ML20116B448

Results of Acute Toxicity Test W/Daphnia Magna & Pimephales Promelas on Samples from Calgon Corp

ML20116B448
Person / Time
Site:	Perry
Issue date:	10/20/1992
From:	Goodfellow W, Sohn V EA ENGINEERING, SCIENCE & TECHNOLOGY, INC.
To:
Shared Package
ML20116B444	List: ML20116B440 ML20116B448
References
NUDOCS 9211020213
Download: ML20116B448 (32)

Text

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hiTAC H DT EA EbCLVEER1NG, SClENG, AND TECHNOLOGY, L4C EA Project 70C03.10 RESULTS OF ACUTE TOXICITY TESTS

WITH Daphnia magna AND Pimephales promelas ON SAMPLES FROM CALGON CORPORATION

[Y Preparedfor:

Calgon Corporation Elyria, Ohio Prepared by:

EA Mid Atlantic Regional Operations EA Engineering, Science, and Technology, Inc.

151.oveton Circle Sparks, Maryland 21152 mw o_

VirginiyK; Sohn O Ifm ~

do Or r c) , ,

Date

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Project Manager t M~

William L. GoodfeUod, Jr./

. fk- 0kAlt b$U Date Senior Scientist October 1992 R por: Number 1168 l

9211020213 921028 0 PDR ADOCK 0500 P

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l. INTRODUCTION

!e l['i At the request of Calgon Corporation, EA Engineering, Science, and Technology performed

I' acute toxicity screening tests with Daphnia magna (water flea) and Pimephales promelas (fathead minnow) on three samples from the Perry Nuclear Power Plant. Each species was exposed to 1

100 percent samples of (1) influent, (2) effluent prior to treatment, and (3) effluent during

< treatment, i, h.+

Sample collection and receipt data, together with initial water quality data, are reported in

! a Table 1. Test results, test organism source and age, reference toxicant data, and water quality r

! measurements are summarized on pages 3-6. Test protocols, copies of raw data sheets, and the

L Report Quality Assurance Record are included as Attachments I, II, and III, respectively.

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RESULTS

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Results of the heute screening ssays indicate that the three samples from Perry Nuclear Power Plant were not acutely toxic to Daphnla magna or Pimephales promelas. There was 100 percent survival of D. magna after 48 hours5.555556e-4 days <br />0.0133 hours <br />7.936508e-5 weeks <br />1.8264e-5 months <br /> of exposure to ti.*, influent, effluent prior to

treatment, and the effluent during treatment (page 4). In the P. promelas test (page 6), there was a minimum of 95 percent survival after 96 hours0.00111 days <br />0.0267 hours <br />1.587302e-4 weeks <br />3.6528e-5 months <br /> of exposure to the thrm test samples.

! Both tests hau 100 percent survival of control organisms.

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SUMMARY

OF SAMPLF1 TEST INFORMATION e ,.

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, Test: 48-hour Daphnia magna Static Acute Toxicity Test 4 Client Name: Calgon Corporation i/

}/ Test Procedure: EA Protocol ATS SAI DS-04 Acute 48 hour5.555556e-4 days <br />0.0133 hours <br />7.936508e-5 weeks <br />1.8264e-5 months <br /> asszy with Daphnia i (1. '

t Sample

Description:

Influent; ef3uent prior to treatment; effluent during treatment iK EA Accession Numbers: AT2 833, AT2-334, AT2-83S

.b EA QC Test Number: SA-10-02 92-1043

, Test initiation time aad date: 1250,2 October 1992

]. Test co npletion time and date: 1300,4 October 1992 Control Water: Dechlorinated tap water

{

l. Organism Lot Information I

1 o- Lot Number: Not Applicable Source: EA's Culture Facility, Sparks, Maryland 3

Age: <24 hours

!L.

f-c Reference Toxicant (SDS) Test Informali20 Reference Toxicant: Sodium dodecyl sulfate f, ,o. EA QC Test Number: RT-10-05-921046 4 i Dilution Water: Dechlorinated tap water 4

I- . Results:

l 24-hour LC50: 30.4 n.g/L i[

j) Laboratory control chart acceptability range for 24-hour LC50:

.19.4 - 55.5 mg/L

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SUMMARY

OF SELECTED TEST DATA (Cont.)

