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BACK TO MAIN Study Title Acute Toxicity of L - i '492 to the Daphnid, DapJinla magna Data Requirement: Guideline Humber u.s. EPA-TSCA, Guideline 797.1300 Authors Timothy J . Ward Jeanne p. Magazu Robert L. Boeri Study Completed July 13, 1995 Sponsor 3H Company Environmental Laboratory 935 Bush Avenue Building 2-3S-09 St. Paul, Minnesota 55144 Testing Facility T.R. Wilbury Laboratories, Inc. 40 Doaks Lane Marblehead, Massachusetts 01945 T.R. wilbury study Number 840-TH Page 1 of 22 BACK TO MAIN T.R. Wilbury Study Number 840-t h II. QUALITY ASSURANCE STATEMENT Submitted by: T.R. Wilbury Laboratories, Inc. 40 Doaka Lane Marblehead, Massachusetts certification: T.R. Wilbury Laboratories, Inc. study number 840-TH, Acute Toxicity of L-13492 to the Daphnid, Daphnia magna, was audited by the Quality Assurance Unit for compliance with the protocol, standard operating procedures, and applicable Good Laboratory Practices (IJ.S. EPA, 1993). The original raw data and a copy of the final report will be archived at T.R. Wilbury Laboratories, Inc. for at least 10 years. Quality assurance audits were performed and the findings reported to T.R. Wilbury Laboratories management and the study director on the following dates: Aspect Audited Audit Date Reported to Study Director Reported to Management Protocol : In-life: Draft Report: Final Report: 6/ 8/95 6/28/95 7/ 5/95 7/13/95 6/ 9/95 6/28/95 7/ 5/95 7/13/95 6/ 9/95 7/13/95 7/13/95 7/13/95 Arthur P. Paradice Q u a l i t y A s s u r a n c e Officer T.R. Wilbury Study Number 840-TH Page 3 of 22 BACK TO MAIN III. TABLE OF CONTENTS SECTION: I. Good Laboratory Practice Compliance Statement II. Quality Assurance Statement III. Table of Contents IV. Index of Tables and Figures V. Summary VI . General Information VII. Introduction Vili. Methods and Materials IX. Results X. References XI. signature Page XII . Protocol Amendments and Deviations Appendix A. Water Quality Data from Toxicity Test PAGE 2 3 4 5 6 7 8 8 13 17 18 19 20 T.R. wilbury study Number 840-TH Page 4 of 22 BACK TO MAIN IV. INDEX or TABLES AND FIGURES ti i Table 1 Chemical characterization of a representative sample of dilution water used for the toxicityteat with daphnids, Daphnia magma, and L-13492 Table 2 Teat system summary for the toxicity test with L-13492 and daphnids, Daphnia magma Table 3 Survival and sublethal effect data from the toxicity teBt with daphnids, Daphnia magna, and L-13492 Table 4 Median lethal concentrations (LC50s) and median effective concentrations (EC50s) from the toxicity test with daphnids, Daphnia magma, and L-13492 Table A. 1 Dissolved oxygen concentration, conductivity, pH, and temperature measured during the toxicity test with daphnids, Daphnia magna, and L-13492 Figure 1 Survival and sublethal effects of daphnids, Daphnia magna, exposed to L-13492 for 48 hours 11 14 16 21 15 V T.R. Wilbury Study Number 840-TH 1 Page 5 of 22 V. SUMMAAr The acute toxicity of L-13492 to the daphnid, Daphnia magna, is described in this final report. The test was conducted for 3H company for 48 hours from June 27 to 29, 1995, at T.R. Wilbury Laboratories, Inc. in Marblehead, Massachusetts. It was conducted by Jeanne Magazu, Jacqueline Nevius, Peter Kowalski, Keven Stevens, Peter Nevius, Robert Boeri, and Timothy Ward according to the protocol developed for T.R. wilbury Study Number 840-TH. L-13492 was supplied by the sponsor. The test was performed under static conditions with five concentrations of test substance and a dilution