Document YjmLdpZvOY2rv4gODojL8nY6K

B IO D EG R A D A T IO N (301E) TEST SUBSTANCE Identity: A mixture containing perfluorooctanesulfonate, which may a lso be referred to a s P F O S , FC -95, or a s a com ponent of FC -3017. (1Octanesulfonic a d d ) (C A S # 2795-39-3). R e m arks: The 3 M production lot num ber w as 2330. T he test sam ple is FC -30 17 produced in Antwerp, Belgium. Current information indicates F C 3017 is a mixture of 0 .6 7 % P F O S , 2 0 % diethylene glycol butyl ether, 5 5 .4 1 % water, 2 1 % ethylene glycol, 1 .3 3 % Hydroxy foamer, 1 % sodium octyl sulfate, 0 .5 % polyoxyethylene m onooctylphenyl ether, 0 .0 4 % sodium lauryl sulfate, and 0 .0 5 % tolyltraizole. The following summary applies to a mixture with incompletely characterized concentrations o f impurities. Data may not accurately reflect the biodegradation potential o f the fluomchemical component of the test sample. M E T H O D _____________________________________________________________ M ethod: Modified O E C D Screening Test, O E C D 3 0 1 E with D O C Analysis, 1979 version T e st type: Aerobic R e a d y Biodegradation GLP: No Y ear Com pleted: 1980 A nalytical m onitoring: D issolved organic carbon (D O C ) Sta tistical m ethods: R esults were determined by calculation of the % D O C removed a s com pared to the blank controls. T e st o rg a n ism so u rc e : A 50:50 mix of soil extract and secondary effluent. The secondary effluent w as from an activated sludge aeration basin at the Metro W astew ater Treatm ent Plant, St. Paul, M N, while the soil w as garden soil from the town of W hite B ear Lake, R am sey County, MN. T e st condition: D ilution water: Deionized water M ineral N utrient M ed ium : Nutrient m edium per O E C D 30 1E method (1979). Initial pH 7.0. T e st and reference so lu tio n preparation: The test material w as prepared by d issolving 20 8 mg in on liter of mineral nutrient medium. T his solution g ive s a final test concentration of 40 mg DOC/L. The reference substance, hydroquinone, w as prepared by dissolving 30.6 mg in one liter of mineral nutrient medium. T h is solution give s a final test concentration of 20 mg DO C/L T e st v e sse ls: 500 m L Erlenm eyer fla sks containing 250 mL test solution and capped with foam plugs. Incubation co n d itio n s: L ig h tin g : Constant dark conditions. 002747 Temperature: 23.5 - 24C Agitation: Continuously Number of concentrations: 1 plus hydroquinone (reference substand) and blank, all in duplicate. Inoculum condition on test initiation: Not given (No m easurem ents are required per the referenced method). Total suspended solids and pH on day of testing: Not given. Element B asis: D ecrease in dissolved organic carbon com pared to the blanks. Test substance flask conditions: Not given. R E S U L T S ____________________________________________________________ Nominal concentrations: Blank control, hydroquinone at -20 mg DOC/L and at -20 mg DO C/L plus H g C h (inhibited), test material at ~ 40 mg DOC/L and at -40 mg DO C /L plus H gC I2, all in duplicate? Element values: 28 -D a y Degradation = 9 6 .2 % D O C removal in duplicate 1 and 9 4 .6 % D O C removal in duplicate 2. M ean value = 95 % . Remarks: Testing w as conducted on the mixture a s described in the Test Sub stan ce Rem arks field. The values reported apply to that mixture and not the fluorochemical proportion alone. C O N C L U S IO N S ______________________________________________________ Percent biodegradation of FC -30 17 w as based on the m ean D O C removal value w as 95 after 28 days. Submitter: 3M Com pany, Environm ental Laboratory, P.O. Box 33331, St. Paul, Minnesota, 55133 D A T A Q U A L IT Y ______________________________________________________ Reliability: Klim isch ranking = 2. T his study m eets the criteria for quality testing. However, the sam ple purity w as not properly characterized and the study lacks analytical confirmation of the am ount of fluorochemical proportion in solution. R E F E R E N C E S _______________________________________________________ The studies were conducted by the 3M Com pany, Environm ental Laboratory, St. Paul, MN, Lab Request num ber 5541S, 1980. O T H E R ______________________________________________________________ Last changed: 6/26/00 002748 m -m6747-11-A TECHNICAL REPORT S U M M A R Y i ): TECHNICAL COMMUNICATIONS CENTER - 201-2CN Ost* 6/30/8C nportant -- I f report is printed on both sides o f paper, send two copies to TCC J vision - Environmental