ML20076J858

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

ML20076J858
Person / Time
Site:	Perry
Issue date:	10/17/1994
From:	Mcculloch W, Sohn V EA ENGINEERING, SCIENCE & TECHNOLOGY, INC.
To:
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ML20076J856	List: ML20076J853 ML20076J858
References
NUDOCS 9410260398
Download: ML20076J858 (39)

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EA ENGINEERING, SCIENCE AND TEGINOLOGY, INC EA Project 70003.10 RESULTS OF ACUTE TOXICITY TESTS -

WITH Daphnia magna AND Pimephales promelas ON SAMPLES FROM CALGON CORPORATION i

Preparedfor:

Calgon Cogoration Elyria, Ohio Prepared by:

EA Mid-Atlantic EA Engineering, Science, and Technology, Inc.

15 Loveton Circle Sparks, Maryland 21152 J_

I ? O c "L s d w r 19 9 l A.

CL_

Date VirginipA. Sohn Project Manager DM oc Date Wayne L. MgCulloer (

Senior Scientist October 1994 Report Nandrer 1755 9410260398 941020 hDR ADOCK 05000440 PDR

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l. INTRODUCTION 1

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At the request of Calgon Corporation, EA Engineering, Science, and Technology performed acute toxicity screening tests with Daphnia magna (water flea) and Pimephales promelas

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(fathead minnow) on three samples from the Perry Nuclear Power Plant. Each species was j

l exposed to 100 percent samples of (1) influent, (2) effluent prior to zebra mussel treatment, i

and (3) effluent during treatment.

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Sample collection and receipt data, together with initial water quality data, are reported in Table 1. Test results, test organism source and age, reference toxicant data, and water quality measurements are summarized on pages 3-6. Test protocols, copies of raw data sheets, and the Report Quality Assurance Record are included as Attachments I, II, and III, i

respectively.

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i RESULTS Results of the acute screening assays performed on samples from Perry Nuclear Power Plant are reported on pages 4 and 6. There was 100 percent survival of Daphnia magna (page 4) after 48 hours5.555556e-4 days <br />0.0133 hours <br />7.936508e-5 weeks <br />1.8264e-5 months <br /> of exposure to the influent, the efnuent collected prior to treatment, and the laboratory water control (page 4). There was 85 percent survival in the effluent composite I

collected during the zebra mussel treatment. In the Pimephales promelas test (page 6), there

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was 100 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 influent, the effluent collected l

prior to treatment and to the effluent composite collected during treatment. There was 95 percent survival of laboratory water control organisms.

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SUMMARY

OF SAMPLE / TEST INFORMATION Test: 48-hour Daphnia magna Static Acute Toxicity Test Client Name: Calgon Corporation Test Procedure: EA Protocol ATS-SAI-DS-04 Acute 48-hour assay with Daphnia Sample

Description:

Influent; effluent prior to treatment; effluent during treatment EA Accession Numbers: AT4-773, AT4-774, AT4-775 EA QC Test Number: SA-09-24-94-983 Test initiation time and date: 1350,24 September 1994 Test completion time and date: 1350,26 September 1994 Control Water: Dechlorinated tap water Organism Imt Information Lot Number: -Not Applicable Source: EA's Culture Facility, Sparks, Maryland Age: <24 hours j

Reference Toxicant (SDS) Test Information Reference Toxicant: Sodium dodecyl sulfate EA QC Test Number: RT-09-01-94-875 Dilution Water: Dechlorinated tap water Results:

48-hour LC50: 14.1 mg/L SDS l

Laboratory control chart acceptability range for 48-hour LC50:

l 4.0 - 25.0 mg/L SDS l

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SUMMARY

OF SAMPLE / TEST INFORMATION (Cont.)

