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# • .Biological
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# Methods

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## -~-~n\,-.,.~**~~M1'** - .. --."

# •

PART A.

### BIOLOGICALTESTINGMETHODS STATIC ACUTE FISHTOXICITYTEST



# • PART B• 

### ACUTE ORALRAT TOXICITYTEST

Prepared by



## •


# • .Hazardous·Waste Section Washington State Department of Ecology

Revised June 1991

# •
PART A. TABLE OF CONTENTS

STATIC ACUTE FISHTOXICITY TEST I. INTRODUCTION 1 

### 1. 

### 2. 3. General Terminology Health and Safety 1 2 • 2 

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

### 1. 

### 2. 3. 4. 

### 5. Holding Facilities . • . • Test Chambers . . • Cleaning and Disinfection
. Dilution Vater • . • • Vater Quality
. e. • • 4 4 5 6 

### • • 7 

### III. TEST ORGANISMS 9



### IV: SAMPLE PREPARATION 

# • 1. 

### 2. 3.

1. 

### 2.

.3.

4. 

### 5. Care and Handling Holding".
. . Quality
. . . .
General
. . .. Particle Size Reduction
. Sample Extraction: Vrist Action Shaker Method Sample Extraction: Rotary AgitationMethod . Rotary AgitationApparatus 10 

### 11 11 

### 12 12 12' 13 13 

### 14 V. TEST PROCEDURE 

# · ·

# · · · · · · · · 

# ·· · · . · . . 15 1. 

### Conducting the Fish Toxicity Test

# · · · 

# · · 15 

### 2. Adjusting the pH 

# ·

# ·· · 15 3. 

### Replicates and Control 

# · · · · 

# · · · ·

# · . 15 

### 4. Dissolved Oxygen 

# ·

# ·· · 16 5. 

### Test Temperature 

# ·

# · · · · ·

# · · · 16 6. 

### Loading 

# . · · ·

# · · 16 7.


### .Dissolving Toxicant

# · · · · · 17 

## • 8. 

### Feeding

# . · · · · · · · · · 17 

### 9. 

### Duration 

## · 

# · · · · 17 

### 10. 

### Biological Data
. 

# · 17 

### 11. Chemical and Physical Data for Test Tanks 18 

### VI. QUALITY ASSURANCE/QUALITY CONTROL •

# · · ·

# · · · 19 1. 

### Procedures for Quality Assurance/Quality Control 19· 

### 2. Reference Toxicants 

# · · · · · · ·

# · · ·

# · · ·

# · · . .

# ·. . 19 

# •VII. CALCULATION OFRESULTS 

### 1. 

### 2. 3.
APPENDICES Examples • • • • Precision. Reporting • 

# . ... . 

# . . .. 

# . . 20 20

-

# C 22 22

A. Neutralized Fish Toxicity Test Procedure B. Effluent Toxicity Test Procedure C. Good Laboratory Practice Standards D. Recommended Fish Loading E. Data Sheet for Static Basic Acute Fisb Toxicity Test
BIBLIOGRAPHY
PART B. ACUTE ORAL'RAT TOXICITY TEST 1. INTRODUCTION II. STANDARDS

# . .

# · .' III. REPRESENTATIVE SAMPLING

IV. SAMPLE PREPARATION 

# ··'. V. TEST ORGANISMS 

# ·· · · VI. DOSING

..... 

# . . · VII. TESTDURATION

# · · ·· VIII. 'TEST PROCEDURE IX. DATA REPORTING 

# · ·

# ··. 26 

### 27 32 ' 33
34



### B-1 B-1 

# C B-1 B-1



# · B-2



## · B-2



### •

# · B-2 B-2 B-2



## ,

# •

•

-, 

# • PART A 

### STATIC ACUTE FISH TOXICITY TEST
Revised June 1991

# •

•

• PART A 

### STATIC ACUTE**~ISHTOXICITY** 

### TEST I. INTRODUCTION 1. General This acute fish toxicity test was developed specifically for determining whether a waste meets the definition ofdangerous waste as found in Chapter 173-303 WAC. Amodified test**proced~re** 

### for conducting a neutralized fish toxicity test is included in Appendix A. A second modified test procedure for conducting effluent toxicity testing is included in Appendix B. The purpose of the acute fish toxicity test described in this document. is to provide a simple, reliable, low-cost method for testing the toxicity of a material against the requirements of the Washington State.Hazardous Waste Regulation. This test method is strictly a range-finding test and does not result in defining the actualmedianlethal concentration (LC so) . 

### Themedianlethal concentration is defined as the concentration of material that kills 50 percent of the population (fish) within a stated time period, usualIy 96 hours. If the toxicity of a waste material is not known, the waste maybe tested at two concentrations, 100 mg/l and 1000 mg/l. The waste concentrations of 100mg/l and 1000mg/lwere selected to correspond with the definition ofdangerous waste (DW) and extremelyhazardous. waste (EHW) found in the state's Dangerous Waste Regulations, Chapter 173-303 WAC. Wastes that kill fish at 100 mg/l are highly toxic and are classified as extremely hazardous waste. Similarly, wastes that kill fish at 1000 mg/l wouldbe classified as dangerous waste. Material so designated would be regulated and handled as speo!fied in the regulation. The section "Calculation ofResults"**g~es** 

### into greater detail on data interpretation. Wastes being subjected to the acute fish toxicity test must be sampledin accordancewith theprocedures referenced inWAC 173-303- 110, Sampling and Testing Methods. Anyperson whose waste fails the acute fish toxicity test and who believes that mortality resulted from effects other than acute toxicity (e.g.", disease or physical stress) may (a) petition the Department to exempthis waste from designation as a dangerous waste (DW) underWAC 173-303-101, or (b) petitionthe Department tomodify the test procedure. Anyperson whobelieves that his waste fails the acute fish toxicity test because of pH only, may choose to demonstrate thiS factby performinga neutralizedfishbioassay test as found in Appendix A of this manual. The purpose of such demonstrationwouldbe for change ofdesignation as provided inWAC 173-303-910 (5). Any pHwastes containing hazardous constituents (such as ammoniumhydroxide orhydrogencyanide) whichhave inherent toxicity (1.

### e . , ammonia and cyanide) will not qualify for such -1-

### 2. designation change. Petitions mustbe submittedin accordance with 

### WAC 173-303-910. Terminology The words "must," "would," "may," ·can," and "might" have very specific meanings in this guide. '"Must" is used to express an absolute requirement, that is, to state thae ehe eese oughe to be' designed to satisfy the specified condition, unless the purpose of the test requires a different design. "Must" is only used in connection with the factors that direcdy relate eo the acceptability of the test. "Should" is used to state that the specified condition is recommended and ought to be met ifpossible. Although a violation of,one "should" is rarely a serious maeeer, violation of several will often render the results questionable. Terms such as "is desirable," "is often desirable," and "might be desirable" are used in connection with less important factors. "Kay" is used to mean "is(are) allowed to," "can" is used to mean "is(are) able to," and "might" is used to mean "could possibly." Thus, the distinction between "may" and "can" is preserved, and "might" is never used as a synonym for either "may" or "carr." 

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3. Health and Safety Toxicity testing-may involve significant risks to personal safety and health. Personnel conducting toxicity tests should protect themselves by taking all safety precautions necessary to prevent bodily injury. Procedures to prevent inhalation or skin absorption of test materials should be observed. Each laboratory should be

## ~) equipped with all necessary safety equipment prior to conducting toxicity tests. Personal Safety Gear. Personnel should use safety equipment as required, such as rubber aprons, lab coats, respirators, gloves, safety glasses, and safety shoes. Laboratory Safety Equipment. Each laboratory should have safety equipment such as first ,aid kits, fire extingUishers, and fire blankets. Included below is a summary of recommended laboratory procedures which, if followed, will promote a safe working environment and minimize health risks to persons in the lab. While this list contains good suggestions regardinghealth and safety in the lab, it is by no means exhaustive. Testing laboratories are encouraged to implementthese, andother, recommendationswhichpromote thehealth and safety of laboratory persons.
a. Testingofindustrialwastes shouldbeperformedincompliance with accepted rules pertaining to the handling of hazardous materials. It is recommended that personnel carrying out**~oxicity** 

### tests should not work alone. 

# ()

# • b . 

### Because the chemical composition of industrial wastes is generally only poorly defined, such wastes should be considered potential health hazards, and exposure to them· shouldbe minimized.



