 1500 10.20 8.16 6.12 5750 8.75 7.00 5.25 1750 10.11 8.09 6.07 6000 8.67 6.94 5.20 2000 10.03 8.02 6.02 6250 8.58 6.86 5.15 2250 9.94 7.95 5.96 6500 8.50 6.80 5.10 2500 9.86 7.89 5.92 6750 8.41 6.73 5.05 2750 9.77 7.82 5.86 7000 8.33 6.66 5.00 3000 9.69 7.75 5.81 7250 8.24 6.59 4.94 3250 9.60 7.68 5.76 7500 8.16 6.53 4.90 3500 9.52 7.62 5.71 7750 8.07 6.46 4.84 3750 9.43 7.54 5.66 8000 7.99 6.39 4.79

1 Based on Pillard (1996).35 

# Appendix E
Recommended Fish Loading

Prior to use in toxicity tests, fish are usually maintained at the test facility in a circulated holding tank. A minimum level of circula tion is required to ma intain the viability of test fish. The recommended level of circulation varies dependi ng on the species of fish, size of fish, and temperature. For small fish, the size of the fish is usually reported in term s of number of fish per pound. The table below lists the recommended fish mi nute loadings (pounds of fish per gallon per minute of inflow), to promote the health and qua lity of salmonids used in toxicity tests.

Table 10. Recommended Fish Loadings a

SPECIES WATER TEMPERATURE POUNDS FISH PER GPM

o C o F 1000 b 500 b 100 b

Coho Salmon 3 9 14 17 38 48 58 63 3.5 2.7 2.2

---- 5.0 4.0 3.0 2.0 8.0 6.0 4.5 3.5

Fall/Spring Chinook Salmon 3 9 14 17 38 48 58 63 3.0 2.5 2.0

---- 4.0 3.0 2.2 1.2 6.0 5.0 3.5 3.0

a Adapted from Wedemeyer & Wood (1977)
b Fish size (number of fish per pound)36 

# Appendix F

Sample Data Sheet

The Data Sheet for Static Basic Acute Fish Toxicity Test is printed on page 37. Please copy this form to report test data.37LaboratoryAnalystIndustry/Toxicant Beginning Date TimeAddress Ending Date TimeCollector Test OrganismDate SampleCollectedRequired Test TemperatureRangeLaboratoryReferenceNumberTestContainerNo.Conc.(mg/l)Number of CumulativeDeathsDissolved Oxygen (mg/l)pH 25 CTemperature (C)TotalHardness(mg/l asCaCO3)TotalAlkalinity(mg/l asCaCO3)ConductivityμMHOS/cm0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 96 0 96 0 96Sample Description ________________________________________________________________________________________________Average Weight ____________ Mean Length ____________ Longest ___________ Shortest _____________ Ratio (long/short) ______________Number of organisms per chamber ________________________ Ratio of flesh to water _________________Comments___________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________Method on File with theDepartment of EcologyDATA SHEET FOR STATIC BASICACUTE FISH TOXICITY TEST38GENERAL PROCEDURE FOR STATIC BASIC ACUTE FISH TOXICITYTEST Data Verified By _________________________________Date _________________39 

# Appendix G

Recommended Test Conditions and Procedures for Fish Bioassay

Test type • Static non-renewal

Duration • 96 hours
Light quality • Ambient laboratory illumination
Light intensity • 10-20 uE/m 2 /s (50-100 ft-c)

Photoperiod • 16 hours light, 8 hours darkness

Test chamber size • 3.8 L or 1 gal minimum

Minimum depth • 15 cm test solution

Test solution renewal • none

Source of fish • hatchery stock, free of known diseases

Control water • moderately hard water; see Table 1
Fish age • 30 - 90 days

Temperature • 12 o C + 1 o C

Oxygen/aeration • None unless DO<80 percent saturation at altitude; ideal DO is 80 to 100 percent saturation

pH • no pH adjustment
Feeding • not required
Cleaning • not required

Number of fish per test chamber • minimum 10

Number of replicate chamber per concentration • minimum 3

Number of fish per concentration • minimum 30

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

Measurements • temperature, DO, pH, in each tank daily; conductivity test
Endpoints • mortality
Test acceptability • 90 percent survival or greater in controls

Reference toxicant • CuSo 4 , CdC1 2 , KCl, NaCl, or ZnSO 4 (not inclusive); evaluate at least once/month and with each new batch of fish

Solvents • Extraction water is the control water used in test controls40 Concentration • 100 mg/L to determine dangerous waste; 10 mg/L to determine extremely hazardous waste

Control water • Moderately hard water

Sampling date • Date of sample collection; tests done on sample collected more than 45 days prior to extraction must be noted in the report

Transport and storage • Extract within seven days of sample receipt; store in dark at 4 ° C; test within 8 days of sample collection