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Test Organism: D. magna

,. EA QC Test Number: SA 10-02 921043 i Test Material (100 cercenLqoncentration) Percent Survival at 48 Hours

'. Laboratory Control 100 Influent 100 L '

Effluent prior to treatment 100

• Effluent during treatment 100 i

Selected Water Ouality Raiice

! Temperature (C) 19.0 - 21.0

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pH 7.8 - 8.8 i Dissolved oxygen (mg/L) 8.6 - 9.3 Conductivity ( S*) 235 - 340 1

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SUMMARY

OF SAMPLE / TEST INFORMATION y

Test: 96 hour0.00111 days <br />0.0267 hours <br />1.587302e-4 weeks <br />3.6528e-5 months <br /> rimephales promelas Static Acute Toxicity Test Client Name: Calgon Corporation Test Procedure: EA Plc.ocol ATS-SAF-FM-04 Acute assay with fathead minnows (Pimephales promelas)

Sample

Description:

Influent; emuent prior to treatment; emuent dt ring treatment c EA Accession Numbers: AT2-833, AT2-834, AT2-835 EA QC Test Number: 3A-10-02-92-1042 Test initiation time and date: 1320,2 October 1992 Test completion time and date: 1320, 6 October 1992 Control Water: Dechlorinated tap water Organism Lot Informatiott Lot Number: FH 9/22-23 Source: EA's Culture Facility, Sparks, Maryland Age: 9-10 days (hatched within a 24-hour window)

Reference Toxicant (SDS) Test Informati20 Reference Toxicant: Sodium dodecyl sulfate

,, EA QC Test Number: RT-10-13-92-1065 Dilution Water: Dechlorinated tap water

• Results:

24-hour LC50: 28.3 mg/L Laboratory control chart acceptability range for 24-hour LC50:

21,8 - 38.4 mg/L f.

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SUMMARY

OF SELECTED TEST DATA (Cont.)

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1I! Test Organism: P. promelas

, EA QC Test Numbei: S A-10-02-92-1042 J

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i Test Material r (100 oercent concentration) Percent Survival at 96 Hours l Laboratory Control- 100 Influent 100

, Effluent riior to treatment 95

[s ., Effluent during treatment 100 Selected Water Ouality Rance

, Temperature (C) 19.0 - 21,0

.: , pH 7.7 - 8.8 1

p- Dissolved oxygen (mg/L) 8.0 - 9.1 Conductivity (nS*) 208 - 328 i

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t TABLEI SAMPLE COLLECr!ON AND INITIAL WATER QUALITY DATA FOR SAMPLES FROM CALGON

CORPORATION ,

Sample EA Accession Type of Sample Sample AlkaFairy liardness i Description Number Samplc Collection Receipt (me/L CaCOd (mell CACO _d j i

Influent AT2-833 Grab 0840,30 SEPT 92 1040,2 O(X 92 44 72 Effluent prior AT'e-834 Grtb 0928,30 SEPT 92 ' 1040,2 OCr 92 102 124 to treatment Effluent ' AT2-835 Composite 1000,30 SEPT 92 to 1040,2 OCT 92 100 12s 4 during treatment 0100,1 OCT 92 l

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PROTOCOL ATS-SAI DS-04 e

? 3. ACUTE 48-HOUR LC50 ASSAY WITH R thnia sp.

1

[ 1. TEST OBJECTIVE To assess the toxicity of a test material to aphnia and determine the LC50 or p EC50 using mortality or immobilization, respectively, as the test end points

> 2. TES*1 ARTICLE 2.1 Dercription/ Identification rc Unless otherwise specified, the test materia) is supplied by the client. Adequate j? ~

chemical specifications with special reference to hazardous properties and storage conditions ts also supplied by the client, 2.2 Methods of Synthesis in most cases the test article is an effluent sample. Information on the methods of synthesis, stability, and camposition or other characteristics which define the test article are on file with the client.

j 3. EXPERIMENTAL DESIGN 3.1 Test Organinns A species of &phnia (water fleas), as determined by project needs, is the test 4

orgamsm.

3.2 Source

&phnia used for acute toxicity tests are obtained from stock cultures maintained in EA's Aquatic Toxicology Culturing Facility.

3.3 Culturing and Holdinc Conditions

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&phnia cultures are maintained at 20 2 C and a 16-hour light, 8-hour dark photoperiod cycle in an environmentally controlled laboratory.