Test Organism: D. magna EA QC Test Number: SA-09-24-94-983 Test Material l

(100 percent concentrafiqn)

Percent Survival at 48 Hours Laboratory Contrel 100 Influent 100 Effluent prior to treatment 100 Effluent during treatment 85 Selected Water Ouality Rance t

Temperature (*C):

20.3 - 20.9 pH:

6.8 - 8.4 Dissolved oxygen (mg/L):

8.1 - 8.9 l

Conductivity ( S"):

211 - 330 I

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SUMMARY

OF SAMPLE / TEST INFORMATION Test: 96-hour Pimephales promelas Static Acute Toxicity Test l

Client Name: Calgon Corporation l

Test Procedure: EA Protocol ATS-SAF-FM-04 Acute assay with fathead minnows (Pimephales promelas)

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l Sample

Description:

Influent; cffluent prior to treatment; effluent during treatment l

EA Accession Numbers: AT4-773, AT4-774, AT4-775 t

EA QC Test Number: SA-09-24-94-984 Test initiation time and date: 1420,24 September 1994 Test completion time and date: 1425,28 September 1994 Control Water: Dechlorinated tap water Oreanism Lot Information lot Number: FH 4-9/18-19 Source: EA's Culture Facility, Sparks, Maryalnd i

Age: 5-6 days (hatched within a 24-hour window)

Reference Toxicant (CdCl LTest Information 2

Reference Toxicant: Cadmium chloride EA QC Test Number: RT-09-28-94-996 Dilution Water: Dechlorinated tap water Results:

48-hour LC50: 31.4 pg/L Cd Laboratory control chart acceptability range for 48-hour LC50:

13.0 - 50.7 g/L Cd 5

SUMMARY

OF SAMPLE / TEST INFORMATION (Cont.)

Test Organism: P. pmmelas l

EA QC Test Number: SA-09-24-94-984 Test Material (100 percent concentration)

Percent Survival at 96 Hours Laboratory Control 95 l

Innuent 100 l

Effluent prior to treatment 100 l

Effluent during treatment 100 Selected Water Ouality Ranee Temperature ( C):

19.5 - 21.4(*

l pH:

6.8 - 8.1*

l Dissolved oxygen (mg/L):

8.1 - 8.8 Conductivity (pS ):

223 - 343 l

(a)

Temperature of test solutions was greater than target range of 20 1*C at 48 hours5.555556e-4 days <br />0.0133 hours <br />7.936508e-5 weeks <br />1.8264e-5 months <br />.

Test chambers were relocated in laboratory to correct for the temperature deviation.

(b) pH of test solutions was not measured at 48 hours5.555556e-4 days <br />0.0133 hours <br />7.936508e-5 weeks <br />1.8264e-5 months <br />.

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TABLE 1 SAMPLE COLLECTION AND INITIAL WATER QUALITY DATA FOR SAMPLES FROM CALGON CORPORATION Sample EA Accession Type of Sample Sample Alkalinity Hardness Description Number Sample Collection Receipt (me/L CACO _d (me/L CaCOd Influent AT4-774 Grab 0900,22 Sep 94 0925,24 Sep 94 82 128 Effluent prior AT4-773 Grab 0900,22 Sep 94 0925,24 Sep 94 92 132 to treatment Effluent AT4-775 Composite 1030,22 Sep 94 to 0925,24 Sep 94 90 140 during treatment 2330,22 Sep 94

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ATTACHMENT I PROTOCOLS

I PROTOCOL ATS-SAI-DS-04 i

ACUTE ASSAY WITH Daphnia sp.

1. TEST OBJECIT'.'E 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.

2. TEST ARTICLE 2.1 Description / Identification Unless otherwise specified, the test material is supplied by the client. Adequate chemical specifications with special reference to hazardous properties and storage conditions is also i

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, l.

stability, and composition or other characteristics which define the test article are on file with j

the client.

3. EXPERIMENTAL DESIGN 3.1 Test Oreanisms A species of Daphnia (water fleas), as determined by project needs, is the test organism.

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

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

Effective May 1981 Revision 4, April 1992 Page 1 of 9 l

PROTOCOL ATS-SAI-DS-04 I

3.3 Culturine and Holdine Conditions Daphnia cultures are maintained at 20i2 C and a 16-hour light, S-hour dark photoperiod cycle in an environmentally controlled laboratory. Cultures are maintained in 18.9-L all 1

glass aquaria or other appropriate container and are fed algae (Selenastrum capricornutum) and a trout chow / yeast / cereal leaves suspension in the manner described in Weber 1991.

New cultures are initiated on a routine basis to ensure healthy, productive populations.

Organisms from cultures producing ephippia are not used for toxicity tests. Certain regulatory or project specific objectives may require organism acclimation to the dilution water when it is different from the holding / culture water.

