ML20079J234

Results of Acute Toxicity Screening Tests W/Daphnia Magna & Pimephales Promelas on Samples from Perry Nuclear Power Plant

ML20079J234
Person / Time
Site:	Perry
Issue date:	10/01/1991
From:	Black J, Sohn V EA ENGINEERING, SCIENCE & TECHNOLOGY, INC.
To:
Shared Package
ML20079J230	List: ML20079J228 ML20079J234
References
NUDOCS 9110150371
Download: ML20079J234 (33)

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RESULTS OF ACUTE TOXICITY SCREENING TESTS

{' WITH Daphnia magna AND Pimephales promelas

.. ON SAMPLES FROM

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PERRY NUCLEAR POWER PLANT .

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Prepared for-Calgon Coryxation Elyria Dhio

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Prepared by:

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

I 15 Loveton Circle

, 1 Sparks. Maryland 21152

g. October L991 EA Project 70003.10 l

Remn Numwr to 9110150371 9;1011 1 l PDR ADOCK 05000440 I

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EA Project 70003.10 R

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" RESULTS OF ACUTE TOXICITY SCREENING TESTS -

WITH Daphnia magna AND Pimephaless ' ,melas I3 ON SAMPLES FROM f PERRY NUCLEAR POWER PLANT -

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i Prepared for:

Calgon Corporation t Elyria. Ohio

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EA Mid-Atlantic Re

' i-EA Engineering. Science,gional Operations and Technology. Inc.

15 Loveton Circle Sparks. Maryland 21152 li' d / 0e4sJa j c 9/

Virgufa A. Sohn Date Project Manager I

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Jeffret/ #. BlacV. Ph.D Date Projeft Managdr i

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,, INTF.000CTION N

c, At the request of Calgon Corporation EA Engineering, Science, and Technology performed acute toxicity screening tests with Daphnia magna (vater flea) and Pimephales promelas (fathead minnov) on three samples T fsom Perry Nuclear Power Plant. Each species was exposed to 100 percent sample of 1) influent prior to treatment, 2) effluent prior to treatment, and 3) effluent composite during treatment.

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Sample collection and receipt data are reported in Table 1. Table 2 y,

,f7 prosents initial vater quality data. Test results, test organise source and age, reference toxicant data, and water quality meesurements are summarized on pages 3-6. Test protocols, copies of raw data sheets, and the Report Quality Assurance Record are included at Attachments I, II, and III, respectively.

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SUMMARY

OF RESULTS The rosults of the acute toxicity screening tests performed for Calgon Corporation on samples from Perry Nuclear Power Plant are summarised on pages 4 and 6. In the D. Eagna test, 48 hours5.555556e-4 days <br />0.0133 hours <br />7.936508e-5 weeks <br />1.8264e-5 months <br /> of exposure to the 100 percent samples resulted in 85 percent survival in the influent, 100 percent survival in the effluent prior to treatment, and 95 percent survival in the effluent composite collected during treatment. There was ,

95 percent survival of laboratory control organisms. In the P,. promelas test there was 100 percent survival of f athead minnovs in all three y treatments af ter 96 hours0.00111 days <br />0.0267 hours <br />1.587302e-4 weeks <br />3.6528e-5 months <br /> of exposure, and 100 percent surnval of

e. minnovs in the laboratory control.

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SUMMARY

OF SAMPLE / TEST INFORMATION I .

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,. Test: 48-hour Daphnia magna Static Acute Toxicity Test

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s Client name: Calgon Corporation Test procedure or protocol followed: EA Protocol ATS-SAI-DS-04 F Static acute 48-hour LC50 assay with Daphnia Sample description: Perry Nuclear Power Plant: Influent prior to 3- treatment; Effluent prior to treatment; Effluent g, composite during treatment ff; EA accession number: AT1-739, AT1-740, AT1-741 if Time and date of sample collection: See Table 1 Time and date of sample receipt: See Table 1 EA OC test number: SA-09-14-91-759 Test initiation time and date: 1145, 14 September.1991

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Test completion time and date: 1145, 16 September 1991 h

Control vater: Dechlorinated tap water Organism Lot Number: NA Source: EA Culture Facility, Sparks, MD Age <24 hours c:

Reference Toxicant: Sodium dodecyl sulfate

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EA QC test Number: RT-09-10-91-739 Dilution water: Dechlorinated tap vater E

jr. Referance Toxicant (SDS) Results

.m 73 24-hour LC50: 29.5 mg/L Laboratory control chart acceptability range for 24-hour LC50:

14.6 44.8 mg/L f

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SUMMARY

OF SELECTED TEST DATA (Cont.)

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Lt EA Accession Numbers: AT1-739, 740, 741 Sample

Description:

4 Influent prior to treatment; Test Species: ~D. magna Effluent prior to treatmenti Effluent composite during 5* treatment EA QC Test Number: SA-09-14-91-759 1:

Test Material Percent Survival at 48 Bours c;

.[9 Laboratory Control 95

"- Influent prior to treatment 85 Effluent prior to treatment 100 Effluent composite during treatment 95

. Selected Vater Quality , Range Temperature (C) 19.9 - 21,0 f

pH 7.5 - 8.5 Dissolved oxygen (ag/L) 8.3 - 9.0 Cenductivityt (uS*) 236 - 365

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SUMMARY

OF SAMPLE / TEST INFORMATION 9.

