Document JJKbkBe6K3dKnYEYo3wDErewr

A R fc -o ! TOXICITY TO AQUATIC PLANTS (E.G., ALGAE) TEST SUBSTANCE Identity: Perfluorooctanoic acid; may also be referred to as PFOA, FC26, or FX-1001. (Octanoic acid, pentadecafluoro-, CAS # 33567-1) Remarks: The 3M production lot number was 269. The test substance is a white powder. The test sample is FC-26 referred to by the test laboratory as N2803-3. The purity of the sample was not sufficiently characterized, although current information indicates it is a mixture of 96.5 -100% test substance and 0 - 3.5% C6, C7, and Cgperfluoro homologue compounds. Remarks field: The sample preparation directions given to the laboratory were to dissolve the test material in a 50:50 water.isopropanol solution. In the protocol amendment, it was stated that the sample was combined with isopropanol in a 50:50 ratio prior to use. METHOD Method: U.S. EPA-TSCA Guideline 797.1050 Test: Static acute GLP: Yes Year completed: Study completed in 1995. Report completed in 1996. Species: Selenastrum capricornutum Source: Originally, from The Culture Collection of Algae at the University of Texas at Austin, maintained in culture medium at T.R. Wilbury, Inc., Marblehead, MA. Element basis: Reported two ways: number of cells/mL and growth rate. Exposure period: 96-hours Year study conducted: 1995 Year study completed: 1996 Analytical monitoring: Nominal concentrations. pH and temperature. Test organisms laboratory culture: Algae cultures were growing in U.S. EPA-recommended sterile enriched medium for at least 14 days prior to test initiation. Test Conditions: Algal medium: prepared to U.S. EPA recommended concentrations by spiking deionized water with nutrient stocks. The pH of the synthetic algal medium at test initiation was 7.4. Algal medium used for culturing and as diluent. Stock and test solutions preparation: A 1,000 mg/L primary stock solution was prepared in sterile enriched media. Appropriate amounts of this stock solution were added directly to dilution water to formulate the test media. Exposure vessels: 250 mL glass Erlenmeyer flasks containing 50 mL of test solution. Agitation: Shaken continuously at 100 rpm Number of replicates: three Initial algal cell loading: 1.0 X 104cells/mL Number of concentrations: Five plus a negative control Water chemistry: pH range: (0 - 96 hours) 7.4 - 10.3 (control exposure) 2.9 - 3.0 (1,000 mg/L exposure) Test temperature range: (0 - 96 hours) 23.5 - 24.0C Light levels: ( 0 - 9 6 hours) -380 ft-c from continuous cool-white fluorescent lighting Remarks: The pH of the test solutions for the 250, 500 and 1,000 mg/L exposure concentrations were in the range of 2.9-4.0 at test initiation. This low pH would have adversely affected the survival and subsequent growth of the algae. RESULTS Nominal concentrations: Blank control, 63, 125, 250, 500, 1,000 mg/L (Tested as 50:50 mixture of test substance and isopropanol). The concentration of test substance in solution was 32, 63, 130, 250 and 500 mg/L. Element value (as-tested): 72-hour EC50 (cell density) = 180 (125-250) mg/L 72-hour ErCso (growth rate) = 180 (125-250) mg/L 96-hour EC50 (cell density) = 180 (125-250) mg/L 96-hour ErCso (growth rate) = 180 (125-250) mg/L 96-hour NOEC: 125 mg/L Element value (based on concentration of test substance in solution): 72-hour EC50 (cell density) = 90 (63-130) mg/L 72-hour ErCso (growth rate) = 90 (63-130) mg/L 96-hour EC50 (cell density) = 90 (63-130) mg/L 96-hour ErCso (growth rate) = 90 (63-130) mg/L 96-hour NOEC: 63 mg/L (..