Document KGB01Vb9M7Lx8oQ18r7rkgwDK

AR226-3108 Huntingdon CONFIDENTIAL DPT 438/984469 Ij >3 ASSESSMENT OF READY BIODEGRADABILITY MODIFIED STURM TEST Sponsor Dupont Speciality Chemicals, Jackson Laboratory, Chambers Works, Dcepwater, NJ 08023, USA. Research Laboratory Huntingdon Life Sciences Ltd, Eye. Suffolk, IP23 7PX, ENGLAND. Page 1 of 19 Final report issued: 18 December 1998 Company Sanitized. Daes nof contain T3CA CB! DPT 43 8/984469 CONTENTS COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS.................... Page 3 ! QUALITY ASSURANCE STATEMENT............................................................................ 4 ]. CONTRIBUTING SCIENTISTS.......................................................................................... 5 1. 6 SUMMARY.......................................................................................................................... 7 INTRODUCTION................................................................................................................. TEST SUBSTANCE............................................................................................................. 8 EXPERIMENTAL PROCEDURE ....................................................................................... 9 MAINTENANCE OF RECORDS ...............................................:........................................ 13 14 / RESULTS.............................................................................................................................. DISCUSSION AND CONCLUSIONS................................................................................. 16 FIGURE ) I 1. Degradation of the test and reference substances (COz/TCO^)................................... 17 ! TABLE I 1. Blank-corrected cumulative C02 production i and degradation as a percentage of TCOz in reference and test mixtures ................... 18 APPENDIX 1. Preparation of mineral salts medium ............................................................................ 19 : 2 : Company San..-. 'te.,^-oD"-- -"st contain TSCA CBl DPT 438/984469 COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS The study described in this report was conducted in compliance with the following Good Laboratory Practice standards and I consider the data generated to be valid. The UK Good Laboratory Practice Regulations 1997 (Statutory Instrument No. 654). EC Council Directive 87/18/EEC of 18 December 1986 (Official Journal No. L 15/29). OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17. Christine E. Burwood B.Sc. (Hons.), Study Director, Huntingdon Life Sciences Ltd. .L^..^&^J^/...lcl Date Company Sanded. Doe. ^ co^n TSCA CBE QUALITY ASSURANCE STATEMENT The following have been inspected or audited in relation to this study: DPT 438/984469 Study Phases Inspected Protocol Process Based Inspections Formulation and test set-up Collection of activated sewage sludge Determination of CO, by titration " Audit Report Date of Inspection 17.08.98 16.09.98 24.06.98 26.06.98 02.04.98 14.12.98 Date of Reporting 17.08.98 16.09.98 24.06.98 26.06.98 02.04.98 14.12.98 Protocol: An audit of the protocol for this study was conducted and reported to the Study Director and Company Management as indicated above. Process based inspections: At or about the time this study was in progress inspections and audits of routine and repetitive procedures employed on this type of study were carried out. These were conducted and reported to appropriate Company Management as indicated above. Report Audit: This report has been audited by the Quality Assurance Department. This audit was conducted and reported to the Study Director and Company Management as indicated above. The methods, procedures and observations were found to be accurately described and the reported results to reflect the raw data. Helen Comb B.Sc. (Hons.), Principal Auditor, Department of Quality Assurance, Huntingdon Life Sciences Ltd. .' .^L>^^'^5^'. [c^0^ Date ;4 : Company S^ed.D^ot=^nTSCACBl CONTRIBUTING SCIENTISTS Christine E. Burwood, B.Sc. (Hons.) (Study Director) Robert A. Dickinson, Higher National Certificate (Applied Biology) (Senior Laboratory Technician) Faye 0. Shanahan (Laboratory Technician) DPT 43 8/984469 :5: ^co^a>nTSCACB Company San..