water control at a temperature of 20.4 to 20.7C. The dilution water was filtered deionized water collected at Marblehead, Massachusetts and adjusted to a hardness of 164 mg/L as CaCOj. Aeration was not required to maintain dissolved oxygen corcentrations above an acceptable level. Nominal concentrations of L-13492 were 0 (control), 13, 22, 36, 60, and 100 mg/L. Daphnids used in the test were less than 24 hours old at the start of the test. They were produced at T.R. Wilbury Laboratories and acclimated under test conditions for their entire life (cultures that produced te3t specimens were maintained at test conditions for more than 7 days). After 48 hours of exposure the control daphnids had an average wet weight (blotted) of 0.36 mg. All daphnids were in good condition at the beginning of the study. Exposure of daphnids to the test substance resulted in a 48 hour median lethal concentration (LC50) of 77 mg/L L-13492, with a 95% confidence interval of 60 to 100 mg/L and a 48 hour median effective concentration <EC50) of 34 mg/L L-13492, with a 95% confidence interval of 30 to 39 mg/L. The 40 hour no observed effect concentration (NOEC) is 13 mg/L. T.R. Wilbury Study Number 840-TH Page 6 of 22 BACK TO MAIN Material Tested: Sponsor : Test Substance shipped From: Reported Purity: study Initiation Date: Expeiinental Start Date: Experimental Completion Date : Study Completion Date: Archive Location for the Biological Raw Data and a Copy of the Final Report: L-13492 3M Company Environmental Laboratory 935 BuBh Avenue Building 2 -3E-09 St. Paul, Minnesota 55144 3M Company Environmental Laboratory 935 Bush Avenue Building 2-3E-09 St. Paul,. Minnesota 55144 100% June 14, 1995 (protocol signed) June 21, 1995 June 29, 1995 July 13, 1995 T.R. wilbury Laboratories, Inc. Marblehead, Massachusetts T.R. wilbury Study Number 840-th Page 7 of 22 VII INTRODUCTION ThiB study was sponsored by 3H Company, St Paul, Minnesota. The objective of the study was to determine the 24 and 48 hour median lethal concentrations (LCSOs) and median effective concentrations (ECSOr) of the test substance to the daphnid, Daphnia magna,, a freshwater invertebrate, under static conditions. The test protocol was based on U.s. EPA methods {U.S. EPA, 1993). The report contains sections that describe the methods and materials employed in the study, and results of the investigation. The report also contains an appendix that presents water quality data collected during the test. VIII. METHODS AND MATERIALS TEST SUBSTANCE i Tre sample of L-13492 (T.R. Wilbury Laboratories sample Number 502) was delivered to T.R. Wilbury Laboratories on June 14, 1995 in a cardboard box at ambient temperature. It was contained in a 125 ml plastic bottle. The label attached to the bottle included the following information: ~L13492, Hazardous Components/Special Precautions: isopropyl Alcohol, Originator: S.J. Martin -* S.A. Beach, 53-4H-02 - 2-3E-09, 8-5774, 8-7065, 5/4/95, 6/7/95, N2332-1" . L-13492 (a thin, clear liquid) was supplied by 3m Company, St Paul, Minnesota. Prior to use the test substance was stored in the dark at room temperature. Test substance stock solutions were formulated in dilution water without the use of a solvent. The test substance was assumed to have a purity of 100% active ingredient and to lie stable under storage and testing conditions. Unused test substance will be returned to the sponsor. DILUTION WATER: Water used for acclimation of test organisms and for all toxicity testing was deionized water collected at T.R. wilbury