`3 3 3 3 7 All element values based on nominal concentrations. A toxicity test conducted previously with isopropanol at 1,000 mg/L showed no effect on algal growth. Statistical methods: Cell densities, growth rates and percent inhibition values used to estimate the EC10, EC50, and EC90 values and 95% confidence limits were calculated using the computer software of C.E. Stephan. The no observed effect concentration (NOEC) was calculated using one-way analysis of variance (ANOVA). Control response: Satisfactory Biological observations after 96-hours: N om inal C o n c e n tra tio n (a s -te s te d ), m g/L C o n tro l Mean Number o f Cells per mL P ercen t Inhibition via D e n s ity P ercen t Inhibition via Growth R ate __ 1 ,6 7 1 ,0 0 0 63 1 ,7 3 2 ,0 0 0 -4 -2 125 1 ,5 6 0 ,0 0 0 7 0 250 < 10,000 100 100 500 < 1 0 ,0 0 0 100 100 1 ,0 0 0 < 10,000 100 100 Observations: Algal cell counts in each test vessel were determined by means of direct microscope counts with a hemocytometer. After 96 hours of exposure, there were no signs of aggregation, flocculation or adherence of the algae to the flasks in the control or any test treatment group. In addition, there were no noticeable changes in cell size, color or morphology when compared to the control. Reversibility of Growth Inhibition: Effect of the test substance was determined to be algistatic based on the results of the post-definitive test exposure. CONCLUSIONS The test sample 96-hour EC50 and 95% confidence interval for Selenastrum capricornutum was determined using two calculation methods. The as-tested test substance 96-hour EC50 for Selenastrum capricornutum was determined to be 180 mg/L with a 95% confidence interval of 125 - 260 mg/L, when calculated using either the cell density or growth rate. The as-tested test substance 96-hour no observed effect concentration (NOEC) was 125 mg/L. The 96-hour EC50 for Selenastrum capricornutum based on the concentration of test substance in solution was determined to be 90 mg/L with a 95% confidence interval of 63 - 130 mg/L using either the cell density or growth rate. The 96-hour no observed effect concentration (NOEC) for the test substance in solution was 63 mg/L. No signs of aggregation, flocculation, or adherence were noted in any of the test solutions. This test substance was determined to be algistatic. Submitter: 3M Company, Environmental Laboratory, P.O. Box 33331, St. Paul, Minnesota, 55133 DATA QUALITY Reliability: Klimisch ranking 3. The study lacks analytical measurement of test substance concentrations in the test solutions and sample purity is not sufficiently characterized. Additionally, there appears to be a discrepancy between the sample preparation directions given to the laboratory and the procedure conducted by the laboratory to prepare the test solutions. Initial low pH values in the higher concentrations may have had an adverse effect on the survival and growth of the algae. REFERENCES This study was conducted at T.R. Wilbury Laboratories, Inc., Marblehead, MA, at the request of the 3M Company. OTHER Last changed: 5/25/00 Study Title Growth and Reproduction Toxicity Test with N2803-3 and the Freshwater Alga, Selenastrua capricornutua Data Requireswnts Guideline Humber U.S. EPA-TSCA, Guideline 797.1050 A . , . "<. Authors Timothy J. Ward Jeanne P. Magazu Robert L. Boeri Study Coagpleted January 24, 1998 Sponsor 3M Company Environmental Laboratory 935 Bush Avenue Building 2-3E-09 St. Paul, Minnesota 55144 V#''V., i-V' s. ; t *. ,,L *i'"t*r - Av I. GOOD LABORATORY PRACTICB COMPLIANCE STATEMENT Thia study waa conductad according to EPA Good Laboratory Practice Regulations (40 CFR 792), with tha following axcaptiona. The stability of' tha teat subatanca undar taat conditions waa assumed but not verified at t .r . wilbury Laboratories. Deviations from the study protocol are discussed in Section XII of this report. V I im W Y V ftlliB L E XIX. T A B U OP CONTENTS SECTIO 1 PAGE I, Good Laboratory Practice compliance statement II. Quality Asaurance Statement III. Table of Contents IV. Index of Taj2le3 and Figures V. Summary VI. General information VII. Introduction vili. Methods and Materials IX. Results X. References XI. Signature Page XII. Protocol Deviations 2 3 4 5 6 ? 8 8 13 . 