Laboratory (EE & PC) :>Jact Commercial Chemicals Division port+itla Biodegradation of "LIGHT WATER" Products in OECD Test-6/80 Oapt.Number 0535 ProjectNumber 9970012600 ReportNumber 040 John A. Pignato - Commercial Chemicals Division - 236-2A (01) jthoWtl EmployeeNumberls) Eric A. Reiner - Environmental Lab (EE&PC) - 21-BW (63 47816 itsbookReference No.ofPagetIncludingCovershest None - See 3M Environmental Lab Request No. 5541S 31 iC U RITY^ E ] Open Closed 3M C H EM IC A L ^ R EG ISTRY * " ~ New Chemicals Reported Q Yes S No EYW O RDS: elect terms from 3 M lesaurus. Suggest other plicable terms.) vironmental boratory E & PC) C U R R EN T OBJECTIVE: To use the internationally recognized OECD Screening Test with DOC analysis to show that ''LIGHT WATER" products are highly biodegradable. FFF R E P O R T A B S T R A C T : (200-250 words) This abstract information is distributed by the Technical Communications Center to alert 3M'ers to Company R& D . This study used the "Modified OECD Screening Test with DOC Analysis" and supplemental parallel sterile controls to conclusively demonstrate the extensive biodegradability of "LIGHT WATER" Brand AFFF products: FC-203, FC-206, and FC-3017. In 14 days, the dissolved organic carbon (DOC) levels of FC-206 degraded by 90% and FC-3017 was 94% degraded. FC-203 showed 93% DOC-degradation in 21 days. The parallel sterile controls proved that this DOC loss was not due to chemical or physical, processes such as adsorption, volatilization, or precipitation of the parent material. "formation Liaison V \a O 002749 a. TECHNICAL REPORT SU M M A R Y Oat* 6/30/8C TO: TECHNICAL COMMUNICATIONS CENTER - 201-2CN im p o rta n t -- I f rep o rt is prin ted on both sides o f paper, send two copies to TCC.) Divitlon Dept.Number Environmental Laboratory (EE & PC) Project 0535 ProjectNumber Commercial Chemicals Division ReportTitl* 9970012600 ReportNumber Biodegradation of "LIGHT WATER" Products in OECD Test-6/80 To 040 John A. Pignato - Commercial Chemicals Division - 236-2A (01) Author(s) Employe*Number!*) Eric A. Reiner - Environmental Lab (EE&PC) - 21-BW (63 47816 Notebook Reference No.ofPeg**IncludingCoversheet None - See 3M Environmental Lab Request No. 5541S 31 SEC U R IT Y ^ 0p*n Closed 3 M C H E M IC A L REG ISTRY New Chemicals Reported Yes H No KEYW ORDS: (Select terms from 3 M Thesaurus. Suggest other applicable terms.) Environmental Laboratory (EE & PC) C U R R E N T OBJECTIVE: To use the internationally recognized OECD Screening Test with DOC analysis to show that "LIGHT WATER" products are highly biodegradable. ) ff TOC R E P O R T A B S T R A C T : (200-250 words) This abstract information is distributed by the Technical Communications Center to alert 3M 'ers to Company R& D . This study used the "Modified OECD Screening Test with DOC Analysis" and supplemental parallel sterile controls to conclusively demonstrate the extensive biodegradability of "LIGHT WATER" Brand AFFF products: FC-203, FC-206, and FC-3017. In 14 days, the dissolved organic carbon (DOC) levels of FC-206 degraded by 90% and FC-3017 was 94% degraded. FC-203 showed 93% DOC degradation in 21 days. The parallel sterile controls proved that this DOC loss was not due to chemical or physical processes-such as adsorption, volatilization, or precipitation of the parent material. JO c t v . rut" f C ' 203 t O f f v',r7 S'I- 1It f.S' C " CC^ Irv -fKj She*? tS *]~\ ) Inform ation Lijaaiisson Initials: 002750 2- - BIODEGRADATION OF "LIGHT WATER" PRODUCTS IN OECD TEST INTRODUCTION The 3M. Environmental Laboratory chose to attempt to demonstrate the biodegradability of "LIGHT WATER" products using the OECD screening method because the test has wide international acceptance, and the Environmental Lab has had experience using the OECD test as a participant in an international ring test. These results will complement the existing BOD data that show these products to have a high degree of biodegradability. The OECD test is run at 20-25C and it measures only dissolved organic carbon. For these water-miscible "LIGHT WATER" products, which are not expected to volatilize, precipitate, or be adsorbed during testing, the OECD test is an appropriate me.thod for measuring % total degradation. The TOC analysis method even measures the perfluorinated portion of these products. METHODS AND MATERIALS Methods and Conditions - Wade A. Scheil performed this testing following the September 19, 1979. version of the Modified OECD Screening Test with DOC Analysis(l). A copy of