$ Test: 96-hour Pimephales promelas Static At.u.te Toxicity Test

1. Client names Calgon Corporation 1

Test procedure or protocol folloved: EA Prt. .scol ATS-SAF-FM-03 Static acute 96-hour LC50 assay with fathead sinnovs I. (Pimephales promelas)

Sample description: Perry Nuclear Power Plants Influent prior to g treatment;_ Effluent prior to treatment; Effluent composite during treatme.nt b EA accession number: AT1-739, AT1-740, AT1- ai d.

Time and date of sample collection: See TabL 1

- Time and date of sample receipt: See Table 1 EA QC test number: SA-09-14-91-760 Test initiation time and date: 1200, 14 September 1991 Test completion time and date: 1200, 13 September 1991 Dilution vater: Dechlorinated tap vater

. Organism Lot Number: FH 1-7/28-8/1 a

'[ Source: EA Culture Facility, Sparks, MD

. Age: 41 44 days Length- 16.22 mm S.D.. 2.38 mm Veight: 0.06 g S.D.: 0.03 g Reference Toxicant: Sodium dodecyl sulfata EA QC test Number RT-09-10-91-725

• Dilution water: Dechlorinated tap water p

y Reference Toxicant (SDS) Results 24-hour LC50: 32.2 mg/L

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

.1.2-45.2 mg/L k..

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SUMMARY

OF SELECTED TEST DATA (Cont.)

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.l  !' EA Accession Numbers: AT1-739, 740, 741 Sample Descri}, tion:

Influent prior to treatment; Test Species: P. proselas Effluent prior to trestment;

} Eff',uent composite during treatment EA QC Tes: Number: SA-09-14-91-760 Test Material Percent Survival at 48 Hours Laboratory Control 100 Influent prior to treatment 100 p..

C' Effluent prior to treatment 100

't Effluent composite during treatment 100 Selected Vater Quality Range Temperature (C) 19.5 - 21.0 pH 7.4 - 8.3 Dissolved oxygen (mg/L) 7.3 - 9.0 Conciuctivity: (uS*) 234 - 362 a.

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i TABLE 1 SAMPLE COLLE 7 ION DATA FOR SAMPLES FROM PERRY '4UCLEAR F0VER PLANT Sample Description EA Accession Number Sample Collection Sample Receipt Type of Sample 0920, 14 SEP 91 Influent prior to AT1-739 0800, 12 SEP 91 Grab

. treatment Effluent ptfor to AT1-740 0900, 12 SEP 91 0920, 14 SEP 91 Grab treatment

. Effluent compocite AT1-741 0952, 12 SEP 91- 0920, 14 SEP 91 Composite l during treatment 0005, 13 SEP 91 ,

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3 7 ,g, - w -m p r e TABLE 2 INITIAL VATER QUALITY DATA FOR SAMPLES FROM PERRY NUCLEAR POWER PLANT EA Accession Alkalinity Hardness Coiductivity Description Number (ag/L as CACO,) (mg/L as CACO3 ) (pS*)

Influent prior AT1-739 96 116 292 to treatment

. Effluent prior AT1-740 112 128 343 to treatment Effluent composite AT1-741 112 128 345 during treatment i

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ATTACHNDrf 1 PROTOCOLS s

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J PROTOCOL ATS-SAI-DS-04

.- 3. ACUTE 48-HOUR LC50 ASSAY WITH &phnia sp.

(f r L TEST OBJECTIVE To assess the toxicity of a test material to Daphnia and determine the LC50 or EC50 using mortality or immobilization respectively, as the test end points.

I 2. TEST ARTICLE .

l 2.1 Descrintion/ Identification i

L:

Unless otherwise specified. the test materialis supplied by the client Adequate e chemical specifications with special reference to hazardous properties and storage

? conditions is 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 composition or other characteristics which define the test article are on file with the client.

3. EXPERIMENYAL DESIGN P

3.1 Test Orennisms A species of &phnia (water fleas) as determined by project needs, is the test

{; orgamm 3.2 Source e &phnia used for acute toxicity tests are obtained from stoch cultures mamtained in EA's Aquatic Toxicology Cultunng Facility.

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$,., 3.3 Culturing and Holding conditions

&phnia cultures are maintained at 20 2 C and a 16-hour light.

0 S hour dark photoperiod cycle in an environmentally controlled laboratory.

Cultures are maintained m 18.9-L all glass aquana or other appropriate container and are fed algae (Selenastrum capricornurum) and a trout chow / yeast / cereal

,I leaves suspension in the manner described in Peltier and Weber (1985). New

'I cultures are initiated on a routine basis to ensure healthy, productive populations. Orgamsms from cultures producing ephippm are not used for to:.. .y tests.

c ATS-102 EA Engineering. Science, o and 'fechnology. Inc i

Effective May 2.981 '

Revision 4. April 1991 Page 1 of 7 i

" PROTOCOL ATS-SAI DS-04 N-g.