19 20 21 Appendix A. ater Quality Data from Toxicity Teat -a . ' 22 ' s i c ^ r TM a ... : - . ;;.-v lE ST W SVAll ABLE orXV. XMSEX TABLES AMD rICUBES PACE Table 1. chemical characterization of a representative sample of teat media and water used to formulate teat media for the toxicity teat with the freahwater alga, Selenaatrua capricornutum, and N2803-3 9 Table 2. Teat system summary for the toxicity test with N2803-3 and the freahwater alga, Selenastrua capricornutum 11 Table 3. Cell growth data from the toxicity test with N2803-3 and the freshwater alga, Selenastrum capricornutum 14 Table 4. Historic cell growth data from a toxicity test with isopropyl alcohol and the freshwater alga, Selenastrum capricornutum (from Ward, et.al., 1995) 1! ! Table 5. Average specific growth rate and percent change from the control from the toxicity test with the 17 freshwater alga, Selenastrum capricornutum, and N2S03-3 Table 6. Effective concentrations (EC50s) from the toxicity test with the freshwater alga, Selenastrum capricornutum, and N2803-3 18 Table A.l. Temperature measured during the toxicity test with N2803-3 and the freshwater alga, Selenastrum capricornutum 'I* , 23 IK T S B P Y f ill ABLE I$ :* f SUMMARY Th# toxicity of N2803-3 to the freshwater alga, salaaatrua ctprlcornutua, la daacrlbad in thia final report. Tba teat waa conducted for 3H Company for 96 hour from August 8 to 12, 1995, at T.R. Wilbury Laboratories, Inc. in Marblehead, Maaaachuaetta. It waa conducted by Jeanne Magazu, Peter Kowalski, Jacqueline Nevius, Raven stavena, Peter Nevius, Robert Boeri, and Timothy Hard according to the protocol developed for T.R. Wilbury Study Number 893-TH. N2B03-3 was supplied by the sponsor. The teat waa performed under static conditions with five concentrations of test substance and a dilution water control at a temperature of 24 t 2*C. The sample of N2803-3 supplied by the sponsor was combined with iBopropanol in a 50:50 ratio prior to use, and this mixture in defined as the test substance as-tested. A toxicity teat was previously conducted with 1,000 mg/L isopropanol (Hard et.al., 1995) and algal growth waa not affected at 1,000 mg/L, indicating thit the concentration of isopropyl alcohol in the sample of N2803-3 can not, by itself, account for the toxicity of N2803-3 to algae. Results of the toxicity test are reported twice, once based on nominal concentrations of teat substance as-tested and a second time based on concentrations of test substance as-received (N2803-3 without iBopropanol). Nominal concentrations of N2803-3 astested ware 0 (control), 63, 125, 250, 500, and 1,000 mg/L and nominal concentrations of N2803-3 as-received were 0 (control), 32, 63, 130, 250, and 500 mg/L. The dilution water was sterile enriched media. Exposure of algae to the test substance for 96 hours resulted in a median effective concentration (EC50) of 180 mg/L N2803-3, -with 95 confidence limits of 125 and 260 mg/L, when calculated using either the number of cells per ml or the average specific growth rate, baaed on test substance as-tested (50* N2803-3 and 50* isopropanol). : The 96 hour no observed affect concentration (NOEC) is 125 mg/L and the lowest observed effect concentration (LOEC) is 250 mg/L, based on test substance astested. The 96 hour EC50 is 90 mg/L N2803-3 (95* confidence limits - 63 and 130 a^/L,` when calculated using either the number of cells per ml or the average specific growth rate and the 96 hour NOEC is'63 mg/L and the LOEC is 130 mg/L, based on test substance as-received (N2803-3 without isopropanol). tm mi t 4- 1 <' i V4' 11 <k 5k fcT.R. Wilbury Study Number 893-TH frrp m i >* ^ Page $ of 24 . ... *X ' <vb -asi i"yw/lf*'. m i:*-' *,$ 0 3 9 4 5  /\.k\ ,v -.n< V . . K S T COPY AVAR ABU VII. INTRODUCTION Thi study was aponaored by 3M Company. The object!vs of tbs study was to determine the 24, 48, 72, and 96 hour effective concentrations (EClOs, EC50s, EC90a) and