- ^^dA.CPO^SS fOtC-'-" SUMMARY DPT 438/984469 The ready biodegradability '^j^^^^H^^I^H^^^^^^^^^^Ij^^ assessed in the CO^ Evolution test (Modified Stunn test. Procedure C.4-C oftheAnnextoOTRctive 92/69/EEC; OECD Procedure 301B). IJH^I^^----vas added to two vessels containing mineral salts medium inoculated with activated sludge (30m^olids/l) to give a nominal test substance concentration of 10 mgCarbon[C]/l. Control vessels comprised two containing inoculated mineral salts medium alone, and one containing inoculated mineral salts medium plus the reference substance^odium benzoate (10 mgC/1). Afl, additional mixture containing sodium benzoate (10 mgC/1) anc^^^^H^^H------------^^------| was established in order to assess the potential inhibitory enectsofthetestsubstanceontn?1 microbial inoculum. Test; control- and reference mixtures were aerated for 29 days with-air that had been treated to remove carbon dioxide (CO^). The CC>2produced by each culture was trapped in a series of Dreschel bottles containing barium hydroxide which were connected to the outlet from each test vessel. The residual barium hydroxide was determined at intervals by titration. The pH of control, reference and test mixtures was measured at the start of the test and after 28 days. The pH of the test plus reference mixture was measured at the start of the test and on the day of its termination (Day 9). D Sodium benzoafe had been biodegraded by 60% after 5 days and 85% after 2'^days in the afa&enceof f HHBHIIHand by 72% after 9 days in its presence which confirmed thatjlH|^IBBvas not inhibitory to fne microbial inoculum. ^-- Cumulative levels of CO^ production in the controls after 29 days (43.2 and 44.3 mgCC>2) were within the acceptable range for this assay system (recommended maximum = 120 mgC02 for a three-litre culture). These results confirm that the inoculum was viable and that the test was valid. Mean cumulative'CO^ production by mixtures containingHI^^HMjwas equivalent to 10% of the theoretical value (TCC>2, 109.6 mgCO^) after 5 days ofIncubation any 16% by the end of the test on Day 29. Substances are considered to be readily biodegradable in this test if CO^ productionJgj:Qual to greater than 60% of the theoretical value within 10 days of the level achieving 10%.^ cannot, therefore, be considered to be readily biodegradable. V Company.SSaannitSiz^e"d. .Doss^^t^7SCACB! INTRODUCTION DPT 438/984469 The objective of this study was^o assess the biotic degradability OJ^^HB^BI It was conducted in accordance with Procedure C.4-C 'C02 Evolution test' of the^nnextoOn-ective 92/69/EEC (OECD Procedure 301B). The parameter deterguned in thi^jest is the cumulative amount of carbon dioxide produced by mixtures containingfl^HBH|H|mineral salts medium and activated sludge inoculum after 29 days of incubation at temperatures nominally in the range 20 to 24C. The study was preceded by a preliminary formulation trial. Owing to the formulation procedure employed, actual test concentrations were slightly higher than {i. the .-value given in the protocol at 10.3 mgC/1 in both test vessels and 10.4 mgC/1 in the inhibition vessel. i The protocol was approved by Huntingdon Life Sciences Management on 7 July 1998, by the | Sponsor on 17 July 1998, and by the Study Director on 13 August 1998. The experimental phase of the study was conducted between 18 August and 19 September 1998. . ^^^^'^ CoETlpsry oann- Identity: Chemical name: TEST SUBSTANCE DPT 43 8/984469 Lot number / Batch number: Expiry date: Purity / Composition: Appearance: Storage conditions: Date received: 2 years from date of receipt 25% Pale yellow slurry Room temperature 23 June 1998 Company Sanit^ed. Dass *c6 co^ta'^ TSCA CB6 EXPERIMENTAL PROCEDURE DPT 438/984469 REFERENCE SUBSTANCE Sodium benzoate (AR grade, product number S/2^11/53) was obtained from Fisher ScientifiCpUK. ^ -* STANDARD SOLUTIONS USED IN THE DETERMINATION OF CARBON DIOXIDE PRODUCTION The following solutions were obtained from BDH chemicals: Hydrochloric acid - Concentrated volumetric solutions (0.5N/1000 ml), product number 18077. Sodium hydroxide - Concentrated volumetric solutions (0.5N/500 ml), product number 18024. Barium hydroxide - Analytical volumetric solutions (0.1N), product number 192356P. INOCULUM A sample of activated sludge was collected on the day before the test from Oakley sewage treatment works which treats predominantly domestic waste. Aliquots (25 ml) of a homogenised sample were filtered through dried (115C) and pre-weighed Whatman GFC Filter papers. The filters were dried for one hour, allowed to cool and re-weighed. The solids level in the sludge was