3.4 Age of Test Organisms 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 initiated. On the day of the test, neonates are segregated from the parent organisms.

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 detergents, and hexane-soluble organic materials. This water source has proven safe for aquatic organism toxicity testing as evidenced by maintenance of the multigeneration Daphnia and Pimephales promelas cultures, with no evident loss of fecundity. If requested, reconstituted fresh water or an appropriate receiving water can be used as the dilution water.

3.6 Test Concentration Series 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).

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

Effective May 1981 Revision 4, April 1992 Page 2 of 9 1

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PROTOCOL ATS-SAI-DS-04 t

j 3.7 Test Concentration Preparation Test concentrations are prepared with Class A glassware.

3.8 Test Vessels and Test Volume Test vessels are 400-ml glass beakers or 200-ml culture bowls; the final test volume of each replicate is 200 ml. Depending on the study requirements, other test vessels and test volumes may be used.

3.9 Test O'reanism Number Tests are conducted using two replicates per concentration, with ten organisms per container.

Neonates are randomly assigned to each replicate test container. More replicates can be added, if appropriate.

l 3.10 Test Environment The test vessels are maintained at 2011 C or 25il C in an environmentally controlled l

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

l 3.11 Analysis of Test Concentrations for Test Article If required, test solutions may be analyzed for verification of chemical concentrations. The analytical method and number of analyses are determined after consultation with the client.

When chemical analyses are necessary, both nominal and actual measured test solution i

concentrations are reported.

3.12 Test Observations 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.

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

Effective May 1981 Revision 4, April 1992 Page 3 of 9

PROTOCOL ATS-SAI-DS-04 Each sample received is analyzed for temperature, conductivity, alkalinity, and hardness.

Measurements of water quality taken daily include dissolved oxygen, pH, temperature and conductivity from a minimum of one replicate of every concentration. Aliquots of effluent and receiving water may be gently aerated (100 bubbles / min) if dissolved oxygen is less than 4 mg/L or greater than 105% saturation. After test initiation, if the dissolved oxygen in any test chamber is less than 4 mg/L, all test chambers are gently aerated or other corrective action is taken. Analytical determinations are conducted according to APHA et al. (1992) and EPA (1979).

3.13 Solution Renewal (When Anolicable)

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 using a wide bore pipet or glass tube. The number of live organisms is recorded. 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 discarded.

3.14 Data Analysis The. LC50 or EC50 values and associated statistics are calculated using the probit, moving average, and binomial methods as described 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 Litchfield and Wilcoxon (1949), SAS probit analysis (SAS Institute 1985) or graphical interpolation using the log concentration vs. percent mortality as described by APHA et al. (1989). The methods used are specified in the final report.

3.15 Test Accentability An individual test may be conditionally acceptable if temperature, DO, and other specified conditions fall outside specifications, depending on the degree of the departure and the objectives of the tests.

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

Effective May 1981 Page 4 of 9 Revision 4, April 1992

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PROTOCOL ATS-SAI-DS-04

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

Objectives and procedures stated in the approved protocol, including any changes made to the original protocol Identity of the test article (s) by name or code number and the strength (in quality / purity and a description of any pretreatment) s I

S9mc of the dilution water, its chemical characteristics, and a description i

of ar.y petreatment I

Test concentration series used and duration of the assay a

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

Any unforeseen circumstances that may have affected the quality or i

i integrity of the study I

Signature of the project manager, senior technical reviewer, and quality control officer, authorizing release of the report i

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 organisms (if applicable); morbidity and mortality; presentation of water quality characteristics; and survival data.

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

Effective May 1981 Revision 4, April 1992 Page 5 of 9

I PROTOCOL ATS-SAI-DS-04

5. QUALITY ASSURANCE l

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

i 5.2 Standard Ooerating Procedures L

Unless otherwise specified, all procedures specified in the protocol are subject to detailed Standard Operating Procedures (SOPS) which are contained in the SOP manuals of the l

participating departments. These SOPS and protocols generally follow the types of requirements as outlined in the U.S. EPA's Good Laboratory Practice Standards (GLPs)

(EPA 1989).