no observed effect concentration (NOEC) of the test substance to the freshwater alga, Stlanastrvm capcicornutum, under static conditions. The test protocol was baaed on U.s. EPA methods (EPA, 1993). The report contains sectiona 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 AMD MATERIALS TEST SUBSTANCE I The sample of N28Q3-3 (T.R. Wilbury Laboratories Sample Number 521) was delivered to T.R. Wilbury Laboratories on August 14, 1995 in a cardboard box at ambient temperature. It was contained in an approximately 50 ml glass jar. The label attached to the bottle included the following information! "N2803-3". N2803-3 (a white solid) was supplied by 3M Company, St Paul Minnesota. Prior to use the sample of N2803-3 was stored in the dark at room temperature. The sample of N2803-3 supplied by the sponsor was combined with isopropanol in a 50:50 ratio prior to use. This 50:50 m a t u r e was then considered to be 100* test substance during the performance of the toxicity test. All results are reported both on the basis of test substance as-tested (50% N2803-3 and 50% isopropanol) and test substance as-received (N2803-3 without iaopropanol). The H2803-3 was assumed to have a purity of 100% active ingredient and to be 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 sterile enriched media (U.s. EPA, 1978; T.R. Wilbury standard Operating Procedure number 6) adjusted to a target pH of 7.5 with 0.1 H HCl prior to use. characterization of a representative sample of media is presented in Table 1. - Tabi 1. Chemical characterization of a reprasentativa aampia of taat - nadia and watar uaad to formulata taat madia for tha toxicity - -|4, taat vith tha fraahwatar alga, Selenaatrum capricornutua, and ' r< Vi N2803-3. <-' h' Parameter1 Unit of Haaaurement V* .., PH Total Phosphorus Nitrate pH units mg/L mg/L as N Chloride Total Organ_p Carbon Metals ^ mg/L mg/L Arsenic mg/L Cadmium mg/L Copper Fluoride mg/L mg/L Boron mg/L Aluminum Chromium Cobalt mg/L mg/L mg/L Iron Lead Mercury mg/L mg/L mg/L Nickel Silver y Zinc * 1"<' Organochlorine Pesticides mg/L mg/L mg/L pg/L plus Toxaphene pg/L Organophosphorus . Pesticides > pg/L PCB3 * : r . P9/L . . "' iV'^V **j O o Detection Limit .. 0.03 0.05 1 1 0.0002 0.005 0.1 Meaaured Value 7.4 0.46 VVS <vSiv-**/jsr;-' '4:.> r-;:;;j' . .;.y 0.08 14 v ND1 .' -: '-V'f>-W.*v?*wi>tv.-'' NO * 0.0002 ND `':: W\. t ND Notes: l.ThepH'vas'measured in a control test veasel at'the atartjfo `the' taat-J^ Total organic carbon, total phosphorus,-nitrate? 1-: f -;v *chlor.i-de,'--fluoride, maiala pesticide, and PCB data varaii s. ^collected during.February/il995, aa part of routine:biannuiail N watar^quality .testing (th#seranalysia vara performed 5^f Inci^Ha^toh|fiNev^BMpahira --'abova.'.the detection slimlt1 ^V.V^.'VP'Z P4- r' ajr' -v`:j,'`4 T 'R *, Hilbury study Number'|8.9933-TH ' ' Jy !5;M< fr* 4.-t m ^ ii tlii, * \ I B I W W W AHABI i* TOXICITY TESTI Results of a rang finding test war* usad to aalact tha concentration* for tha definitive teat. Tha range finding test was performed from August 25 to 29, 1995. At tha conclusion of the test tha number of cells/ml equalled the following percent of tha control) 0.050, 0.50, 5.0, and 50 mg/L a >1001 end 500 mg/L <1, based on test substance as-received. The definitive toxicity test was performed from September 8 to 12, 1995, according to T.R. Wilbur: Protocol 893-TH (Growth and Reproduction Toxicity Test with N2803-3 and the Freshwater Alga, jSelenastrum caprlcornutua). The protocol, which was signed by the study director on August 16, 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 24 1 2C with five concentrations of test substance and a dilution water control. Nominal concentrations of tha test substance as-tested were: 0 mg/L (control), 63, 125, 250, 500, and 1,000 mg/L. A 1,000 mg/L stock solution of test substance as-tested was formulated on September 8, 