# •

• c. It is advisable to immediately wash with soap· and water any body parts which maycontact the waste. d. All containers shouldbe adequately labeled to indicate· their contents.
e. Goodhousekeeping contributes to safety and reliable results. f. Only electrical equipment having Underwriters Laboratories approval shouldbe used. Ground-fault interrupters shouldbe installed in all "wet" labs where ·electrical equipment is 

### used. g. Staff training in basic first aid and cardio-pulmonary resuscitation is strongly recommended. Disease Prevention. Personnel handling samples which are known or

.suspected
.to contain hUDian wastes should be immunized against

tetanus,. typhoid fever, and polio. Safety Manuals. For further guidance on safe practices when: conducting toxicity tests, see permittee and general industrial safety manuals, including USEPA, 1977, and Walters and Jameson, 1984
.

-3-II.

- METHODS 

### AND MATERIALS The methods andmaterials used iJl this procedure were taken from numerous 

# 0

-sources (see Bibliography).· This method is an attempt to balance scientific principles against practical considerations without compromising the accuracy or precision of the test. Although more stringent test procedures may be used (1.e., using more fish, longer acclimationtimes), the requirements presentedwillusuallybe sufficient. The purpose for establishing a standard laboratory method is to ensure that results obtained-fromdifferent testing laboratories are comparable. 1. Boldine Facilities The facilities shouldinclude tanks forholdingandacclimating test fish anda constant-temperature roomorrecirculatingwaterbathfor the test chambers.

- Holding tanks mustbe operated ona flow-through basis. Holding, acclimation, anddilution-water tanks andheadboxes should be equipped for temperature control and aeration. Air used for aeration shouldbe free ofoil and fumes; filters to remove oil and water may be used. During holding, acclimation, and testing, test fish should be shielded from disturbances. The test facility shouldbe well ventilated and free- offumes. Aphotoperiod of 8-16 hours oflightper 24hours shouldbe provided during the test. The light iritensityshould be in the range of natural outdoor light. High intensity and extreme lighting conditions should be avoided. Tanks find utility in the lab as holding tanks, stock solution tanks, or test solution tanks. Whether fabricated in the lab or 

# -0, purchased commercially, tanks should not be constructed of materials, or have surface coatings, which will leach or dissolve into aqueous solutions. In addition, tanks andotherequipment that contact stock solutions or test solutions should be chosen to minimize sorption oftoxicants from water. Glass, stainless steel, and fluoroplastics should be used whenever possible to minimize leaching, dissolution, and sorption. Concrete and rigid**(~lasticized)** plastics may be used for-holding and acclimation tanks and in the water supply system. Cast ironpipe maybe used in freshwater supply systems, but colloidal iron will be added to the dilution water and strainers will be needed to remove rust particles. Natural rubber, copper, brass, galvanizedmetal, solder, and lead shouldnot come in contactwith dilutionwater, stock solutions, or test solutions before or during exposure of test fish. 2. Test Chambers The test chambers most commonly used in static tests for fish are wide-mouth, 0.95 L (1 qt), 3.8 L (1 gal) or 19.0 L (5 gal) softglass bottles or aquaria. Special glass or stainless steel test chambers can be constructed to accommodate test fish requiring particularphysicalconditions (largesurface-area-to-volumeratio). Test chambers may be constructed of 3-mm (0.125-in.), double strength, or 6-mm (0.25-in.) temperedplate glassheld togetherwith clear silicone adhesive. Silicone adhesive absorbs some -4- 

# .. 

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# •

•

• 

# • 3. organochlorine andorganophosphorus pesticides. -whichare difficult to 

### remove. Therefore. as little ofthe adhesive, as possible should be in contact with water; extra-,beads of adhesive inside the containers shouldbe remOved. Stainless steel (No. 304 orNo,. 316)

.canbe used in the construction oftest chambers. but shouldbe of welded, not soldered. construction. Commercially**,availabl~** 

### test chambers maybe used ifthey meet the above requirements. The tank configuration should. at a minimum. satisfy the following criteria: The test solution tank 

### shouldbe at least 15 cmdeep for fish over 0.5 g each. and at least five emdeep for smaller fish. Chambers filled to within 15 emof the top need to be covered to prevent the test fish fromjumping out. Cleaning and Disinfection Test chambers should be cleaned before use. New test chambers should be washed with detergent and rinsed first with water. then withreagent grade acetone. followedby copious rinsing withwater. then with acid (such as 5'percent concentrated nitric acid). and finally copious rinsing with tap or other cleanwater. At the end ofevery test; the chambers shouldbe (1) emptied. (2) immediately rinsed with water. (3),cleaned by a procedure appropriate for removing'the toxicanttested (e.g.. acidto remove metals andbases; detergent. organic solvent. or activated carbon to remove organic compounds), (4) disinfected to remove fish pathogens. and (5), copious rinsing with water. Acid is useful for removing mineral deposits. and 200 mg hypochlorite/liter for one hour is useful for removing organic matter and for disinfection.' (A solution containing 200mghypochlorite/liter is convenientlymade by adding: 6 ml of liquid household chlorine bleach to 1 liter of water.) However. acid andhypochlorite should not be used together because hazardous chlorine fumes may be produced. Tanks may also be' sterilizedwith an iodophor. Commercially prepared iodofors using polyYinylpyrrolidone-iodine (PVP-I) as the active ingredient. are availableasVescodine. Betadine. Argentyne. orotherproductnames. For disinfection ofequipment such as fish tanks. prepare a 50 ppm 'solutionoftitrateable iodineby diluting a commercially availabie product. The equipment should be immersed in or washed with this solution for at least 10 minutes followed by copious rinsing with cleanwater. Iodophors andhypochlorites are very acutely toxic to ,most aquatic organisms. Hypochlorite can be removed by flushing with water or by reacting it with an equivalent concentration of sodium thiosulfate or sodium sulfite.**Alternatively~** 

### after a tank is washed with hypochlorite and rinsed. the equipment may be air dried overnight to,allow dissipation ofresidual chlorine
. When fish are first brought into a facility. they should be quarantined for at least 14 days. To maintain test fish in good condition during holding and acclimation. crowding should be avoided. The dissolved oxygen concentration must be maintained between 60 and 100 percent saturation; gentle aerationmaybe used ifnecessary.
-5-4. Dilutionyater An adequate supply of a 4i1ution water that is acceptable to the test fish and the purpose of the test must be available. The minimal criterion for an acceptable dilution water for acute toxicity tests is thathealthy test fish will survive in it for the durationofacclimationandtestingwithout showing signs ofstress, such as discoloration or unusual behavior. Abetter criterion for an acceptable freshwater dilution water is that test fish will survive, grow, and reproduce satisfactorily in it. Freshwater dilution water is considered to be of constant quality if the monthlyranges ofthehardness, alkalinity, and specific conductance are less than 10 percent of their respective averages and if the monthly range ofpH is less than 0.8 unit. Chlorinated water should never be used for dilution water because residual chlorine is toxic to aquatic organisms. Dechlorinated water shouldonlybe used as a lastresort, because removal ofresidual chlorine is often incomplete. In addition, municipal water supplies ·often contain unacceptably high concentrations of copper, lead, zinc, and fluoride. Toxicity tests canbe adversely affected by the use of dilution water containing these materials. When necessary, .water containing the above materials can be treated to make such water suitable for bioassay testing. Metals can usually be removed with che1ating agents.
. Chlorine may be removed by filtering through activated carbon then treating with a sodium thiosulfate or sodium sulfite drip. Sodium thiosulfate is better for dechlorinating water than sodium sulfite. Both are more reliable than a carbon filter, especially for removing chloramines. The dilution water should be intensively aerated by such means as air stones, surface aerators, or screentubes before introductionof the toxicant. Adequate aeration wi11.bring the concentration of dissolved oxygen and other gases into equilibrium with air and minimize oxygen demand and the concentration of volatiles. The concentration of dissolved oxygen in the dilution water should be between 90 and 100 percent saturation. Test chambers should be rinsed with dilution waterjust before.use. The hardness of the dilution water can affect the outcome of the fish toxicity test. The hardness of the dilution water used in fresh water toxicity tests must not exceed 100 mgll (expressed as CaCo,) and should not be less than 40 mg/l. (Irregular toxicity test results may occur when the hardness falls below this level.) Use reagent-grade chemicals to adjusthardness and alkalinity. The lab report should include informationwhichconfirms the final water qualityparameters are within the ranges found inTable 1 for ·soft· to -moderately hard· water.