this method is attached (Appendix 1). Reductions in the scale of the test were necessary to adapt it to available shaking equipment. Five hundred-ml Erlenmeyer flasks replaced 2-liter flasks, 250 ml of inoculated nutrient solution replaced 900 ml, and the sample size was 25, not 30 ml. As in the prescribed procedure, the analyst used only the last 10 ml of sample filtrate for DOC analysis. A calibrated chart recorder monitored the temperature which stayed within the 20-25C range during the 28-day test, except for a 3-4 hr. period when it reached 25.5C. The temperature was most frequently near the high end of the range (23.5-24C). Chemicals - All chemicals were reagent-grade unless otherwise noted. The 3M "LIGHT WATER" products used were samples of commercial material of Antwerp manufacture. Their label identified them as FC-203 NFP, FC-3017 Lot 2303, and FC-206 (FM 3824) Lot 2330 10/79. The hydroquinone used as the calibration compound was Aldrich reagent grade hydroquinone 98.5%, Lot 100147. Four chemical supply houses were contacted in an attempt to obtain the 99.5% pure hydroquinone prescribed by the Modified OECD Screening Test, but this purity-is apparenty not readiy available in the USA. We expect that this reduced purity will have no signficant effect on results. The impurities have no apparent inhibitory effect on microorganisms and their presence could make only a trivial change in TOC measurements. 002751 3- - Water used in the preparation of all solutions, except the inoculum, was St. Paul city water passed through a carbon bed, macroporous anionic resin bed, and 2 mixed bed deionizers, and then filtered through a 0.2-um filter. This water was also used to make up losses to evaporation. Stocks of water collected at the start of the experiment ensured consistent water quality. In preparing nutrient solutions, 26.4 g of anhydrous Na2HPC>4 replaced the prescribed 33.4 g of NI^HPO^* 211^0, 36.3 g of CaCl2*2ll20 replaced 27.5 g CaCl2. These replacements yielded solutions and concentrations identical to those required by the OECD Method. The optional yeast extract solution was used in place of the vitamin solution (1.6.1.2(f)). The 100 mg of Fe-chelate used in the trace element solution consisted of equimolar amounts of FeCl3 and EDTA (30 mg FeCl3 and 70 mg EDTA). Test Concentrations - DOC measurements of s_ample stock dilutions served as the basis for adjusting initial test concentrations to approximately 40 mg of carbon per liter. This is the highest concentration in the range recommended by the OECD test. Use of this high initial concentration increased the sensitivity of % DOC removal measurements. The low toxicity of "LIGHT WATER" products to microorganisms made significant inhibition at this concentration unlikely. Adjustment of hydroquinone, the calibration compound, to 20 mg of carbon per liter was done by weight based on its known carbon concentration of 65.4%. Two blank^controls, instead of 1, were utilized in making blank DOC corrections. Inoculum - The inoculum was 50-50 mixture prepared according to steps 1.6.2.1 and 1.6.2.2 of the OECD test using fresh soil from E. A. Reiner's garden and the supernatant of a sample from an activated sludge aeration basin at the St. Paul Metro Plant. The filter paper used was WhatmanR 54, and the water for soil extraction was chlorine-free well water. - Sterile Controls - Replicate vessels containing 400 mg/1 HgCl2 served as sterile controls for each test material and the calibration compound. Handling of these sterile controls was identical to the handling of the viable cultures. They had the same innoculum and sampling frequency, but the analyst only made DOC measurements on the 0- and 28-day samples. Sample Preservation - The sample preservation method used was that prescribed by the TOC manufacturer(4 ). The method involves adding 1 drop of concentrated HC1 to the 10 ml filtered samples, bringing ~the sample to <pH 2, and storing the samples under refrigeration in vials with aluminum foil-lined caps. The method is an effective preservation method. The manufacturer indicates that the method can stabilize calibration solutions made from potassium hydrogen phthalate, a readily biodegradable material, for several months. 