1995 by adding l.u g N2803-3 as-tested to sterile enriched media and adjusting the final media volume to 1,000 ml. Appropriate amounts of this stock solution were added directly to dilution water to formulate test media. A test was performed from June 26 to 30, 1995 with isopropyl alcohol (Mallinckrodt, Lot 3035), a component that represents 50* of test substance (Ward, et.al., 1995). This test was conducted at 4.4 mg/L and at 1,000 mg/L. A 1,000 mg/L stock solution was formulated on June 26, 1995 by adding 0.5 g isopropyl alcohol to sterile enriched media and adjusting the final media volume to 500 ml. Algae were distributed among three replicates of each treatment at the rate of 10,000 cells/ml. The test was performed in 250 ml glass Erlenmeyer flasks that contained 100 ml of test solution. Test vessels were randomly arranged on a rotary shaker adjusted to 100 rpm in an incubator.during the teat (a random numbers table was used to select the .{..-i .r-v A- ' location of each vessel). A 24 hour light and 0 hour dark photoperiod was automatically maintained with cool-white fluorescent lights that provided a light intensity of approximately;380 footcandles. . .v "Vi :v j- number of algal cells/ml i n e a c h test vessel and the occurrence relative size'differences, unusual cell shapes, colors, flocculations, h ':adherence ;.of/ cells^ to test ' containers, ,or aggregation* of cells was ^determinod^.visually^ by ;mean*.,'.,of ;direct microscopic examination with ytoMtir e l'CelV counts test." :made*, and r e c ordeddaily the ; -a.! v WV/S * m Page 10 of .-./.Si1 .'0 ,- -`H- h [? ** ."i^Vrrf1: '/; C 0 3 9 .A f-**' ;<*** Tabi* 2. T*at system summary for tba toxicity taat with N2803-3 and tha fraahwatar alga, salanastrua caprlcornutua. Species: Salanastrua caprlcornutua Cultura Acclimation Period: >14 days Taat Duration: 96 hours Taat Vaaaalai volum* oi Taat Madia> 250 ml glass flasks 100 ml v: Numbar of Replicata Test Vessels : 3 Inoculum: 10,000 cells per ml Oscillation Rate: 100 rpm Incubator Temperature Range: 23.5-24.0 #C Photoperiod: 24 hours light and 0 hours dark. Light Intensity: -380 footcandles Water Quality Measurements: pB in each test vessel at the beginning and end of the test. Incubator temperature daily. Dilution water analysis is summarized in Table 1. End Points: Acceptability criteria: ; -r , ,, } V.r!,' V/ 24, 48, 72, and 96 hour EC10, EC50, and EC90; no observed effect concentration. .. '^'\i .`"".''i.-T ' *s t .' Logarithmic growth at'96 hours'and acceptable temperature range. ^ ,\ , t1' , Vrt ' -X -`. A ifA' .t 'iA'. ''ft , `, 4 >l>fk . 5 r'r'i?*K)W -* * ' * * -' A *''<J Th determination of whathar toxic affact* vara algistatic or algicidal vas performed at tha conclusion of tha toxicity tast (O.S ml of madia frcai aach 250 mg/L vassal vas transfarrad to a vassal containing fraah siadia vithout M2803-3 and incubatad for 21fi hours undar tast conditions). Temperature of tha incubator vas maasurad and racordad daily (tharmomatar numbar 2968), and pH (Beckman modal pBI 12 mater; instrument number 124) vas determined in each test vessel at tha beginning and and of tha test. Tha temperature in a representative vessel of vater incubated vith the test vessels vas continuously recorded. STATISTICAL METHODS I The average specific grovth rate vas calculated as the natural log of the number- of cells/ml at 24, 48, 72, and 96 hours minus the natural log of the number of cells/ml at 0 hours divided by the exposure period. The percent change from the control vas calculated by subtracting the treatment average specific grovth rate from the control average specific growth rate, dividing the difference by the average specific grovth rate in the control, and multiplying that value by 100. Aesults of the toxicity test were interpreted by standard statistical techniques. The binomial/interpolation method (Stephan, 1984) was used for