Laboratories in Marblehead, Massachusetts. Water was adjusted to a hardness of 160 180 mg/L as CaCO^ and stored in 500-gallon polyethylene tanks where it was aerated and continuously passed through a particle filter, ultraviolet sterilizer, and activated carbon. Results of chemical analysis of a representative sample of water are presented in T a b l e 1. TEST ORGANISM: Juvenile daphnids, Daphnia magna, employed as test organisms were less than 24 hours old at the start of the test. They were from a single source and were identified using an appropriate taxonomic key. Test specimens were produced by adult daphnids that were maintained under test conditions at the T.R. Wilbury facility for more than 7 days. T.R. Wilbury Study Number 840-TH Page 8 of 22 BACK TO MAIN Table 1. Chemical characterization of a representative sample of dilution water used for the tonicity test with daphnids, Daphnia magna, and L-13492. Parameter Unit of Measurement Detection Limit Measured Value pH Hardness Alkalinity Residual Chlorine Total Organic Carbon Total Phosphorus Nitrate Chloride Metala Arsenic Cadmium Copper Fluoride Boron Aluminum Chromium Cobalt Iron Lead Mercury Nickel Silver Zinc Organochlorine Pesticides plus Toxaphene Organophosphorus Pesticides PCBs pH units mg/L as CaCOa mg/L as CaCOj mg/L mg/L mg/L mg/L as N mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L pg/L pg/L pg/L pg/L -- -- 0.01 1 0.03 0.05 1 0.01 0.0002 0.005 0.1 0.5 0.1 0.01 0.03 0.03 0.005 0.0003 0.03 0.02 0.02 0.5 2 0.52 0.5 8.5 164 131 ND ND 0.46 0.08 14 ND 0.0002 ND ND ND ND ND ND 0.00 ND ND ND ND ND ND ND ND ND Notes: 1. T h e p H w a s m e a s u r e d in a control teat vessel at the start Of the test. Hardness, alkalinity, total organic carbon, and residual chlorine were measured in dilution water at the beginning of the toxicity test. Total phosphorus, nitrate, chloride, fluoride, metals, pesticide, and PCB data were collected during February, 1995, as part of routine biannual water quality'testing (these analysis were performed by Pace, Inc., Hampton, New Hampshire). 2. ND not detected at or above the detection limit. T.R. Wilbury Study Number 840-TH Page 9 of 22 BACK TO MAIN The original culture wae obtained on June 1, 1994 from Aquatic Research Organisms, Hampton, New Hampshire. Measurements made during the 7 days before the start of the test indicated that the culture temperature ranged from 20.5 to 21.0*C and the dissolved oxygen concentration was always at least 8.0 mg/L. The culture produced young prior to day 12 and a aubsample of adults produced at least 3 young/daphnid/day for 7 days before the start of the test. During acclimation daphnida were not treated for disease, they were free of apparent sickness, injuries, and abnormalities at the beginning of the test and there was no mortality during the 48 hours preceding the start of the test. The culture was supplied with a yeast/trout chow mix (Loti DC02) and the freshwater alga, Selenastrua capricornutum, daily during acclimation. Daphnids were not fed during the test. Control daphnids, blotted and weighed at the conclusion of the toxicity test, averaged 0.36 mg and the loading rate during the definitive toxicity test was approximately 14 mg/L. TOXICITT TESTING: A static screening test was conducted from June 14 to 16, 1995 with five concentrations of test substance and a dilution water control. Nominal concentrations of L-13492 were 0.10, 1.0, 10, 100, and 1,000 mg/L. After 48 hours of exposure there was 1r*0% survival in the control and at 0.Z0, 1.0, and 