002752 4- - Since the acidification in this preservation method was also a ; necessary step in the TOC analysis protocol, its use was the most practical and allowed elimination of the IIgCl2 preservation technique described in the OECD method. All TOC analyses were made within a month of the preservation of the samples. Instruments and equipment - The organic carbon analyzer used was a Dohrman DC-52A. Its sensitivity limit is between 1 and 2 mg of carbon per liter. The stoppers for the reaction flasks were clean, porous, plastic foam plugs. Gelman 0.2 urn membrane filters, (Part Ho. 64814) boiled 3 times and stored in deionized water, served to filter samples for DOC analysis. Glassware cleaning involved soaking in chromic acid cleaning solution (ChromergeR ) followed by 6 deionized water rinses. RESULTS AND DISCUSSION Table 1 summarizes the results of this study. Appendices 1 and 2 contain the actual data sheets and plots of the degradation as a function of time. All 3 "LIGHT WATER" AFFF products degraded nearly completely. TABLE 1 Percent Degradation of Eiydroquinone and "LIGHT WATER" AFFF Products FC-203, FC-206, and FC-3017 With Time in Modified OECD Screening Test % Degradation at Day: Product 7 14 21 27 28 FC-203 13 85 93 91 _ 93 FC-206 23 90 94 93 92 FC-3017 37 94 96 96 95 Hyd ro- 89 92 97 94 93 quinone In the case of the highly water soluble "LIGHT WATER" products, it is very unlikely that physical means such as adsorption, volatilization, or precipitation caused the loss of soluble TOC (DOC). This is substantiated by the relatively high oxygen demand observed in BOD~ tests of the 3M Environmental Laboratory (FC-203 BOD5/COD = 0.5, FC-206 BOD5/COD = 0.5). The sterile control data in Table 2 provide further substantial J evidence that the soluble TOC loss from the test samples containing "LIGHT WATER" products is not due to adsorption, volatilization, or precipitation. These samples were handled identically to the test samples except that they contained HgCl2 to prevent microbial growth. 002753 5- - At the end of the 28-day test period, they still contained nearly all the initial DOC. Thus loss of soluble TOC by these physical modes was not a major factor, at least for nonmetabolized "LIGHT WATER" components. This control doesn't prove that physical processes did not remove "LIGHT WATER" metabolites in the test runs, since no metabolism occurred in these sterile controls, but the catabolic* formation of less water soluble materials is unlikely because catabolic products are usually more polar and smaller. TABLE 2 DOC Loss from Sterile Controls Product % DOC Remaining at -Day 28 FC-203 FC-206 FC-3017 Hydroquinone 91.3 94.2 88.1 77.5 An off-white precipitate formed in the hydroquinone sterile control on day 0 prior to filtering for DOC analysis. The precipitate was slightly brownish, as was the solution in the hydroquinone sterile control after 21 days. This suggests that hydroquinone is not stable, at least in the presence of HgCl2> throughout the course of the 28-day OECD experiment. This apparent tendency of hydroquinone to spontaneously form insoluble humus-like material makes it an inappropriate control compound for the OECD test. Its partial precipitation with HgCl2 also casts some doubts about the appropriateness of using the HgCl2 preservation procedure in the OECD method. CONCLUSION The present study conclusively demonstrates that "LIGHT WATER" products, FC-203, FC-206, and FC-3017, are nearly completely degraded in 21 days under the conditions of the modified OECD Screening Test. REFERENCES (1) Organization for Economic Co-operation and Development Chemicals Testing Program Expert Group on Degradation/Accumulation, Dec., 1979, Test Guideline for the Modified OECD Screening Test with DOC Analysis (Level I) (H. G. Nosier) Revision of Sept. 19, 1979. (2) Dohrman DC-52A Operating Manual, 4th Ed., 1978, p. 3-1. Appendices: 1 - OECD Test Guidelines (Sept. 19, 1979) 2 - Data Sheets 3 - Graphs Catabolism is biologically facilitated breakdown to less complex molecules. 002754 ) "** **- , . ) APPENDIX 1 ' Tokyo, December 1st, 197^ C 118/79/Int. OECD - Chemicals Testing Programme Expert Group C, Degradation/Accumulation ) Mbdified OECD Screening Test with DOC Analysis 002755 Test Guideline C 118/79/Int. Test Guideline for the Modified OECD Screening Test with DOC Analysis Date of last revision; Sept. 19,1979 Level I test for ready biodegradability 1. Prerequisites It has to be known whether the test material is soluble in the concentration range employed in the test (corresponding to 5 - 40 mg DO C / 1 ) 2. Guidance Information Knowledge of the bacterial toxicity or inhibitory properties of the test material is not unequivocally required but constitutes useful information for the conduction of the test. 3. Qualifying Statements The method is suited for the measurement of the aerobic ultimate biodegradability of water soluble, non-volatile organic compounds. It is unsuited for the biodegradabiiity evaluation of mixtures. 