calculating 72 and 96 hour values. All calculations were performed using the number of cells/ml and the average specific grovth rates and nominal concentrations of the test substance. The no observed effect concentration (NOEC) vas determined using a parametric one-vay analysis of variance (ANOVA) and the number of cells/ml and the average specific growth rate in each test vessel at the end of the test. t< `t" y:y<->' >':-f" % BEST COPY AVAll ABLE IX. RESULTS No insoluble material was noted in any teat vessel during the test. The algal population in the control test vessels grew well, resulting in an average of 1,671,000 cells/ml after 96 hours (Table 3). Water quality throughout the test was within acceptable limits (Appendix A ) . The range of incubator temperatures was 23.5 to 24.0*C (Table A.l). The pH of test media (Table A.2) was affected by the test substance at the beginning of the test. Test substance concentrations above 125 mg/L reduced pB values to 4.0 and lover. Biological data generated by the acute toxicity test with N2B03-3 are presented in Tables 3 and 4. The growth curve for algae exposed to the test substance for 96 hours is presented in Figure 1 (the slope of the dose-response curve could not be calculated from this data set). The 72 and 96 hour EC50 values for algae exposed to N2803-3 are presented in Table 6. The 96 hour EC50 (and associated 35 percent confidence limits) is 180 mg/L (125 and 260 mg/L), based on test substance as-tested (50 N28033 end 505 isopropanol) and the 96 hour EC50 (and associated 95 percent confidence limits) is 90 mg/L (63 and 130 mg/L), based on test substance as-received (N2803-3 without isopropanol). No effects (size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells) were noted. The 96 hour no observed effect concentration (NOEC) is 125 mg/L and the lowest observed effect concentration (LOEC) is 250 mg/L N2803-3, based on test substance astested and the 96 hour NOEC is 63 mg/L and the lowest observed effect . concentration (LOEC) is 130 mg/L N2B03-3, based on test substance asreceived. The determination of whether toxic effects were algistatic or algicidal was performed at the conclusion of the toxicity test. A 0.5 ml aliquot of media from each 250 mg/L (as-tested) vessel vs transferred to a vessel containing fresh media without N2603-3 and incubated for 216 hours under test conditions. Algae increased from <10,000 cells/mL to t 4,320,000 cells/mL, indicating that the effect of N2803-3 at this concentration was algistatic rather than algicidal. * if**!.. 4 . [ ' \- --K`*''- ".V"' < :. /.&* Biological data generated by a previous exposure of algae to , !" " . / isopropyl alcohol (Ward, et.al., 1995) are presented in Table 4. Algal t 4,growth was not affected at.sl,000 mg/L, indicating that the concentration ,\ > ^ o f ,isopropyl alcohol in N2803-3 can not, by itself, account for the v ttoxicity.,of N2803-3 to algae V .a? '-k V? 4g/. T,R#`WJLIbury Study`Number 893-TH tv* 5^-4 v4 v' v- >.'.*V .i'fVj * t7r Page 13 of 24?' m r * * lX M /'Jty %Jrf'st> BESTCOPlfii 4BU f Tabla 3. call growth data from tha acuta toxicity taat with N28Q3-3 and tha fraahwatar alga, Balaaaatrum caprlcornutua. Nominal Concantratlon of N2803-3 (mg/L) aa-Taatad aa-Racaivad Rap. 0 (control) -- 1 2 3 mean % control 63 32 1 2 3 mean t control Mumbar of Calla/ml x 10' (hour) . 0 24 .. Y - 48 72 96 10 22 10 20 10 22 10 21 -- -- 10 28 10 26 10 18 10 24 100 114 168 360 1,800 146 402 1,612 154 510 1,600 156 424 1,671 -- -- 134 532 1,720 152 520 1,810 160 410 1,666 149 487 1,732 *::.v" 96 115 10.' Table 4. Hiatorio call growth data from an acuta toxicity taat with laopropyl alcohol and tha fraahwatar alga, tfalanaatrua capricocnutua (iron ward, at.al., 1995). Homlnal Concentration of laopropyl Alcohol (g/L) rap. 0 (control) 4.4 1,000 1 2 3 mean * control 1 2 3 mean control l 2 3 mean t control Number of Calla/ml x 10J (hour) 0 24 48 72 96 10 22 134 538 1,132 10 20 132 568 1,260 10 23 114 614 1,356 10 22 127 573 1,249 ---- -- -- 10 14 112 584 1,236 10 32 128 554 1,270 10 16 144 584 1,064 13 21 128 574 1,190 100 95 101 100 95 10 31 136 608 1,370 10 26 144 580 1,338 10 24 106 S 12 1,334 10 27 129 567 1,347 100 123 ;102 . 