determined and then an appropriate volume used to inoculate control and test vessels to give a final suspended solids concentration of 30 mg/1. TEST PROCEDURE The day before the start of the test, air-saturated ultrapure water was added to each of six, five litre glass culture bottles followed by the volumes of each of the stock solutions required to prepare three litres of mineral salts medium (MSM, Appendix 1). Each culture bottle was then inoculated with activated sludge (30 mg solids/1) and the mixtures aerated overnight with a supply of air obtained by passing oil-free compressed air through cylinders containing fused calcium chloride and Carbosorb AS followed by traps containing 0.025N nominal (0.0125M) barium hydroxide solution, to remove carbon dioxide. The test substance was provided as an aqueous slurry containing 25% active ingredient (A.I.), which was known to be poorly soluble in water at ambient temperature. To promote dissolution of the active ingredient, the slurry was heated in a waterbath at 39.5C until a clear solution was obtained. An approximate amount of the test substance was then transferred to pre-weighed volumetric flasks (50 ml). The flasks were allowed to cool and a final weight recorded once a stable balance reading could be obtained. Ultrapure water was then added to each flask and the mixtures treated with ultrasound for 30 minutes and added to the appropriate test vessels. The preparation flasks were then rinsed with further 50 ml portions of ultrapure water and the rinsings added to the appropriate vessels. An allowance was made for the concentration of the active ingredient during the formulation :9: Company Sanded, ^s not contain T3CA CB DPT 43 8/984469 procedure. The t.heoretical car^n dioxide production of each test mixture was calculated from the actual weights of|BUUHadded. A magnetic follower was added to each bottle followed by the three-litre volumes of inoculated, aerated mineral salts medium. Each bottle was stoppered, the contents were mixed by swirling and placed on an electrically-operated magnetic stirrer fitted with a perspex insulating cover. The reference substance sodium benzoate was added to one bottle containing the test substance and to one containing inoculated mineral salts medium alone; no additions were made to the remaining two control vessels containing inoculated mineral salts medium alone. The mixtures prepared for the test are summarised in the following table: Bottle No. Contents 1, 2 Controls - mineral salts medium plus inoculum (30 mg solids/I) 3 Reference - inoculated mineral salts medium plus sodium benzoate (10.0 mgC/1) 4, 5 Test substance (10.3 mgC/I) plus inoculated mineral salts medium 6 Sodium benzoate (10.0 mgC/1) plus test substance (10.4 mgC/1) plus inoculated mineral salts medium Following these preparation of test and control mixtures, samples (100 ml) were removed for the determination of pH. Each vessel was then fitted with a stopper holding an air inlet tube reaching approximately 10 cm below the liquid surface and an air outlet just below the stopper. The vessels contents were continuously flushed for 29 days with treated air. The air outlet from each vessel was connected to three Dreschel bottles in series, each containing 0.025N, nominal barium hydroxide (100 ml). The residual concentrations of barium hydroxide in the bottles nearest to the test vessels were determined at intervals by duplicate titration of 20 ml samples with hydrochloric acid (0.05N), using phenolphthalein indicator. Following the removal of the first Dreschel bottle in a series, the second was connected to the test vessel, and a bottle containing fresh barium hydroxide was connected to the outlet of the bottle at the end of the series. The degradation of the reference substance in the test plus reference mixture was calculated to confirm that the test substance was not inhibitory to the microbial inoculum. When the level of ; degradation reached 60% or more, the pH was measured and the treatment terminated. j On Day 28 of the test, titrations were undertaken and samples (approximately 100 ml) removed from ! each test vessel for pH determination. Concentrated hydrochloric acid (1 ml) was then added to each vessel to drive off dissolved inorganic carbon. The contents of the vessels were aerated overnight and the final titrations carried out on Day 29. The rate of air flow through the apparatus was determined at intervals during