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li 5.3 Reference Toxicant

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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 on organisms which are cultured in-house. The results of each test are compared with historical, species-specific toxicological t

information from reference toxicant tests performed at EA, to determine if the results are l

within acceptable limits. Limits are established using the control charts outlined in Weber (1991).

5.4 Ouality Assurance Evaluation I'

Studies conducted under this protocol may be subject to internal audit by EA's 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

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

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

Effective May 1981 Revision 4, April 1992 Page 6 of 9

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PROTOCOL ATS-SAI-DS-04 5.5 Inspection by Reculatory Authorities In the event of an inspection of EA by an outside authority during the course of 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.

5.6 Archives Copies of project-specific records shall be transferred to the client promptly after the project is completed or as negotiated and budgeted. Original primary data are retained at EA for

. - 5 years. Pdmary data include chain-of-custody records, laboratory data sheets, records, memoranda, 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.

5.7 Location Studies are conducted at the Aquatic Toxicology Laboratory of EA Engineering, Science, and Technology, Inc. at the Loveton Office in Sparks, Maryland.

6. SPECIFICATIONS OF THE Daphnia ACUTE TOXICITY TEST i

6.1 Basic References l

American Public Health Association (APHA), American Waterworks Association, Water Environment Federation. 1992. Standard Methods for Examination of Water and Wastewater,18th edition or most recent version. APHA, Washington, D.C.

EA Manual ATS-102.1992. Aquatic Toxicology Studies, Quality Control and Standard Operating Procedures Manual. Fourth Revision. Internal document prepared by Aquatic Toxicology Services, EA Engineering, Science, and Technology, Inc.

Litchfield, J.T., Jr. and F. Wilcoxon. 1949. A simplified method of evaluating dose-effect experiments. J. Pharm. Exp. Ther. 96:99-113.

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

Effective May 1981 Revision 4, April 1992 Page 7 of 9

PROTOCOL ATS-SAI-DS-04 SAS Institute Inc.1985. SAS* User's Guide: Basics, Version 5 Edition. Cary, NC: SAS Institute Inc. 1290 pp.

Stephan, C.E. 1977. Methods for calculating an LC50, in Aquatic. Toxicology and Hazard Evaluation (F.L. Mayer and J.: Mamelink, eds.), pp. 65-84. ASTM STP 634.

American Society for Testing ano Materials, Philadelphia.

U.S. Environmental Protection Agency. 1979. Methods for Chemical Analysis of Water and Wastes. EPA-600/4-79-020. U.S. EPA, Washington.

Weber, C.I.1991. Editor. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms. Fourth Edition.

! 7 EPA /600/4-90/027. U.S. Environmental Protection Agency. Environmental Monitoring Systems l2boratory, Cincinnati, Ohio.

U.S. EPA. 1989. Toxic Substances Control Act (TSCA); Good l2boratory Practice Standards. Title 40 CFR Part 792. Erfl. Regist. 54(158):34034-34074.

1 6.2 Test Snecifications Test organism:

Daphnia magna or D. pula; species to be specified in the study plan and final report i

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:

20il C or 25il C Light quality:

Wide-spectrum fluorescent light Light intensity:

50-100 f.c.

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Photoperiod:

16-hour light, 8-hour dark l

Aeration:

None, unless dissolved oxygen falls below 4 mg/L i

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

Effective May 1981 Revision 4, April 1992 Page 8 of 9

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l PROTOCOL ATS-SAI-DS-04 Dilution water:

Dechlorinated municipal water, reconstituted water, or appropriate receiving water l

l Test container:

400-ml beaker or 200 mi culture bowl Test volume:

200 ml per replicate No. of concentrations:

Minimum of five test concentrations and a control I

No. o'f 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 Mortality-defined as cessation of all movement for a period of at least 5 seconds even when the test container is tapped or rotated, or the organism is gently prodded with glass rod l

Test acceptability:

90% or greater survival in the control solution.

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

Effective May 1981 Revision 4, April 1992 P::ge 9 of 9

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4 PROTOCOL ATS-SAF-FM-04 ACUTE ASSAY WITH FATHEAD MINNOWS (Pimephales promelas)

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

2. TEST ARTICLE l

2.1 Descrintion/ Identification l

l Unless otherwise specified, the test material is supplied by the client. Adequate chemical i

specifications with special reference to hazardous properties and storage conditions are 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. EXPERIMENTAL DESIGN 3.1 Test Organisms The test species is the fathead minnow, Pimephales promelas.

