ML20086Q256

Forwards Annual Rept to Maintain Approval for Use of Calgon H-130 as Cooling Water Additive for Zebra Mussel Control, Including Rept on Ecotoxicity Study of H-130 Microbiocide & Daphnia Magna.Repts Withheld

ML20086Q256
Person / Time
Site:	Perry
Issue date:	12/20/1991
From:	Lyster M CENTERIOR ENERGY
To:	Magni R OHIO, STATE OF
Shared Package
ML19351A982	List: ML20086Q256
References
PY-CEI-OEPA-013, PY-CEI-OEPA-13, NUDOCS 9112300081
Download: ML20086Q256 (2)

Text

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PERRY NUC' LEAR POWER PLANT und Asdress:

po oox gr Michael D. Lyster OA0 pg ngy, c:llO 4 081 yENT RY, y VICE PnESIDE NT . NUCL EAR g (216) n s4rv December 20, 1993 PY-CE1/0 EPA-0143 L Hr. F.ichard Hagni OEPA Division of Vater Pollution Conttol P.O. Box 1049 1800 JaterHark Drive Columbus, Ohio 43266-0149 Rei Director's Final Findings and. Orders f or use of 11-130 and CL-37 Cooling Vater Additives for the Perry Nuclear Power Plant OEPA Permit No. 31500016*DD

Dear Mr. Magni,

?n accordance with the Director's Final Findings and Orders, this letter and attachments comprise the annual report to maintain approval for use of Calgon 11-130 as a cooling water additive for rebra mussel control at the Perry Nuclear Pove; Plant.

The results of monitoring and toxicity testing done during the application of 11-130 are included in Attachment 1. Reports on progress Aning siade in the areas of research specified in Section 4.a. - e. of the orders are in Attacha.ents 2-7. Reporta on anticipated future research, problems encountered, and any additional information is in Attachment 8. The reporta from Calgon Corp. stamped " Confidential" contain proprietary information. Any release cf these documents should be approved through Calgon Corp.

Please call Louise Barton at (216) 259-3737 ext. 5S12 if you should have any questions or need additional information.

1. ' Sincerely '

i r

JG Michael D. Lyste g HDL:LKBisec o vr a QgA Attachments

-o M cc NRC Document Control Desk NRC Project Manager G,_C NRC Resident Inspector Office 88 NRC Region III g4 B. Hall. OEPA - Northeast District Office

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s PY-CE1/0 EPA-0143L ATTAcilHEllT 1 11-130 Honitoring and Toxicity Testing Results tiott This information was previously submitted ihe OEPA as required per Section 3 of the Orders.

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s CENTEQ PERRY NUCLEAR POWER PLANT WiM*m PO box 9? Michaal D. Lyster 10 CENTER ACAC PEnRY. OHO 64001 ya pgg ,

PEnAY. OHIO 44081

-(218) 2 4 3 5 October 11. 1991 PY-CE1/0 EPA-0137L Mr. Richard Hegni OEPA Division of Vater Pollution Control P.O. Pox 1049 1800 Vatermark Dr.

Columbus, Ohio 43266-0149 Ret Director's Pinal Findings and Orders for use of H-130 and CL-37 Cooling. Vater Additives for the Perry Nuclear Power Plant OEPA Permit No. 31800016DD

Dear Mr. Magni,

In accordance with the Director's Final Findings and Orders, the following is a report of the monitoring and toxicity data obtained during the Perry Nuclear Power Plant's H-130 treatment, conducted on September

12. 1991.

Grab &amples of the ef fluent vere collected every two hours during the treatment-from a point dovn7tream of the detoxification site. Analysis of these samples demonstrated that the concentra:lon of active H-130 discharged into Lake Erie vas maintained at <0.02 mg/1.

f A composite sample of_the effluent was collected during the H-130 treatment and used to conduct acute toxicity tests. Control samples vere collected at both the intake and discharge prior to treatment. Mortality was not found to be at a statistically significant level for any test.

Test results are attached.

The use of CL-37 to reduce fosa vas not required.

Please call Louise Barton (216) 259-3737 ext. 5512, if you have questions or need additional information.

Sincerely.

// 'f' '

Michael 1). Lyster Attachment HDL 1XB cc: U5tRC Document Control Desk NPJL Project Manager

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.ESULTS OF ACUTE TOXICITY SCREENING TESTS WITH Dapnia magna AND Pimephales promelas

" ON SAMPLES FROM PERRY NUCLEAR POWER PLANT Pergusred for-Calton Corporation klyna. Ohio Prepared by:

EA Mid Atlantic Re EA Engineerine. Science,gional Operations and Technology. Inc.