Cultures are maintained in 18.9-L all glass aquaria or other appropriate container J. and are fed algae (Selenattrum capricornutum) and a trout chowlyeast/ cereal leaves suspension in the manner described in Peltier and Weber (1985). New cultures are initiated on a routine basis to ensure healthy, productive populations. Organisms from cultures producing ephippta are not used for to: ..ytests.

ATS-102 EA Engineering, Science.

and Technolog), Inc.

Effective May 1981

, Revision 4. April 1991 Page 1 of 7 i

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PROTOCOL ATS SAI-DS-0/

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).) 3.4 Age of Test Organisms at Test Initiation 5-Neonates of known age (i.e., less than 24 hours2.777778e-4 days <br />0.00667 hours <br />3.968254e-5 weeks <br />9.132e-6 months <br /> old) are obtained for testing by segr,egating adult females from the mass cultures on the day before a test is imtiat,ed. On the day cf the test, neonates are segregated from the parent or gumsms.

3.5 Djiution Watu

The source of dilution water is the City of Baltimore Municipal Water System.

.. Upon entry to the laboratory, the water is passed through a high-capacity, activated-carbon filtration system to remove chlorine and other possible e contaminants such as ,tergents, and hexane soluble organic materials. This h water source has proven safe for aquatic orj;anism toxicity testing as evidenced by i maintenance of the multigeneration Dbphma and Pimephales promelas cultures.

with no evident loss of fe

uncity. If requested, reconstituted fresh water or an appropriate receiving water can be used as the dilution water.

, 3.6 Iest Concentration Series The test concentration series consist:; of a minimum of five dilutions and may be determined from a prior screening of the test material (e.g., 1, 3.10. 30. and 100

j. percent effluent: or 6.25,12.5,25,50, and 100 percent effluent p!us a control).

3.7 Test Concentration Precaration Test concentrations are prepared with Class A glassware.

3.8 Test Vessels and Test Volume Test vessels are 400-ml glass beakers or 10-cm culture bowls: the final test volume of each replicate is 200 ml. Depending on the study requirements, other test

. vessels and test volumes me.y be used.

3.9 Test Organism Number Tests are conducted using two replicates per concentration, with ten organisms wr container, Neonates are randomly m:gned to each replicate test container.

, More replicates can be added, if ap},rupriate.

3.10 Test Environment The test vessels are maintained at 20 2 C in an environmentally comrolled laboratory with a 16-hour light, S-hour dark photoperiod m

ATS-102 EA Enginee ing, Science, and Technology. Inc.

Effective May 1981 Revision 4. April 1991 Page 2 of 7

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PROTOCOL ATS sal DS-04 7

- 3.11 Analysis of Test Concentrations for Test Article '

1 1, If required. test s,olutions may be analyzed for analytical verification of chemical y concentrations. The analytical method and numbar of analyses are determined i af ternominal consultation with measured the client. test When chemical analyses are are reporte necessary, d.

. both and actual solution concentrations 3.12 Test Observations

h. Each test day, test organisms are observed to record the number of surviving

@ organisms, the study terminates after completion of the 2-day observation

! period. The study may be exterded, however, at the request of the client.

Ar Fach sampic and receiving water areofanalyzed for taken temperature, conductivity, ion,

[i" alkalinity, and hardness. Measurements water quahty 24 hours2.777778e-4 days <br />0.00667 hours <br />3.968254e-5 weeks <br />9.132e-6 months <br />, and test termination include dissolved oxygen, pH, temperature and at test initiat i[

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conductivity from a minimum of one replicate of the control, low, medium, and be high gentlydilutions.

aerated, ifAliquots necessaryof(dissolved effluent oxygen and receiving less thanwater 40% or and test than greater solutions 105 may%

q saturation). Analytical dela.rminations are conducted according to APHA et al.

h (1989) and EPA (1979).

! 3.13 Solution Renewal (When Acolicable) i When static renewal testing is required, the test solutions are renewed at

, 24-hours. New test solutions are prepared on the day of renewal and dispensed according to Section 3.7. After the new solutions have reached test temperature,

] the test organisms are transferred from the old test vessels to the new test vessels i using, a wide bore pipet a glass tube. The number of live organisms is recorded.

I Caution is given not to stress the test organisms during transfer. After water quality measurements (temperature, pH. dissolved oxygen, and conductivity) are completed, the old solution is disard d.

3.14 Data Analysis 6.

3 The LC50 or EC50 values and associated statistics are calculated using the probit,

@~ moving average, and binomial methods as descrbed by Stephan (1977).