-6- 

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# •

• 5. 

### -Adapted from Methods for Measuring the Acute Toxicity ofEffluents .to Freshwater and Marine Organisms, 1985, by EPA. bAdd reagent grade chemicals to distilled or deionized water. cApproximate equilibrium pHafter aerating 24 h. dExpressed inmg/L as**CaCO,~** 

### Water OUality Waterqualityparameters inadditiontopH, hardness, andalkalinity can adversely affect the outcome ofacute fish toxicity tests. Even sub-lethalconcentrations ofcertainmaterials inthe dilutionwater may contribute to fish mortality because of the additive effect of similarmaterials inthewastebeing tested, orbecause ofsynergism' resulting from dissimilar materials. Some'of the more common materials and parameters ofconcern are presented in Table 2 along with recommended ranges or recommended maximum concentrations. If erratic results are experienced in the toxicity test" the dilution water should be checked for the materials included in Table 2, or for other materials which may be present in· the dilution water source.

-7-

### PARAMETER pH Alkalinity Ammonia (NH3) Cadmium Chloride Copper 'TABLE 2. VATER QUALITY PARAMETERS

### RECOMMENDED ENVIRONMENTAL LIMIT 7.2-7.8 30-70 (as CaCO,) 0.02 ppm (un-ionized form) 0.4 ppb in soft water 2-3 ppm is 80ftwater { 100 ppm) less than 

### 5 ppb 

# C)

HarClness Lead Mercury (organic or inorganic) Nickel Nitrogen Nitrite Oxygen Ozone Residual PCB (Polychlorinated Biphenyls as Arochlor 1254) Total Suspended and settleable solids Zinc 40-100 mg/l (as CaCO,) 0.03 ppm 0.2 ppb
88 ppb Maximum 

### total gas pressure 110' of saturation 100 ppb At least 8 ppmat l2-C 3 ppb 0.002 ppm·

80 ppmor less
65 ppb 

# C)

-Adapted from Required Hatchery Environment for Smoltificationby

G. Wedemeyer, et ale (1981)

-8- 

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# •• III. TESTORGANISMS The following table lists the .test organisms that can be used in the static fish toxicity test. TABLE 3. SPECIES AND TEST TEMPERATURES Species· Test Temperature (OC> Freshwater Vertebrates Coho salmon, Oncorhynchus kisutcb Rainbow trout,
.Oncorhynchus (formerly gairdneri) Brook trout, Salyelinus fontinalis
·The species must be verified. 

### 12 *.1 12 * 1 12 * 1



# • All fish used in a test should be of the same age, and from the same source since organisms ofthe same**spec~es** from different sources mayproduce different results in an acute toxicity test
.. Usual sources offreshwater fish are private, state, and federal hatcheries. All fish used in a test should be as uniform as possible in size. Fish that are between 30 and 120 days old shouldbe used. The age ofa fish is reported from the time the fish is at the sWim-up, orbutton-up stage, and not from the time the egg hatches. Unless there is a specific reason for doing so, very young (not yet actively feeding) fish should not be used. In any single test, all fish should be from the same year class, and the standard length.:(tip of snout to end of cauda:l peduncle) of the longest fish should be no more than twice that of the shortest fish (Figure 1)
.



# • Figure 1

-9- 

# ......

### All efforts shouldbemade to ensure that testfish aredisease-free prior to using them in a toxicity test. To this end, one may: a) Select fish from a source (hatchery) which will certify that all fish are disease-free. b) Treat all fish which arrive on-site using treatments commonlyused by sources (hatcheries) whichproduce'certifieddisease- free fish. c) Discrimlnately select test fish from a reputable source, and/or on the observations andjudgment ofknowledgeable individual. All test fish shouldbe obtained from a hatchery thathas been certified disease-free for the following diseases; bacterial kidney disease (RenibacteriUJDsalmoninarum); Costia (Ichythobodo): bacterialgilldisease (Myxobacteria species); and FUrunculosis (Aeromouas salmonicida). 

### As an alternative to obtaining certified disease-free fish, one maytreat fish on-site to control externalparasites andlatentbacterial infection. The treatment requires two
.steps: (1) Immerse the fish in an aerated solution of 200 ppmFormalin for 60 minutes, and (2) 24 hours after the Formalin treatment, begin and continue a commercial feed medicated diet (e.g., Terramycin) for 10 days. Ifthe fish are severely diseased, it is better to destroy the entire lot immediately. Between use with different groups of test fish, holding and acclimation tanks should be sterilized according to the procedures in Chapter 11.3, Cleaning and Disinfection. 

### 1. Care and Handlin& Test fish must be cared for and handled properly so they are not wmecessarily stressed. To avoid unnecessary stress, test fish should not be subjected to rapid changes in temperature or water quality. In general, aquatic organisms should notbe subjected to**mor~** 

### than a 3-C. change inwater temperature in any l2-hourperiod. The test fish mustbe maintained in the dilution water at the test temperature for at leastthe last48 hours before they areplacedin the test chambers. Testfish shouldbehandledas little as possible. Whenhandling is necessary, it should be done as gently, carefully, and quickly as possible, so that the fish are not unnecessarily stressed. Fish that
.touch dry 

### surfaces or are dropped or injured during handling should be discarded. Dip nets are best for handling fish. Such nets are commercially available, or can be made from small-mesh nylon netting, nylon or silk bolting cloth, plankton netting, or similarmaterial. Equipmentusedtohandle aquatic organisms should be 

### sterilized between uses by autoclaving or by treatment with 

### an iodophor, 200 mghypochloriteperliter, or 30percentformalinplus one percent benzalkonium chloride for at least one hour; Hands shouldbe washed or sterilizedbefore handling test fish.

-10- 

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•

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# • 2.

3. Boldine After collection or transportation. fish should be acclimated to laboratory conditions for 14 days before they are used in tests. However. fish whichhavebeen acclimatedfor only 7 days JBIXbeused in tests. Fish should be held in uncontaminated. aerated water 'of constant temperature and quality in a flow-through system with a flow rate ofat least one volUllle exchange perhour. llhenpossible. the fish should be ,held in dilution water and as close to the temperature at which they are to be tested (Table 3) as possible. During long holding periods. however. it is generally easier and safer toholdfish at temperatures lower than 12·Cbecause themetabolic rate and the number and severity of disease outbreaks are reduced. The recommended fish loading for holding tanks. is prOVided for various water temperatures. fish size. and species. in Table'Dl in Appendix D. If stock fish have 

### 5' mortality between 24 hours after arrival onsite and the end of the 14 day acclimation period. the entire lot should not be used for testing. If at ariy time during the holding period there is an abnormal number ofmortalities. the fish should not be used for testing. Duality , All fish in a test must be ofacceptable quality. Test fish should: be carefully observeddaily duringholding andacclimationfor signs'_ of disease. stress. physical damage. abnormal**beha~ior** and mortality. Dead. injured. and abnormal individuals must be discarded. Visual examination of the behavior and external appearance ofthe fish shouldcover the following:, Are they eating? Is flipping.' flashing. or erratic swi.amling evident? Are they gasping at the surface? Is their color normal? Is hyperventilation. hemorrhaging. or excessive mucus production evident? Are "fin rot. fungus. or external parasites present? Are test oqanisms emaciated? Ifthe organisms show any signs ofdisease or. stress. appropriate action should be taken
.