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i RESULTS OF ACUTE TOXICITY SCREENING TESTS WITH Daphnia magna AND Pimephales promelas ON SAMPLES FROM PERRY NUCLEAR POWER PLANT

, Prepared for-Calgon Corporation Elyria. Ohio Prepared by:

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

15 Loveton Circle Sparks. Maryland 21152 October L991 LA Project 70003.10 Arget N mkr a60 i

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

Sample collection and receipt data are reported in Table 1. Tab'.e 2 presents initial vater qvality data. Test results, test orgsnism source and age, reterence toxicant data, and vater quality measurements are summarised on pagas 3-6. Test protocols, copies of rav data sheets, and the Report Quality Assurance Reentd are included as Attachments I, II, and III, respectively.

S 4

1

SUMMARY

OF RESULTS 5 .

1 The results of the acute toxicity screening testa performed for Calgon Corporation on samples from Perry Nuclear Power Plant are summarcized on pages 4 and 6. In the D. magna 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. prornelas test there was 100 percent survival of fathead sinnovs in all three

- treatments after 96 hours0.00111 days <br />0.0267 hours <br />1.587302e-4 weeks <br />3.6528e-5 months <br /> of exposure, and 100 percent survival of minnovs in the laboratory control.

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SUMMARY

OF SAMPLE / TEST INFORMATION G.

If, Test 48-hour Daphnia magna Static Acute Toxicity Tsst Client names Calgon Corporation Yest procedure or protocol followed EA Protocol ATS-SAI-DS-04 Static acute 48-hour LC50 assay vith Daphnia Sample Jescription: Perry Nuclear Power Plants Influent prior to treatment: Ef fluent prior to treatment: Effluent

. composite during treatment ,

{, EA accession 7. umber AT1-739, AT1-740, AT'.-741

! Time and date of sample collection: See Table 1 Time and date of sample receipts See Table 1 EA Oc test number: SA-09-14-91-759 Test initiation time and date: 1145, 14 September.1991 Test completion time and date: 1145, 16 September 1991 Control vater: Dechlorinated tap vater

' Organism Lot Number NA Source: EA Culture Facility, Sparks, KD Age (24 hours2.777778e-4 days <br />0.00667 hours <br />3.968254e-5 weeks <br />9.132e-6 months <br /> Reference Toxicant Sodium dodecyl sulfate EA OC test Number: RT-09-10-91-739 Dilution Vater Dechlorinated tap vater 3 Reference Toxicant (SDS) Results c

3 24-hour LC50: _ 29.5 mg/L Laboratory control chart acceptrbility range for 24-hour LC50:

14.6 - 44.8 mg/L Cr

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SUMMARY

OF SELECTED TEST DATA (Cont. )

El Accession Numbers: AT1-739, 740, 741 Sample

Description:

Influent prior to treatment Test Species: -D. magna Effluent prior to treatment Ef fluent composite during trea tmen t EA QC Test Nutabert SA-09-14-91-759 Test Material Percent Survival at 48 Hours ---

Is Laboratory Control 95 Influent prior to treatment 8.5 Effluent prior to treatment 100 >

Ef fluent composite during treatment 95 Selected Vater Quality Range Temperature (C) 19.9 - 21.0 pH 7.5 - 8. 5 Dissolved oxygen (ag/L) 8.3 - 9.0 Conductivityt (uS') 136 - 365 d

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SUMHARY OF SAMPLE / TEST INFORMATION ya Test: 96-hour Pimephales gromelas Static Acute Toxicity Test Client name: Calgon Corporation t

Test locedure or protocol followed: EA Protocol ATS-SAF-FM-03 htstic acute 96-hour LC50 assay with f athead minnovs (Pimephales promelas)

Sasple description: Perry Nuclear Power Plant: Influent prior to l treatment Ef fluent prior to treatment: Effluent composite during treatment

- E.A accession number AT1-739, AT1-740, AT1-741 Time and date of sample collection: See Table 1 Time and date of sample receipt: See Table 1 EA QC test number SA-09-14-91-760 Test initiation time and datet 1200. 14 September 1991 Test completion time and date: 1200. 18 September 1991 Dilution water: Dechlorinated tap water Org nism Lot Number: F3 1-7/28-3/1 Source: EA Culture Facility, Sparks, MD Aget 4144 days Length: 16.22 mm S.D.: 2.38 na Veight: 0.06 g S.D.: 0.03 g Reference Toxicant Sodium dodecyl sulfate 3