the test. : 10: Company Sanitized. Doss no1; contain TSCA CBl DPT 438/984469 The minimum and maximum temperature of the test area and of a three-litre volume of water held in a culture bottle under test conditions was recorded daily using a thermometer. CALCULATION OF RESULTS Theoretical carbon dioxide production (TCOz) The theoretical amount of C02 that can be generated by a 2.9-litre mixture containing the test or reference substance at concentrations of 10 mgC/1 and 10.3 mgC/1, was calculated in the following way: TC02 /mg carbon = mw of carbon dioxide == 3.67 mg C02/mg mw carbon Reference mixture TC02 = Volume (2.9 1)x conc.QO mgC/1) x 3.67 =106.4 mgC02 Test mixture TCC>2 = Volume (2.9 1)x conc.(10.3 mgC/1) x 3.67 = 109.6 mgC02 C02 production Carbon dioxide production by control, reference and test mixtures was calculated in the following way. Since 1 mmol of carbon dioxide is produced for every 2 mmol of hydrochloric acid titrated, the amount ofC02 precipitated as BaC03 in a Dreschel bottle containing 100 ml Ba(OH)2 is: mgC02 = fN x (Vs - VQ1 x jjOO x mw(C02) ~ ' 2 V i where: ' N = normality of hydrochloric acid. Vs [ ! Vt = volume of hydrochloric acid used to titrate the stock solution of barium hydroxide. == volume of hydrochloric acid used to titrate test samples. V = volume of sample titrated. i mw(C02) = molecular weight of carbon dioxide. i On each occasion when C02 levels were determined, the mean C02 levels in the control group was | . subtracted from that in reference or test groups to give the blank-corrected value. The cumulative ! production of C02 in each group was calculated by adding successive estimates of blank-corrected C02 production. 11 : Company Sanitized. Dcss noi contain 73CA CBE DPT 438/984469 Estimates of the percentage degradation of test and reference substances at intervals during the test were calculated from blank-corrected cumulative C02 production in the following way: % degradation = cumulative C0;(mg) x 100 TC02 of test mixture In the mixture containing both sodium benzoate and the test substance, the mean C02 levels in the control mixtures and mean blank-corrected CC>2production by mixtures containing the test substance alone were subtracted from the measured C02 production of the test plus reference mixture to give an estimate of the amount of carbon dioxide derived from the degradation of sodium benzoate. The resultant estimate of carbon dioxide production was expressed in cumulative production ofCC>2 was calculated by adding successive estimates of blank-corrected CC>2production. 12: Company Sanitised. Does not csntain TSCA Ci: DPT 43 8/984469 MAINTENANCE OF RECORDS All specimens, raw data and study related documents generated during the course of the study at Huntingdon Life Sciences, together with a copy of the final report will be lodged in the Huntingdon Life Sciences Archive. Specimens and records will be retained for a minimum of five years from the date of issue of the final report. At the end of the five year retention period the Sponsor will be contacted and advice sought on their future requirements. Under no circumstances will any item be discarded without the Sponsor's knowledge. : 13: Company Sanitized. Daes r;ot contasn TSCA CBE RESULTS DPT 43 8/984469 CALCULATION OF THE THEORETICAL ORGANIC CARBON CONTENT r" ^ The theoretical organic carbon content o^^^^^^^Rnd sodium benzoate were calculated to be: ^ r^___--\ T^)8H635Fi3i'6S03No ' 45 Carbon content = 22.0% MW= 439.75 . Sodium benzoate C7H502Na MW = 144 Carbon content = 58.3% MODIFIED STURM TEST The weights of the test substance added to test vessels and the actual concentrations achieved are given below: Vessel Nominal Concentration (mgC/I) Weight Added (mg) Actual Concentration (mgC/1)* 10.0 561.18 10.3 10.0 563.11 10.3 10.0 568.08 10.4 *AIlowing for an A.I. content of 25% The blank-corrected CO^ production and percentage degradation in test and reference mixtures at intervals during the test are given in Table 1. Degradation of the test and reference substances are illustrated graphically in Figure 1. Cumulative CO^ production in the controls (43.2 and 44.3 mgCO;?) was within the acceptable range for this assay system (recommended maximum for a three litre culture = 120 mgCO^). The degradation of sodium benzoate was rapid and achieved 60% of its TCOz after 5 days and 85% after 29 days. These results confirm that the inoculum was viable and that the test was valid. : 14: Company Sanitized. C33S n^ contain TSCA CBS DPT 43 8/984469 cQH^HI^B^ The degradation of sodium benzoate was also rapid in the presence achieved 67% of its TC02 after 7 days. This test vessel was terminated on Day 9~olF the study; Tne final pH of the culture was 7.5. Mean cumulative CC>2production by the mixtures containing^l^M------Bvasequivalent to 10% of the TC02 after 5 days and 16% by the eriff of the test onDay29.