002756 Sept. 19. 1979 Modified OECD Screening Test with DOC Analysis Preamble It has to be realized that the following procedure for the modified OECD Screening Test has to be regarded in some points as provisional since se veral important features such as the calibration compound and the new test duration of 28 days are completely untried yet. However, since the test constitutes a modification the official, widely practised and well accepted OECD Screening Test for the biodegradability evaluation of surfac tants there should exist a good prospect for success. 002757 September 19** 1979 OECD Chemicals Testing Programme Test Guideline for the Modified OECD Screening Test with DOC Analysis 0. The test procedure constitutes a modification of the OECD Screening Test (OECD Environment Directorate, Proposed Method for the Determination of the Biode gradability of Surfactants Used in Synthetic Deter gents, Paris 1976, and council directive of Nov. 22, 1973, on the approximation of the laws of the member states relating to methods of testing the biodegrada bility of anionic surfactants (73/405/EEC), Official Journal of the European Communities No. 3^7/53 of Dec. 17, 1973) for the application of the dissolved organic carbon (DOC)analysis. 1. Method 1.1 Introduction: Purpose, scope, relevance, and applica tion of test and explanation of"limits. The purpose of the method is the measurement of the ultimate biodegradability of water soluble, non volatile organic compounds in an aerobic, aqueous medium at a starting test concentration corresponding to 5 - mg D0C/1 ( In order to avoid inhibitory effects it is in the investigator's own interest to choose as low a starting concentration as his analytical capability permits ) 1.2 Definitions and units 1.2.1 Definition of biodegradability D= t 1 where Dt = degradation in.percent DOC-removal at time t C Q = starting DOC concentration of the culture medium (mg DOC/1) = DOC concentration of the culture medium at time t (mg DOC/1) Cfel= starting DOC concentration of the blank (mg DOC/1) Cbi= DOC concentration of the blank at ^ time t (mg DOC/1) 1.2.2 Units The degradation is stated as the percentage DOC-removal within 28 days with respect to the test material ( % DOC-removal ) 002759 3 -3- 1.3 Reference compounds 1.3.1 Calibration compound The calibration compound used in this test is hydroquinone at a concentration corresponding to 20 mg DOC/1. Hydroquinone has to exhibit a DOC-removal o f ^ 6 0 % within 28 days, otherwise the test is regarded as invalid. 1.4 The principle of the method A predetermined amount of the compound is dissol ved in an inorganic medium (mineral nutrient solu tion, fortified with a trace element and essential vitamin solution), providing a concentration corres ponding to 5 - ^0 mg DOC/1. The solution is inocu lated with a small number of microorganisms from a mixed population and aerated at 293 ~ 298 (20 - 25C) in the dark or at least in diffuse light only. The degradation is followed by DOC analysis over a 28 day period. The procedure is checked by means of a standard (hydroquinone). A control with inoculation but without either test material or standard is run parallel for the deter mination of DOC blanks. 1.5 Quality criteria 1.5.1 Reproducibility The reproducibility of the method is appropriate for a screening test which has solely an accep tance but no rejective function. -4- 002760 1.5.2 Sensitivity The sensitivity of the method i3 largely deter mined by the sensitivity limit of the organic carbon analysis which is 0.5 mg C/l at the present state of the art. 1-5.3 Specificity, applicability Applicable for the biodegradability evaluation of water soluble, non - volatile organic compounds. 1.5.4 Possibility of standardization The test version* with specific analyses for anionic and nonionic surfactants is standardized as "OECD Screening Test". 1.5.5 Possibility of automation Parts of the test, e.g., the analysis,can be automated, although hardly the total procedure. The procedure is, though, well suited for being operated with whole series of test materials. 