"

-11-IV. SAMPLE PREPARATION General A sample of the waste to be tested shall be obtained using an appropriate samplingmethodapprovedbythe Department. All samples must be extracted using the method and equipment described below. In addition, solid samples must be reduced in size prior to extraction unless

- the sample meets the particle size criteria described in IV.2. below. Samples should be mixed prior-to opening the sample container. A

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

Iurbula,

-or other appropriate

-device may be used for miXing. Viscous samples should be stirred after opening to make the sample as homogeneous as possible. Two methods of sample extraction are presently allowed for samples being testedusing the fish toxicity test. Samples maybe extracted using a wrist-action shaker or a rotary agitation apparatus. In eachmethod, the sample is allowed to extract over a six-hourmixing period. The extractionwater (solution) will be**th~** stock dilution water used in the fish toxicity test. 

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t 2. The rotary agitation apparatus must be capable of rotating the extractionbottles in an end-over-end fashion at 30 t 2 rpm. Alist of known suitable rotary extraction devices is included in IV, 5. Particle Size Reduction Prior**t~** conducting the extraction, a determination must be made regarding the need to reduce the particle size of the sample. Particle reduction is required, unless the solidhas a surface area per gram ofmaterial equal to or greater than 3.1 cm 2
, or is smaller than

### -1 em in its narrowest- dimension (1.e., is

-capable of passing thx.ough a 9.5 mm (0.375 incb) standard sieve). If the surface area is smaller or the particle size larger than described above, prepare the solid portion of the waste for extractionby crushing, cutting, or grinding the waste to a surface area or particle-size as described above.**~:** Surface area criteria are meant for filamentous (e.g., paper,

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

area is not required, nor is it recommended. For materials that do not obviouslymeet the criteria, sample-specific methods would need to be developed and employed to measure the

-surface area. Such methodology is currently not available.



### -12- 

# -0



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# • 3 . Sample Extraction:· Wrist Action Shaker Method' '
a. Weigh out the**corr~ct** amount of a well-mixed sample and

,transfer into a 500 ml screw-top Erlenmeyer flask. b. Add 200 ml of extraction water (solution) to the Erlenmeyer flask.
c. Place a teflon cap liner over the mouth of the Erlenmeyer flask and screw the cap on. d. Mix the sample on the wrist-action shaker for six hours, remove the cap andpour the extractionwater into the dilution water in the fish test 

### tank. Rinse all loose material from the flask into fish test tank with a 200 ml aliquot of stock dilution water Place the flask into the fish tank so that the flask is full of water and laid on its side on the tank bottom. Drop the cap liner into the fish test tank.
e. Begin the test as described in

### V. Test Procedure.

,f. Care should be exercised throughout the course of the extraction so as not to contaminate the exterior of the extraction flask. 4. Sample Extraction: Rotary Aa;1tation Method 

# '. a. 

### b.
C 

### c. d.

• e. Weigh out the correct amount of a well-mixed sample and transfer into a 1000 ml extractor bottle'. Add 200 ml of extraction water (solution) to the extractor bottle. Place a teflon cap liner over the mouth of the extractor

- bottle and screw the cap on.

Kix the sample on the rotary agitation apparatus for six hours, remove the cap and pour the extraction water into the dilution.water in the fish test tank. Rinse all loose material from the flask into the fish test tankwith a 200 ml

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

fish tank so that the bottle is full o'f water and laid on its side on the tank bottom. Drop the cap liner into the fish test tank
. Begin the test as described in

### V. Test Procedure.


## -. 

# • f. Care should be exercised throughout the course of the· extraction so as not to contaminate the exterior of the extraction flask:



### -13-

### 5. Botary A&itatiQD Apparatus The following rotary agitation devices are suitable for extracting samples prior to**con~ucting** 

### a fish toxicity text. This is not an exhaustive list ofrotary agitationdevices suitable forperforming sample extractions. Anydevice that rotates the extractionvessel in an end-over-end fashion at 30 • 2 rpm is acceptable. luiS,'I.

## "5'" "'tHip Apptnbl' 

# ()

Mo4tl 10,

...- --- 1, Azaal7tloal t.niDa**"a=~,** PA**4-1at~1. Eat~ac~o~**  (DC205) aIIIll**e-.ul~iDa** (215) '''-4490 '-Ioul.**Eat~ac~o~** (DC20)**Se~,** IDa.**12-~U. Eat~~o~**  (DC201) 2,**.u.oo1&~ed** De.laD 61--=1&. VA 2-V••••l (3740-2) aIIIll Mfa" Co, (70S) 549-5999 4-V••••l (S740-4) 6-V
....l (S740-6) S,**Blwl_~ ~,VA**  4-V
....l (04-00-00) Mach1JM
.. ('04) 845-6424 .-V••••l (08-00-00) De.laD, IDa, 4. IRA Hach1De SUp**&.D~,** PR 8-V••••l (OliOO1) aIIIll**1.ebo~.UZ7** (80') 752-4004 5, L&r. LeDda, Mfa,**1lh1~** Lab, 111 5-V
....l (5VIlE) (31S) 449-4116 10-V ••••l (lOVRE) 6. M11110n Cozp, ladfozd, teA**4-1o~~la Eat~ac~or**  (YTSOOJWN) (aGO) 225-SS84



### 1Federal

### Register, Vol. 55, No. 61, p. 11867 (Mar. 29, 1990). -14- 

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o

# • v. TEST PROCEDURE 1. Conductins the Fish Toxicity Test Proper conduct of the toxicity test requires that the test include all of the steps listed below. It is important that steps (a) through (e) be done in the sequence shown.
a. Fill the test and control tanks with dilutionwater, allowing for 400 ml of water from the sample extraction step. The dilution water must have hardness. alkalinity, and pH within the range for -soft- to -moderately hard- water as described in Table 1. b. Introduce the waste into the test 

### tank. The waste includes 

### any undissolved sample, the extract, the rinse, and the extraction flask or bottle
.

.c. Collect a sample for measuring hardness, alkalinity, and conductivity. d. Measure and record D.O.• pH. and temperature
..

h. Terminate the test after 96 hours. f. Observe and record data as indicated on the data sheet
. e. Introduce the fish into the test tank. 

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

i. Complete the data reporting sheet. 2. Adjustins the pH .**Th~** pH of the test solution KAY NOT be adjusted. Any adjusting of thepHwill render the testnull andvoid. there are provisions for demonstrating that the waste kills fish only because of the pH. Appendix A includes a procedure for conducting such toxicity test. I t is not uncommon for the pH to drift during. the 96 -hour test period. This drift is credited, inpart, to fish respiration which increases the CO2 content of the water. which in turn tends to buffer the solution. Do not adjust the pH when this' drift is observed. Record the pHat the time intervals indicatedon the data sheet. 

# • 3.. Replicates and Control At least 10 fish in each of three replicate test tanks must be exposed to each treatment. Replicates must be true replicates with no water connections between the replicate test chambers. Treatments should be randomized. Random assignment of one test chamber for each treatment in a row, followed by random assignment 

### -15-of a second test chamber for each treatment· 'in another or an extension of the same row, is more desirable than total 

# 0 randomization. A represet\tative sample of the test fish should be impartially distributed to the test chambers by adding to each test chamber no more than 20 percent of the number of test fish to be placed in each test chamber 

## and repeating the process until each test chamber .contains the desired number of test fish. Alternatively, the fish canbe assigned either by random assignment ofone fish to each test chamber, random assignment of a second fish to each chamber, etc., or by total randomization. It is often convenient to assign fish to other containers and then add them to the test chambers all at once. 

### Every test requires a control that consists of,the same dilution water, conditions, number of replicates, procedures, and test species used in the test 

### tanks, except that none of the toxicants being tested is added.· 5. Dissolved OXygen The dissolved oxygen in each test chamber shouldbe 80 percent (8.5 mgll at l2·C) of saturation at the begirming ofthe test. The D.O. in each test chamber must not fall below 60 percent (6.4 mgll at l2·C) of'saturation during the first 48 hours ofthe test, andmust not fall below 5.0 mgll during the remainder of the test. If the D.O. falls below 5.0 mgll during the test and the mortality exceeds the pass/fail threshold, the test must be voided. Test solutions may be gently aerated ifthe D.O. does not meet the above criteria. 

