Document 91xwaGjRQdrg2g87aBVBEjpXe

M w- m 485 Accession No. Study No. S03-4101 14101 M M C- M Study Report .I I I (Study off] H l l ^ e g r garaddation by Microorganisms (non-GLP study) October 16, 2003 Chemicals Evaluation and Research Institute, Japan --Kumme Laboratory- Company Sanitized. Does not contain TSCA CBI Tables Table 1. Table for calculating percentage biodegradation by BOD Figures Fig. 1 Chart of BOD Fig. 2 Mass fragmentograms of LC-MS analysis for calibration curve, and calibration curve Fig. 3 Mass spectra of test substances Study No. 14101 2 Company Sanitized. Does net contain TSCA CB1 Table o f Contents T i tle ......................................................................................................................................... 4 Commissioned by.................................................................................................................. 4 Testing Facility............................................................................................ 4 Purpose of Study................................................................................................................... 4 P ro c e d u re s .............................................................................................................................. 4 Schedule.......................................................... 4 Personnel........................................ 4 Approval of Study R eport................................................................................................... 4 S u m m a ry ................................................................................................................. 5 1. Test Substance...................................................................................................................... 6 2. Activated Sludge...................................................................................................................7 3. Conducting the Study........................................................................................................... 8 4. Confirming the Study Conditions..................................................................................... 12 5. Study R esults...................................................................................................................... 13 Study No. 14101 3 Company Santifeed. Does not contain TSCA CBT Tide: Study ow study) Degradation by Microorganisms (non-GLP Commissioned by: DuPont KK 1-8-1 Shimo-meguro, Meguro-ku, Tokyo 153-0064 Testing Facility: Kurume Laboratory Chemicals Evaluation and Research Institute, Japan (CERI) 19-14 Chuo-machi, Kurume-shi, Fukuoka Prefecture 830-0023 Purpose of Study: To determine the degree to w h i c h H R H H H H H p degradable by microorganisms. Procedures: . This study was carried out in accordance with the following test procedures. (1) `Testing the Degradability of Chemicals by Microorganisms" in Test Methodsfo r New Chemicals (Notification No. 5 of the Planning and Coordination Bureau, Environment Agency; Notification No. 615 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare; 1974 Notification No. 392 of the Basic Industries Bureau, Ministry of International Trade and Industry; dated July 13,1974) (2) "Ready Biodegradability: Modified MOT Test (T) (Guideline 301C, July 17,1992)," in OECD Guidelinefo r Testing o f Chemicals Calculations of biodegradability were carried out using only oxygen demand. Schedule: Study began on: Testing period: Study completed on: April 30, 2003 July 2, 2003 to July 30,2003 October 16,2003 Personnel: Coordinator: Researchers: Tomohiko YOSHIDA* Testing Section 1 Tomohiko YOSHIDA Yasuko MATSUNOBE Masanori TARUKI Approval of Study Report: [signed] Tomohiko YOSHIDA Study Coordinator October 16, 2003 * Translator's Note: Spellings o f personal names may need to be verified. StudyNo. 