99 , 108 - * + - Controls 250 mg/L Hours ---- 63 mg/L 125 mg/L. 500 mg/L - * * - i, 000 mg/L II lillE Tabla 5. Avaraga apecifia growth rata and parcant changa from tha control iron tha toxicity taat with tha fraahwatar alga, sal ana* tnun caprlcornu tum, and N2803-3. Nominal Concentration of N-2B03-3 (mg/L) Avaraga specific Growth Rata Parcant changa Prom control aa-Teated aa-Received 24hr 48hr 72hr 96hr 24hr 48hr 72hr 9Ghr 0 (control) 63 32 125 63 250 130 500 1,000 250 500 0.031 0.057 0.052 0.053 0.036 0.056 0.054 0.054 -16 2 -4 -2 0.031 0.050 0.055 0.053 0 12 -6 0 0.000 0.000 0.000 0.000 100 100 100 100 0.000 0.000 0.000 0.000 100 100 100 100 0.000 0.000 0.000 0.000 100 100 100 100 Tabla G. Effective concentration* (ECSOa) from tha toxicity taat with tha fraahwatar alga, Stlmntttrua caprlcornutua, and N2B03-3 Time EC Value (mg/L) 95 percent Confidanca Limits (mg/L) Calculated basad on Test Substanca as-Tasted (50% M2S03-3 and 50% Isopropanol) 1. Calculated Using the Number of Cells/ml 72 hours -- 96 hours EC50 - 180 EC50 - 180 125 - 250 125 - 250 2. calculated Using the Average Specific Growth Rate 72 hours 96 hours EC50 - 180 EC50 - 180 125 - 250 125 - 250 Calculated based on Test Substance as-Received (N2803-3 without Isopropanol) 1. calculated Using the Number of Cells/ml 72 hours 96 hours EC50 90 EC50 9 0 63-130 63 - 130 2. calculated Using the Average Specific Growth Rate K S T COPY A V .il S i i z. Rzrziixjicis Bruca, R.D., and Versteeg. X992. A Statistical Procedura for Modeling Continuous Toxicity Data. Environ. Toxicol, and Chest. Voi. 11. No. 10, pp. 1,485-1,494. Stephan, C.E. 1983. Computer Program for Calculation of LC50 values. U.S. EPA. 1978. The Selenastrum capricornutum prints Algal Assay Bottle Test. EPA-600/9-78-018. Environmental Research Laboratory, Corvallis, Oregon. U.S. EPA. 1993. 40 CPR Part 797. Toxic substances Control Act Test Guidelines; Final Rules. Section 797.1050. U.S. EPA. 1993. 40 CFR Part 792. Toxic Substances control Act (TCCA); Good Laboratory Practice Standards; Final Rule. Nard, T.J., J.H. Magazu, and R.L. Boeri. 1995. Growth and Reproduction Toxicity Test with L-13492 and the Freshwater Alga, Selenastrum capricornutua. Study conducted for 3n Company. T.R. Wilbury Laboratories final report number 841-TH. V - 1BEST COPY AVAI AB '.;.V ..-.V.,^*,-;- ,'. " .a, xv'- ,.> r .',,**f1j .?i*-'1 ,>v^.ju,.. - ' a* '- ^ f i ',.'ts,-- `..V *- .-. . *&'. ': >; )V 4 ' ,, '.t* .V*V y ,`^ ' ' V*5/> - ' fv V \ ' * J. i' *I*v$$ > p 4 ',$ r.V 1 BEST -%V z n . womwiimprotocol axd deviations PROTOCOL AMENDMENTS: The protocol waa amended to specify that tha teat aubatanca would ba combined In a 50:50 ratio with iaopropanol and thla aolution would be conaldered the teat aubatanca at 100% active ingredient and to H a t nominal concentrationa for tha definitive study. h o other amendmenta were made to the protocol. PROTOCOL DEVIATIONS: Tha 24 and 48 hour EC10, EC50, and EC90 values were not calculated. The 72 and 96 hour EC10 and EC90 values and the slope of tha dose response curve could not bo calculated with the data from this test. The pH of test media was 7.4 rather than 7.5 at 0 hr. These deviations did not affect tha outcome of this test and no ottar deviations were made from the protocol. y'J.V 'vi> C`O-3 ,S-W lyiiH K S T COPY AYM lABli M S tU P Y V A im P I 1 S* 5; , ; (control) 63 J2 125 63 250 130 500 1,000 250 500 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 7.4 10.3 7.4 10.3 7.4 10.3 6.8 10.3 6.8 10.3 6.8 10.3 6.4 10.3 6.3 10.3 6.3 10.3 4.0 4.1 4.0 4.1 4.0 4.1 3.4 3.4 3.4 3.4 3.4 3.4 3.0 3.0 2.9 3.0 2.9 3.0