• Depending on the nature of the data, other methods may be used, including the Trimmed Spearman-Karber method, the probit approximation method of 1 Litchfield and Wilcoxon (1949) SAS prcbit analysts (SAS Institute 1979) or 1 graphical inter %1ation usms the log tencentration vs. percent mortality as described by APHA et al. (1989). The methods used are specified ir. the final report.

ATS-102

~ EA Engineering. Science, and Technology. Inc.

Effective Mavl981 3

Revision 4 April 1991 Page 3 of 7 1

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PROTOCOI. ATS-sal DS-04

4. FINAL REPORT j The final report is prepared to contain at a minimm the following information:

. Objectives and procedures stated in the approved protocol, including t any changes made to the original protocol

! . Identity of the tekt article (s) by name or code number and the ,

strength (i.e., quality / purity and a description of any pretreatment)

. Source of the dilution water, its chemical characteristics. and a description of any pretreatmt.nt r

JI . Test concentration series used and duration of the assay 5 . Water quality characteristics of dilution water and selected test concentrations during testir ;(pH dissolved onygen,- temperature.

e tc.)

. Any unforeseen circumstances that may have affected the quality

or mtegrity of the study d

i . Signature of the oroject manager, senior technical reviewer, and f

goality control officer, authorning release of the report >

l . Location of all archived data and the original copy of the final

{, report at EA i Items of data to be included in the report consist of experimental design and test 1 performance; effects on general appearance of test organisms (if applicable):

morbidi'y evaluation ofand mortality:ity characteristics: and survival data. tabular presenta j water qual

h. 5. QUALITY ASSURANCE 5.1 Amendments to Protqspl lh,,

2 Amendments to the authorized protocol established by EA or by the client are

! made only after proper authorization. Such authorization is achieved by f completion of the Amendment to Protocol Form by EA after consultation with the client.

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v. EA Engineering. Science,

'f and Technology. Inc. -

Effective May 1981 ,

Revision 4. April 1991 Page 4 of 7 '

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PROTOCOL ATS sal DS-04 1e w 5.2 Standard Ooerating Procedures a

4i Unless otherwise specified, all procedures specified in the protocol are subject to J' detailed Standard Operating Procedures (SOPS) which are contained in the SOP manuals of the participating departments. These SOPS and protocols follow the

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U.S. EPA's Good Laboratcny Practice Standards (GLPs)(EPA 1989).

U 5.3 Reference Toxicant A reference toxicant test, utilizing sodium dodecyl sulfate (SDS), cadmium chloride, or another appropriate chemical is used as an internal quality check of j the sensitivity of the test organisms. Tening is conducted at least once monthly 4 on organisms which are cultured in-house. The results of each test are compared f with historical, species specific toxicologicalinformation fr an reference toxicant tests performed at EA, to determine if the results are within ecceptable limits.

1.imits are established tu ug the control charts outlined in Peltier and Weber

(1985).

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SA Onality Assurance EvaluAlinn

[ Studies conducted under this protocol may be subject to internal audit by EA's Quality Assurance Unit. A quality control officer is responnble for monitoring a each study to assure the client that the facilities, equipment, personnel, methods,

[' practices, records, and controls are in conformance with EA's QC program and, if applicable. EPA's GLPs.

i 5.5 Insrection by Regulatory Authorities f

In the event of an inspection of EA by an outside autho.ity during the course of 1I the study, the client whose study is being inspected will be consulted before examiners are permitted access to any of the project records or the experimental areas.

k 5.6 Archives

,s Copies of project-specific records shall be transferred to the client promptly after

, m the project is completed or as negotiated and budgeted. Origi ml psurv data

• will be retained at EA for 5 years. Primary data Mclude cinin of-custody records, laboratory data sheets, records, memoranda, notes, photographs,

!!: microfilm, and computer printouts that are a result of the original observations 1- and activities of the study and which are necessary for the reconstruction and evaluation of the study report.

1 ATS-102 p EA Engineering, Science, and Technology, Inc.

Effective May 1981

. Revision 4. April 1991 Page 5 of 7

PROTOCOL ATS-SAI-DS-04 5.7 Location Studies are conducted at the Aquatic Toxicology Laboratory of EA Engineering.

Science, and Technology, Inc. at the Mid Atlantic Regional Offices. Sparks Mary-land.