10 mg/L, and 0% survival at 100 and 1,000 mg/L. The definitive toxicity test was performed from June 27 to 29, 1995, according to T.R. Wilbury Protocol 840-TH (Acute Toxicity of L-13492 To The Daphnid, Daphnia magna). The protocol, which was signed by the study director on June 14, 1995, is based on procedures of the U.S. Environmental Protection Agency (1993). a summary of the test system is presented in Table 2. The test was conducted at a target temperature of 20 t l*c with five concentrations of test substance and a dilution water control. Nominal concentrations of the test substance were: 0 mg/L (control), 13, 22, 36, 60, and 100 mg/L. A 100 mg/L stock solution was formulated by combining 0.2 g test substance and dilution water in a Class A volumetric flask and adjusting the final water volume to 2,00Q ml. A p p r o p r i a t e amounts of this stock solution w e r e a d d e d to dilution water in test vessels to formulate test media without the use of a solvent. Twenty daphnids were indiscriminately and equally distributed among two replicates of each treatment. The test was performed in 300 ml glass beakers that contained 250 ml of test solution (water depth was approximately 9 cm). Test vessels were randomly arranged during the 48 hour test (a random numbers table was used to select the location of each vessel) and they were loosely covered. T.R. Wilbury Study Number 840-TH Page 10 of 22 BACK TO MAIN Table 2. Test system summary for the toxicity test with L-13492 and daphnids, Daphnia magna Species: Daphnia magna Age/Size of Test organisms: <24 hours old Test Duration: 48 hours Test Vessels: 300 ml glass beakers Test Media Volume: 250 ml Depth of Test Media: 9 cm Number of Replicate Test Vessels: 2 Organisms per Replicate: 10 Temperature Range: 20.4-20.7C , Minimum Dissolved Oxygen: 8.5 mg/T. Photoperiod: 16 hours light and 8 hours dark. Light Intensity: 20 footcandles Water Quality Measurements: Dissolved oxygen, pH,, temperature, and conductivity daily in each test vessel. Hardness, total organic carbon, and alkalinity once in the control. Residual chlorine daily. Dilution water analysis is summarized in Table 1. End Points: 24 and 48 hour LC50 end EC50 (based on immobilization), 43 hour no ooserved effect level. Acceptability Criteria: 510% control mortality and acceptable temper2iture range. j T.R. Wilbury Study Humber 840-TH Page 11 of 22 BACK TO MAIN A 16 hour light and 8 hour dark photoperiod with a 15 minuta transition period was automatically maintained with cool-white fluorescent lights that provided a light intensity of 20 footcandlea. Aeration was not required to maintain dissolved oxygen concentrations above acceptable levels and daphnids were not fed during the test. The number of surviving organisms and the occurrence of sublethal effects (immobilization, loss of equilibrium, loss of reflex, excitability, discoloration, or change in behavior) were determined visually and recorded initially and after 24 and 48 hours. Dead test organisms were removed when first observed. Dissolved oxygen (YSI Model 57 meter; instrument numbers 1), pH (Beckman model pHI 12 meter; 'instrument number 124), conductivity (Cole Parmer meter, instrument number 94>, and temperature (Beckman model pHI 12 meter; instrument number 124, prooe pHT-1) were measured and recorded daily in each test chamber that contained live animals. The temperature in a beaker of water incubated among the test vessels was recorded continuously during the test. STATISTICAL METHODS: Results of the toxicity