# 0 Once aeration is initiated, it must be continued throughout the
. . duration ofthe test. Turbulence shouldbe avoidedbecause itmight.! stress the fish, resuspend fecal matter, and greatly increase volatilization.. Because aeration readily occurs at the surface, efficient aeration canbe achievedwithminimum turbulence by using an air lift to tranSfer solution from the bottom to the surface. Aera&ion should be the same in all test chambers, including the controls. If aeration is used, it is desirable to conduct a stmultaneous testwithout aeration to determine ifaeration affects the results of the test. A test is not a valid toxicity test ifmore than 10 percent of the organisms die in the control test chambers. 5. Test Temperature The test temperature for the test organism must be l2·C (see Table 3). The actual test temperature must not deviate from lrC by more than

## * 1.0·C at**an~** time during the**~est.** 6. 

### Loaeline The glliter of test fish (wet weight, blotted dry) to. solution in the test chambers mustnotbe ao high that itaffects the results of the test. Therefore, the loadingmustbe limitedto ensure that (1) the concentrations'of dissolved oxygen and toxicant are not -16- 

# o**--~------------,**

# •

,7. decreased below acceptable levels, (2)'the' 'concentration of metabolic products does not increase above acceptable levels, and (3) the fish are not stressedbecause of crowding. The loading in test chambers must not exceed 

### 0.8 glliter. Lower loadings may be used ifthe concentrationofdissolved oxygen does not remain above 

### 60 percent saturation for the first 

### 48,hours of the test and above 

### 40 percent saturation after 

### 48 hours. Pissolvin& Toxicant The toxicant shouldbe added to the dilutionwater, at 

### 100 mg/liter or 

### 1000 mglliter, without the use of

### any solvents or other carriers, 'exceptwater. Agentle swirl with a glass rod is usually sufficient to disperse the t:0xicant., 8. Feeding, Test fish should not be fed during acute toxicity tests or for a minimum of

### 48 hours before the test. Fecal matter anduneaten food will decrease the dissolved oxygen concentration which may affect the biological activity of some toxicants. 9. Duration 

# •

•


# • 10. The testbegins whenthe test fish are first exposedto the toxicant. and ends 96 hours later
. Bio1odca1 Data Death is the adverse effectmost often'used in acute toxicity tests: with aquatic organisms. The criteria for death is lack ofmovement,. especially the absence ofrespiratory movements in fish and lackof,' reaction to gentle prodding. The loss of equilibrium, defined as the inability'to make coordinated movement and maintain a normal upright position, is not to be 'considered' a mortality. The latter responses canbeuseful ininterpretationoftestresults and should be noted in the comments section of the data reporting sheet. It is extremely important that procedures used to determine whether test fish are dead" immobilized, or otherwise affected, and to remove dead fish do not stress the live test fish. Movement oftest chambers and prodding must be done very gently. The number of dead or affected fish in each test chamber must be counted ,every 24 hours after the beginning of the test. More observations are desirable, especially near the beginning of the test. A suggested schedule is to count the number of dead or affected fish in each chamber 3, 6, 12, and 24 hours after the beginning of the test and twice a day thereafter to the end of the test. Dead fish must be removed as soon as they are observed, or once every 24'hours. Weights and lengths of test fish may be determined by weighing and measuring and discarding either; (1) a 

### -17-representative group ·of fish before the test;· or (b) the control fish that are alive at the end of the test. The length should be 

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

### dry.
. Fish used in a test, including those used in a control treatment. must not be used in a later test. 11. Chemical and Physical Data for Test Tanks The dissolved oxygen concentration and the pH must be measured in all test tanks at the beginning, every 24 bours (as long as live fish are present), and at the end of the test or at the time the last fish dies in the test tank. Hardness. alkalinity. pH, and conductivity must be measured in the test and control tanks at the start of the test. It is desirable to measure the same parameters at the end ofthe test. Measurement ofcalcium. magnesium, sodium, potassium,**ch~oride,** sulfate, particulate matter. and total organic carbon (TOC) orchemical oxygendemand (COD) is also desirable. For all tests, continuous temperature monitoring in at least one test tank is desirable. As an alternative, the maximumandminimumtemperature must be recorded every 24 hours. As a second alternative. temperatures in at least one test tank must be recorded at least every six hours. 

# o

-18- 

# ()

# •

•**VI~** QUALITY ASSURANCE/QUALITY CONTROL 1. Procedures for Quality**~surancetouality** Control Because reliable laboratory data is important to making good environmental decisions, a good quality assurance/quality control (QA/QC) program is essential to ensure the reliability ofthe data. Quality assurance (QA) is the total integratedprogram for assuring the reliability ofmonitoring andmeasurement data. Acomprehensive quality assurance programwould address the entire system affecting data quality, from collecting a representative sample in the field "to preparing the final report. The QA program should address: (a)"sample collection and sample preservation; (b) laboratory services; (c) use andcare ofinstruments, glassware, andchemicals; (3) datamanagement; and (e) laboratory personnel. Quality control (QC) is the routine applicationofstatisticallybasedprocedures to evaluate and control the accuracy of results from analytical measurements. Acomprehensive quality control program"includes procedures for estimating and controlling precision and bias (e.g., accuracy of results) and for determining lower reporting limits (e.g., detection limits). Acomprehensive quality control program wouldaddress specific topics relatedto sample analysis, including; (a) defining data quality objectives; (b) choosing analytical methods"whichwill meet those objectives; (c) estimatingwithin-lab precisionandbias; (d) settingup control charts; (e) participating in inter-lab tests; and (f) analyzing samples
. For specific recommendations regarding the conduct of a fish bioassay, refer to Appendix A, GoodLaboratory Procedure Standards.



# • 2. Reference Toxicants Areference toxicant is used to establish the validity of toxicity data generated. The reference toxicant measures every aspect of toxie-ity testing, including: The condition of the test fish; the pusence and effects of stressors (other than the compound being tested) inholding andtesting; and, the analytical techniques being used. Ifa reference toxicant LC 50 differs significantly from that previously established in a laboratory, a full review of test conditions and procedures is indicated. Test fish must be evaluated with a reference toxicant within the fourteen days immediately preceding a toxicity test, orpreferably, concurrently"with the test. More frequent testing with a reference toxicant is appropriate when situations occur which may affect test results. For example, a change in dilution orholding water conditions, new equipment or piping in the laboratory, or a change of analysts, all indicate the need for additional use of a reference toxicant. A variety of compounds are commonly used as reference toxicants. Three reference toxicants are available from EPA; including sodium dodecylsulfate, sodium pentachlorophenate, and cadmium chloride


### -19-(EnvironmentalMonitoringandSupportLaboratory"(EKSL). Cincinnati. Ohio 45268). VII. CALCULATION OFRESULT As mentioned in the introduction. this test procedure does not determine the LC so• but rather provides information -about- the LC so of a test toxicant, specifically. whetherthe LC sois above orbelowa preestablished concentration. Therefore. this method is classified as a range finding test. Data collected during the testwill be analyzed according to the binomial distribution. Data from the three replicates will be pooled-for several reasons: (1) ease of interpretation. (2) to increase the number of organisms (n) from 10 to 30. and (3) variation between the three replicates will provide information on sample representativeness and on laboratory methodology. 1. Examples For a waste material to be designated 

### an extremely hazardous waste (EHW). greater than ten cumulative deaths out of 30 test organisms

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

is said

-to have an LC so less than 100 mgll at the 95 percent confidence level (example 1). 