6. SPECIFICATIONS OF THE pat.hnia ACUTE TOXICITY TEST 6.1 Basic References American Public Health Association (APHA).1989. Standard Methods for Examination of Water and Wastewater,17th edition or an updated edition.

? APHA AWWA. and WPCF. Washington, D.C.

EA Manual ATS-102,1981. Aquatic Toxico;ogy Studies, Quality Control and Standard Operating Procedires Manual. Revision 2 - August 199L (Internal document prepared by Aquatic Toxicology Services, EA Engineering. Science, and Technology, Inc.)

Peltier, W.H. and C.I. Weber.1985. Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine Organisms. EPA-600/4-85

-013.

U.S. En'vironmental Protection Agency, Cincinnati, Ohio.

Weber, C.I., et al.1989. Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. Second Edition. EPA /600/4-89/001. U.S. Environmental Protection Agency, Cindanati, Ohio.

U.S. EPA 1989. Toxic Substances Control Act (TSCA): Good Laboratory Practice Standards. Title 40 CFR Part 792. Eed. Regist. 54(158):34034-34074.

l 6.2 Test Soecifications

n Test organism: Daohnia magna or D. pulex: species to be specified in the study plan and final report Age: Less than 24 hours2.777778e-4 days <br />0.00667 hours <br />3.968254e-5 weeks <br />9.132e-6 months <br /> old Temperature: 20 2 C 4

Light quality

Wide spectrum fluorescent light Light intensity: 50-100 f,c.

l ATS-102 .

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Effective May 1981 '

Revision 4. April 1991 Page 6 of 7 >

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PROTOCOL ATS SAI-DS-04 4 i Photoperiod: 16-hour light 8-hour dark Aeration: None. unless dissolved oxygen falls below 40 percent saturation Dilution water: Dechltarinated municipal water, reconstituted water, or appropriate receiving water Test container: 400-m1 Pyrex beaker or 10-cm culture bowl

Test volume

200 ml per replicate ,

No. of concentrations: Minimum of five test concentrations and a control .

No. of replicates: Two No. of animals per replicate: Ten Feeding regime: Animals will not be fed during test Test type and duration: Rangefinding-24 to 48 hours5.555556e-4 days <br />0.0133 hours <br />7.936508e-5 weeks <br />1.8264e-5 months <br /> Defmitive--48 hours Endpoints: Mortality or immobilization Immobilization--defined as cessation of

! movement except for minor activity of

appendages i

Mortality--defined as cessation of

. all movement for a wriod of at least 5

,' seconds even when tae test container is j *. tapped or rotated, or the organism i ia gently prodded with glass rod Test acceptability: 90% or greater survival in the control solution.

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ATS-102 t EA Engineering. Science, and Technology. Inc.

Effective May 1981 Revision 4. April 1991 Page 7 of 7

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. PROTOCOL ATS SAF FM 04

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11. ACUTE ASSAY WITH FATHEAD MINNOWS (i'imechales oromelas)

1. TEST OBJECTIVE To assess die toxicity of a test material to Pimenhnles nrnmeins and determine the LC50 or EC50 using mortality and/or immobilization. respectively, as the test endpoint (s).

/ 2. TEST ARTICLE

• 2.1 D2 crintion/ Identification U

Unless otherwise specified. the test material is supplied by the client. Adequate chemical specifications with special reference te hazardous properties and storage conditions are also supplied by the client.

2.2 Methods of Syntheds In most cases the test article is an effluent sample. Informt. tion on the methods of synthesis, stability, and composition or other characteristics which define the test 6rticle are on file with the client.

3. EXPERIMENTAL DESIGN 3.1 Test Orcanisms The test species is the fathead minnow. Pimenhales promel t u.

3.2 Source E. oromebs used for toxicity tests are usually obtained from stock cultures maintamed at EA's Aquatic Toxicology Culturin Facility. However, organisms may be obtained from a scientific orgamsm venc;or. if necessary.

7 3.3 Culturing and Holdine conditions E. promelas stock are maintained at 20 2 C and a 16-hour light. 8-hour dark

[ photopenod cycle in an enn.snmentally controlled laboratory. Test organisms are maintaine'd in 18.9-L or 30 L all glass aquaria in a recirculating system of dechlorinated municipal tap water. If organisms are obtained from a scientific vendor, the fish, upon receipt, are transferred to static recirculating holding tanks of an aparopnate size containing dec11orinated tap water at the temperature ( 2 C) of the water F

ATS-102 EA Engm' eering. Science.

and Technology. Inc.

