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BACK TO MAIN INHIBITORY EFFECTS OF L-13492 ON ACTIVATIED SLUDGE RESPIRATION ~ jIl REPORT COMPLETED: July 26,1995 Prepared by: SusrvlA. Beach Advanced Environmental Biologist 3M Environmental Laboratory Bldg. 2-3E-09 935 Bush Ave. St. IPaul, MN 55144 BACK TO MAIN I t P Activated Sludge Reeplration Inhibition Page 1 July 26, 1QQ5 23JMWiY The purpose of this stu& was to evaluate the inhibitory effect of L-13492 on aerobic sludge system inaccordance with the 'Activated Sludge Respiration Inhibition Telst', OECD No. 208. The median effective concentration, 3-hour EC50, and the 95% confidence interval were reported as > 1000 (-) mg/L. lMmtuau Toxicity tests with aerobic sludge provide a rapid screening method and are relieble predictors of the inhibitory effects of potential contaminants on the operation of activated sludge treatment systems. This toxicity study measured the respirat;oo rate of activated sludlge obtained from a municipal wastewater treatment system and followed decreases in Uie rate of oxygen consumption over a relatively short period (30-minutes to 3-hours) in the presence of different concentrations of L-13492. WTFRIAI S AND bEIHQQs Jest Substance: Appearance L-13492, Lot 2, 45% solids, 5/4/95 Clear, coiorlass 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 rocim temperature (22 2 2OC) Stock Solution Reportedvalues . MA; direct weights were used The reported values were based on nominail concentrations in the lest media et the beginning of !he microbial assay. Controls: Functional Control (Reference substance) phenol (3.5-DCP). Aldrich Chemical Company, P.O. Box 355, Milwaukee, WI, 5320. Lot 00410K2, Received 9/28/94. Blank control (untreated) Two test vessels containing inoculum and nutrient medium, but no test or reference chemicals, one at the start and Uie othor at the end of the test series. BACK TO MAIN Activated Sludge Respiratior1 Inhibition Page 2 July 26, 1995 Microbial Inoculum Activated sludge mixed liquor was obtained from the St. Paul, Mn - MetropolitanWastewater Treatment Plant on test day. The suspended solids level was adjusted to 3.8 f .148 fl.; pH 6.0 7.7; and the dissolved oxygen concentration at or near saturation. Nutrient Medium Synthetic sewage feed prepared on test day as outlined in the method. Exoosure: ScreeningTest Datos: 5/22/95, 611195, 6115.195, 6/22/95 Final Test Date 6/29/95 Contact Time 30-minutes and 3-hour while aeration takiss place. Vessels 1200 mL Borosilicaie glasf;Fleakersw vessels Test Solution Vol. 500 mL Starting Test Conc. Test Substance 1000 mgL, in duplicate ReferenceSubstance 10 mgL Limit Test: I I/ A limit test may be performed at 1000 m#L (in duplicate) in order to determine that the EC50 i:; greater than this concentraticrl. If 2 5 0 % inhibition in respiration is seen, then a full test should be conducted. Mixed Liquor Suspended Solids Level in Test Media 1.6 g L Millipore Milii-OTMwater 18.6 - 21.0 O C t (I Mixin-s and Aeration ' Measurements /I , 3 1 Procedure nthually aerated with clean, oil-free air. ations (using dissolved oxygen meter and BOD flasks) were measured after 30-minutes end 3-hours contact time. The test was conducted in accordancewith the "Activated Sludge Respiration Inhibition Test', OECD No. 209 ('1. Values were reported to different levels of significaiice depending on the accuracy of the measuring devicesinvolvedin any one process. BACK TO MAIN Activated