3.4 Ace of Test Ornnisrns at Test Initiation Neonates of known age (i.e., less than 24-hours old) are obtained for testing by r' segregating adult females from the mass cultures on the day before a test is initiated. On the day of the test neonates are segregated from the parent orgamsms.

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

i, Upon entry to the laboratory. the water is passed through a lugh-capacity, i activated-carbon filtration system to remove chlorine and other possible contaminants such as detertents, and hexane-soluble organic materials. This p', water source has proven sale for aquatic organism toxicity testing as evidenced by d maintenance of the multigeneration Dzphma and Pimephales promelc cultures. ,

with no evident loss of fecundity. If requested, reconstituted fresh water or an n appropriate receiving water crat be used as the dilution water.

3.6 Test Concentration Serica

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The test concentration series consists 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 percent effluent: or 6.25,12.5, 25, 50, and 100 percent effluent plus a control).

-e 3.7 Test . Concentration Frenaration Test concentrations are prepared with Class A glassware.

l' 3.3 Test Vessels and Test Volume Test vessels are 400-m1 glass beakers or 10-cm culture bowls: the final test volume h of each replicate is 200 ml. Depending on the study requirenims, other test vessels and test volumes may be used.

6 3.9 Test Orvnniet Number

, fc Tests are conducted using two replicates per concentration. with ten organisms D per container. Neonates are randomly assigned to each replicate test container.

More replicates can be added, if appropriate.

3.10 Test Envirnnmnt L, - The test vessels are maintained at 20 2 C in an environmentally controlled

, laboratory with a 16-hour light. 8-hour dark photoperiod.

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ATS-102 '

EA Engineering. Science, and Technology. Inc.

p-Effective May 1981 Revision 4, April 1991 Page 2 of 7 4

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PROTOCOL ATS-SAI-DS-04 3.11 Analys s of Test Concentrations for Test Article '

i If required, test solutions may be analyzed for analytical verification of chemical concentrations. The analvi:al method and number of analyses are determined af ter consultation with the client. When chemical analyses are necessary.

} both nominal and actual measured test solution concentrations are reported.

[ 3.12 Test Obrervations 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 extended, however, at the request of the client.

g Each sample and receiving water are analy:ed for temperature, conductivity, fV alkalinity, and hardness. Measurements of water quahty taken at test initiation.

24-hours and test termination include dissolved oxygen. pH. temperature and conductivity from a muumum of one replicate of the control. Iow medium, and be high dilutions. Aliquots of effluent and receiving water and test solutions may%

gently aerated, if necessary (dissolved oxygen less than 40% or greater than 105 saturation). Analytical determinations are conducted according to APHA et al.

L (1989) and EPA (1979).

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3.13 Solution Renewal (When Acolicable)

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 orgamsms are transferred from the old test vessels to the new test vessels using, a wice bore pipet or glass tube. The number of live o amsms is recorded.

Caution is given not to stress the test or amsms during trans er. After water quality measurements (temperature, p dissolved oxygen. and conductivity) are b completed. the old solution is discarded.

- 3.14 Data Annivsis The LC50 or EC50 values and associated statistics are calculated using the probit.

,r* moving average, and binomial methods as described by Stephan (1977).

p Depending on the nature of the data, other methods may be used including the Trunmed Spearman-Karber method, the probit approximation method of Litchfield and Wilcoxon (1949). SAS probit analysa (SAS Institute 1979) or e graphical interpolation c-ing the log concentration vs. percent morta.uty as U described by APHA et al. (1989). The methods used are specfied in the final report.

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-[v. ATS-102

. A Engineering. Science, and Technology. Inc.

f Effective May 1981 Revision 4. April 1991 Page 3 of 7

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3 PROTOCOL ATS-SAI-DS-04 e . 4. FINAL REPORT v

i*' The final report is prepared to contain at a mmimum the following information:

j~ . Objectives and procedures stated in the approved protocol including any changes made to the original protocol I . Identity of the test 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 pretreatment g., . Test concentration series used and duration of the assay e ..

C . Water quality characteristics of dilution water and selected test concentrations during testing (pH. dissolved oxygen, temperature, etc.)

. Any unforeseen circumstances that may have affected tiie quality or uitegrity of the study -

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. Signature of the project manager, senior technical reviewer. and n

quality control officer, authorning release of the report i . Locition of all archived data and the original copy of the final report at EA

'h Items of data to be included in the report consist of experimental design and test

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

! morbidity and mortality: tabular presentation and appropnate staustical

}- evaluation of water quality characteristics: and survival data.

5. QUALITY ASSURANCE

.i 5.1 Amendments to Pmtocol Ei Amendments to the authorized protocol established by EA or by the client are h made only after proper authorization. Such authorization is achieved by completion of the Amendment to Protocol Form by EA after consultation with

[ the client.

