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BACK TO MAIN 3M ENVIRONMENTAL LABORATORY INHIBITORY EFFECT OF L-13492TO MICROBICS* MICROTOXTM TOXICITY ANALYZER SYSTEM REPORT COMPLETED: 7/26/95 Prepared by: Susan A. Beach Adv. Environmental Biologist 3M Environmental laboratory Bldg. 2-3E-09 935 Bush Ave. St. Paul, MN 55144 l BACK TO MAIN MicrotoxTM Inhibition Pagel July 26, 1995 Microbica' MicrotoxTM Analyzer is a temperature controlled photometric device which measures the reduction in bioluminescence of a naturally occurring marine bacterium, Photobactarium phosphoreum, in response to a chemical toxicant The MicrotoxTM System provides a simple and inexpensive method for the rapid screening of microbial toxicity of chemical substances and waste streams. It requires only a small sample size and permits duplicate determinations of one sample in 30-m inutes. Toxicity of a chemical compound is reported as a percent decrease in light output. Similar to conventional bioassay toxicity data (e.g., LC50). EC50 is used for MicrotoxTM data. EC50 is defined as the concentration of a toxicant causing a 50% reduction in light output from the base-line level. MATERIALS AND METHODS Test Substance: L-13492, Lot 2, 45% solids, 5/4/95 Appearance Clear, colorless liquid Aqueous Solubility Apparently completely miscible with water. Physicochemical Properties: See MSDS. This sample contains 44.9% product, 27.2% isopropyl alcohol and 27.9% water. Date Received Not recorded Storage In the dark at ambient room temperature (22 +. 2 C ) Stock Solution Reported values BlanLconlfais: Exposure: Test date 200 mg/100 mL Millipore Milli-QTM Water. No color was apparent. Initial stock solution pH = 4.7; adjusted to pH 6.7 with 0.1 N NaOH. The osmotic strength of the stock solution was adjusted to 2% NaCI by adding 200 mg NaCI to 10 mL stock solution. The reported values were based on nominal concentrations in the test media at the beginning of the microbial assay, Two controls containing bacterial inoculum, but no test substance. it J 5 /1 7 /9 5 ' ' " " ' '' ' ' Contact time 5, 15, and 30-minutes . ;.'! :;W:i:l I|:i!|' m. 11 iii V: j. i; ,!:1!j 1: lfitv ifc ll BACK TO MAIN MicrotoxTM Inhibition Page 2 July 26, 1995 Exposure fcont.t: Starting test concentrations No. replicates Dilution water Temperature Procedure 125, 250, 500, and 1000 mg/L 2 Microbics' 2% NaCI solution 15C (controlled) The test was conducted in accordance with Microbics' MicrotoxTM Toxicity Analyzer, Model 500, Operating manual (1). The concentrations tested and the corresponding response data (percent reduction in light output) derived from duplicated determinations were used to calculate the median effective concentration, "ECso". and the 95% confidence limits by least-squares regression statistics for log log data using a computer program provided by Microbics. RESULTS; ' The percent reduction in light output and the EC50 values obtained after 5, 15, and 30-minutes exposure to the test substance L-13492 