14101 4 Company Sanitized. Doss nef confsfn TSCA C- Summary Study of. 'egradation by Microorganisms (non-GLP study) Study Conditions (1) Concentration of test substance: lOOmg/L-. (2) Activated sludge concentration: 30m g/L l| (3) Amount of test liquid: 300 mL (4) Test liquid incubation temperature: 251C (5) Test liquid incubation time: 28 days Measurement The biochemical oxygen demand (BOD) was measured using a closed oxygen demand analyzer. Test Results Percent degradation, based on BOD: 18%, 16%, 17% Average: 17% Study No. 14101 5 Company Sanitized. Does not contain TSCACB'i 1. Test Substance Lot No. The test substance was regarded as having a purity o: n Study No. 14101 6 CoWpfty Sanitized. Doss not contain TSC CE; 2. Activated Sludge 2.1 Places and Times of Sludge-Collection (1) Places of Collection: Sludge was collected from the following ten places throughout Japan. Fushiko River Treatment Plant (City of Sapporo in Hokkaido) Nakahama Treatment Plant (City of Osaka in Osaka Prefecture) Kitakami River (City of Ishinomaki in Miyagi Prefecture) Yoshino River (City of Tokushima in Tokushima Prefecture) Hiroshima Bay (City of Hiroshima in Hiroshima Prefecture) Fukashiba Treatment Plant (Kashima-gun, Ibaraki Prefecture) Ochiai Treatment Plant (Shinjuku-ku, Tokyo) Shinano River (Niigata City, Niigata Prefecture) Lake Biwa (Otsu City in Shiga Prefecture) Dokai Bay (City of Kitakyushu in Fukuoka Prefecture) Samples taken from streams, lakes and the sea were topsoil at the water's edge in contact with both the surface of the water and the atmosphere. Samples taken from wastewater treatment plants were return sludge. (2) Time of Collection: March 2003 2.2 Processing of Activated Sludge In order to maintain the uniformity of the activated sludge, 5 liters of filtrate from a mixture of the above sludges collected from various sites was mixed with 5 liters of filtrate from activated sludges that were cultured for about 3 months.*1 The mixture was adjusted to a pH of 7.01.0, then aerated in the culturing tank by passing air through a prefilter. *1: Activated sludge obtained by culturing, in accordance with section 2.3 below, 10 liters of filtrate from a mixture of the above sludges collected from various sites. 2.3 Culturing Aeration of the culture tank was stopped for about 30 minutes, following which supernatant accounting for about one-third of the total amount was removed. To this was added dechlorinated water such as to bring the total volume up to 10 liters. Next, aeration was again carried out (for at least 30 minutes), after which 50 g/L synthetic wastewater*2 was added so as to set the concentration of synthetic wastewater within the added dechlorinated water to 0.1 wt%. Activated sludge was obtained by culturing in which the above operations were repeated once a day. The culturing temperature was set to 252C. *2: Glucose, peptone and potassium dihydrogen phosphate were dissolved to respective concentrations of 50 g/L in purified water, and adjusted with sodium hydroxide to a pH of 7.0+1.0. Study No. 14101 Company Sanitised. Does nc? contain 7SCA CBI 7 3. Conducting the Study 3-1 Initial Preparations for the Study (1) Measurement o f Suspended Solids in Activated Sludge: The suspended solids concentration was measured in order to determinate the amount of activated sludge to be added. Measurement method: Carried out in accordance with "Plant W astewater Test Methods - Suspended Solids" (US K 0102-1998, section 14.1) Measurement date: June 30,2003 Measurement results: The activated sludge had a suspended solids concentration o f 4,600 mg/L. (2) Preparation o f Basal Culture Medium: Purified water (produced by Takasugi Pharmaceutical; The Pharmacopoeia of Japan) was added to a mixture of 3 mL each of liquids A, B, C and D