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,. ATS-102 EA Engineering. Science, w and Technoloev. Inc.

( Effective May '.?81 Revinon 4. April 1991 Page 4 of 7

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f PROTOCOL ATS-SAI-DS-04 5.2 Standard Overating Procedures Unless otherwise specified, all procedures ied in the protocol are subject to detailed Standard Operating Procedures (S Ps) which are contained in the SOP manuals of th articipating partments. These SOPS and protocols follow the

[ U.S. EPA's O Laboratory ractice Standards (GLPs) (EPA 1989).

i- 5.3 Reference Toxicant i .-

A reference toxicant test, utilizmg sodium dodecyl sulfate (SDS). cadmium p chloride, or another appropriate chemical is used as an internal cuality check of g the sensitivity of the test orgamsmx Testing is conducted at least once monthly on organisms which are cultured in-house. 'llte results of each test are compared y with historical, species-specific toxicologicalinformation from reference toxicant V tests performed at EA. to determme if the results are within acceptable limits.

Limits are established using the control charts outlined in Peltier and Weber (1985).

5.4 Onnlity Assurance Evaluation ,

Studies conducted under this protocol may be subject to internal audit by EA's 1 Quality Assurance Unit. A quality control officer is responsible for monitoring each study to assure the client that the facilities, equipment. personnel. methods.

practices, records. and controls are in conformance with EA's QC prr., gram and, if

,- applicable. EPA's GLPs.

g 5.5 Insoection by Repilnterv Authorities In the event of an inspection of EA by an outside authority during the course of the study, the client whose study is bein ' ted will oe consulted before L examiners are permitted access to any o e project records or the experimental

}. areas.

p 5.6 Archives Copies of project-specific records shall be transferred to the client p'romptl after ,

the project is completed or as negotiated and budgeted. Onginal ata bo will be retained at EA for 5 years. Primary data mclude cham o -cust y b records, laboratory data sheets, records, memoranda, notes, phot phs.

microfilm. and computer printouts that are a result of the ori observations i

and activities of the study and which are necessary for the reconstruction and O evaluation of the study report.

I h ATS-102 EA Engineering. Science.

.p and Technology. Inc.

c Effective May 1981 Revision 4. April 1991 Page 5 of 7 j l

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, PROTOCOL ATS-SAI-DS 04

g. 5.7 Location

< y 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 Dachnin ACUTE TOXICITY TEST e

b. 6.1 Basic Refer nces

American Public Health Association (APHA).1989. Standard Methods L for Examination of Water and Wastewater.17th edition or an updated edition.

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

N EA Manual ATS-102.1981. Aquatic Toxicolo5y Studies. Quality Control and Standard Operating Procedures Manual Revision 2 - Auntst 199L (Internal document prepared b and Technology. Inc.)y Aquatic Toxicology Services. EA Ungineering.

Peltier. W.H. and C.I. Weber.1985. Methods for Measuring the Acute Toxicity of '

l.- Effluents to Freshwater and Marine Orgamsms. EPA-600/4-85

-013.

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

f. 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 /600/4-89/00L U.S. Environmental Protection Agency. Cincmnati.

Ohio.

U.S. EPA.1989. Toxic Substances Control Act (TSCA): Good Laboratory Practice Standards. Title 40 CFR Part 792. Esd. Engig. 54058h34034-34074.

6.2 Test Soecifications Q Test organism: Daphnia magna or D. puins species to be

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specfied in the study plan and final y,- report be.

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

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Effective May 1981 Revision 4. April 1991 'ag - of 7 l'

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f PROTOCOL ATS-SAI-DS44

. Photoperiod: 16-hour light. 8-hour dark Aeration: None unless dissolved oxygen falls below 40 percent saturation Dilution water: Dechlorinated municipal water.

recons,tituted water, or appropriate h receiving water I

Test container: 400-m1 Pyrex beaker or 10<m culture

. bowl 3

Test volume: 200 mi per replicate N No. of concentrations: Minimum of five test concentrations and it a control

!/ No. of replicates: Two 1-No. of animals per replicate: Ten Ammals will not be fed during test

! Feeding regime:

, 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 /> Defuntive-48 hours Endpoints: Mortality or immobilization Immobilization-defined as cessation of movement except for minor activity of appendages r

Mortality-defined as cessation of

- all movement for a wriod of at least 5 seconds even when tae test container is f^.i tapped or rotated, or the orgamsm is gently prodded with glass rod 3

Test acceptability: 90% or greater survivalin the control solunon.

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and Technology. Inc.

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• PROTOCOL ATS-SAF-FM-03 1 ,.

y. 11. STATIC ACUTE 96-80UR LC50 ASSAT VITH FATHEAD MINNOVS

.,j (Pimephales proselas)

1. TEST OBJECTIVE To assess the toxicity of a test article to fathead ainnov and determine 1 the 96-hour LC50 using lethality as the test end point.