EA QC test Labar RT-09-10-91-725

's Dilution water: Dechlorinated tap vater Reference Toxicant (SDS) Results 24-hour LC50: 32.2 mg/L Laboratory control' chart accept ' Y tange for 24-hour LC50:

11.2-45.2 mg/L 5

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SUMMARY

OF SELECTED TEST DATA (Cont. )

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

Description:

Influent prior to treatment:

Test Species: P. proselas Effluent prior to treatment; Effluent composite during i treatment EA QC Test Number: SA-09-14-91-760 Test Material Percent Survival at 48 Hours

^

Laboratory Control 100

c. Influent prior to treatment 100 y' Effluent prior to treatment 100 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 - 90 conductivityt (uS') 234 - 362 h

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._ , . , . .> 'e TABLE 1 SAMPLE COLLECTION DATA FOR SAMPLES FROM FERRY NUCt. EAR POVER PiltTT EA Accession Number Sample Collection Sample Receipt Type of Sample Sample Description 0800, 12 SEP 91 0920, 14 SEP 91 Grab Influent prior to AT1-739 treatment 0900, 12 SEP 91 0920, 14 SEP 91 Grab Effluent prior to AT1-740 taeatment ]

0952, 12 SEP 91- 0920, 14 SEP 91 Composite l Effluent composite AT1-741 '

during. treatment 0005, 13 SEP 91 f

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TABLE 2 INITIAL VATER QUALITY DATA FOR SAMPLES FROM PERRY NUCLEAR POWER PLANT EA Accesslen Alkalinity Hardness Conductivity Number _(ag/L as CACO,) (ag/L as CaC01) (pS*)

Description 96 116 292 l Influent prior AT1-739 to treatment 112 128 343

. Effluent'prfor AT1-740 to treatment 112 128 345 Fffluent composite AT1-741 during treatment i

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

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

2. TEST ARTICLE 2.1 Descrirtion/Identifientien Unless otherwise specified, the test material is supplied by the client. Adequate chemical specificat: ens with special refsrence to hazardous properties and storage

,( _ conditions is also supplied by the client.

2.2 Methods of Syntheris 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. EXPERIMENTAL DESIGN

, 3.1 Test Orvsnisms

p A species of &phnia (water fleas), as determined by project needs, is the test organism.

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3.2 Source

! Da shnia used for acute toxicity tests are obtained from stock cultures maintained in ;iA's Aquatic Toxicology Culturing Facility.

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y;. 3.3 culturine and Hntaine cnnditinns Daphnia cultures are maintained at 20 2 C and a 16-hour light.

..h,. 8-hour dark photoperiod cycle in an environmentally controlled laboratory.

Cultures are maintained m 18.9-L all glass aquaria or other appropriate container and are fed algae (Selenastrum capricornurum) and a trout chowlyenneereal i leaves suspension in the manner described in Peltier and Weber 0985). New cultures are initiated on a routine basis to ensure healthy productive

, , populations. Orgamsms from cultures producing ephippta are not used for-to:.. .y tests.

I

• E ATS-102 EA Engineering. Science, o and Technology. Inc.

i-Effective May 1981 '

Revision 4. April 1991 Page 1 of 7 I

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l PROTOCOL ATS SAI DS 04 9 '

3.4 Age of Test Ortmnisms at Test Initiation Neonates of known age (i.e., less than 24-hours old) are obtained for testing by segregating adult females from the mass cultures on the day before a test is imttsted. On the day of th. 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.

Upon entry to the laboratory, the water is passed through a high capacity, activated carbon filtration system to remove chlorine and other possible contaminants such as detertents, and hexane-soluble organic materials. This

? water source has proven sale for aquatic organism toxicit testing as evidenced by maintenance of the multigeneration Odphma and Pimeph) ales promelas cultures.

with no evident loss of fecundity, if requcsted, reconstituted fresh water or an appropriate receiving water can be used as the dilution water. j

- 3.6- Test Concentrstion L ..es

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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 6.25.12J. ?.5. 50, and 100 percent effluent plus a control).