Extraction • Extract using rotary agitation device (30 + 2 rpm) for 18 hours + 2 hours at 23 ° C + 2 ° C41 

# Glossary

o C degree(s) Celsius
CaCO 3 calcium carbonate
CaSO 4 calcium sulfate

CaSO 4 • 2H 2 O calcium sulfate dihydrate (gypsum)
cm centimeter
CO 2 carbon dioxide COD chemical oxygen demand d day DO dissolved oxygen DW dangerous waste EHW extremely hazardous waste

o F degree(s) Fahrenheit

g grams gal gallons gpm gallons per minute
h hour HCL hydrochloric acid

H 2 O water in inch(es) KCL potassium chloride
L liter

m meter mg milligram mg/L milligram per liter MgSO 4 magnesium sulfate min minute(s)
mL milliliter mg/kg milligram per kilogram
NaHCO 3 sodium bicarbonate NaOH sodium hydroxide ng/L nanograms per liter
NH 3 ammonia QA/QC quality assura nce/quality control qt quart rpm rotations per minute TOC total organic carbon42 WAC Washington Administrative Code > greater than
< less than

> greater than or equal to

< less than or equal to43 

# Terminology

Grammatical Terms

Can ...means “is/are able to”
May ...means “is/are allowed to” Might ...means “could possible;” never used as a synonym for “may” or “can”

Must ...expresses an absolute requirement

Should ...means the specified condition is recomm ended and ought to be met if possible



### Technical Terms

Acclimatize ...to become physiologically adapted to a partic ular level of one or more environmental variables, such as temperature or water chemistry.

Acute toxicity ...a discernible, adverse effect (lethal or subl ethal) induced in the test population within a short period of exposure to a test material, usually constituting a non-substantial por tion of their life span.

Conductivity ...is a numerical expression of an aqueous solution’s ability to carry an electrical current. This ability depends on the concentrations of ions in solution, their valence and mobility, and on the solution temperature. Conductivity is normally repor ted in the Système Intern ational d’unités (SI) unit of millisiemens/meter, or as micromhos/centimeter (1 mS/m = 10 μ mhos/cm).

Control ...a treatment that is essentially free of cont aminants and is used routinely to assess the acceptability of a test. In Salmonid tests , the control must duplicate al l conditions of the exposure treatment(s), but must cont ain no test material.

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

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

Distilled water ...water passed through a distil lation apparatus of borosilicate glass or other material, to remove impurities.

Extremely hazardous waste ...those dangerous and mixed wastes designated per WAC 173-303-100 as extremely hazardous. Also, those dangerous wastes s howing statistically signifi cant mortality in test concentrations of 10 mg/L.

Flow-through ...continuous renewal of holdi ng water or test solution.

Hardness ...a measure of the concentration of calcium and magnesium ions in water, expressed as mg/L calcium carbonate (CaCO 3 ) or equivalent.44 LC 50 ...the median lethal concentration (i .e., the concentration of material in water estimated to be lethal to 50 percent of the test organisms). The LC 50 and its 95 percent confidence limits are usually derived by statistical analysis of mortalitie s in several test concentrations, af ter a fixed period of exposure. The duration of exposure must be specified (e.g., 96 hour LC 50 ).

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

Lethal ... means causing death by direct ac tion. Death of fish is defined as the cessation of all visible signs of movement or other activity, su ch as no response from gentle prodding.

Loading ...the weight of organisms per liter of test soluti on; this is limited to minimize the decrease in dissolved oxygen (DO) or toxicant below acceptable le vels, the accumulation of injurious concentrations of metabolic waste products, or stress induced by crowding.

Lux ...a unit of illumination equal to 1 lumen per square meter. One lux = equals 0.0929 foot-candles and one foot-candle = equals 10.76 lux.

Observations ...routine checks of biological and water qua lity variables, which may include fish survival and behavior, water temperature, dissolved oxygen, pH, hardness and conductivity.

pH ...the negative logarithm of the hydrogen ions activity in gram equivalent s per liter. The pH value expresses the degree of intensity of both acidic and alkaline solutions on a scale from 0 to 14, with 7 representing neutrality. Numbers less than 7 signify increasingly greater acidic solutions, and numbers greater than 7 indicate increasingly gr eater basic, or alkaline, solutions.

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

Reconstituted water ...standard, synthetic water prepared with de-ionized water and reagent grade chemicals or mineral water; to be used for culturing of organisms, as control water, and as dilution water for test solutions.

Reference toxicant ...a known toxicant used to measure all aspects of toxicity testing, including organism quality, presence and eff ects of stressors other than test compounds, and the quality of analytical techniques. Data from regular reference toxi cant testing is used to establish the precision of bioassay results generated by the laboratory.

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

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

Swim-up ...stage at which Salmonids emerge from the gravel and begin to actively fee d. It is the point at which to begin marking the age of the fish.

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