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2. TEST ARTICLE 2.1 Description / Identification

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. Unless otherwise'specified, the test article vill be supplied by the

. $.. client. Adequate chemical specifications with special reference to

'b' hazardous properties and storage conditions also vill be supplied by the client.

2.2 Methods of Synthesis Inic.rmation on the eethods of synthesis, stability, and data on composi-tion or other characteristics which define the test article are on file with the client.

h 3. EXPERIMENTAL DESIGN 3.1 Test Organisms i

Fathead sinnov (Pimephales proselas) vill constitute the test species.

3.2 Source

' Fathead sinnov stocks are lab reared, obtained from a scientific orga-nism vendor, or field collected using techniques to be specified in the

{ report.

3.3 Holding Conditions i? Upon receipt, fish are transferred to holding tanks which are continu-

! ously replenished with dochlorinated municipal tap water. The fish are acclimated to laboratory conditions for a minimum of 7 days prior to Li" testing. The laboratory is maintained on a 16-hour light, 8-hour dark

'i photoperiod cycle by a programmable electrical timer. All fish stocks are examined regularly throughout the work veek. Dead fish, or those displaying abnormal svimming behavior, discoloration, or pronounced

} lethargy are removed as observed, and recorded on appropriate log sheets.

i l u Jr - RTC-T-101 EA Engineering, Science, p and Technology, Inc.

'* Effective May 1981 Revision 3, August 1986 Page 1 of 9 l-9 n --sgw+ s-w -- w - , - i9.e-.-y--.,, <w- c 9 e4g , , - =gr9, T PT w-v-Tr

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. PROTOCOL ATS-SAF-FM-03

s Fish stocks are fed a commercial fish food a minimum of once daily throughout the work veek. They are not fed for 48 hours5.555556e-4 days <br />0.0133 hours <br />7.936508e-5 weeks <br />1.8264e-5 months <br /> prior to initi-

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ating a test.

r. f 34 Dilution Vater

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The source of dilution water is the City of Baltimore Municipal Vater System. Upon entry to the laboratory, the water is passed through a high-capacity, activated-carbon filtration system to remove chlorine. I detergents, and hexane-soluble organic _aaterials. This vater source i has proven safe for aquatic organism toxicity testing, as evidenced by maintenance of the multigeneration Daphnia cultures, with no evident loss of fecundity. If requested, reconstituted fresh vater or other dilution vater any be used.

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6 3.5 Test Concentration Series The test concentration series to be used for definitive toxicity tests i vill be selected following preliminary tests to bracket appropriate i concentration ranges. A minimum of five concentrations and diluent water controls vill be used. If it proves necessary to use a carrier solvent to disperse the test article in aqueous solution, an additional set of solvent controls vill be used. All test concentrations and controls vill normally be conducted with tvo replicates. Additional replicates may be used at the client's request.

3.6 Test Concentration Preparation

' Tesc concentrations vill be prepared by-standard serial dilution (APHA et al. 1985) using Class A volumetric glassvare and dilution Vater.

Test organisms will be added to prepared test solutions.

3.7 Test Vessels and Test volume

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Test vessels vill consist of 19- or 38-L all glass aquaria, with the

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final test volume of each replicate 15 or 30 L, respectively. Other test vessels any be used depending on the study's requirements.

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S' 3.8 Test Organism Number r A minimum of 10 test organisms vill be used in-eech test replicate, constituting a minimum number of 20 fish per treatment gorup. - Fish -

vill be randomly assigned to each test replicate.

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EA Engineering, Science, p and Technology, Inc.

Effective May 1981 Revision 3, '.qust 1986 Page 2 of 8

l-PROTOCOL ATS-SAF-FM-03

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y . 3.9 Test Environment

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The test vessels vill be maintained at a temperature similar to that at which the fish have been acclimated, but within the recommended test range. Test temperature vill not vary more than 32 C from the specified

[ acc11mation temperature. The tests vill be conductes on a 16-hour light, 8-hour dark photoperiod cycle.

3.10 Analysis of Test Concentrations for Test Article

c If required, test solutions replicates may be analyzed for chemical analytical verification of achieved test concentrations. The analytical method and number of determinations vill be formulated af ter consultation with the client. Where chemical analyses are necessary, both nominal and p,9 actual measured test solution concentrations vill be reported.

3,11 Test Observations l-b Test organisms vill;be observed at a minimum of 24, 48, 72, and 96 hours0.00111 days <br />0.0267 hours <br />1.587302e-4 weeks <br />3.6528e-5 months <br /> af ter test initiation to determine number of animals killed by test article concentrations. The study vill terminate after completien of the 4-day observation period. The study may be extended, however, at the request of the client.

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Routine water quality measurements are performed on the test solutions and diluent vater controls according to the following schedule: tempera-ture is measured daily from all test. chambers; dissolved- oxygen and pH

{, are measured initially from all test chambers as well as at test termina-

' tion, and from the control, lov, medium, and-high concentration at 24 ,

48 , and 72-hour intervals. Specific conductance, total alkalinity, and

[' total hardness are measured from the diluent vater and sample. Specific

[: conductance may also be used for concentration monitoring at the request of the client. Test solutions vill not be aerated unless dissolved e oxygen concentrations fall below 40 percent of saturation. Analytical methods vill be conducted according to APHA et al. (1985) and EPA (1979).