are summarized in Table 1. The 30-minute median effective concentration, "ECso" was derived from duplicate determinations and was reported as 260 mg/L with a 95% confidence interval of 220 300 mg/L. The data reported herein are based on tests performed by Gary W . Jorgensen. All raw data generated are archived in the Environmental Laboratory test facility, Bldg. 2-3E, St. Paul, Mn. ; ACCEPTABILITY CRITERIA fc ': 1 ) The control correction factors were > 0.7 at 5-minutes and > 0.6 at 15-minutes of exposure. |! 1:'. f f f :: r ' . ' '. ^ ' ,' T iff 2) The calculated 95% confidence factor for the EC<jo calculation for all three exposure periods i . was < 1.3. ' f 'if;;,'"''" i f ; ). - H if iif ii ' . f 3 ) The correlation coefficient for the EC50 calculation for all three exposure periods was > 0.9. 4 ) The coefficient of determination for the EC50 calcualtion for all three exposure periods was > 0.8. ... 5 ) The reported values were based on nominal concentrations in the lest media at the beginning of the microbial assay. , ti IS : :'U BACK TO MAIN MicrotoxTM Inhibition Page 3 July 26, 1095 GOOD LABORATORYPRACTISES This study was not conducted under strict Good Laboratory Practices, but was conducted under the standard operating procedures for this laboratory. The raw data was reviewed by S A Beach prior to reporting. Copies of all raw data are included with this report This study was conducted under 3M Environmental Lab Request number N2169. REFERENCES 1 ) Microbics Corporation. 1989. How to run toxicity tests using the MicrotoxTM Model 500. Carlsbad, California. f TABLE.1 L-134.9 2 PERCENT LIGHT LOSS(^ Test Cone.. ma/L 125 V 250 : 500 ; 1000 EC50 (95% C.l.) % by Voi. 5-min Exposure 13 19 37 75 630 (590-665) 15-min Exposure 29 43 ' ; 65 `r ' 92 H 300 (270-330) 30-min Exposure 34 si 75 97 260 (220-300) (1) Mean light loss of two replicates. i l i s t ( BACK TO MAIN ENVIRONMENTAL LABORATORY: MICROTOX PREP SHEET TEST SUBSTANCE: A rL V ftZ - 1. Preparation of Stock Solution Mass/Vol. Test Substance: //drw. Final Dilution Volume: 2a IL Diluent: M lL U L A r ^ ~ Final Concentration o f Stock Solution: JdL- 2. pH Adjustment of Stock Solution InitialpH ofStock Solution: f. ~7- *pH Adjusted to: Qjf- pH Adjusted with: ,,-fQlV A Off, Idrof of C.Q5AJ ) c o e c $ O .O Z ^ }^ L O ff 3. Osmotic Adjustment of Stock Solution * Osmotic Adjuster: jm Comments: / U 2 \ L A r i 5rfo -<-a 2 C O ^ MolCJ 4. Appearance of pH and Osmotically Adjusted Stock Solution ^CLEAR^r OPAQUE (circle one) Color: ; ; Comments: i l l " 1- H O : 5. Comments S t a c i S o l a o 2 a jL (eaMeA u p to.lw m .v tiy e rf jg .-tf/e t/ do.u.\J BACK TO MAIN N lCEGTOX v n Th NERCr* r:> 2c f 10 LR K U lffi FILE NAME i N21A9SJ.KS 11*vstiga tor :_ J & U L > ___ .Samle Descriotion: L-13492 uiauid Procedure: BASIC Initial Concentration : 1000 ma/L Teat Time: 3 minutes NUMBER 10/IT TEST DATE: J i o / a s L . TEST TIME:. - U I 3 0 P i Q _ Arjoroved bv : Gamoti.c Adjustment: NaC Dilution Factor : 2 Concentration Unita in o /L CONC. GrtMMA 1 101.53/ 79.33 2 104.71/ 80.56 102.56/ 73.40 4 107.47/ 79.24 5 103.48/ 57.90 6 1O2 2o/ 5o 55 7 99.23/ 21.23 8 99.24/ 22.9o jNTROL IT/10 = 84.34/ 94.33 RATIOS = 0.3994 CORRECTION FACTOR = 0.6939 125.0000 125.0000 250.OOOO 25C'.0000 500.oOOO 500.0000 1000.0000 1000 *oooo 91.24/10 1.23** 0.3925* 0.14595 0. 