# o

Example 1. Number of Cumulative Deaths 0 24 48 72 96 (hours) 

# 0 Tank A 2 3 5 6 6 Tank 

### B 3 3 4 4 4 Tank C 1 2 3 3 

### .:.,1- 13 EHW 

### @100 mgll Similarly. a waste materialwouldbe designateda dangerous waste if greater the 11 cumulative deaths out of 30teBt organisms occurred

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

said to have an LC so of less than 1000 mgll at the 90 percent confidence level (example 2). Example 2. Number of Cumulative Deaths 0 24 48 72 96 (hours) Tank D 

### 3 4 5 5 "6 Tank E 2 4 4 5 5 Tank F 3 3 "3 5 

### • 16 DY

### @1000 mg/l

-20- 

# o

# •

•



# • Normally, 

### any control deaths should be cause for concern with the test conditions. For those borderline tests, corrections for up to 10'percentmortality in cOJ\trol canbe achievedby Abbott's Formula (Equation 1).
. (Equation 1) p.-C P=

....**_-~** 

### l-C P - corrected proportion for control mortality p*

- observed test fish mortality as a proportion

(i.e., 

### .3, .4, etc.)
. C - observed control fish mortality as a proportion 

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

### any preselected confidence interval (Equation 

### 2). (Equation 2)



### Z - test statistic Po - 0.50 (i.e., LC 50) N - 30 (pooled organisms) If 

### the test statistic 

### Z is less than the break point value for a c}wsen confidence interval (Table 4), then the hypothesis that the LC50 is less than 100 or 1000mg/l is rejected. An applicationusing Abbott's formula appears in Example 3.
. Table 4. Confidence Interval

'.. Confidence Interval 90' for 1000 mg/l 95' for 100 mg/l Example 3. Break Point 

### -1.28 -1.64 

# • A waste material has been tested at 100 mg/l and killed 11 fish within 96 hours. It appears the material is an EHW. However, one control deathwas observed (less than or equal to 10 percent), does this change the test conclusions?
. 

### -21-

### Step 1. Input variables into Equation 1: Where p*

- 11/30

- 0.31 C - 1/10

- 0.10 P=0.37-0.10 

### 1-0.10
P=0.30 

### Step 2. Input P into Equation 2: Z= . P-Po

# Po(~Po) Where Po - 

### 0.50 .N - 

### 30 

### P - 0.30 

### Z 0.30-0.50

= 

# I .50(1-.50) 30
Z= 

### .30-.50 .09128 

### Z=-2.19 Since Z is 

### less than the breakpointof -1.64 

### the material is not an 

### EHW at the 95 

### percent confidence interval. 2. 

### Precision The results of acute toxicity tests conducted in different laboratories using the same species, dilution water, toxicant, and**pr~cedures** 

### will usually differ by no more than a factor of two. 3. Reportinl A 

### report of the results of a test must include the following:
a. Name 

### of test, iuvestigator, and

### laboratory, and date testwas. begun;

-22- 

# o



# o



# o

# • b • 

### Adetailed description ofthe toxicant, includingits source, lot number, composition (identity and concentration ofmajor ingredients and maJor impurities), and known physical and chemical propertie·s;
c. The source of the dilution water, its chemical characteristics, and a description of any pretreatment, including any additives to the dilutionwater; d. 

### Detailed information about the test organisms, including scientific name and how verified (and strain for salmonids whenappropriate), weight (wet, blotteddry), 

### length (standard length for fish), age, life stage, source, history, observed diseases, treatments, acclimation procedure, and food used;
e. A description ofexperimental desim and the test chambers, the depthvolume ofsolutionin the chambers, the waythe test wasbegun, thenumber oforganismsper-treatment, the-loading, and the lighting. f. A description of 

### any aeration perfOrmed on test solutions before or during the test; g. Definition of the criterion used to determine the effect and a summary of general observations on other effects or symptoms; 

# • h. Number and percentage of fish that died in the test and the control tanks; 

### i. Identification and concentration of reference toxicant used and the resulting mortality and LCso• 

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

- of, all chemical analyses of water quality and toxicant**concentration~** 

### including validation studies and reagent

blanks;

- k , The average and range of the followin& parameters: acclimation temperature, test temperature, - pH, hardness, alkalinity, dissolved oxygen, and conductivity; 1. Anything unusual about the test, any deviation from these procedures, and any other relevant information.



### ., II. 

### The volume and concentration ofneutralizing solutionused to adjust the pHof the test solution
. 

# • A sample reporting form is attached. Any alternative forms should provide, at a minimum, the information required on the attached sample form•



### -23-

# • (h) (1) Observe and'record data as indicated on the data sheet. Adjust the temperature 

### an4 D.O. ·as necessary.



# •

• (j) Terminate the test after 96 bourse (k) Complete the data reporting sheet. 3. Recording the Data When conducting a neutralized toxicity test. record the following information on ·the data reporting sheet: (a) The neutralizing material (i.e.• NaOH or HC1). (b) The normality of the neutralizing solution. (c) The volume of the neutralizing solution. (d) Indicate whether' readjustment ofpH was necessary 30 minutes after initial pH adjustment
.



### -25-APPENDIX·A NEUTRALIZED FISH TOXICITY TESTPROCEDURE 1.
.General The neutralizedfish toxicity testprocedure is similar in all respects to the test procedure described in the main body of this manual except as noted in this appendix. The only difference is that aquarium water is neutralized the waste is introduced into the aquarium, and before the fish are placed into the aquarium. The aquarium water may be neutralized using only aqueous solutions of hydrochloric acid or sodium hydroxide. Add the neutralizing solution dropwise while stirring the contents of the test tank, and measuring .the pH continuously. The·pH of the aquarium water should be adjusted to 7.5 and allowed to stand 30 minutes to detect any pHdrift. Ifthe pHdrifts outside the range 7.2

- 7.8, the aquariumwater shouldbe readjusted to pH .7.5, and the fish introduced immediately after. Further pHadjustment is not allowed. 

# o

2. The neutralizing·solutions should be as concentrated as practicable to minimize diluting the aquariumwater. Under no circumstances shouldmore than 50 m1be Used to adjust the pH. The actual volume and normality of neutralizing solution used should be reported on the. data reporting sheet. Conducting the Neutralized Fish Toxicity Test Proper conduct of the Neutralized Fish Toxicity Test requires that the test include all of the steps listed below. It is important that.steps (a) through (e) be done in the sequence ,shown. (a) Fill the test tankand the control 

### tankwith dilutionwater allowing for 490 m1 ofwater from the sample extraction step. The dilution w"er must have hardness, alkalinity, and pHwithin the range for ·soft-to -moderately hard· water as described in Table 1. (b) Introduce the waste into the test .tank. The waste includes any wtdisso1vedsample, the extract, the.rinse, andthe extractionflask or bottle. (c) Measure and record the pH. (d) Adjust the pH to 7.5 andwait 30 minutes. Ifthe pHdrifts outside the range 7.2-7.8 re-adjust the pH and proceed immediately to (e), (f), and (g).
. (e) Collect a sample for measuring hardness, alkalinity, and conductivity. (f) Measure and record D.O., pH, and temperature. 

# o

(g) Introduce the fish into'the test 

### tank. -24-

# •

•

• 1.



### 2. APPENDIX B EFFLUENT TOXICITY TESTPROCEDURE General The effluent toxicity test procedure is similar in all respects to the test procedure described in the main body of this manual except as noted in this appendix. This procedure shall be used for the salmonid toxicity limit required in NPDES permits. The purpose ofthe static effluent toxicity test required by the Department ofEcology is to evaluate the effect of an effluent on the resident andmigratory**salm~nid** fishes in state waters. The effluent "tOXicity standards for Washington State require that 80 percent of the salmonid fish tested survive in at least 65 percent concentration of effluent for 96 hours. The sample shall not be dechlorinated prior to conducting .the test. Samples shall be maintained at a 4·C in the dark during shipping and storage. The sample"should be collected with minimum head space. The recommended holding time is 72 hours. Conducting the Effluent Toxicity Test Proper conduct of the Effluent Toxicity Test requires that the test include all of the steps listed below
. (a) Fill the test tank with a solution of effluent and dilution water. The solution strengthmustbe as specified in the NPDES permit. The: dilution water must have hardness. alkalinity. and pH within the "ranges specified in Table 1. (b) Collect a sample for measuring hardness, alkalinity• and conductivity. " (c) Measure and record D.O., pH, and temperature. (d) Introduce the fish into the test tank. (e) Observe and record data as indicated on the data sheet. (f) Adjust the temperature and D.O. as necessary. (g) Terminate the test after 96 hours. (h) Complete the data reporting sheet
.