1.5.6 Costs ( in 1978 Swiss Francs ) 1.5.6.1 Equipment Glassware 2000,- shaking machine 6000 - 20000,-__ Carbon analyzer 42000,- Miscellaneous (pH meter, balance, provision for air donditioning) 5000,- -5- 002761 1.5.6.2 Person - hours 10 1.5.6.3 Approximate total cost per test 750,- - 1000,(investments assumed to be amortized) 1.6 Description of the method 1.6.1 Reagents and materials 1.6.1.1 Deionized water Deionized or distilled water free of toxic substances (copper in particular), for general use as a solvent. Water which has been deioni zed by distillation or ion exchange is suitable. A high purity of this test water is necessary in view of the DOC analyses in the concentra tion range of 0 - *0 mg/1. The contaminations result from inherent impurities but also from the ion exchange resins and microbial devel opments (bacteria, algae under the influence of light etc). Only one water charge must be used for one test series which is to be' controlled beforehand by DOC analysis. If necessary, suitable water may be gained by UV irradiation or other means. 1.6.1.2 Nutrient solution Mix 1 ml each of the following solutions (a) to (f) and make up to a volume of 1 1 with water 1.6.1.1 ) 6 002762 KH2P04 A.R. K 2HPOu A.R. Na2HPOl4 . 2 H20 N H UC1 A.R. A.R . 8.5 g 21.75g 33.4 g 20*0 S in 1000 ml of water 1.6.1.1 the pH value should be 7.2 (b) 22.5 g of MgSOi, . 7 H 20 A.R. dissolved in 1000 ml of water (3.3.1) (c) 27.5 g of CaCl2 A.R. dissolved in 1000 ml of water (3.3.D (d) 0.25 g of FeClj . 6H20 A.R. dissolved in 1000 ml of water (3.3.1) This solution is prepared freshly immediately before use. (e) Trace element solution MnSO. . ^ H,0 h 3b o 3 ZnS04 . 7 K20 (NH4 )6Mo ?0 24 39.9 mg (30.23-mg MnSO^. . H 20) 57,2 mg 42.8 mg 34.7 mg (36.85 mg (NH,,)6Mo?0 2J4.4K20) Fe - chelate (FeClj,EDTA) water 1.6.1.1 100 mg 1000 ml Sterilisation of the trace element stock solution at 393 (X) (120C), 2 atm., 20 min. 7 002763 ') - 7- (f ) Vitamin solution Biotin 0.2 mg Nicotinic acid 2.0 mg Thiamine 1.0 mg p-Aminobenzoic acid 1.0 mg Pantothenic acid 1.0 mg Pyridoxamine 5.0 mg Cyanocobalamine 2.0 mg Folic acid 5.0 mg water 1.6.1.1 100 ml The solution is filtered sterile (0.2 yum). Instead ^ of solution 1.6.1.2 (f) 15 mg of yeast extraxt may be used per 100 ml of water 1.6.1.1. 1.6.1.3 Biodegradability standard Hydroquinone 99.5 % DAB Erg. B. 6 1.6.1.4 Mercuric chloride solution 1 per cent of HgCl2 in water - 1.6.1.5 Shaking machine accomodating 2 ltr. Erlenmeyer flasks either with automatic tempera ture control or used in a conrtant temperature room at 293 - 298 0 0 (20 - 25C) 1.6.1.6 Marrow neck - 2 ltr. Erlenmeyer flasks. (Creased flasks are recommended) The flasks must be care fully cleaned with, e.g., alcoholic hydrochloric fluted- 8 - 002764 13 r C' .:e use, rinsed and dried in order to avoid ; jnation with residues from previous tests. .lasks also have to be cleaned before their '.rst use since they may be contaminated. 1.6.1.7 Membrane filtration apparatus 1.6.1.8 Membrane filters 0,2 jam 1.6.1.9 Carbon analyzer 1.6.2 Inoculation Either of the following three alternatives m a y be used a.s inoculum or a composite sample thereof. ) 1.6.2.1 Inoculum from secondary effluent : 'i The inoculum is gained preferentially from a secondary effluent of good quality collected from a treatment plant dealing with a predominantly domestic sewage. The effluent must be kept under aerobic conditions in the period between sampling and use. To prepare the inoculum- the sample is filtered through a coarse filter, the first 200 ml >eing discarded. The filtrate is kept aerobic until ased. The inoculum must be used on the day of collec tion. 1.6.2.2 Inoculum from soil 100 g of soil (fertile, not sterile) are suspended in 1000 ml of chlorine-free drinking water (soils with an extremely large content of clay, sand or organic carbon are unsuited). After stirring the suspension is allowed to settle for 30 minutes. 9 002765 -9- The supernatant i3 filtered through a coarse filter paper, the first 200 ml being discarded. The filtrate is aerated immediately and until use. The inoculum must be used on the day of collection. 1.6.2.3 Inoculum from a surface water An inoculum is drawn from a suitable surface water. The sample is filtered through a coarse paper, the first 2oo ml being discarded. The filtrate is kept aerobic until used. The inoculum must be used on the day of collection. 1.6.2.4 Composite inoculum Equal volumes of the 3 inoculum samples are united, mixed well, and the final inoculum drawn from this mixture. The suitability of the inoculum is checked by means ) of the standard hydroquinone. 