{

Dead fish are removed when observed. At the end of the 96-hour test (f - period, a minimum of 30 surviving fish are removed from the test con-tainers, placed in labeled specimen bags, and frozen. The fish are subsequently thaved, measured to the nearest as (standard length), patted dry,' and veighed to the nearest 0.01 g. The average length and veight of the test fish are reported, along with their respective ranges and stan-dard deviations.

i-1 ETC T-101 EA Engineering, Science, P and Technology, Inc.

ffective May 1981 Revision 3, August 1986 Page 3 of 8

fi PROTOCOL ATS-SAF-FM-03 C l'

[. f

3. g ation Renewal (Vhen Applicable)

't j -

.Then static-cenewal testing is reouired, the tast solution is renewed g, ince every 24 or 48 hours5.555556e-4 days <br />0.0133 hours <br />7.936508e-5 weeks <br />1.8264e-5 months <br />. Tne solution renewal may be pecformed by e & T. attring the organises from one test chamber to acother or by

{e. ' ,

y + 1r.cing the test solution. If the test solution replacement method 3,Qyy sed, caution must be given not ts stress the test organisms while M.(*~

e s. -

• i . :., t .-- r! :r is siphoned. Usually 75 percent of the old solution iplaced; however, the amount is dependant on the size f[g[f ~ y is rem . u.

of i e tot, organisms.

gp L13 Data J.nalysis The LC50 values and ensociated statistics vill be derived by the probit gc 9 [h analysis method, moving average < and binomial test methods as described Ui V Ly Stephan (1977). Depending on the nature of the datr, other methods may be used including the probit approximation method of Litchfield and Vilcoxon (1949), SAS probit analysis (SAS Institute 1979), oc graphical

, y*

interpolation using the log concentration vs. percent lethality as described by APRA et al. (1985). ihe methods used vill be specified -

i in the report.

l 4. FINAL REPORT 9e final .;epcrt vill be prepared to contain the following information:

. Objectives and proc v!ures stated in the approved protocol, including any changes ande to the original protocol ,

I

. Identity of the test articles by naeat or code numbet and their strength (i.e. , qualit'// purity) and a escription of 8

{ - any pritre.atment 4

Source of the dilution vater, its chemical characteristics, and s description of any pretreatment

[

. Test concentration series used and duration of the assay 0 . Mean standard lengths ad ver weights of test fish, the respective ranges, and approximate 1.or. dings of fish co te+t g volume (g/L)

[

\

. Routine vater quality characteristics of dilution vater and j

selucted test concentrations ]

l 9

C ETC-T-101 EA Engineerir.g, Science, and Technology, Inc.

2ffective May 1981 Revision 3 August 1986 Page 4 af 8 i

\

,9 ,

I y- .

PROTOCOL ATS-SAF-FM-03

$- . Any unforescer circumstances that may have affected the T quality or integrity of the study l . Signatur the project coordinator, program manager, and I quality control officer as authorization of the report

. Location of all erchived data and the original copy of the

, >s final report at EA 4

Irems of data to be included in the report consist of: experimental

[O design and test perfc mance effects on general appearance of test organisms morbidity and mortality; tabular presentation and appropriate statistical evaluation of water quality characteristics, and survival

, p data where n me sary.

b "' 5. QUALITY As.;UP#lCE  ;

l- 5.1 Amendments to Protocol L Amadments to the authori:ed protocol instigated by EA or by the client vill be made only after' proper authori:ation. Such authorization is

[- achieved by completion of an Amendment to Protocol form by EA and the

'l:

l ' client.

l i .- 5.2 :tandard Operating Procedures _

Unless ctherwise specified all procedures mentioned in the protocol are subject to detailed Sta.1dard Operating Procedures (SOPS) which are l contained in the SOP manurls of the participating departments. These ,

i SOPS and protocols follow the U.S. EPA's Good Laboratory Practice {

l Standards (GLPs) (EPA 1983).

5.3 Reference Toxicant A reference toxicant bionssay, utilizing sodium dodecylsulfate (SDS -

other appropriate reference toxicant, vill be used as an internal quality i

l check of the sensitivity of esch batch af test organisms. Testing vill l>= prior to or concurrent with the test or following the acclimation lhw period for newly acquired organisms. The results of each test vill be 1 compared si.th historical torkological information of reference toxicant l! tests ptrformed at EA on the species, to determine if the results are vithir. seceptable limits using the control charts outlined in ?eltier and i 1

';eber (1985).

lk l l >[ .

FOC-T-101 {

EA Engineering, Science, l

[', and Technology, Inc.