3.7 Test concentration Frenarstion Test concentrations are prepared with Class A glasrware.

3.3 Test Vesseis nnd Test Voliime l

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Test vessels are 400 m1 glass beakers or 10-cm culture bowls: the final test volume of each replicate is 200 ml. Depending on the study requireesnts. other test vessels and test volumes may be used.

,1 3.9 Test Orvantum Niimber L Tests are conducted using two replicates per concentration, with ten organisms b.

per container. Neonates are randomly assigned to each replicate test container.

More replicates can be added,if appropriate.

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3.10 Test Envirnement

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! - The test vessels are maintained at 20 2 C 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. B-hour dark photoperiod.

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' 3 ATS-102 EA Engm' eering. Science.  !

and Technology. Inc. -

Effective May 1971 . .

j- Revision 4. April 1991. Page 2 of 7 g me e g. p.g,y q.g.'8 9 "T Y7 6W -8WT" TWT' T T't$F Tu'*8d M'WM e-g t 8-S" m y VW % W $ W p W W'FW Q S'M

PROTOCOL ATS-sal DS 04 g, 3.11 Analyris of Test Concentrations for Test Article '

I If required, test solutions may be analyzed for analytical verification of chemical concentrations. The analytical 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.

3.1 Im_Observatiens Each test day. test organisms are observed to record the number of surviving organisms, the study terminates af ter completion of the 2 day observation period. The study may be extended, however, at the request of the client.

Each sample alkalinity, and receiving and hardness. water areofanaly:ed Measurements for temperature, water quahty cunductivity, ion.

taken at test initiat 24-hours. and test termination include dissolved oxygen, pH temperature and conductivity from a mmimum of one replicate of the control, low, medium, and high dilutions. Aliquots of effluent and receiving water and test solutions may be gently aerated, if necessary (dissolved oxygen less than 40% or greater than 105%

saturatien). Analytical determinations are conducted according to APHA et al.

(1989) and EPA (1979). -

3.13 Solution Renry11 Nhen Anplicabid When static renewal testing is required, the test solutions are renewed at 24-hours. New test solut; ; 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 wide bore pipet or glass tut ><. The number of live organisms is rec rded.

Caution is given not to stres1 the test organism.s during transfer. After water quality measurements (temperature, pH. dissolved oxygen. and conductitity) are completed, the old solution is discarded.

3.14 Dam.Jnalysis The LC50 or EC50 values and associated statistics are calculated using the probit, s moving average, and binomial methods as described by Stephan (1977).

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 prob.it :nalysu (SAS Institute 1979) or graphical interpolation using the log concentration vs. percent mortality as described by AFHA et al. (1989). The methods used are specified in the final

, report, ATS-102 EA Engineering. Science.

and Technology. Inc.

Effective May 1981 Revision 4, Aprt11991 Page 3 of 7 WTdNii$$$$  %

PROTOCOL ATS-sal-DS 04 w . 4. FINAL. REPURT

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The final report is prepared to contain at a minin um the following inforTnation:

. 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 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 h

. Test concentration series used and duration of the assay

. Water quality characteristics of dilution water and xlected test concentrations during testing (pH. dissolved oxygen, tr.mperature, etc.)

. Any un/vaseen circumstancas that may have affected the quality or mtegmy a de study -

. Signature of the aroject manager, senior technical reviewer, and quality control officer, authorizing release of the report

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

report at EA Items of data to be included in the report consist of experimental design and test performance: effects on general appearance of test orgsnisms (if applicableh morbidityofand evaluation water mortality:ity qual characteriset.s; and survival data. tabular pre.wntat;

5. QUALITY ASSURANCE 5,1 Amendments to Pmtocol Amendments to the authorized protocol established by EA or by the client are made only af ter proper authorization. Such authorization is achieved by completion of the Amendment to Pmtocol Fmm by EA after consnitauon with the client.

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

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

a PROTOCOL ATS sal DS 04 3 .

5.2 Standard omrating Prxedurra b

Unless otherwise specified, all procedures specified in the protocol are subject to detailed Standard Operating Procedures (SOPS) which are contained in the SOP inanuals of the participating departments. These SOPS and protocols follow the U.S. EPA's Good Laboratory Practice Standards (GLPs)(EPA 1989).

5.3 Reference Toxiant 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. Testme is conducted at least once monthly on orgamsms which are cultured in-house, the results of each test are compared

- with his'orical, species-specific toxicologicalinformation from reference toxicant 2

tests performed at EA. to determine if the results are within acceptable limits.