16453 0.25188 0.21514 0.601254 0.56481# 3.18770# 2.372354 10 ZDVDDDDVDDDVDDDDDDDCDDDODOVDVDDDDODDDDDDDDPOODDDDDDDOCDDDDDD'- 53LOPE -- Z.37P : 3; ' 3 - _ 3: 37 - M A j 3 1 3 ,10 `WDDDDDDDDOODOBO IO 0 . .3 -3 EC50 3 3 3 ODDDDDDDDZVDDDDDDODDDD-DODDDODPDDDDDZ/DDDDDY 1000-'. CONCENTRATION 10E4 ,' 10E5 1` 1 QNF i .9::.'->CE RANGE : TO .4 1 9 1 4 Used tor ca .1-atxon ; 3 ! Cf BACK TO MAIN ht \0 im i sTATSTICAL REPORT FGR DATA RILE N2169GJ.KS E3 ftMATING SQUAT ION t LOO C 0.41 To LOO b -2.747S NUMBEh OR DATA PAIRS USEDJ 4 RESIDUAL VARIANCE: 0.OOO10 9371 CONFIDENCE FACTOR: i.05999 FOR EC50 CORRELATION COEFFICIENT: R a 0.9t So 3 COEFFICIENT OF DETERMINATION: fir2 = 0.99727 EFFECTIVE CONCENTRATIONS <937. CONFIDENCE RAN GEs EC50 =s 627 *7610 mo/l. ECCO SX 350.8651 mo /:. EC IO St 249 *6539 m /L e c o ; as 91.2698 mo/L ( 592.23.T9 TO 1 309.8203 TO < 209.6549 TO < 3.3324 TO 665.4 ;*3) 3?7.34'"6 )**i 297,2960>** 127.010= :*' *** EXTRAPOLATED RESULTS BACK TO MAIN <4IT I mi. DATA NUMBER CONCENTRAIION t 12 3.000 2 1 2 5 . `.`00> ) 2 5 0 . 000 > 4 2 5 o .><_(!.) 5 5 0 0 0 0 0 1< 6 500000i 7 1 0 0 0 ,0 0 0 1i a 1000 OOOt GAMMA . 14 0 0 o o 0 .18a 2 Me. 9 1 C u j^ S N 2 1d - *:-o . }5 EFFECT `HHrhin:-: 1 2 . 7 4* 14- 13 2 0 . 12 L7 .7 0 Z7 , 5 5 7o . 09 7 6 ,1 2 7 4 ,1 6 1 18.9 1 3c*. B2 T ' 5 .15 CiC'ic.uch FACE o m R to continue.... CCCCCCCCCCCCCCCCCCCCl CCi CCCCCi . FINAL RESULTS N2-A.; .Kt NUMBER CONG. GAMMA GAMMA EST. EFFECT 5 500 -0000 0.6013 0 *5828 . 37.54'- 6 GOO,0000 0.5648 0.5*328 ,044 7 1000,0000 3>18/7 3.0260 7n .1,21 6 1000.0000 2.8725 3.0260 74.177 EC50 627.7610 ma/L (937. CONFIDENCE RANGE : 592.2339 TO 665.4193? H,1,!, IH H , i , 1,11 1HH H , , l i l <1l i i l H i l H H H H , l l i m i l l H i l H UH H U - ' - - 1 7 K.*JL_ BAK 1 1j** - '.rlljt; . . . . . , . 7,; , r ; ;iHr!H, u n , I hlH rlH H : :, ' BACK TO MAIN S V fl lO/ROTO/ DATA REr'GRT L% N 21M investigator Samole Descnotions Liauid P r o c e d u r a ! BASIC Initial Concantration 100 0 ilio / L Test Time: 13 minuta* NUMREfi 1 0 /I TEST DATE : TEST TIMEs Aoorovwd ov; Osinotic Ad iy*tmon ts N*C l Dilution Factor : 2 Concentration Unita* mu/L roN C . GAMMA ' 1 101.23/ 63.92 2 104.71/ <t>Z.37 3 102.50/ 50-.39 4 107.47/ Ci'J3w 5 103.48/ 32.23 102.26/ :i.44 7 99.23/ 2.78 3 99.24/ 7.07 wGNTKGL IT'IO 84.08 / 94.3.' RATICS -= tj .;69 i7- uGRRECTION rACTOIR - 0..BS20 1 2 3 .0 0 0 0 :i. 0000 .(500C . 000)0 jU >. o o o t 50'0i . 000" 100)0>. 0 0 0 10 0 . uooc* 01.. 