^A?%radation plateau was achieved after approximately 12 days. The pH of all test and control mixtures ranged from 7.4 to 7.6 at the start of the test, and from 7.4 to 7.7 on Day 28. The rate of air flow during the test was typically in the range 30 to 80 ml/minute. On six occasions during the test the rate of air flow fell below the recommended minimum for this type of test (30 ml/min) to a minimum of 20 ml/min. This deviation is not considered to be significant, nor to have. afTected.-the-integrity of the test. The temperature of a three-litre volume of water, held under test conditions ranged from 20.8 to 23.9C over the test period. : 15: Company Sanitized. Doss not cor.ia^ TSCA G3S DISCUSSION AND CONCLUSIONS DPT 43 8/984469 The biodegradation of sodium benzoate in the presence of the test substance was monitored in order to assess whether any inhibitory effects were exerted on the inoculum. No inhibitory effects were observed. The mean cumulative C02 production by mixtures containingH^|^B|^pat 10 mgC/1 was equivalent to 10% of the TC02 after 5 days and 16% by the end of the test on Day 29. A degradation plateau was achieved after approximately 12 days. Substances are considered to be readily biodegradable in this test if C02 productions equal to greater than 60% of the theoretical value within ten days of the level achieving 10%J cannot, therefore, be considered to be readily degradable. Owing to the stringency of this type of test failure to achieve more than 60% degradation does not necessarily mean that the substance is not biodegradable under environmental conditions. More favourable conditions such as a higher concentration of sewage microorganisms, a more diverse microbial population, or a longer exposure period may be necessary to establish biodegradability. The results obtained for the degradation of sodium benzoate (60% of its TC02 after 5 days and 85% after 29 days) and for cumulative C02 production by the control mixtures (43.2 and 44.3 mg) fulfil the validity criteria for this test. 16: Company Sanitized. Dees nst contain 7SCA C3! DPT 438/984469 TABLE 1 Blank-corrected cumulative CO; production and degradation as a percentage ofTC02 in reference and test mixtures Day sodium benzoate (lOmgC/1) CO:, (mg) 2 23.3 4 48.9 5 64.3 7 73.9 9 78.3 12 : 82.7 15 84.8 20 : 87.6 25 ' 89.2 28 90.6 29 90.6 %TGa 22 46 60 69 74 78 80 82 84 85 85 0 -- -- B sqdiumbenzoatfcul (10.4 mgC/1) + sodium benzoate(10mgC/l) mixture "' CO; (mg) % TCO; 22.8 21 47.2 44 61.2 58 71.6 67 76.5 72 ' - - - - - - - - - - - Culture 1 C0,(mg) 3.5 7.1 10.4 12.3 14.5 16.7 17.7 18.8 18.8 19.6 19.6 %TCO; 3 6 9 11 13 15 16 17 17 18 18 Culture 2 C0;(mg) 4.1 8.2 10.9 11.7 11.7 13.3 13.8 13.8 13.8 13.8 13.8 %TCO:, 4 7 10 11 11 12 13 13 13 13 13 : 18: Company Sanitized. Soes net contain TSCA CBf DPT 43 8/984469 APPENDIX 1 PREPARATION OF MINERAL SALTS MEDIUM DILUTION WATER The dilution water used to prepare stock and test solutions of mineral salts and aqueous preparations of the test substance was tap water that had been softened and treated by reverse osmosis (Elga Ltd, Prima 4 reverse osmosis unit) and then purified (Elga Ltd, UHP) to give a resistivity of 18 Megohm/cm. This water complies with the relevant standards (British Standard (BS) 2486, 1978; American Society for Testing and Materials (ASTM) D 1193-77) for classification as ultrapure water. MINERAL SALTS MEDIUM The mineral salts medium (MSM) for the final test was prepared by mixing 10 ml of stock solution 1 with 800 ml dilution water and then adding 1 ml of solutions 2 to 4 to each litre of water required for the test. Stock 1 g/1 Potassium dihydrogen phosphate di-Potassium hydrogen phosphate di-Sodium mdnohydrogen phosphate dihydrate Ammonium chloride 8.50 21.75 33.40 0.50 Stock 2 Magnesium sulphate heptahydrate 22.50 Stock 3 Calcium chloride dihydrate . 36.40 Stock 4 Iron (Hi) chloride hexahydrate 0.25 Minor variations in the weights taken are riot considered to be significant. : 19: Compaq Sanitized. Eoes r.ot contain TSCA C3I