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

5.4 Quality Amrsnee Ev11uaban Studies conducted under this protocol may be subject to internal audit by EA's Quality Assurarce Unit. A qualirf control officer is responsible for monitoring nca study to assdre the client that _ the facilities, equipment, personnel, methods, practices, recordt, and controls are in confonnance with EA's QC program and. if applicable. EPA's GLPs.

5.5 inmerien by R; m btery Authojijta In the event of an inspection of EA by an cutside authority during the course of the study, the client whose study is being mspected will be consulted before e.uminers are psrmitted acce:s to any of the project records of the expenmental areas.

5.6 Archives Copies of project-specific records shall be transferred to the client promptly af ter

, the projec;t is completed or as negotiated and budgeted. Origmal primary data

will be retained at EA fcr 5 years. Pnmary data mclude cham of-custody records, laboratory data sheets, records, memomnda, notes, photographs.

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

ATS-102 EA Engineering. Science.

. snd Technology. Inc.

Effective May 1981 Revision 4. Apnl 1991 Page 5 of 7 1

PROTOCOL ATS sal DS-04

. 5.7 Locatien 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 Darhnia ACUTE TOXICITY TEST 6.1 Baric References American Public Health Association (APHA).1989. Standard Methods for E.tamination of Water and Wastewater.17th edition or an updated edition.

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

< EA Manual ATS-102.1981. Aquatic Toxicology Studies. Quality Control and Standard Operating Procedures Manual. Revision 2 - August 1991. (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

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Effluents to Freshv .r and Marine Orgamsms. EPA-600/4-C5

-013.

U.S. Environmental Protection Agency Cinemnati. Ohio.

Weber. C.I.. et al.1989. Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Orgamsms. Second Edition. EPA /600/4-69/00L U.S. Environmental Protection A',:ncy. Cincuman.

Ohio.

U.S. EPA.1989. Toxic Substances Contml Act (TSCAh Ocod Laboratory Practice Standards. Title 40 CFR Part 792. Eed. Rent. 54(158):340w34074.

6.2 Ten Snecifientions Test organism: Daphnin magna or D. pulex: species to be specfied m the study plan and final

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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.

, .; ATS-102 i

EA ens ncering. Science.

and Technology. Inc. .

Effectis N.ay 1981 Revision 4. April 1991 Page 6 of 7 1

1 PROTOCOL ATS-SAI DS-04

. Photoperic<t 16 hour1.851852e-4 days <br />0.00444 hours <br />2.645503e-5 weeks <br />6.088e-6 months <br /> light. S-hour dark l Aeration

None. unless diuolved oxygen falls below 40 petcent saturation I

Dilution water: Dechlorinated municipal water.

reconstituted water, or appropriate receivmg water Test container: 400-m1 Pyrex beaker or 10<m culture ,

bowl 1

Test volume: 200 mi 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 Immobili.ation--defined as cessation of movement except for minor activity of appendages Mortality-,:lefined as cessation of all movement for a wriod of at least 5 seconds even when tie test container is tapped or rotated, or the organism

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is gently prodded with glass rod Test acceptability: 90% or greater survivalin the control solution.

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Effective May 1981 Revision-4, April 1991 Page 7 of 7 y ,pm, - - - + ww -r y -w----- ,,y --

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4 PROTOCOL ATS-SAF-TM-03

. 11. STATIC ACUTE 96-BOUR LC50 ASSAY VITH FATHEAD MI!MOVS N; (Pinephales proselas)

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

2. TEST ARTICLE 2.1 Description /Identi7ication Unless othervise specified, the test article vill be supplied by the client. Adequate chemical specifications with special refererce to hazardous properties and storage conditions also vill be supplied by the client.

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

3. EIPERIMENTAL DESIGN 3.1 Test orrenisms

. Fr.theed sinnov-(Pinephales proselas) vill constitute the test species.

3.2 Source Fathead sinnov stocks are lah reared, obtained from a scientific orga-nisa vendor, or field collected using techniques to be specified in the y report, e .

3.3 Holding Conditions Upon receipt, fish are transferred to holding tanks which are eintinu-ously replenished with dechiorinated municipal tap water. The fish are accliented to laboratory conditions for a sinimum of 7 days- prior to testing.- The laboratory is maintained on a 16-hour light, 8-hour dark -

photoperiod cycle bv a programmable electrical timer. All fish stocks are examined regularly throughout the work veek. Dead fish, or those displaying abnormal svisning behavior, discoloration, or pronounced lethargy are removed as' observed, and recorded on appropriate log sheets.