2 3 * * 3727** 0 .40>102# .41 283# 0.79323# 0<. i; 9 7 7# 1.82733# 1.S6SS7# 11.89023 11.04037 i 0 BDDDVODBPBBDDBDBBDO,D O 'OOO, JSLGPE -= 1.091- i 3 3 3 .10 OODBSOCODODOOODODOBVDDDOOVD.DTjOOBUjOOD''DDOPVO'DDODDDDODDDVDY 1/- 1;`i. CONCENTRA T1014 1000 10'E) lONFIOENCE fi.AMi3E 3.71.480i>3d '.'f - c u i a t t o r i t BACK TO MAIN (c 1 6 L g M Z j 6f L STa TISTICAi. REPORT FOP DATA FILE N21o?Gi.Kli ESTIM A TIN G EQUATION . LOG C 0.9002 x LOG o +2.4735 NUMSER OF DATA PAIRS USED: o RESIDUAL VARIANCE 0.00157 957. CONFIDENCE FACTOR: 1.11 +16 FOR EC50 CORRELATION COEFFICIENT: R: - 0.99133 COEFFICIENT OF DETERMINATION: R-'2 = 0.96273 EFFECTIVE CONCENTRATIONS (95>1 CONFIDENCE R A N G E ): EC50 - 297.5171 mo/L EC20 = S5.413I ma/L EC 10 = 41-1607 ma/L EC01 = 4.7533 mq/L ( 267.0336 TO t 66 34 19 TO 29.0734 TO < 2.2759 TO 311.4304' 10c-.74oc. /*** 53.2 701 )**.i 9.9277)** *** EXTRAPOLATED RESULTS BACK TO MAIN I N I f * Hi- CATh NUMBER CONCENTRATION 1 125.0000 2 I25.0000 3 250.0000 4 250.0000 cr 500.0000 O 300.0000 7 1000.0000 'i l0 0 0 0 0 0 0 GAMMA 0 .401 0.413 0.775 0 .677 1.328 1 .36 7 1 1 .890 1I.040 f`j 7 o f 1C L R AjgjgL tC l W .K it 7. EFFECT 28.62 27.22 44.30 4 i.OC 64.63 0-5-14 72.24 9 ..67 "3. '7 3 42 * ? 64 ,*3C ?i. 'itTouch SPACE BAR to continue.... CCCCCCCCCCi.'CCCCCCCCCCCCCCCCCC'.'il' FINAL RESULTS N2167GJ. K i5 NUMBER CGNC. GAMMA GAMMA EST. 7. EFFECT -, . . 1 A*-% 3 4 o 6 125.0000 125.0000 250. 0000 250. 0000 500. 0000 500. 0000 0 . 4C10 0.4127 0 . 7952 0 6973 1. 3276 1.8689 >: O 3S6o 0 . 33t 0 . oZ-^.T 0.3242 1.736.1 28. 624 2''. 31 -4,27 7 . :-83 4, o34 EC50 277.S17I mq/L <757. CONFIDENCE RANGE; 2*7.0336 TG 331.48<>4> ?.* i - -inni*-< >li/1 i l '<-1 >< / , i / . / : nv. i .Wj-f,-;' ' i. < (''. ;'>* iK, . . ... ; ^ J%>, L e t i i. . * a BACK TO MAIN P 3 S' o f 1 0 MICRO Tj Oh Th R^r'Or * LW -i LE NAME ! N2169SJ .K30 lovescia * lor ! Q>tO"3 Samoie DescriotianI L-1 3492 Lioe-.d ProciJur?i BASIC Initial Concentration < 1000 mq/L Ted?. Time: 30 minutes NUMBER IO/IT test d/ t e s t rn 'c : J 2 - J K > P (V1 Approved d v : ____ _ Osmotic Adjustment: NaCi Dilution Factor : 2 CONIC. GAMMA ! 101.53/ 61.47 104." 1/ 60.5* *% - 102.56/ 43.2-. 4 107.47/ 49.49 103.48/ 23.36 6 102.26/ ,72.39 7 99.23/ 2.91 6 99.24 i 3.1c CONTROL IT/IO - 83.59/ 94.33 RATIOS = 0.83-3 1 CORRECTION FACTOR = 0.3940 125.0000 123.0000 2;30.0000 230 .00010 500.0000 300.0000 lv 0.0000 .1000.OOOv 92, .7 102 23411 0.9-319*4! 047o65 0.54o24 1.10762 0.941404 2.7C3 i4 3.04 7024 29.4Eo'-i 2 * (>7o3 iO ' ' l ' l lDDDL'DDWD0lyDDDDDl>DDDDV0u~'''- `0 D D D r i : ~.`DDD0DU0:iU D 0 0 0 B B 0 D r>Vr) p `''0 33l_0AG = ( . i l e ___ ' ' - 3 3- __L' 2 J 1! I .3 1 ZZZZZZZZZZZZZZZZZZZZZZZZZZ 3 3 r \ , 3 3 s' ; 3 3 3 .EC50 . 10 ODDUDDDDDWZOVODDDDly'ODDQDDU .DOOPIDOVDDD-DDPdDOOODDDODl'V-:.DDD-` . tO . 1 0 0 .CONCENTRA f I ON 1 0 0 0 10E4 ' ' To 7.0 t . 777n 1 5>. C X r IDEMCE RAN'.-.'