### -26-

# •

. '

e,



# • APPENDIX C GOOD LABORATORY FRQCEDUU

0STANDARDS1 

### The information includedbelowwas adapted from EPA's GoodLaboratory Practice (GLP) Standards. These standards are intendedtoberecommendedstandardsrather than

### requirements. Laboratories are encouragedto develop QA/QC programs which address these recommended standards• A. ORGANIZATION AND PERSONNEL 

### 1. Personnel Each individual engaged in the conduct of or responsible for the supervision of a study shall have education, training, and experience, or combination thereof, to enable that individual to perform the assigned functions. Each testing facility shall maintain a current summary of training andexperience and

### job descriptionfor each individualengagedinor supervising the conduct of a study. There shall be a sufficient number ofpersonnel for the timely and proper conduct ofthe study according to the protocol. Personnel shall take necessary personal sanitation and health precautions designed to avoid contamination of test and control substances and test systems. Personnel engagedina study shallwearclothingappropriate for the duties they perform. Such clothing shall be changed as often as necessary to prevent microbiological, radiological, or chemical contamination oftest systems and test and control substances. Any individual found at any 

### time to have an illness that may adyersely affect the quality and integrity of the study shall be excluded from direct contact with test systems, test and control

,substances and any other operation or function that may adversely

affect the study until the condition is corrected. All personnel shall be instructed to report to their immediate supervisors any health or medical conditions that may reasonably be considered to have an**adver~e** 

### affect on a study. 

### 0., 2. Biolo&istin Char&e


# • For each study, a biologist, or a scientist, or other professional of appropriate education, training, and experience, or combination thereof, shall be identified as the biologist in charge. The biologist in charge has overall responsibility for the technical conduct ofthe study, as well, as for the interpretation, analysis,

lFederal 

### Register, 

### Vol. 48, No. 230, p. 53922ff (Nov. 29, 1983). -27-documentation, and reporting ofresults, and represents the single point of study control. The biologist in charge shall assure that: (a) Theprotocol, including

### anychange, is approvedasprovidedby Ecology and**~s** followed. (b) Allexperimentaldata, includingobservations ofunanticipated responses of the test system are accurately recorded and verified. 

# o

(c) Unforeseen circumstances that may affect the quality and integrity of the study are noted when· they occur, and corrective action is taken and documented. 

## " (d) Test systems are as specified in the protoc91. (e) All applicable good laboratory practice regulations are followed. (f) All raw data, documentation, protocols, specimens, and final reports are transferredto the archives during or at the close of the study. 3. Protocol' Each study shall have an approved written protocol that clearly indicates the objectives and all methods for the conduct of the study. Theprotocol shall containbutnotbe necessarilybe limited to the following information: 

# 0
(a) Adescriptive title and statementofthepurpose ofthe study. (b) Identification of the test and control substance by chemical name, Chemical Abstract Service Registry Number (CAS), or

- other established identification system. ' (c) The name and address of the sp::,nsor and the name and address ofthe testing facility atwhich thestuciy is beingconducted. (d) The proposed,starting and completion dates. (e) Justification for selection of the test system. (f) Where applicable, the number, body weight range, sex,**s~urce** of supply, species, strain, substrain, and age of the test system. (g) The procedure for identification of the test system. (h) A description of the experimental design, including the methods for the control ofbias. (i) A description'and/or identification of the diet used in the study as well as solvents, emulsifiers and/or othermaterials 

### -28- 

# o

# •

.. (j) (k) used to solubilize or sUspend the test or ·control substances· before mixing with the carrier.2 The description shall include**specificati~ns** for acceptable levels of contaminants that are reasonably expected to be present in the dietary materials and are known to be capable ofinterfering with the purpose or conduct of the study ifpresent at levels greater 

### than establishedby the specifications. The route of administration and the reason for its choice. Each dosage level, expressed in milligrams per kilogram of bodyweightorother appropriate units, ofthe test or control substance to be administered 

### and the method and frequency of administration. (1) Method by which the degree of absorption of the test and control substances by the test system will be determined if necessary to achieve the objectives of the study. (m) The type andfrequency oftests, analyses, andmeasurements to be made•. (n) The records to be maintained. (0) The date of approval of the protocol by the sponsor and the signature of the biologist in charge
. 

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



## ...


# • (q) All changes in or revisions of an approved protocol and the reasons therefor shallbe documented, signedby the biologist in charge, dated, and maintained with the protocol. 4. Conduct of a Study The study shall be conducted in accordance with the protocol. The test systems shallbe monitored inconformitywith theprotocol. Specimens shall be identified by test system, study, nature, and date of collection. This information·shall be located on the specimen container or shall accompany the specimen in a manner that precludes error in the recording 

### and storage of data
. Records of gross findings for a specimen from postmortem observations. shall be available to a pathologist wllen examining that specimen histopathologically.

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

### -29-All data generated during the conduct ofa study, except those that· are generated as direct computer input. shall be recorded directly. 

# 0 promptly. and legibly in .ink. All data entries shall be dated on the day ofentry and signed or initialedby the person entering the data. Any change in entries shall be made so as not to obscure the original entry. shall indicate the reasonfor such change. and shall be dated and signed or identified at the time of the change. In computer driven data collection systems. the individual responsible for direct data input shallbe identifiedat the time ofdata input. Any change in computer entries shall be made so as not to obscure the original entry. shall indicate the reason for change, and shall be dated and the responsible individual shall be identified. 

### 5. Maintenance and Calibration of Equipment Equipment shall be adequately inspected. cleaned, and maintained. Equipment used for the generation, measurement, or assessment of data shall be adequately tested. calibrated, and/or standardized. Awritten standard operating procedures shall set forth. in sufficient detail the methods. materials, and schedules to be used in the routine inspection, cleaning, maintenance, testing, calibration, and/or standardization of equipment, and shall specify remedial action
.to be taken in the event of failure
.or malfunction of equipment. The written standard operating procedures shall

.designate the person responsible
.for the performance of each operation, and copies ofthe standard operating procedures shall be made available to laboratory personnel. Written records shallbe maintained ofall inspection, maintensnce, testing. calibrating, and/or standardiZing operations. These records, containing the date of the operation, shall describe whether the maintenance operations were routine and followed the written standard operating procedures. Written records shall be**kep~of** non-routine repairs performed on equipment as a result of failure and malfunction. Such records shall document the nature of the defect, how andwhen the defectwas discovered, andany remedial action taken in response to the defect. 

## •



# o

B. TESTING FACILITIES OPERATION 1. Standard Operating Procedures A testing facility shall have standard operating procedures in writing setting forth studymethods thatmanagement is satisfiedare adequate to ensure the quality and integrity of the data generated in the course of a study. All deviations in a study from standard operating procedures shall be authorized by the study director and**~,** shall be documented in the raw data. Significant changes in established standard operating procedures shall be properly authorized in writing by management.

-30- 

# o

# •• Standard operating procedures shall be established for. but not limited.to. the following: (a) Animal roompreparation. (b) Animal care. (c) Receipt. identification. storage. handling. mixing. andmethod of sampling of the test and control substances. (d) Test system observations• 

# • (e) 

### Laboratory tests. (f) Handling ofanimals found moribund or dead during study. (g) Necropsy of animals or postmortem examination of animals. (h) Collection and identification of specimens. (i) Histopathology. 