Effective May 1981 Revision 4. August 1991 Page 1 of 8 1

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> :. PROTOCOL ATS-SAF-FM 04 k

m which the orcanisms were shipped. All fish stocks are examined regularly.

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Dead fish or those displaying abnormal swimming behavior, discoloration, or pronounced lethargy are removed as observed, and recorded on appropriate log sheets. Fish stocks are fed a commercial fish food a minimum of once daily.

3.4 Agt af .T211 OIranies al .Tssi Initiation The age of the test organisms is dependent on the objectives of the study and the specific guidelines being followed. but in general, fathead minnows used for acute

. toxicity testing are <90 days upon test initiation.

3.5 Dilution Water i

J The source of dilution water is the City of Baltimore Municipal Water System.

Upon entry to the laboratory, the water is passed through a high-capacity, activated-<:arbon filtration system to remove chlorine and other possible

contammants such as detergents and hexane-soluble organic materials. This water source has proven safe for aquatic organism toxicity testmg. as evidenced by

o maintenance of the multigeneration Daohnin and fathead minnow cultures. With no evident loss of fecundity, if requested. reconstituted fresh water or other dilution water may be used.

3.6 Test Concentration Series The test concentration series consists of a minimum of five dilutions and may be determmed from a prior screening of the test material (e.g.. 1, 3,10, 30. and J0 percent effluent: or 6.25,12.5. 25,50, and 100 percent effluent plus a control).

. 3.7 Test Concentration Precaration i

Test concentrations are prepared with Class A glassware.

[ 3.8 Test Vessels and Test Volume Test vessels are 8-in glass culture bowls: the final test volume is 1 L. Other test

,, vessels may be used depending on the study's requirements.

3.9 Test Organin Number l

Tests are conducted using 2 replicates per concentration, with 10 organisms per container. Fish are randomly assigned to each replicate test container. More replicates can be added. if appropriate.

b l ATS-102 EA Encineering. Science, and Technology, Inc.

i Ef.fective May 1981 1

Revision 4. Ahgust 1991 Page 2 of 8

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,r-a PROTOCOL ATS SAF F.h04 m

k 3.10 Test Environment 1 The test vessels are maintained at 20 2 C (unless a different project-specific 9 temperature is required)in an environmentally controlled laboratory with a 16 hour1.851852e-4 days <br />0.00444 hours <br />2.645503e-5 weeks <br />6.088e-6 months <br /> light. 8-hour dark photoperiod. Temperature within the envirotunental y room is momtored contmuously usmg temperature recorders.

1 3.11 Annivsis of Test Concentrations for Test .Artic!g H If required, test solutions may be analyzed for verification of chemical

J concentrations. The analytic:i1 method'and number of analyses are determined '

af ter consultation with the client. When chemical analyses are necessary, both nominal and actual measured test concentrations are re' ported.

B; 3.12 Test Observations f: Each test day, test organisms are observed to record the number of surviving organisms. Dead organisms are removed when observed. The study terminates af ter completion of the observation period (24 to % hours). The study may be extended, however, at the request of the client.

3-Each sam and y hardness.ple receivedof is Measurements analyzed water for temperature, quality taken at test initiation conductivity, and term alkalinity,ina

1. include dissolved oxygen pH. temperature, and conductivity from a minimum of one replicate of the control. low, medium, and high dilutions. Aliquots of effluent and recciving water, and test solutions may be gently aerated, if

]. necessary (dissolved oxygen less than 40% or greater than 105% saturation).

4 Anal tical determinations are conducted according to APHA (1989) and EPA (1979.

At the end of the test period, a minimum of 30 sursiving fish are removed from the test vecels placed in labeled containers, and frozen for later measurements.

The fish are subsequently thawed, measured to the nearest mm (standard length).

j.' patted dry, and weighed to the nearest 0.01 g. The average length and weight of the test fish are reported, along with their respective ranges and standard

, deviations.

3.13 Solution Renewal (When Acolicable) 1 '

When static-renewal testing is required, the test solution is renewed daily. New solutions are prepared on the day of renewal and dispensed n

ATS-102 g EA Engineering. Science, j and Technology. Inc.