Ef f active May 1981 l

Revfsion 3, August 1986 Page 3 of 8 l l

. f, PROTOCOL ATS-SAF-FM-03 s

G. 5.4. Quality Assurance Evaluation 6

The study described in this protocol any be subject to internal audit by EA's Quality Assurance Unit. A quality control officer vill be responsi-ble for monitoring each study to assure the client that the facilities, equipment, personnel, methods, practices, records, and controls are in conformance with EA's OC program and EPA's GLPs.

i -5.5 Inspection by Regulatory Authorities in the event of an inspection of EA by an outside authority during the

.(i t course of the study, the client vill be consulted before inspectors are ,

7ermitted access to any of the project records or the experimental areas.

u-5.6 Archives.

E{l ,

Copies of project-specific records shall be transferred to the client promptly af ter the project is completed or as negotiated and budgeted with each client. Original primary data vill be retcined at EA for 5 years. Primary data include' laboratory data sheets, racords, memo-randa, notes, photographs, microfilm,- and computer printouts that are a

. result of the original observat w r and activities of the study and.whien are necessary for the reconstruction and evaluation of the study report, g 5.7 Location This study vill be conducted by the Aquatic Toxicology Services

~j- Department of the Scientific Operations Division of EA Engineering,

~

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

6. FATHEAD MINH07 ACUTE TOIICITT TEST SPECIFICATIONS 6.1 Basic References

.American Public Health Association et al. 1935. Standard Methods

. for Examination of Vater and Vastevater,: 16th editior- APHA,

' k. Washington, D.C.. 1,268 pp. >

-l American~ Society for Testing and Materiah (ACIM). 1980. Standard l _: Practice for Conducting Acute Tests with Fishes, Macroinvertebrates, t- and Amphibians. ASTM Designation: E729-80, Fhiladelphia. Pa.

pp. 285-309.-

L ,

l l.

l l ETC-T-101 EA Engineering, Science, f and Technology, Inc.

fffective May 19d1 l 1e', cion 3. Auguct 1986 Page 6 of 8

). . . . . .,.-.,;. .- , . . , - . . _, . . - . . . .

1 PROTOCOL ATS-SAF-FM-03 9* EA Manual T-101. 1981. Aquatic Toxicology Studies, Quality Control and "a Standard Operating Procedures Manual. Revised, August 1986. (Internal document prepared by Aquatic Toxicology Services, EA Engineering, Science, and Technology, Inc.)

Peltier, V.H. and C.I. Veber, 1985. Methods for Measuring the Acute

,- Toxicity of Effluents to Freshwater and Marine Organisms. EPA-600/

4-85-013. U.S. Environmental Protection Agency, Cincinnati, Ohio.

U.S. EPA. 1983. Toxic Substances Control; Good Laboratory Practice Standards, Title 40 CFR Part 792. Fed. Regist. 28(230):53922-53944.

{.

L.

6.2 -rest Specifications 4

E' Test organism: Fathead mic.nov (Pimeobales promelas)

,i Temperature: 2032 C Fish age All fish for any one test vill be selected from the same age-class Fish length: The largest fish vill not be more than 50 percent longer than the smallest I tish l

Loading ratio: In general, test containers msy not contain greater than 0.8 git biomass .

Aeration: None, unless dissolved oxygen f alls belov 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 belov 40 per- #

cent saturation thereafter Light quality: Vide-spectrum fluorescent light

{w Light intensity: 50-100 f.c.

p.' 16-hour 1.4ght, 3-hour dark Photoperiod:

()

Dilution water: Dochlorinated municipal tap vate ,

b reconstituted fresh water, or appropriate receiving water 4 Test containers: 19- or 38-L all glass aquaria I

~

Test volumet 15 or 30 L y

ETC-T-101 g EA Enginearing, Science, and Technology, Inc.

• Effective May 1981 Revision 3, August 1)B6 Page 7 of 8 .

. [a .

6 PROTOCOL A75-SAF-FM-03

.>i

'No. of concentrations: Minimum of five - te :-cancentratiorr.

+($ A, 3 ' - and a control in duplicate series i

' Test-animals _per container: 10 (if loading is exceeded, more replicates or larger aquaria may i be used)

Feeding rr .me Animals are not fed during tests unless- fish are too small to survive 96 hours0.00111 days <br />0.0267 hours <br />1.587302e-4 weeks <br />3.6528e-5 months <br /> without feeding. Feeding 1(? is termu.ated 48 hours5.555556e-4 days <br />0.0133 hours <br />7.936508e-5 weeks <br />1.8264e-5 months <br /> before-test--

le initiaticn in the lot _ from which organisms are obtained.

k*' '

3& Test type and. durations- Rangefinding--24-96 hours Definitive--96 hours Reference toxicant--24 hours s'

End point: Lethality--defined as cessation of

.- opercular movement and inability to

. respond to external stimuli-(gentle prodding with glass rod)-

.. l 5 R -

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ETC-1-101 1,'

EA .f.ngineering, science, and Tec. : logy, Inc.