### (j) Data handling. storage, and retrieval. (k) Maintenance and calibration ofequipment. 

# • (1) (m) Transfer, proper placement, and identification ofanimals. Each laboratory area shall have immediately available laboratorymanualsandstandardoperatingprocedurespertinent to the laboratory' procedures being performed, e.g." toxicology, histology, clinical chemistry, hematology., teratology, necropsy. Published literature maybe used as a supplement to standard operating procedures.

f' 

# • 2. (n) Ahistorical file of standard operating procedures, and all revisions thereof, including the dates of such revisions, shall be maintained. ReSlents and Solutions All reagents and solutions in the laboratory areas shall be labeled to indicate identity, titer orconcentration, storage requirements, and expiration date. Deteriorated or outdated reagents and solutions shall not be used.

-31-

# •

• APPENDIX D**RECOMMEND~** FISH LOADING Prior to use in a toxicity test, the fish are Usually maintained at the test facility ina circulatedholding tank. Ithas beendeterminedthroughexperience that a minimum level ofcirculation is required to maintain the viability ofthe test fish. The recommended level ofcirculationvaries depending on the· species offish, the size offish, and the temperature. For small fish, the size ofthe fish is usually reported in terms ofnumber offish per pound. The table below lists the recommendedfish minute loadings (pounds offish per gallonperminute of inflow), to promote the health and quality of salmonid fish used in toxicity tests.



# • Species
Coho Salmon

Fall and Spring Chinook Salmon TABLE Dl. RECOMMENDED FISH LOADINGS·
Water Temperature Pounds of Fish per GPMb ·C 

### ·F loooe sooe looe 

### 3 38 3.5 5.0 8.0 9 

### 48 2.7 4.0 6.0 .14 58 2.2 3.0 4.5 -17 

### 63 2.0 3.5

3 38 3.0 4.0 6.0 9 

### 48 2.5 3.0 5.0 14 58 2.0 2.2 3.5 17 63 1.2 3.0

f' 

# • • Adapted from Fish Diseases Leaflet 

### 38 (see bibliography). b GPM means gallons per minute. e Fish size (number of fish per pound).



### -32-

# • • • 11I11 11 111
.. 1 11111 

## '"1''1''' " [COL 0 GY DATASHEETFORSTATICBASICACUTE FISHTOXICITYTEST laboratory

_

. Analyst --:-

_ IndustrylToxlcant Beginning Date T1me

_ Address Ending Date Time



# --------------- Collector Test Organlsm**~---~** Date Sample Collected Required Test TemperatureRange,

_ Test 

# , To1III To1III laboratolyReference Cone. tMnberofCllntil1lve

## Dea'" DissolvedOxygen(mgll) pH25C TlWllper81Ur1 (C) Hanlness AIkaIInItr 

### ConcludIvItr Number ner (mgll) I (mgn. (mgnes uMHOSIan No. CaCOsl 

### caco,l 0 24 48 72 

### ge 0 24 48 72 

### ge 0 24' 48 72 98 0 24 48 72 98 0 

### Ie 0 18 0 

### Ie

Sample Description

_ Average Weight Meanlength longest

__---'- Shortest Ratio(Ionglshort)

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

_

__________ EWH> 10/30

_______ __OW> 11/30 Data Verified By**~=~** Date

_ ECY 030-1-40 MllhodonFilewtth\heDIpattmIntofEClIlIDgy GENERALPROCEDURE FORSTATICBASICACUTE FISHTOXICITYTEST

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# • u.S. Environmental Protection Agency, Karch 1985, Methods for Measuring the Acute Toxicity ofEffluents to Freshwater and Karine Organisms. (EPA/600/4-85/0l3) 2. Federal Register Vol. 48 No. 230, November 29, 1983, Good Laboratory Practice Standards, p. 53946 ff. 3;**Wedem~yer,** 

### G.A., and J.W. Wood, 1977. Stress as a Predisposing Factor in FishDisease. U.S. Fish and Wildlife Service, FishDiseaseLeaflet 38 (FDL-38), Washington, D.C. 4. Wedemeyer, G.A., D.J. McLeay, C. Phillip**Goodyea~.** 

### 1984. Assessing the Tolerence ofFishand FishPopulations to Environmental Stress: The Problems and Methods ofMonitoring. 5. Wedemeyer, G.A., R.L. Saunders, and W.C. Clarke, 1981. The Hatchery Environment Required to Optimize Smoltification in the Artificial Propagation ofAnadromous Salmonids. Pages 6-20 in E. Kineyand L. Allen, eds., Bio-Engineering in Fisheries. Special Publicationby American Fisheries Society, Washington, D.C.

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# • PART B ACUTE ORALRAT TOXICITY TEST
1980

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• PART B ACUTE ORAL R4T TOXICITY TEST I.
.INTRODUCTION The purpose of oral rat testing is to determine the relative biological riskofpotentiallyhazardous waste to man andwildlife. The results will

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

dangerous waste under VAC 

### 173-302 -The Hazardous Vaste Regulation.

- Test analyses arenotbasedupon leachate extractionprocedures andas such, do not attempt to simulate changes that ,the waste might undergo after disposal. The toxicity test is to be construed'as an index of acute single exposure ofbiological risk to the generator, transporter, treater and disposal site operator. The test is not intended to determine what changes in toxicity might occur, after disposal due to environmental reactions. These conditions are difficult to predict and quantify especially for the sanitary landfill scenario. The Departmentuses oral rat tests whenever a generatorhas not or cannot adequately characterize the toxicity of a waste because of:
a. A lack of knowledge of the chemical composition and presence of toxic constituents. b. Alackofknowledge of the biological hazards oftoxic constituents or the waste as a whole
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# • c. The inability to book designate a waste's biological toxicity.



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# • 11. STANDARDS To determine whether a waste is extremely hazardous, a representative sample ,of the waste shall be tested at 

### 500 milligrams of waste per kilogram 

### of rat body weight. Tests showing significant toxicity as definedherein shallbe deemedextremelyhazardous wastes (1.e., LDsoS 

### 500 mg/kg). To determine whether a waste is a' dangerous waste, a representative sample shall be tested at 

### 5,000 milligrams of waste per kilogram ofrat body weight (1.e., LDsoS 

### 5,000 mg/lcg). The death of two or less test animals per dose will be statistically evident that the LD50 is greater than the ,standard at the 95 percent confidence level. The death of any control animal will invalidate the entire test. 111. REPRESENTATIVE SAMPLE Arepresentative sample ofthe waste to be tested shall be obtainedusing methods established by the Department of Ecology. The need to obtain a truly representative waste sample is as important as the biological testing itself, and its inadequacy may be grounds for rejecting an otherwise acceptable result. ' ,IV. SAMPLE PREPARATION



### B-1 ,- i
IThe waste shall be tested in the form it is to be'disposed with the' following exceptions:
a. Waste may be ground and coarsely filtered to allow its oral administration by stomach tube. b. Liquidand solidpllases shouldbehomogenizedbefore administration. 

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c. Upon review and approval on a case-by-case basis by the Department of Ecology.' V. TESTORGANISMS Male albino rats weighingbetween 200 

### and 300 grams shallbeused for test and control animals. Ten rats shall be used at each dose level, and ten rats for each positive control test. (See lbelow)

.VI. DOSING All animals shall be dosedby gavage. All animals shall receive the same concentrationandapproximatevolume ofdosing solution, not·to exceed4-5 mls per animal'. VII. TESTDURATION The animals shall be observed for 14 days. VIII
.. TESTPROCEDURE Food shall be withheld from animals the night prior to dosing. Observations for mortality and clinical pharmacological or toxicological signs will**~e** recorded immediately afterdosing at one andfourhours, and daily thereafter for 14 consecutive days. All surviving animals will be sacrificed at termination on the 14th day. Gross necropsies will be performed on animals that die and those ·sacrificed at termination. IX. DATA REPORTING The test report shall include the following information:

a. Tabulation of response data by dose level (Le.·, number of animals dying per"number of animals exposed).
lNOTE: Ecology may require a positive control group for particular wastes or toxicants when the sensitivity ofthe test animal is indoubt. Whenused, the positive control group should serve as an internal quality control to ascertain whether the test animals are sensitive or resistant to the test material by testing their response to a known toxic agent•. B-2 

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### c. d. 

### Tille ofdeath after**do~ing.** 

### Clinical pharmacological•.and/or toxicological signs. Gross necropsy findings.

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