Effecti"e May 1981 Revirion 4. August 1991 Page 3 of 8

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ya according to Section 3.S. After the new solutions have reached test temperature and water quality measurements tiemperature. pH. dissolved oxygen, and I

4 conductivity) are completed, the solution renewal may be performed by tramferrmg the orgarusms from one test chamber to another or by replacing the test solution. If the test solution replacement method is used, caution must be e given not to stress the Wst organisms while the test chamber is siphoned. Usually j 75 percent of the old solution is removed and replaced: however, the amount is dependent on the size of the test organisms.

, 3.14 Data Analvris 1'

The LC50 and/or EC50 values and associated statistics are calculated using the

'fl 3robit, moving average, and binomial methods as described by Stephan (1977).

"li Npending on the nature of the data, other rnethods may be used including the Trmuned S; :arman Karber Method, the probit approximation method 4l. of Litchfield and Wilcoxon (1949). SAS probit analysis (SAS Institute 1979). or

) graphicalinterpolation using the log concentration vs. percent mortality and/or percent affected as described by APHA (1989). The methods used are specified m the final report.

4. FINAL REPORT h The final report is prepared tu t.cnrain, at a minimum, the following f informatton:

. Objectives and procedures stated in the approved protocol including

} any changes made to the origmal protocol

. Identity of the test article (s) by name or code number and their strength (i.c... quality / purity), and a description of any pretreatment

. . Source of the dilution water, its chemical characteristics, and a 3 description of any pretreatment k

. Test concentration series used and duration of the assay f <-

. Mean standard lengths and wet weights of test fish; the respective standard deviations, and approximate biomass load.ings (g fish /L of

. test solution) 4' i . Water quality characteristica (pH. dissolved oxygen, temperature.

etc.) of dilution water and selected test concentrations durmg testing.

ATS-102

1. EA Engineering. Science, and Technology. Inc.

Effective May 1981 Revision 4. August 1991 Page 4 of 8-5

i f <, .;

1,

j 1

hq PROTOCOL ATS SAF FM 04

/ . Any unforeseen circumstances that may have affected the quality I or 'mtegrity of the study

. Signature of the project manager, senior technical reviewer. and j quality control officer authortzmg release of the report o

. Location of :11 archived data and the original copy of the final report at EA i

.s items of data to be included in the report consist of experimental design and test j

'^

performance: effects on general appearance of test organisms (if applicableh

,,. morbidity and mortality: tabular presentation and appropriate statistical -

'; ,Q evaluation of water quality characteristics. and survival data.

5. QUALITY ASSURANCE -

5.1 Amendments to Protocol .

)

Amendments to the authorized protocol established by EA or by the client are f: made only after proper authorization. Such authorization is achieved by 4 completion of the Amendment to Protocol Form by EA after consultation with

the client.

p

j. 5.2 Standard Ooeratina Procedures t

.. Unless otherwise specified, all procedures given in the protocol are subject to i ' detailed Standard Operating Procedures (SOPS) which are contained in the SOP manuals of the participating departments. These SOPS and protocols follow the

, U.S. EPA's Good Labora:ory Practice Standards (GLPs)(EPA 1989).

p 5.3 Reference Toxicant L.

A reference toxicant test, utilizing sodium dodecyl sulfate (SDS) cadmium -

chloride, or another appropriate chemical is used as an internal quality check of the sensitivity of the test organisms. Testing is conducted at least once monthly n^ on organisms that are cultured in-house. The results of each test are comparad with historical species specific toxicological information from reference toxicant tests performed at EA. to determine if the results are within acceptable limits.

Limits are established using the control charts outlin6d in Peltier and Weber f- C985).

4 s

h 4

[1.

ATS402

. EA Engineering. Science, and Technology. Inc.

Effective May 1981 Revision 4. August 1991 Page 5 of 8

a '. <

4 r- PROTOCOL ATS-SAF-FM-04

" 5.4 Ouality Aesurance Evaluation Studies conducted under this protocci mnv be subject to internal audit by EA's Quality Assurance Unit. A quality control efficer is responsible for monitoring each study to assure the client that the faci ~.ities, equipment, personnel, methods, practices, records, and controls are in conformance with EA's QC program and, if applicable. EPA's GLPs.

5.5 Insnection by Reculatory Stthorities

, In the event of an mspection of EA by an outside authority during the course of the study, the client whose study i being inspectsd will be consulted before

% examiners are permitted access '.o any of the project records or the experimental areas.