Sludge Respiration Inhibition Page 3 July 26, 1995 I2uulxm The reportedvalues are based on nominalconcentrationsin the test media at the beginning of the test, The concentrations tested and the corresponding response data derived from the toxicity test are used to calculate, if possible, the medial effective concentralion, 'EC50' a d the 95% confidence interval by standard logistic regression methods (Le.. Probit Analysis, Trknmed Spearman-Karber). The EC5o is defined as the concentration estimated to inhibit the rate of respirationby 50% as compared to the controls, after 30-minutes and 3-hours of exposure to the test substance. 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 concentraticns. The second study shcwed low toxicity. The third stcL, was invalid due to unacceptable differlances in control respiration rates. The fourth study also ii.c%ated 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. The definitive percent reduction in respiration rate and the median effective concentration, 'EC50'. obtained after 30-minutes and 3-hours exposure to the test subsktnce L-13492 and reference substance 3,5-Dichlorophenol are summarized in Table 1. The median effective concentration, 3-hour 'EC50' of L-13492 to activated sludge was determined and was reported as z 1000 mgL. A 95% Confidence Intervalcould not be calculated. ACCFPTABlLllY C R r m This toxicity test meets quality criteria provided in test guidelines: 1) The two blank control respiration rates were within 15 percenl of each other. 2) The 3-hour percent inhibition induced by the reference substance, '3.5-DCP', was within the range of values historically obtained using thils mixed liquor source in this laboratory. -- 3 ) The reportedvaiues were based on nominalconcentratioris in the test media at the eginning of the microbial assay. This test was conductedby G.W. Jorgensen as requestedby Steven J. Martiri of 3M SCD Lab, SI.Paul, MN. This study was not condircted under strict Good Laboratory Practices, but was conducted under the standard operating procedures for this laboratory. The [rawdote was reviewed by SA. Beach prior to reporting. Copies of all raw data are included with this report. This study was conducted under 3M EnvironmentalLab Request number N2169. BACK TO MAIN Activated Sludge Respiration Inhibition Page 4 July 26, 1995 EEEEEam 1. OECD (1984). Activated Sludge Respiration lnhbition Test, Test No. 209. In: Guidelines for Testing of Chemicals. Orpfkation for Economic Cooperation and Development, Paris, France. 2. OECD. 1981. Principles of Good Laboratory Practice. Annex 2, C(B1)30 (Final):7-28. OECD Guidelinesfor Testing of Chemicals. Organization for Economic Cooperah and Development, Paris, France. -1000 Duplicate EC50 FXDQSULB none 7 1000 3 B > 1000 Not calculable Not ciilculable ACTNATED SLUDGEMIXED UQUOR BACK TO MAIN I PERRENCE SUBSTAN= Test Temperature Range: Comments f x (c10210 OC <"y {t'lv\r` \ I . - BACK TO MAIN ENVIRONMENTAL LABORATORY OECD ACTNATED SLUDGE RESPIRATION INHIBITIONTEST #209 TEST SOLUTIONS PREPARATION L- Test Substance: c I . . Sample Description: I3491 en r lb !AIJ Y BACK TO MAIN ENVIRONMENTAL LABORATORY OECD ACTIVATED SLUDGE RESPIRATION INHIBITION TEST 11209 RAW DATA 7 i. 30-MINUTE EXPOSURE DISSOLVED OXYGEN c I o N c m n a s ,mgrl DISSOLVED OXYGEN c m c m n m s , m s / ~ BACK TO MAIN i CA wt _ Son l FE Eitle Se e e NSeL is To e ouhn es eo e use per Th spee ee epe eR sTPenene E PrmRaaO) Pe)) J pbpee em