described in "Plant Wastewater Test Methods --Biochemical Oxygen Demand" (JIS K 0102-1998, section 21.) so as to bring the total volume up to 1 liter, and the pH was adjusted to 7.0. (3) Reference Substance: Aniline was used as the reference substance to verify that the sludge has a sufficient level o f activity to carry out the study. The reference values for percent degradation of aniline after 7 days and after 14 days, as determined from the BOD, were set at more than 40% and more than 65%, respectively. Study No. 14101 Company Sanitized. Does not contain TSCA CBT 8 3.2 Preparation of Test Liquids Six test vessels were provided and the test liquids were prepared using the methods described below. These test liquids were cultured under the conditions given subsequently in section 3.3. (1) Addition of Test Substance and Aniline: (a) (Water + test substance) system (one: test vessel [6]) Purified water (300 mL) was placed in a test vessel, then 30 mg of the sample furnished was precisely weighed out on an electronic balance and added so as to give a test substance concentration of 100 mg/L. (b) (Sludge + test substance) systems (three: test vessels [2], [3] and [4]) The basal culture medium [in an amount obtained by subtracting the amount of activated sludge added (1.96 mL) from 300 mL] was placed in a test vessel, then 30 mg of the sample furnished was precisely weighed out on an electronic balance and added so as to give a test substance concentration of 100 mg/L. (c) (Sludge + aniline) system (one: test vessel [1]) The basal culture medium [in an amount obtained by subtracting the amount of activated sludge added (1.96 mL) from 300 mL] was placed in a test vessel, then 29.5 fiL [amount added of 30 mg = 29.5 xL x 1.022 g/cm3 (density)] was collected and added so as to give an aniline concentration of 100 mg/L. (d) Sludge blank system (one: test vessel [5]) The basal culture medium [in an amount obtained by subtracting the amount of activated sludge added (1.96 mL) from 300 mL] was placed in a test vessel. (2) Inoculation of Activated Sludge: The activated sludge prepared under the conditions in section 2. above was inoculated into above test liquids (b), (c) and (d) in such amounts as to give suspended solids concentrations of 30 mg/L. Study No. 14101 9 Company Sanitized. Does not contain TSCA CBf 3.3 Test Liquid Culturing Apparatus and Ambient Conditions (1) Closed Oxygen Demand Analyzer: Constant temperature tank and measurement unit: Okura Electric Data processor: Asahi Techneion Test vessel: 300 mL incubation bottle (modified incubation bottle) Carbon dioxide absorber: Soda lime, No. 1 (for carbon dioxide absorption; made by Wako Pure Chemical Industry) (2) Ambient Conditions: Test liquid culturing temperature: Test liquid culturing period: Stirring method: 251 C 28 days rotary stirring with a magnetic stirrer (3) Conducted at: 511 Kuuro [phonetic] Room 3-4 Observation. Measurement. Etc. (1) Observation: The general condition of the test liquid was visually examined on a daily basis during the culturing period. Operation of fee equipment was inspected as appropriate. (2) Measurement of Biochemical Oxygen Demand (BOD): Changes in the BOD of the test liquid were automatically recorded and measured with a data processor during the culturing period. The temperature within the tank was measured and recorded on a daily basis. Study No. 14101 Company Sanitized. Doss not contain TSCA CBS 10 3.5 Establishment of Conditions for Quantitative Determination of Test Substance bv liq u id Chromatoaraphv-Mass Spectrometry Conditions for the quantitative determination of the test substance were investigated using liquid chromatography-mass spectrometry (LC-MS). However, because this study was terminated on July 28, determinations of the test substance remaining in the test liquids were