Effective.May 1981

- Revision 3.-\ugust 1986 Page 8 ol 8 lt ,

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ATTACHMENT II DATA SHEETS f

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- AQUATIC ORGANISMS' ACUTE TOXICITY TEST DATA SHEET -

Psojed No.: Td3DS 8o Fiow-through. Heneral TEST ORGANISM: Beginninp Date: 9-/ -9f Time: il9 f ' .

3EST:

Test Article:181vt I. rDT EdocAelner:M T *did &.e4 haa f Common Name: 14hhr Dm Ending Dsse: 9-ll il Tine: //l/T '*

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Sanple No.: All- TN '7'/o 'h/' Sour 6o: 5/l ., liar <tness:

Dilution No.: bc(Idc[ AccInnation:M or Age ^.3 /M Conduct!vity-

. SALT OR BRACKISH ALL TESTS t/ATER TESTS Test Dissolved Oxygan Con-Nuinber of Terrporature (C)

(mgdites) pH Sal;sery (ppi)p , Mgd ,

Con. tainer I hre Ornanisms -

4 t.

96 I or % tJo 0 24 48 72 If6 0 24 48 72 96 0 24 48 72 96 0 24 46 72 0 24 48 72 96 0 24 48 72 96 l'o ' 4 80 le .10 _

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AQUATIC ORGANISMS ACUTE TOXICITY TEST DATA S* FEET Beginnog Date: 9-t'[ V/ Tine: I#

Pacir,cs tJa : ~70D1th pST.qQWw tim @, Renewal TEST OHGAtitSM. .

8 2 co TeuAn w A M 8"*"*" ' un connahuir:J%4 /.h bcu / conwnon tJarne.lisha /** **"4 Ending Date. 9//f/// Tan =: Dauen '

cn3a: Ool'Er* Iestvoiwne /t ScientJe teame: Duw.h Sanr$

OC Test iJo kT ~ F7-r/4/ ~X0 Test Dusation: 9/o Au Lol tJo.: l '7 d d/s fil Anatury:

Sanple flo:dD .239 7/c 7h Source: Cri Hardness:

Odution th : [Y clf*r' Acchmetion- d c, Age ^60 days Corukovay-I SALT OR BFiACKisit ALL TESTS WATER TESTS q

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Sandard individual Lengtn No. Weyht 10) (mtm i' t C. / / 9 /7,oJ'

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a Chain-of-Custody Accord Fon omce use onty EA ENGINEERING. Species to be tasted:

SC:ENCE. ANO -g D. cara Metmaa a TECHNOL.CGY, INC.

- u.smana

• _ gasam Acuatic Toxter.,6ogy Laboratory i _ g,'7 f*. ,

15 Lovaton Circle .o o..

Sparts (Baltimore). Marytand 21152 am- w.crwermo a eae cxxemum O"'

g 4 '- W -g- 35#

C! lent: OC M Project No.;

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(L ATTACHMENT III

- REPORT OtlALITT ASSURANCE RECORD t*' 3 4

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. REPORT QUALITT ASSURA' ICE RECORD I CLIENT b . / n ,., , b_, ,n t EA REPORT NO. [bO l J

p. , PROJECT NUMBER 9 cec; jo _

TYPE ANALISIS .4c .: , f ,s I 3: x AUTBoR } i~,,,n $~] 8,. ),,,, TEST ORGAN 1SMS :.D n,ee , ,  ? . i , a ),~, e f J '

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REPORT CHECKLIST r QA/0C ITEM FEVII'VER DATE

1. Saciples collacted, transpor ed, t.

and received according to study plan -) .jwl's .

E k '7 /~),

requirements, f c'c i r u- .a_. s ,

N7

2. Sam ales prepared and processed accord. -:l hY ing to study plan requirements, c_ i_c .<- n_ h, . / . a u M 7h/

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3. Data collected using calibrated equipment. ~~(.' . ' ,_. <._cc ,- . . o _ b/ .N_.f'.1, v /,' > /- <

V '

4. Calculations checked:

l' .

Band en1culations checked ^??lm

/ 0.lsy.x.J) ~ Rf3rle/

_ Documented and verified statistical ,, j .

procedure used. f h .v ,.a h_,, dM', - v/ nib

5. Data input / statistical analyses completa and correcr. 'd< j& . b,s. d 7!3.[f/

1

6. Reported results and facts checked g < <

v aga. inst original sources. N /d. [.sna N. 7[3'[4/

7. Data presented in figures and tables l correct and in agreement with text. _7b.ed,e M_0v _

, kJ/[#/ ,

j 8. Results reviewed for compliance with - l ., r,

h. *f .%

l

. study plan requirements. /' . , , z w,,n o Y 'l.' ' ' < /

l.  :

./

lN AUTHOR DATE

9. Commentarv revieved and resolved. <f ,_s a _ <6 [. /G /

y , .

10. All study plan and quality assurance / control requirements have been met an ' the report is approved:

I- PR04E(I MANAGER DATE

^/ Y $ . [r v Y 3 0 ]ke#/Q'n f lh 0.C. OFFICER ,

[ dATE

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SEN DATE

/ECXNICAL2E'/IEVER

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