1.6.3 Conduction of the test 1.6.3.1 Procedure The test materials are evaluated simultaneously in duplicates together with the standard (1.6.1,3) and a control test with inoculation, but without either test or standard material for the determination of DOC blanks. The standard material has to attain - 60 % DOC re moval within 28 days at a starting concentration corresponding to 20 mg DOC/1. If <60 % DOC removal are achieved the whole series has to be discarded +) +) This limit is based on present experience with the 19 day version of the test. A revision of this limit or even of the standard might have to be considered after the accumulation of experien J ce with the new 28 day version of the test. 10 002766 A stock solution of the te3t material in water (1.6.1.1) is prepared. So much of this stock solution is added to the nutrient solution (1.6.1.2) that a carbon concentration of 5 - **0 mg DOC/1 is attained. The starting concentration of the standard hydroquinone is, though, 20 mg DOC/1. Two reaction vessels (1.6.1.5) are each filled with 900 ml of the nutrient solution and inocula ted with 0,5 ml/1 of the inoculum (1.6.2). The opening of the vessel is covered with, e.g., alumi num foil in such a way that the exchange of a'ir between the flask and the surrounding atmosphere is not unduly impeded (Cotton wool is unsuited because of the DOC analysis). The vessels are then inserted in the shaking machine. The tempe rature of 293 -298K ( 20 -25C) must be maintained unchanged during the test, and the vessels should be shielded from light. The air should be free, of pollutants and toxic materials (chlorinated sol vents etc) . In the course of the biodegradation test the DOC concentrations are determined in duplicate (1.6.U.2) at the beginning (day 0), and on the 27. and 28. day. Three additional analyses have to be performed - 11 - 002767 ) - 11 - in rather regular time intervals ( *>^7. ^l^., and **21. day) The analyses are registered in the attached form sheet and evaluated. Only the necessary volumes of culture medium may be drawn for each determination; however, they have to be large enough for the membrane filtration or centri fugation preceeding the carbon determination. The latter requires differing volumes for the different instruments. Evaporation losses of the culture medium are to be made up by adding water (1.6.1.1) in the required amounts. ) The culture medium is to be mixed well before withdrawing a sample. Material adhering to the wall of the vessel has to be dissolved or suspended before sampling. The membrane filtration or centrifugation has to be done immediately. The filtered or centrifuged samples., have to be analyzed on the same day, otherwise they must be preserved with 0,05 ml of the HgCl^ solution (l..l.^L) for each 10ml of nutrient medium or by storing them at 2 - *4C.* The biodegradability test is valid provided the standard exhibits a degradation rate within the specified range. The test can be finished before the 28. day if complete mineralization is accomplished. All steps require great care and cleanliness of the vessels, pipettes etc. but not sterility. j 12 002768 (/>:'/ . * 1 - 12 - 1.6.3.2 Calculation of results The degradation at the time t is calculated from the determinations of the DOC concentra tions at the beginning (CQ ) and the time t (C ) according to v D_ 1 x loo ) where D,, = degradation in per cent at time t CQ = measured starting DOC concentration of the inoculated culture medium (mg DOC/1) Ct = DOC concentration of the culture medium at time t (mg DCC/1) C^-i = starting DOC blank of the mineral nutrient 0 solution with inoculation but without test material (mg DOC/1) C. . = DOC blank of the mineral nutrient solution with inoculation but without test material at the time t (mg DOC/1) The degradation rates are calculated to the nearest 0,1 S. The means of the D t values are calculated ar.d reported to the nearest full per cent. Results 13 0 0 2 7 6 9 ending in 0,5 are rounded up to the nearest whole number. The course of the degradation test is followed graphically in a diagram as shown in the attached example. The results are reported on the attached data sheet. The results of the degradation test are valid if the condition is met that in the same test series the standard yields ^ 60 % DOC-removal. 