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Fish stocks are f ed a con ** cial fish food a sinimum of once daily Jf throughout the work veek. Jhey 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-ating a test.

, 3.4 Dilution Vater i

The source of dilution vater is the City of Baltimore Municipal Vater System. Upon entry to the laboratory, the vater is passed through a high-capacity, activated-carbon filtration system to remove chlorine, detergents, and hexane-soluble organic materials. This vater source has proven safe for aquatic organism toxicity testing, as evidenced by

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maintenance of the multigeneration Daphnia cultures, with no evident loss of fecundity. If requested, reconstituted fresh water or other

,,, dilution vater may be used.

s 3.5 Test Concentration Series

! The test concentration series to be used for definitive toxicity tests l vill be selected following preliminary tests to bracket appropriate concentration ranges. A minimum of five concentrations and diluent vater controls vill be used. It 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 nornally be conducted with tvo replicates. Additional replicates may be used at the client's request.

3.6 Test ConcentratAon Preparation I -

Test concentrations vill be prepared by standard serial dilution (APEA et al.1995) using Class A volumetric glassvare and dilution water.

' Test organisms vill be added to prepared test solutions.

f 3.7 Test 7essels and Test Volume i

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

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

y:.

> 3.8 Test Organism Number A minimum of 10 test organisms vill be used in each test replicate, constituting a minimua number of 20 fish per treatment gorup. Fish vill be randomly assigned to each test replicate.

~

i t

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"2 ETC-T-101 ma Enginearing, Science, F and Technology, Inc.

Effective May 1981 Revision 3. August 1986 Page 2 of 8

.e a A& &h8I$D$%$h$hb806bS$6 M $it!bh %

PROTOCOL ATS-SAF-FM-03

, 3.9 Test Environment The test vessels vill ta maintained at a temperature similar to that at which the fish have been acclimated, but vithin the recommended test range. Test temperature vill not vary more than +2 C f rom the specified acclimation temperature. The tests vill be conducted on a 16-hour light, 8-hour dark photoperiod cycle.

3.10 Analysis of Test Con,centrations for Test Article If required, test solutions replicates may be analy:ed for ehemical analytical verification or 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 actual measured test solution concentrations vill be reported.

3.11 Test obse rva tions Test organisms vill .be observed at a minimum of 24, 48, 72, and 96 hour-after test initiation to determine number of aniemis killed by test article concentrations. The study vill terminate af ter completion of the 4-day observation period. The study nay be extended, however, at the request of the client.

Routine water quality seasurements are performed on the test solutions and diluent water controls according to the following schedules tempera-ture is measured daily from all test chambers dissolved oxygen and pa 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 mik miinity, and total hardness are seasured from the diluent water 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 oxygan concentrations fall belov 40 percent of saturation. Analytical methods vill be conducted according to APEA et al. (1985) and EPA (1979).

Dead fish are removed when observed. At the and of the 96-hour test 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 am (standard langth), patted dry, and veighed to the nearest 0.01 g. The average length and veignt of the test fish are reported, along with their respective ranges and stan-dard deviariaa.

ETC-T-101 3

EA Engineering, Science, and Technology, Inc.

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

l .

. PROTOCOL ATS-SAT-FM-03 4

3.12 Solution Renewal (7 hen Applicable) tj .

Vhen static-rencval testing is required, the test solution is renewed once every 24 or 48 hours5.555556e-4 days <br />0.0133 hours <br />7.936508e-5 weeks <br />1.8264e-5 months <br />. The solution renewal may be performed by transferring the organisms free one test chamber to another or by replacing the test solution. If the test solution replacement method

^

is used, caution must be given not to stress the test organisms while the test chamber is s4 phoned. Usually 75 percent of the old solution is removed and replaced: hovever, the amount is dependent on the sise of the test organisms.

g 3.13 Data Analysis The LC50 values and associated .itattitics vill be derived by the probit i.1 analysis method moving average, rad binomial test methods as described by Stephan (1977). Depending on the nature of the data, other methods any be used ine'Auding the probit approximetion nethod of LJ',chfield and l e Vilcoxon (1949), SAS probit analysis (SAS Institute 1979), or graphical interpolation using the log ::encentration vs. percent lethality as i

described by APEA et al. (1985). The metLods used vill be specified in the report.