E + e + mpE eaS EEE p e uf ss oe pT e me ntpe.pr epp raoeEmEaRE) cy=soaisl==F=EE = E Eee=b=-eE F ==b_e -- H= - re-- e e eF ----R --e e--e e ------ = e i et, iE i Pe = + am BACK TO MAIN I ------ : { LpeySNet2/8 i i; ENVIROMENTAL LABORATORY LAT HRT i pA rSounnplesoDoms.er:3M.i5 DcihckClhcaorpihceesCea! RReofdoLcaesbceelSLuobbsheacyeickcz Any: owe: sicksoon es Snir 03th Re L e Hien R e -- e a-------- coms ratios. mio)wa olor semis chy Touveka peg t a Te2m74p WO7R3 e 200 T $20 = _ I -- _ = : i __-- -- = T _ V--__--s-- WR an py : || BACK TO MAIN Test Substance: ENVIRONMENTAL LABORATORY OECD ACTIVATED SLUDGE RESPIRATION INHIBITION TEST #209 RAW DATA .1 3 ?-D,hL/ hN d r o,_o 0 NCP Date ~ t ~ ~ i 6'~ eZLd7: Analyst: >i3 e be 2mi0o8d = gn Bm B83 BEBeagd) i=EEA = E=e BACK TO MAIN P S RS tpe et A fom R! Eeine rrRew os f ee EP EEEr EE ee e e e eer e efoen | + SE bbetet oy -- ent a Eh e h eee fetesfoTeToo asTaSeTSasTeaatsaTor oeToe] [RERee) a E === E b Demmnme Ae E p-- aTs 2x| BACK TO MAIN LFvr Po b ex F pJfise. wot p( g Ge REEee R=] LpRES SREwoia as| 5 ------ senda As asl [TTTTNT 7 jE es(Vomit CCT x fais Soda ala | [CTC Bas@wens mesmeylems wlTTT LL i i ES0 | fer TT ia 1 mods. gpilin Ef Trrrr wha LTTr 1 B dees sg CHE AA A eae ee LL LLL pL Er NE | BRE EINECe Naa| ge (Oa BLE--Ido (1FuckJaa 450 poslel 75 | OS oa jem oT) [OJ TT TTT `wa i ORNS fate | ian eo |saws | oes wwe: EE | >oo Ey ||| 04/01 /2001 ENVIRONMENTAL LABORATORY ARCHIVED FINAL REPORT 1BACK TO MAIN LAB REQUEST NO. N2169 REQUESTOR NAME: SAB/S.J. MARTIN DEPARTMENT: 3048 PROJECT NO: BI703004UQ DATE RECEIVED: 05/17/1995 DESC: L-13492 ECOTOX TESTING CONTRACT LAB(!$) : EXP COMP DATE: 07/03/1995 DATE COMPLETED: 01/30/1996 PROJECT LEAD: PHONIE NO: 3M FAX NO: 65'1 -778-6176 Results from Microtox Test: Test Conc. 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 (590 - 630 665) 300 (270 - 330) 30-min exp. 34 51 75 97 260 (220 - 300) Activated Sludge Respiration I n h i b i t i o n Test Results: There were some d i f f i c u l t i e s encountered i n o b t a i n i n g t e s t r e s u l t s . The i n i t i a l screening ( l i m i t ) t e s t i n d i c a t e d t o x i c i t y and t h e need ,for a study w i t h lower concentrations. The second study showed low t o x i c i t y . The t h i r d study was i n v a l i d due t o unacceptable d i f f e r e n c e s i n c o n t r o l r e s p i r a t i o n rates. The f o u r t h study a l s o i n d i c a t e d low t o x i c i t y . The f i n a l study was i n i t i a t e d t o confirm t h e second and f o u r t h study r e s u l t s . I t i s p o s s i b l e t h a t i n t h e f i r s t study an e r r o r i n t e s t s o l u t i o n preparation r e s u l t e d i n l o a d i n g t h e t e s t system w i t h more t e s t substance than planned. This could have induced a g r e a t e r i n h i b i t i o n i n r e s p i r a t i o n r a t e . SAB 7/31/95 Test Conc. mg/L 1000 Duplicate Percent I n h i b i t i o n i n Respiration Rate 30-min Exposure 3 -Hour Exposure 0 18 0 22 EC50 >loo0 >loo0 SAB 7/31/95 NOTE: 3-HR r e s u l t s updated 2/19/96. An e r r o r was discovered i n c a l c u l a t i o n s of percent i n h i b i t i o n . sab 2/19/96 SEE LR N2332 FOR FISH, DAPHNIA, AND ALGAE TOXICITY TEST RESULTS. SAB 7/31/95 1 05/04/95 L-13492 LIQUID MICROTOX MICROTOX - EC50 260 MG/L. 04/01 /2001 ENVIRONMENTAL LABORATORY ARCHIVED FINAL REPORT (220 - 300) 2 1 05/04/95 L-13492 LIQUID SLUE-RESP SLUDGE RESPIRATION - EC50 >lo00 MG/L BACK TO MAIN