3.2 Source P. promelas used for toxicity tests are usually obtained from stock cultures maintained at j

EA's Aquatic Toxicology Culturing Facility. However, organisms may be obtained from a scientific organism vendor, if necessary.

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

Effective May 1981 Revision 4, April 1992 Page1 of11

PROTOCOL ATS-SAF-FM-04 3.3 Culturine and Holdine Conditions P. promelas stock are maintained at 20i2 C and a 16-hour light, 8-hour dark photoperiod cycle in an environmentally controlled laboratory. Test organisms are maintained 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 appropriate size containing dechlorinated tap water at the temperature (t2 C) of the water in which the organisms were shipped. All fish stocks are examined regularly. 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 or Artemia nauplii a minimum of once daily. Certain regulatory or project specific objectives may require organism acclimation to the dilution water when it is different from the holding / culture water.

3.4 Age of Test Organisms at Test 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 1-14 days old (with a 24-hour range in age) upon test initiation.

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 detergents and hexane-soluble organic materials. This water source has proven safe for aquatic organism toxicity testing, as evidenced by maintenance of the multigeneration Daphnia and fathead minnow cultures, with no evident loss of fecundity. If requested, reconstituted fresh water or other dilution water may be used.

ATS-102 EA Engineering, Science, i

and Technology, Inc.

Effective May 1981 Revision 4, April 1992 Page 2 of 11

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PROTOCOL ATS-SAF-FM-04 3.6 Test Concentration Series The test concentration seU,s 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).

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i 3.7 Test Concentration Prenaration Test concentrations are prepared with Class A glassware.

3.8 Test Vessels and Test Volume Test vessels are 2 L 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 Oreanism Number 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.

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1 3.10 Test Environment The test vessels are maintained at 20il C or 25 1 C (unless a different project-specific temperature is required) in an environmentally controlled laboratory with a 16-hour light, 8-hour dark photoperiod. Temperature within the environmental room is monitored continuously using temperature recorders.

3.11 Analysis of Test Concentrations for Test Article If required, test solutions may be analyzed for verification of chemical concentrations. The analytical method and number of analyses are determined after consultation with the client.

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

Effective May 1981 Revision 4, April i992 Page 3 of 11

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PROTOCOL ATS-SAF-FM-04 When chemical analyses are necessary, both nominal and actual measured test concentrations are reported.

3.12 Test Observations Each test day, test organisms are observed to record the number of surviving organisms.

Dead organisms are removed when observed. The study terminates after completion of the observation period (24 to 96 hours0.00111 days <br />0.0267 hours <br />1.587302e-4 weeks <br />3.6528e-5 months <br />). The study may be extended, however, at the request of l

the client.

Each sample received is analyzed for temperature, conductivity, alkalinity, and hardness.

Measurements of water quality taken daily include dissolved oxygen, pH, temperature, and l

conductivity from a minimum of one replicate of every concentration. Aliquots of effluent l

and receiving water may be gently aerated (100 bubbles / min) if dissolved oxygen is less than 4 mg/L or greater than 105% saturation. After test initiation, if the dissolved oxygen in any i

test chamber is less than 4 mg/L, all test chambers are gently aerated or other corrective l

action is taken. Analytical determinations are conducted according to APHA et al. (1992) and EPA (1979).

At the end of the test period, a minimum of 30 surviving fish are removed from the test vessels, placed in labeled containers, and frozen for later measurements. The fish are subsequently thawed, measured to the nearest mm (standard length), 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. For fish between 1-14 days old and within a 24-hour age range, weights and lengths usually will not be measured.

3.13 Solution Renewal (When Aeolicable)

When static-renewal testing is required, the test solution is renewed daily. New solutions are prepared on the day of renewal and dispensed according to Section 3.8. After the new solutions have reached test temperature and water quality measurements (temperature, pH, dissolved oxygen, and conductivity) are completed, the solution renewal may be performed by transferring the organisms from one test chamber to another or by replacing the test ATS-102 EA Engineering, Science, and Technology, Inc.