4. FINAL REPORT ,

The final report vill be prepared to contain the following information:

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

. Identity of the test articles by name or code number and their strength (i.e., quality / purity) and a description of

[, any pretreatmant

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

[-

. . Test ccacantration serias - used and duration of the assay ,

3 f2 . Mean standard lengths and vet veights of test fish, the respective ranges, and approximate loadings of fish to test volume (g/L)

. Rou. a vater quality characteristics of dilution vater and selected test concentrations 1

1 7 ETC-T-101 l EA Engineering, Science, lt and Technology, Inc.

I; Effecti e May 1981 Revision 3, Augus t 1986 Page 4 of 8 l

l

y PROTOCOL ATS-SAF-FM-03 P . . Any unforeseen circumstances that may have affected the quality or integrity of the study

. Signature of the project coordinator, program manager, and quality control of ficer as authorization of the report

. Location of all archived data and the original copy of the final report at EA Items of data to be included in the report consist of t experimental design and test performances ef f ects on general appearance of test organismst morbidit'i and aortality; tabular presentation and appropriate statistical evaluation of vater quality cha acteristics, and survival

- da ta where necessary.

5. QUALITY ASSURANCE

$.1 Amendments to Protocol Amendments to the authorized protocol instigated by EA or by the client vill be made only after proper authori:ation. Such authori:ation is achieved by completion of an Amendment to Protocol form by EA and the client.

5.2 Standard operating Procedures Unless othervise specified r,11 procedures mentioned' in the protocol are subject to detailed Standard Operating Procedures (SOPS) vtie.h are contained in the 50P manuals of the participating departments. These SOPS and protocols follov the U.S. EPA's Good Laboratory Practice Standards (GLPs) (EPA 1983).

5.3 Reference Toxicant

A reference toxicant bicassay, utilizing sodium dodecylsulfate (SDS) or other appropriate reference toxicant, vill be used as an interal quality check of the sensitivity of ee.ch batch of test orgsnisas. Testing vill be prior to or concurrent with the test or following the acclimation period for newly acquired organisms. The results of each test vill be compared with historical toxicological information of reference toxicant tests performed at EA on the species, to determine if the results are within acceptable limits using the control charts outlined in Peltier and

. Veber (1985).

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

Et:ww;iaa May 1981 F.avision 3 Augu.t 1086 Page 5 : 3 Ambb ti h (.h I

• l .

PROTOCOL ATS-SAF-FM-03 j

- 5.4 Ouality Assurance Evaluation 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.

5.5 Inspection by Regulatory Authorities In the event of an inspection of EA by an outside authority during the

' course of the study, the client vill be consulted before inspectors are permitted access to any of the project records or the experiuental areas.

5.6 Archives Copies of project-specific're 3rds 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 retained at EA for 5 years. Primary data include laboratory data sheets, records, memo-randa, notes, photographs, microfilm, and computer printouts that are a result of the original observations and activities of the study an6 vhich are necessary for the reconstruction and evaluation of the study report.

5.7 Location This study vill be conducted by the Aquatic Toxicology Services Department of the Scientific Operations Division of EA Engineering, Science, and Technology, Inc. at the Mid-Atlantic Kegional Offices, Sparks, Maryland.

6. FATHEAD MINHOU ACVTE TOI! CITY TEST SPECITICATIONS 6.1 Basic References American Public Health Association et al. 1985. Standari Methods for Examination of Vater and Vastevater,16th edition. APEA, Vashington, D.C.. 1,268 pp. .

American Society for Testing and Materials ( ASTM). 1980. Standard Practice for Conducting Acute Tests with Fishes, Hacroinvertebrates, and Amphibians. ASTM Designation: E729-80, Philadelphia, Pa.

pp. 285-309.

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

Effective May 1981 Revision 3, August 1986 Page 6 af 8 ,

l a t ]

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

Peltier, k.H. and C.I. Veber. 1985. Methods for Measuring the Acute Toxicity of Effluents to Freshvater and Marine Organisms. EPA-600/

4-d5-013. U.S. Environmental Protection Agency Cincinnati, Ohio.

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

6.2 Test Specifications Test organism: Fathead minnov (Pimephales promelas)

Temperatures 20:2 C Fisa aget 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 fish Loading ration In general, test containers may not contain greater than 0.8 g/L biomass Aeration: None, unless dissolved orygen 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 thereaf ter Light quality: Vide-spectrum fluorescent light Light intensi ty: 50-100 f.c.