not carried out following the end of the culturing period. (1) Quantitative Determination Conditions: Instrumentation: Liquid chromagraph-mass spectrometer High performance liquid chromatograph: Alliance 2690 (Waters) Mass spectrometer: ZMD (Waters) Liquid Chromatography Conditions Column: L-column ODS (CERI); 16 cm x 2.1 mm I.D. Column temperature: 35C Eluant: A: 10 nxmol/L acetic acid/ammonium/methanol solution B: 10 mmol/L acetic acid/ammonium/water solution A/B (8/2 V/V) Flow rate: 0.2 mL/min Injection rate: 3 fiL Mass Spectrometer Conditions Ionization method: Detected ions: Detection method: Selected ions (m/z): Ion source temperature: Desolvation system temperature: Cone voltage: Electrospray (ESI) Negative ions Selected ion monitoring (SIM) 549, 569, 649, 669, 749, 769 (see Fig. 3) 110C 400C 30 V (2) Preparation of Calibration Curve: A 1,000 mg/L solution of the test specimen was prepared by precisely weighing out 100 mg of the specimen furnished, then dissolving it in methanol. This was diluted with methanol and methanol/purified water (7/3 V/V) to form 1.50, 3.00 and 6.00 mg/L standard solutions. Quantitative determinations were carried out under the conditions in (1) above, and a calibration curve was prepared using the concentrations and the total peak areas in the respective mass fragmentograms (see Fig. 2). Study No. 14101 Company Sanitized. Does not contain TSCACBI 11 3.6 Computinu the Percent Degradation H ie percent degradation of the test substance was computed from the following formula. H ie results were expressed as integers by rounding the first decimal place to the nearest integer. Percent degradation as determined from the biochemical oxygen demand (BOD) Degradation (%) = [(BOD - B)/TOD*3] x 100 where BOD: Biochemical oxygen demand of (sludge + test substance) system; . measured value in milligrams (mg) B: Biochemical oxygen demand of sludge blank system; measured value in milligrams (mg) TOD3) Theoretical oxygen demand required to completely oxidize the test substance; calculated value in milligrams (mg) *3^C alculate^ssum ingatestsubstance purity o f ^ j j |^ [ and using the frm the molecular formulas and contents of the respective components in the test substance. 4. Confirming (he Study Conditions The degrees of aniline degradation after 7 days and 14 days, as determined from the BOD, were respectively 59% and 76%, thereby confirming that the test conditions used in this study were appropriate. * TN: Due to poor legibility o f the Japanese original, subscripts in formula may need to be verified. Study No. 14101 12 Company Sanitized. Does not contain TSCACBI 5. Study Results 5.1 Condition of Test Liquids The conditions of the test liquids are shown below. At start o f culture At end o f culture Test liquid (water + test substance) system (sludge + test substance) system (water + test substance) system (sludge + test substance) system Condition Test substance did not dissolve. Test substance did not dissolve. No insolubles were confirmed. Sludge growth was confirmed. Insolubles other than sludge were not confirmed. 5.2 BOD Measurement Results for Test Liquids BOD and degradation after 28 days are shown below (see Table 1 and Fig. 1). BOD 4 mg Degradation % (water + test substance) system [61 0.3 -- (sludge + test substance) system T21 T31 T41 6.1 5.3 5.6 18 16 17 Theoretical value 33 9*3 isicj -- *4: Values for the (sludge + test substance) systems are values obtained after subtracting the value for the sludge blank system. Study No. 14101 13 Company SanFffzed. P oes not contain TSCA (W T&b.Ls C alculation ta b le fo r the p ercentage biodegradation by BOD. Test No. 14101 C u ltiv a tin g Durations 28 days Vessel No. a s n Jl m 7th day BOD Deg. (eg) <K> 55.1 59 .... 