Effective May 1981 Revision 4, April 1992 Page 4 of 11

PROTOCOL ATS-SAF-FM-04 4

solution. If the test solution replacement method is used, caution must be given not to stress the test organisms while the test chamber is siphoned. Usually 75 percent of the old solution l

is removed and replaced; however, the amount is dependent on the size of the test organisms.

3.14 Data Analysis The LC50 and/or EC50 values and associated statistics are calculated using the probit, j

moving average, and binomial methods as described 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 Litchfield and Wilcoxon (1949), SAS probit analysis (SAS Institute 1985), or graphical interpolation using the log concentration vs.

percent mortality and/or percent affected as described by APHA et al. (1992). The methods used are specified in the final report.

i 3.15 Test Anolicability An individual test may be conditionally acceptable if temperature, DO, and other specified conditions fall outside specifications, depending on the degree of the departure and the objectives of the tests.

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

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

• Identity of the test article (s) by name or code number and their 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 ATS-102 EA Engineering, Science, and Technology, Inc.

Effective May 1981 Revision 4, April 1992 Page 5 of 11

PROTOCOL ATS-SAF-FM-04

• Test concentration series used and duration of the assay

• Mean standard lengths and wet weights of test fish, the respective standard deviations, and approximate biomass loadings (g fish /L of test solution), if applicable

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

• Any unforeseen circumstances that may have affected the quality or integrity of the study

• Signature of the project 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 organisms (if applicable); morbidity and mortality; presentation 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 made only after proper authorization. Such authorization is achieved by completion of the Amendment to Protocol Form by EA after consultation with the client.

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

Effective May 1981 Revision 4, April 1992 Page 6 of 11

PROTOCOL ATS-SAF-FM-04 5.2 Standard Operatine Procedures Unless otherwise specified, all procedures given in the protocol are subject to detailed Standard Operating Procedures (SOPS) which are contained in the SOP manuals of the participating departments. These SOPS and protocols generally follow the types of requirements outlined in the U.S. EPA's Good Laboratory Practice Standards (GLPs)

(EPA 1989).

5.3 Reference Toxicant A reference toxicant test, utilizing sodium dodecyl sulfate (SDS), cadmium chloride, or another appiopriate chemical is used as an internal quality check of the sensitivity of the test organisms. Testing is conducted at least once monthly on organisms that are cultured in-house. The results of each test are compared 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 outlined in Weber (1991).

l 5.4 Ouality Assurance Evaluation Studies conducted under this protocol may be subject to internal audit by EA's Quality Assurance Unit. A quality control of5cer is responsible for monitoring each study to assure l

the client that the facilities, equipment, personnel, methods, practices, records, and controls l

l are in conformance with EA's QC program and, if applicable, EPA's GLPs.

l 5.5 Insoection by Regulatory Authorities l

In the event of an inspection of EA by an outside authority during the course of 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.

l l

l ATS-102 l

EA Engineering, Science, and Technology, Inc.

Effective May 1981 Revision 4, April 1992 Page 7 of 11 l

s

PROTOCOL ATS-SAF-FM-04 5.6 Archives Copies of project-specific records shall be transferred to the client promptly after the project is completed or as negotiated and budgeted. Original primary data are retained at EA for 5 years. Primary data include chain-of-custody records, laboratory data sheets, records, memoranda, 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.

5.7 Location l

Studies are' conducted at the Aquatic Toxicology Laboratory of EA Engineering, Science, and l

Technology, Inc. at the Loveton Office in Sparks, Maryland.

6. SPECIFICATIONS OF THE FATHEAD MINNOW ACUTE TOXICITY TEST 6.1 Basic References t

American Public Health Association (APHA) American Water Works Association, Water Environment Federation. 1992. Standard Methods for Examination of Water and Wastewater,18th or most recent version. APHA, Washington, D.C.

American Society for Testing and Materials (ASTM). 1991. Standard Practice for Conducting Acute Tests with Fishes, Macroinvertebrates, and Amphibians. ASTM I

Designation: E729-80, Philadelphia, Pa.

EA Manual ATS-102.1992. Aquatic Toxicology Studies, Quality Control and Standard Operating Procedures Manual. Fourth Revision. Internal document prepared by Aquatic Toxicology Services, EA Engineering, Science, and Technology, Inc.