Photoperiodt 16-hour light, 8-hour dark Dilution vater: Dechlorinated municipal tap vater, reconstituted fresh water, or appropriate receiving vater Test containers: 19- oc 38-L all glass rauarta Test volume 15 or 30 L ETC-t-101 EA Engineering, Science, and Technology, Inc.

• Effective May 1981 Revision 3, August 1986 Page 7 of 9 i

Ohh AhwhkI b.

PROTOCOL ATS-SAF-FM-03 No. of concut.trations: Minimum of five test concentrations

[e[ and a control in duplicate series

• Test animals per container: 10 (if loading is exceeded, core replicates or larger aquaria nay be used)

Feeding regime: 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 is terminated 48 hours5.555556e-4 days <br />0.0133 hours <br />7.936508e-5 weeks <br />1.8264e-5 months <br /> betore' test initiation in the lot from which organisms are obtained va

/

Test type and duration: Rangefinding--24-96 hours

. Definitive--96 hocrs Reference toxicant--24 hours End point: Lethality--defined as cessation of

. cpercular movement and inability to respond to external stimuli (gentle prod /f ag with glass red) 3 T*

Th3 x

ETC-T-101 EA Engineering,- Science, and Tec-nolor . Inc.

Effective 'a/ .41 Revision 3, August 1986 Page 8 of 8 l

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- Chain-of-Custody Record - FOR OmCE USE ONI.Y EA ENGINEERING. Speces to be tese 3:

SCIENCE, ANC TECHNCLCGY, INC- d - Q.masca temau an.

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ATTACERENT III REPORT 00ALITT ASSURANCE RECORD 4

4

REPORT QUALITT ASSURANCZ RECORD CLIENT __ t 6 m , __ b, . EA REPORT NO. [

.1

, PROJECT NUMBER " cec 7 /o TYPE ANALYSIS Mn m f 7:.

AUTHOR 2, '. , , -, , a N .$e_ L w_ TEST ORGANISMS.3 m..cr,c, k e ~ , uf , e

./

REPORT CHECKLIST OA/0C ITEM REVIEVER DATE

1. Samples collected, transported, and received according to study plan _.

)

( j, requirements. G d. u . -c_. Y . -d(

'i M*7/U.

./

?' 2. Samples prepared and crocessed accord- [ -

ing to study plan requirements. ( -ft_. ._ps_ ,u et b.'

t'- c. , /?Mh c

~

3. Data collected using calibrated equipment.  ? e_ wm o _ b/ M._d'. "i/J >/_,

/ -

I. . Calculations checked:

- Hand calculatior's checked Nfu d.bl[ _

~

9 /3e/ar /

- Documented and verified statistical ,, r f 4 procedure used, f bu a. mo / j _ / M .._ ~ / i.'.re

5. Data input / statistical analyses complete and correct. 1,/G. 9 b//9/

6. Reported results and facts checked g i i against original sources. , M. [/. dh 7 [3 #[#/

7. Data presented in figures and tables /

correct and in agreement with text. [/ .0v., 7b [#/

8. Results envieved for compliance with r

f, f ,

study plan requirements. (, .O , . 4~ a tr . e -b Y J ' * <, /

il AUTHOR DATE

9. Commentary reviewed and resolved. ae > = -o. k , e [. / C '

7

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

PR04E " hANAGER DATE l0. % 30 & lzn/

Q.C. OFFICER JATE

&)/,

SE24105r JEC'r!NICAL

' blw(V 1CIIEVER

/AOMfTf CATE

/ /

1

PY-CEI/0 EPA-0143L ATTACilHENT 2 l

Research Required per Section 4.a. [

Conduct research to determine if the 11-130, the detoxification material or a (

combination of the two have the potential to cause chronic effects in the  ?,

receiving vater at representative concentrations.

These reports vere compiled by Calgon Corporation, d 1

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_ - - - - - _ _ _ _ _ _ - _ - _ _ _ _ - _ _ _ _ _ _ _ _ - - - - _ _ _ _ _ _ - - _ _ - _ _ _ _ _ - _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - _ _ _ _ _ . _ - - _ _ _ _ _ _ _ . _ _ _ _ - _ _ _ - _ _ _ _ _ _ _ _ _ _ . _ _ _ _ _ . _ _ _ _ _ .