1 .4 - 6 .5 15 5 .8 13 6 .3 14 0.0 - 14th day BOD Deg. (ng) <) 71:5 78 2.9 T 5.5 17 7.9 15 3.5 17 0.3 - 2 1 st day BOD (ag) Dmeg. 74.2 78 4.0 - 10.0 18 9 .3 16 9 .7 17 0.3 - 28th day BOD (mg) Dmeg. Mean DH eg. 74.3 78 4.6 10.7 18 17 9 .9 16 10.2' 17 ' 0.3 - Deg. : Percentage biodegradation V essel no. HJ Vessel no. V essel no.jH HJ S3 V essel ao. S3 : Sludge + A n ilin e : Control blank [BJ : Sludge * T est substance : Water + T est substance T est substance o f 30 mg was added. Chart o f BOD : F ig . 1 Deg. [ BOD - B 3 / [ TOD 3 X 100 (*> TOD^of 'test" su b sta n ce s' '33.9 (mg) ia 689.57 /' 610.37 1 . 1 3 TOD o f a n ilin e : 9 0 ,3 (ag) CbBtN * 8 .7 8 0 -+ 6 CO* * $ .5 H*0 NO# 8 .7 5 Oa / CaHi-N - 2 7 8 .9 9 / 9 3 .1 3 3 .0 1 TOD => 3 0 .0 X 3 .0 1 9 0 .3 (a g ) . 2 0 0 3 .0 7 .3 1 Name / --T Study No. 14101 14 Comoanv Sanitized. Does not contain tscac P lg.l Chart o f BOD T est No. 141OJ. ( T est substance Apparatus ....................................... * ' C ultivatic? conditions: Concentration T est substance ............. Reference substance( aniline A ctivated sludge ...................... Temperature ............................ : ` 1 Duration ............................................ Note: _ No. CM-12 loo (mm 100 (m m 2S30 (BVg/c) 28days(Jul 2 - J u l .3 0 ,,2003) V essel Sample d e s c r ip tio n no. 0 Sludge * A n ilin e B Sludge * T est substance 3 Sludge + T est substance Sludge * T est substance m Control blank [3] HI Water * T est substance 100' 7thday S S .l S 15 5.S 3.3 1 .4 0.0 B O D (mg) 14thday 71.5 8.5 7.9 8.5 ' 2.9 0.S . S lsid a y 74.2 10.0 9.3 9.7 4.0 0 .3 28thday 7 4 .9 ID .7 9.9 10.2 4.6 0.3 a so& 30- 3 40 33 20- -- --Hj 0J ---------- l "*T "f-T" ` T T " T...I....I--J--I - r - r 14 21 t *i --1--2r8~ C u ltiv a tio n Time ( 0~28thday) 2003.07.31 Name Study No. 14101 15 Company Sanitized. D oes not contain TSCA CB Study No. 14101 Study No. 14101 16 CoWpliy Sanitized. Doss noi contain 7SCACB J' Study No. 14101 Cmeeatedtoa (rag/L) y - 308?Lx i 1,00 CoaeentrettMi iW V ) 130 3.00 6.00 Pesfeir <-) 45654.84 91489.66 186111.78 Fig. 2 - 2 Calibration curve oftest item. October6*2003 Nome Study No. 14101 Company Sanitized. C oes not contain 7SC& CBl 17 Instrument MS : Water ZMD, HPLO t Waters At[lanoe25SO Sample Standard solution fl0.0 mn/L Study No. T4101 HPLO Conditions Inlet system Column Sample size 2 uL {Solvent Methanol / purified water {2/3 V/V ) Column L-oolumn OPS (OERI) Column size 15 cm x 2.1 mm IX). Eluent A; IQmmoI/L Ammonium estate In msthanal B: lOrnmol/L-Amroonium asetata In water A/B f8/2V/V) Row rate 02 mL/min MS Conditions ionization mods ESI Detaotiorc mode Negative Function MS Probe ESI Capillary 3 kV. Desolvatton temp. 400 *C. Desalvation gas 400 L/hr APol Corona - kV, APol probe tamp. - C Souroe Cone 30 V. Extraotor. 2_V. RF Lens 0.2 V Source block temp. 110 "0 MSI Ion energy 0.2 V. Multiplier S50 .V Mass range (m/z) 400 - 1000 Set ton m/z Fig. 3-1 Kurame Laboratory! Chemisais Evaluation end Research Institute. Japan Mass spectra of test item (analytical conditions). Study No. 14101 Company Sanitized. Does not contain TSCkCSI 18 Study No. 14101 Study No. 14101 Cotttpsny Sanitised. B oss noi contain TSCA. CBI Study No. 14101 Study No. 14101 20 Company Sanitised. Does not contain TSCA CB? Peak 1 . m /z 549 569 Peak 2 Study Nq. 14101 M .W . 570 Fragment ion (M -H F -H T (M -H )" M W . 670 m /z Fragment ion 649 ( M - H F - H ) " 669 ( M - H ) " Fig. 3*4 Mass spectra of test item (presumed fragments o f peak 1 and peak 2). Dite : `Mei, /g. P Naro:.. A Study No. 14101 21 Ctpsny Sanfffeeii. Does nef contain TSCA CEI Peak 3 Study N o . 14101 M.W. 770 ml z Fragment ion 749 ( M - H F - H ) ' 769 ( M - H ) ' Pig, 3-5 Mass spectra of test item (presumed fragments o f peak 3). Date; 2oe?> fa. ? Name; Translation: Language Services F. Metreaud December 3,2003 Study No. 14101 22 Ccttipany Sanitized Doss not contain TSCCBfc