Litchfield, J.T., Jr. and F. Wilcoxon. 1949. A simplified method of evaluating dose-effect experiments. J. Pharm. Exp. Ther. 96:99-113.

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

Effective May 1981 Revision 4, April 1992 Page 8 of 11 j

~

PROTOCOL ATS-SAF-FM-04 SAS Institute Inc.1985. SAS* User's Guide: Basics, Version 5 Edition. Cary, NC: SAS Institute Inc. 1290 pp.

Stephan, C.E. 1977. Methods for calculating an LC50, in Aquatic Toxicblogy and Hazard Evaluation (F.L. Mayer and J.L. Hamelink, eds.), pp. 65-84. ASTM STP 634.

American Society for Testing and Materials, Philadelphia.

U.S. Environmental Protection Agency. 1979. Methods for Chemical Analysis of Water and Wastes. EPA-600/4-79-020. U.S. EPA, Washington.

U.S. EPA. 1989. Toxic Substances Control Act (TSCA); Good Laboratory Practice Standards. Title 40 CFR Part 792. Ecd. Regist. 54.

Weber, C.I.1991. Editor. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms. Fourth Edition.

EPA /600/4-90/027. U.S. Environmental Protection Agency. Environmental Monitoring Systems Laboratory, Cincinnati, Ohio.

6.2 Test Soecifications Test organism:

Fathead minnow (Pimephales promelas)

Temperature:

20i1 C or 25 iC Fish age:

Dependent on test guidelines being followed, but in general,1-14 days with a 24-hour range in age 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 0.65 g/L biomass ATS-102 EA Engineering, Science, and Technology, Inc.

Effective May 1981 Revision 4, April 1992 Page 9 of 11

PROTOCOL ATS-SAF-FM-04 Aeration:

None, unless dissolved oxygen falls below 4 mg/L Light quality:

Wide-spectrum fluorescent light l

Light intensity:

50-100 f.c.

Photoperiod:

16-hour light, 8-hour dark Dilution water:

Dechlorinated municipal tap water, reconstituted fresh water, or appropriate receiving water i

Test containers:

2 L glass culture bowl Test volume:

1 Ureplicate l

l No. of concentrations:

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

2 l

Test animals per container:

10 (if loading is exceeded, more replicates or larger test containers may be used) l 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.

Test type and duration:

Rangefinding--24-96 hours Definitive--24-96 hours l

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

Effective May 1981 Revision 4, April 1992 Page 10 of 11

PROTOCOL ATS-SAF-FM-04 Endpoints:

Mortality and/or Immobilization Immobilization--defined as cessation of movement except for minor activity of appendages Mortality--defined as cessation of opercular movement and inability to respond to external stimuli (gentle prodding with glass rod)

Test acceptability:

90 percent or greater survival in the control solution 1

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

Effective May 1981 Revision 4, April 1992 Page 1I of1I o,

l ATTACHMENT II DATA SHEETS l

l I

l

I AQUATIC ORGANISMS ACUTE TOXICITY TEST DATA SHEET NIN /353 T ST:

Flow-through, Renewal TEST ORGANISM:

Beginning (Time, Date):

fb b

6'dk 7lb (L Ending (Timo, Dato): (/-N-9 V /3';T

{ilffMkT{est Container:

Project No.:

^/ " /Y2td Common Namo:

Test ArticleQ!

diebt

.NA /hc en/ /LclerTest Volume:

C'

_ 1 Scientific Name: !O.m#0!t r1 Adults Isolated (Time. Date):'l.? b CFI Do7/

OC Test NoY9M -O@hh/MTest Duration: _4/ rdM 3 Lot No.:

Idb d

Organisms Pulled (Time, Date): #7 9'l-G' f / O/ O-(

CA Acclimation: 0 f A or Age Gd /1,L3 Sampio No.: O Tcl 9 75'77'I A Tc o 1p b5l Source:

/

Cu!!uro Water T/S. @ Transf er (1 ) fd t-2 Dilution No.: (bf1ICC OTHER ALL TESTS Dissolved Oxygen

@ductivity ( S-Test Number of

'9" or %

No.