5.6 Archives Copies of project-specific recceds shall be transferred to the client promptly af ter the project is comnieted or ar. negotiated and budgeted. Original pnmary data will be retained at EA for 5 yeara. Primary data mclude chain-of-custody records, laboratory data sheets records, memoranda, notes, photographs, microfihn and computer printouts that are a result of the original observations and activities of the study t.nd which are nece sary for the reconstruction and evaluation of the study report.

5.7 Location Studies are conducted at the Aquatic Toxicology Laboratory of EA Engineering.

Science, and Technology, Inc. at the Mid-Ath-~- Regional Offices. Sparks.

Maryland.

6. SPECIFICATIONS OF THE FATHEAD lWNNOW ACUTE TOXICITY TEST

, 6.1 Basic References America' Public Health Association (APHA).1989. Standard Methods for Examination of Water and Wastewater.17th or updated edition. APHA.

AWPA. and WPCF. Washington D.C.

American Society for Testing and Materials (ASTM).1980. Standard Practice for Conducting Acute Tests with Fishes, Macroir. vertebrates, and Amphibians.

ASTM Designation: E729-80. Philadelphia, Pa. pp. 285-309.

ATS-102 EA Engineering. Science.

i-and Technolo'gy. Inc.

Effective May 1981 Revision 4. August 1991 Page 6 of 8

?

kf p PP.OTOCOL ATS SAF-FM-04 b

EA Manual ATS 102.1981. Aquatic Toxicology Studies. Quality Control and Standard Operating Procedures Mcnual. Revision 2 August 1991. (Internal document prepared by Aquatic Toxicology Services. EA Engmeeting. Science. O and Technology. Inc.)

Peltier. W.H. and C.I. Weber,1985. Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine Orgamsms. EPA-600/4-85-013. U.S.

Environmental Protection Agency. Cincinnati. Ohio.

" U.S. EPA.1989. Toxic Substances Control Act (TSCA): Good Laboratory Practice Standards. Title 40 CFR Part 792. Ecd. Repst. 54(158): 6 34-34074, f 6.2 Test Soecifications Test orgamsm- Fathead minnow (Pimen%1es oromelas)

Temperature: 20i2 C Fish age: Dependent on test guidelines being fonowed but in general. <90 days old Fish length: The largest fish is not more than 50 percent longer than the smallest fish Loading ratio: In general, test containers may not contain greater than 0.8 g/L biomass Aeration: None. unless dissolved oxygen falls belcw 60 percent saturation in first 48 hours5.555556e-4 days <br />0.0133 hours <br />7.936508e-5 weeks <br />1.8264e-5 months <br /> of the test or below 40 percent saturation thereafter g Light quality: Wide-spectrum fluorescent light e

Light intensity: 50-100 f.c.

Photoperiod: 1Ghour light. 8-hour dark Dilution water: Dechloringard municipal tap water. ,

reconstituted fresh water, or appropriate receivtr water Test containers: 20-cm glass culture bowl f .-

c..:

ATS-102 EA Engineering. Science, and Technology. Inc.

Effective May 1981 Revision 4. August 1991 _

Page 7 of 8 1

_ - _ _ _ _ _ _ _ _ . \

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PROTOCOL / iS-SAF-FM-04 Test volume: 1 L/ replicate No, of cencentrations: Minimum of five test concentrations and a control

!(

1 I: No. of replicates: 2

^

Test animals per container: 10 (if loading is exceeded more replicates or larger test containers may be used)

Feeding regime: Animals are not fed during tests unless

y fish are too small to survive % hours E without feeding. Feeding is termmated 48 hours5.555556e-4 days <br />0.0133 hours <br />7.936508e-5 weeks <br />1.8264e-5 months <br /> before test initiation in the lot

.> from which organisms are obtained l' Test 5fpe and duration: Rangefinding-24-% hours Defmi:ive-24-96 hours Endpoints: Mortality and/or Immobilization

!! Immobilization--defined as cessation of

,. movement except for minor activity of appendages O- Mortality--defined as cessation of

' opercular movement and inability to respond to external stimuli (gentle

!~I prodding with glass rod) 1 Test acceptability: 90 percent or greater survivalin the

- control solution h-I f

Ia ATS-102 lp EA Engineering Science.

i1 and Tt.:hnclogy, Inc.

P Effective May 1981 Revision 4. August 1991 Page 8 of 8 j

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