test were interpreted by standard statistical techniques. Computer methods (Stephan, 1983) were used to calculate LC50a and C50s, when possible. All calculations were performed by the author using nominal concentrations of test substance and the number of live or unaffected daphnids. The no observed effect concentration is the highest concentration of test substance that did not cause any toxicant-related mortality or sublethal effects. Ii T.R. Wilbury Study Number 840-TH Page 12 of 22 BACK TO MAIN IX, RESULTS Insoluble material was not noted during the test. Biological and water quality data generated by the acute toxicity test are presented in Table 3 and Appendix A, respectively. The measured concentrations of dissolved oxygen ranged from 8.5 to 8.8 mg/L (mean = 8.6 mg/L), the temperature ranged from 20.4 to 20.7C (mean = 20.5*C), conductivity ranged from 620 to 640 pmhos/cm (mean = 630 pmhos/cm), and pH ranged from 8.4 to 8.6. One hundred percent survival occurred in the control exposure and these daphnids did not exhibit any sublethal effects. The 24 and 48 hour LC503 and EC50g for daphnids exposed to L-13492 are presented in Table 4. The 40 hour LC50 for daphnid3 exposed to L17 492 is 77 mg/L (95% confidence interval = 60 to 100 mg/L) and the 48 hour EC50 is 34 mg/L (95% confidence interval = 30 to 39 mg/L). The slope of the 48 hour dose response curve (based on mortality and immobilization) is 9.4. The 48 hour no observed effect concentration (NOEC) ia 13 mg/L. T.R. wilbury Study Number 840-t h Page 13 of 22 BACK TO MAIN Table 3. survival and aublethal effect data from the toxicity test with daphnids, Daphnia magna, and L-13492. Nominal concentration of L-13492 (mg/L) Rep. Number alive 0 24 48 hr hr hr Number affected 0 24 48 hr hr hr 0 (control) 13 22 36 60 100 1 10 10 10 2 10 10 10 1 10 10 10 2 10 10 10 1 10 9 9 2 10 10 10 1 10 10 9 2 10 10 10 1 10 10 2 10 10 9 8 1 10 10 2 10 10 3 0 000 000 000 000 000 000 006 004 009 008 063 0e Note: Affected daphnids were immobilized. T.R. Wilbury Study Number 840-TH Page 14 of 22 BACK TO MAIN Figure 1. Survival and sublethal effc:ct3 of daphnids, Daphnia magna, exposed to L-13492 for 48 hours. T.R. wilbury Study Number 840-TH Page 15 of 22 Table 4 Median lethal concentrations (LC50s) and median effective concentrations (EC50s) from the toxicity test with daphnids, Daphnia nagna, and L-13492. Exposure period LC50/EC50 95 percent confidence limit Calculation method LC50 24 hours 48 hours >100 mg/L 77 mg/L -- 60 to 100 mg/L -- Binomial fc.250 24 hours 89 mg/L 60 to >100 mg/L Binomial 48 hours 34 mg/L 30 to 39 mg/L Probit T.R. wilbury study Number 840-TH ' Page 16 of 22 BACK TO MAIN X . REFERENCE.'# Stephan, C.E. 1983. Computer Program for the Calculation of LC50 Values. . U.S. EPA. Duluth, HN. Personal Communication. U.S. EPA. 1993. Toxic Substances Control Act Test Guidelines; Final Rules. Section 797.1300. U.S. EPA. 1993. 40 CFR Part 792. Toxic Substances Control Act (TSCA); Good Laboratory Practice Standards,. Code of Federal Regulations, Title 40, July 1, 1993. T.R. Wilbury Study Number 840-TH Page 17 of 22 BACK TO MAIN XI. SIGNATURE PAOE ^ 'I />'y-r 1-'- Jeanne P. Magazu * study Director * Aquatic Toxicologist - -2_________ /Xj' / Robert L. Boeri Coauthor 7 Peter L. Kowalski Aquatic Toxicologist l-\> si i\ , ''A'O1.-*/ A/., M !v. For Jacqueline U. IJevius Aquatic Toxicologist Keven M. Stevens Biologist ih 1/i Peter C. Uevius Biologisi- ?