0 24 48 72 96 0

24 48 72 96 0

24 48 72 96 0

24 48 72 96 0

24 48 72 96 0

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a AQUATIC TOXICOLOGY BENCH SHEt:.:

SET UP (SMALL VOLUMES) 1b Ib Project No.:

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3/10/93 l

AQUATIC ORGANISMS ACUTE TOXICITY TEST DATA SHEET 9.@/9I /42d ib M

b Renewat TEST ORGANISM:

Beginning (Time, Date):

TEgTQ ? Flow-thro Project No_:

M[

O._ LOG Common Name: IAE/tfild-Ending (Time, Date):

9116 /et hit s' Test Articio:S NIl7NIW'f[stContainer:

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8/l Acchmation 9lA or Age f k/4.g1 Ddution No.: dc~t Itf C Culture Water T/S. @ Transf er (12) /7 3 <-

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SET UP (SMALL VOLUMES)

" b6b I Project No.:

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' \\ *# ' ' ' ' ' ' "

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1 i

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l l

ATS-MS2 3/10/93

Chain-of-Custody Record e,

l EA Engineering, Science, i

and. Technology A

./f6%,.

FOR OFFICE USE ONLY

_=:-

EA A.quatic Species to be tested:

{gg "'jUdes I

F ' *quatic Toxicology Laboratones b

l aveton Circle D. magna Menidia sp.

Sparks (Baltimore), Maryland 21152 T~ ~y D. poler P. pugio Telephone: (410) 771-4950 C. dubia C. vanegatus i

?

Fax: (41 771 4204 l

P. prometas M. bahia hdltrf Other Other l

Client:

yN Project No.:

f I

A = Acute C = Chronic B = Bioaccumulation I

NPDES Number:

Client Purchase Or er Number:

State / City / County Collected:

I

/

//

l PLEASE READ SAMPLING INSTRUCTIONS ON BA CK OFFORM Accession Collection Sample Description Number Start End (including Site, Station Number / Volume

. office use only).

Grab Composite Date/ Time Date/ Time Number, and Outf all Number) of Container

(

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Date/ Time Received By:

Date/ Time 1

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Samplel's Printed Name:

Title:

Relinquished By:

Date/ Time s

Relinquished By:

Date/ Time

~ Received B j

' Date/ Time Y.

CN

• /

M

%g.

Was Sample Chilled During Collection

/No Sample Shipped By: (circle)

Comments:

Fed.Ex.

Puro.

UPS' Airborne 3

s Other:

Air Bill Number.

Sample Temperature at Receipt:

s C

/

gr4-7 7 3 - t oc Pink--Clent/Campler Whde---Report Producion Yellow--Laborstory gy,y7y g

E A 0534 F&B 9/15/92 p

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i l

l l

ATTACHMENT III REPORT QUALITY ASSURANCE RFCORD i

l l

l

l

',.t p

REPORT QUALITY ASSURANCE RECORD Client: Ale ~ b a m tetm~

EA Report No.:

l '7 G' y

Project Number: 9000 3.Ib Type Analysis: bute Oi <-n s J e a b

b b r, Test Organisms: ), mon no d prnmg Author:

g REPORT CHECKLIST QA/QC ITEM REVi!WER DAIE

1. Samples collected. transported, and received

/0, t [9 V according to study plan requirements

M

2. Samples prepared and processed according

._, )

lo [i,/9 t/

to study plan requirements.

W o_

uL b

ro [< /W we

3. Data collected using calibrated equipment.

V

4. Calculations checked:

Hand calculations checked 76_ A ogla(Lislt

- Documented and verified statistical NJ a_o n J. n n 3.dr procedure used.

//.((F.NP ompte e n co ect.

6.

rted results and facts checked against

[l '

[

FN9 n in agree e rth te f

8. Results reviewed for compliance with study plan requirements.

ho

/ o,[i / 9 /

y AUTHOR DAIE O-

/o//7/9[

9. Commentary reviewed and resolved.

we-a o m

g

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

p{

lo ll 7 l9 {

A Ax~~.D PROJECT 4AANAGER DATE' 0' U k I ///P/' RF ITY OLOFFIC

[

' DATE W ww O\\

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SENIORTEC$1CALBEVfB(ER DATE l

ATS-QA12 3/10/93 l

,