. :-o t . ; 4 i-sed o r r .s ic u l io n s X BACK TO MAIN 10 LR . K lz l^ lATInTICAL REPORT FOR DATA FILE N2.169GJ.K30 ESTIMATING EQUATION: LOG C = 0.5972 :< LOG b +2.4157 NUMBER OF DATA FAIRS USED: 3 RESIDUAL VARIANCE: 0.00003 957. CONFIDENCE FACTOR: 1.16209 FOR SCOO CORRELATION COEFFICIENT : R: = 0 .9997c COEFFICIENT OF DETERMINATION: R'"2 = 0.99951 EFFECTIVE CONCENTRATIONS (957 CONFIDENCE RANGE:: ECCO - 259.2361 tT>q/L EC20 - 113.2775 mq/L EC 10 = 69.7942 mq/L ECOl -- 16.635 nq/L ( 223.07 73 TO 301.2560) < 7e.S.".3i TO 162.7922)M < 425430 TO 114.4939) * t ( >8314 *!J 40 .7 10 '! ** * * * EXTRAPOLATED RESULTS BACK TO MAIN ' <i r I Al. OATA IMBEP CONCENTR-h TION i "? 3 4 5 7 8 225.0O00 125.OOOO 250.0000 2 5 u .0000 500.0000 500.0000 1 0 0 0 .0 0 0 0 1000.OOOO CAMMA 0.4 :7 0.54a 1 .108 0.94 L 2 .9,-0 3.04 7 29.48 27.0 7-7 3 io^-f \ o L& Mz i & L Na loSGo . t-0 1 ErPECT 32.23 35.33 52.56 48.49 74.75 '3.29 96.72 96.44 3 -*50 75 '-'3 fTi"Touch SPACE EAfi: i o continue..,. CCCCCCZCCCCCCCCCCCCi CCCCCCCCCCCC rlNAL RESULTS NUMBEn: CONO. 4 2500000 D 500.000C o 500.0000 GAMMA 0.9414 2.9605 3.0470 GAMMA EST. 0.9414 Z .00.'3 .'ji.,.33 N 2 169GJ .k'30 *. crrECT 45.491 74.749 75.2 90 EC50 257.2361 m?/L <95% CCNr .'DEMCE RANEs 22.;... / '3 TQ 301.25o0.< :c T f o u c n P - . ' : : ; . A P n o c o n c i n u * . . . -' E 3 ; : :: c c C l CC 0 L n ' 0 3 /2 9 /2 0 0 1 ENVIRONMENTAL LABORATORY ARCHIVED FINAL REPORT BACK TO MAIN LAB REQUEST NO. N2169 REQUESTOR NAME: SAB/S.J. MARTIN DEPARTMENT: 3048 PROJECT NO: BI703004UQ DATE RECEIVED: 05/17/1995 DESC: L-13492 ECOTOX TESTING CONTRACT LAB(S): EXP COMP DATE: 07/03/1995 DATE COMPLETED: 01/30/1996 PROJECT LEAD: PHONE NO: 3M FAX NO: 651-778-6176 Results from Microtox Test: Test Cone. mg/L 125 250 500 1000 EC50 95% C.I. sab 5/19/95 PERCENT LIGHT LOSS 5-min exp. 15-min exp. 13 29 19 43 37 65 75 92 630 300 (590 - 665) (270 - 330) 30-min exp. 34 51 75 97 260 (220 - 300) Activated Sludge Respiration Inhibition Test Results: There were some difficulties encountered in obtaining test results. The initial screening (limit) test indicated toxicity and the need for a study with lower concentrations. The second study showed low toxicity. The third study was invalid due to unacceptable differences in control respiration rates. The fourth study also indicated low toxicity. The final study was initiated to confirm the second and fourth study results. It is possible that in the first study an error in test solution preparation resulted in loading the test system with more test substance than planned. This could have induced a greater inhibition in respiration rate. SAB 7/31/95 Test Cone. mg/L 1000 Duplicate Percent Inhibition in Respiration Rate 30-min Exposure 3-Hour Exposure 0 18 0 22 EC50 >1000 >1000 SAB 7/31/95 NOTE: 3 -HR results updated 2/19/96. An error was discovered in calculations of percent inhibition, sab 2/19/96 SEE LR N2332 FOR FISH, DAPHNIA, AND ALGAE TOXICITY TEST RESULTS. SAB 7/31/95 SAMPLE DATE CODE DESCRIPTION RESULT MDL or (95% C.I.) 1 05/04/95 L-13492 LIQUID MICROTOX MICROTOX - EC50 260 MG/L (220 - 300) 03/29/2001 ENVIRONMENTAL LABORATORY ARCHIVED FINAL REPORT 2 -AB REQUEST NO. N 2169 SAMPLE DATE CODE CONTINUED DESCRIPTION RESULT 1 05/04/95 L -13492 LIQUID SLUDG-RESP SLUDGE RESPIRATION - EC50 >1000 MG/L BACK TO MAIN MDL on (95% C.I.)