`7 I'' r L,.,:r,.x. T iinothy 1. W ard Author ... T.R. Wilbury study Number 840-TH Page 18 of 22 BACK TO MAIN XIX. PROTOCOL. AMENDMENTS AND DEVIATIONS PROTOCOL AMENDMENTS: The protocol was amended to specify the nominal concentrations for the definitive teat and to correct a typographical error that stated that test vessels would be 300 ml beakers containing 250 liters of solution (rather than 250 ml). PROTOCOL DEVIATIONS: No deviations were made from the protocol. T.R. wilbury Study Number 840-t h Page 19 of 22 BACK TO MAIN Appendix A: HATER QUALITY DATA FROM TOXICITY TEST j \ i T.R. Wilbury study Humber 840-TH BACK TO MAIN Table A 1. Dissolved oxygen concentration, conductivity, pH, and temperature measured during the toxicity test with daphnids, Daphnia magna, and L-13492. Nominal Concentration of L-13492 (mg/L) Dissolved oxygeni (mg/L) 0 24 48 Rep. hr hr hr Conductivity (pmhos/cm) 0 24 48 hr hr hr 0 (control) 11 22 36 60 100 1 8.7 8.6 8.5 2 8.7 8.6 8.6 1 8.7 8.6 3.6 2 8.7 8.7 13.6 i 8.7 8.6 3.5 2 8.7 8.8 3.5 1 8.7 8.7 8.5 2 8.7 8.7 8.5 1 8.7 8.7 8.6 2 8.7 8.7 8.5 1 8.7 8.7 8.5 2 8.7 8.7 8.5 630 630 630 630 630 630 630 6.30 630 630 640 640 620 620 620 620 620 620 620 620 620 620 620 620 630 630 630 630 630 630 630 630 640 640 640 640 T.R* Wilbury study Number 840-TH Page 21 of 22 BACK TO MAIN Table A.l. Continued. Nominal Concentration of L-13492 (mg/L) 0 (control) 13 22 36 60 100 Rep. pH 0 24 hr hr 4 Ei hi- 1 8.5 8.6 8.4 2 8.5 8.6 8.5 1 8.5 8.6 8.5 2 8.5 8.6 8.5 1 8.5 8.6 8.5 2 8.5 8.6 8.5 1 8.6 8.6 8.5 2 8.6 8.6 8.5 1 8.6 8.6 8.5 2 8.6 8.6 8.5 1 8.6 8.6 8.5 2 8.6 8.6 8.5 Temperature C O 0 24 48 hr hr hr 20.6 20.6 20.6 20.5 20.6 20.6 20.6 20.6 20.6 20.6 20.6 20.6 20.5 20.5 20.5 20.5 20.5 20.5 20.4 20.4 20.5 20.S 20.5 20.6 20.7 20.5 20.5 20.5 20.5 20.5 20.5 20.5 20.5 20.4 20.5 20.5 T.R. Wilbury Study Number 840-TH Page 22 of 22 04/01/2001 ENVIRONMENTAL LABORATORY ARCHIVED FINAL REPORT BACK TO MAIN LAB REQUEST NO. N2332 REQUESTOR NAME: SAB/S.J.MARTIN DEPARTMENT: 3048 PROJECT NO: BI703004UQ DATE RECEIVED: 06/07/1995 DESC: L-13492 ECOTOXICITY EVALUATION CONTRACT LAB(S): TRWIL EXP COMP DATE: 08/30/1995 DATE COMPLETED: 09/02/1995 PROJECT LEAD: PHONE NO: 3M FAX NO: 651-778-6176 Tests conducted following 40CFR 797; methods 1050, 1300, 1400. Sample desc.: L-13492, 45% solids, from S. Martin. SAB 6/7/95 Sample is a clear liquid. Lot confirmed as Lot #2 as per S.J. Martin, of 3M SCD Lab on 7/18/95. SAB Results from 48-hr Daphnia magna acute toxicity test: Test Cone., mg/L 0 (control) 13 22 36 60 100 Percent Immobilization and/or Mortality 24-hrs Exposure 48-hrs Exposure 00 00 55 0 55 0 100 -+4-70 100 sab 7/31/95 Results from 96-hr Fathead minnow acute static toxicity test: Test Cone. mg/L 0 (control) 130 220 360 600 1000 24-hrs exp 0 0 0 0 0 50 Percent Mortality 48-hrs exp 72 -hrs exp 00 00 00 00 00 60 70 96-hrs exp 0 0 0 0 0 70 sab 7/31/95 Results from 96-hr algae growth inhibition test: Test cone. mg/L 1.0 2.0 4.0 8.0 16 EC50 95% C.I. sab 9/1/95 Percent change from Control 24-hrs exp. 48-hrs exp. 72-hrs exp. 02 -2 44 17 27 16 17 38 22 42 55 63 51 61 15 14 7.1 (9.8 - >16) (8.2 - >16) (4.7 - 11) 96-hrs exp. 0 30 50 56 80 4.9 (3.5 - 6.7) SAMPLE DATE CODE DESCRIPTION RESULT * = CONTRACT LAB DATA 04/01/2001 LAB REQUEST NO. N2332 SAMPLE DATE CODE ENVIRONMENTAL LABORATORY ARCHIVED FINAL REPORT CONTINUED DESCRIPTION RESULT 1 05/04/95 L-13492 Lot 2 TRWIL 48-HR-EC50 *DAPHNIA ACUTE TEST TRWIL 96-HR-LC50 *FISH ACUTE TEST - 96 HOUR TRWIL IC50-ALGAE *ALGAE ASSAY - IC50 34 MG/L 890 MG/L 4.9 MG/L * = CONTRACT LAB DATA BACK TO MAIN MDL or (95% C.I.) 2 MDL or (95% C.I.) (30 - 39) (600 - >1000) (3.5 - 6.7)