1.6.4 Analytical means 1.6.4.1 Membrane filter 0 : 2 urn, 25 mm 0 (1.6.1.6). Pre paration of the filterms*: membrane filters are impregnated with surfactants for hydrophilizaticn. Thus each filter contains up to several mg of soluble carbon which would interfere in the biode gradability determinations. Therefore the filters are purified from surfactants and other soluble organic interferences by boiling them 3 times 1 hr each in deionized water. These filters may be stored in water (1.6.1.1) for at least one week. Other membrane filters are suitable if it is assured that they neither release carbon nor adsorb the compound in the filtration step. If the samples are centrifuged, this has to be done at 40 000 m sec"2 ( 4000 g) for 15 minutes, preferably in a refrigerated centrifuge, in any case < 40C. (Remark: the differentiation T0C:D0C by centrifuga tion at very low concentrations does not seem to work well since either not all bacteria are removed or carbon as part of the bacterial plasma is redissolved At higher test concentrations ( 10 mg C/l) and the same small inoculation the centrifugation error seems to be comparatively small ). Form Sheet for the Modified OECD Screening Test xp. no. : of start of test: eat / standard material heoretical test cone.: :noculua: .'arbon analyzer: mg DOC/1 Controls: Stock solution of the test material ( 1000 ai(?/l, dilution .../1000 ml of nutrient solution) TOC* DOC** _________ "g/1______ B: Carbon determinations: Culture medium Mineral nutrient solution with test material and with inoculum flask no. Analyses 1 al *2 al + a2 "l " 2 ( ct ) Theor. DCK - concentrations axter x days cone. mg/1 mg/1 0 (CQ ). -.7 . v -14 )- 2 1 ! 2 7 - 1 28 / 1 1 / 2 bl / b2 ' ' / l * bl b2 a2 ~ 2 ( ct .) Mineral nutrient solution without test material but inoculum Blank C1 C2 C1 * c2 "3 " 2 < CM > / / / - - I C : E v a lu a t io n o f raw d ata: DOC - concentrations minus blanks Flask no. " 3 1 D,1 - -- m--x -- . 100 Bm Ba 2 D - -S-- -- i loo 2 2 mean D. e D2 0 . -t-5- for day x DOC removal after x days 7 14 21 27 1 28 iI 1 i 1.6.A.2 The DOC measurement The sample withdrawn from the culture medium (about 30 ml) is centrifuged or membrane filtered immediately in the filtration apparatus (1.6.1.6) using the membrane filters prepared acc. to 1.6.4.1. The first 20 ml of the filtrate are discarded'. The DOC concentration is determined twice in the remaining filtrate (about 10 ml) by means of the TOC/DOC instrument (1.6.1.8). If the filtrate cannot be analyzed on the same day it has to be conserved acc. to 1.6.3.1. The DOC measurements (mgC/1) obtained *are registered on the'attached data sheet and the DOC concentrations of the culture medium and of the blanks calculated for each sampling time. 002772 APPENDIX 2 3M Environmental Laboratory Data Sheet -results of the Modified OECD Screening Test- Lab Request Mo.: 55^1 S Date of S ta r t of T est: 4 - " - B o . A n a ly s t : Wade A. S c h e ll_______________ |est Material : A N T W E R P FC- 9 .o a N F P S tandard M a te ria l; hydroqulnone. 98.5%, Lot 1001A7. A ld ric h T h e o re tica l DOC o f T e st M a te ria l; ftdQ T . c A U C u it - A T C D *3 Measured TOC* of T est M a t e r ia l; 14-7, 0 0 0 mg TOC/kq 1noculum: s o l i and n o n -c h lo rln a te d secondary e f f lu e n t fro n a m u n icip al wastewater treatment p lan t Carbon A n a ly se r: Dohrmann 0C-52A T o ta l O rgan ic Carbon A n a ly z e r A. Determ ination of TOC^ o f T e st M a te ria l; A sto c k s o lu t io n o f the te s t m ate rial was prepared by d i l u t i n g ) I 7 0 mg to a f in a l volume o f 5 0 0 ml with deionized water cone, te st m aterial TOC In stock solution stock solution mg/l mg/l TOC test m aterial mg/kg T h is r e s u lt was used to calc u la te the necessary amount o f t e s t m ate rial to prepare a c u ltu re medium o f kO mg/l DOC' . 3 AO . 332, B Carbon Determ inations: 2 14% , o oo BEST COPY AVAILABLE C u ltu re medium F la s k no. Mineral nutrient solution wlth test material and wlth Inoculum 1 2 Mineral nutrient solu tion wlthout test material but wlth Inoculum Blank 1 Blank 2 . Eva lu a tio n of Raw Data; Cone, o f te s t C a lc u la te d DOC*12 m aterial In of test m aterial f la s k , mg/l In f l a s k , mg/1 2 ,8 2 4 0 .0 J + 2 eoep ct j" n H -o .o c . b l * b2 bn Lt z n DOC DOC - cone, a f t e r x day$ Incubiit lo n , me1/1 A n a l 0 (Co) 7 lk 21 27 28 yses ai . v z - v a, ... </> 6 3 *k . 3 7 .0 159u : . 36.2. e -l fe.o '.t TTg 7 -S ' 4-.2. 1 </./ / Z -7 HUS bl VV.o b? .... b. < J l J h ibp5 4Uk> 37. 4 3 i. 32./ It. 0 aa fc>!T S -r (o. 0 S -4 z.e m .o 24 4.1 3. 2. S-.o V .6 -2. H .'L Z .8 2 .9 2 -6 2,-8 ..Ci .. \ . o .t .c,, .. 9... - 0 o .z o .y / r ~1h9 0 - 4 O. 1 o. / . r . C > + C2 Cb1, n / S ' -.% O .H d. } . & - 0 `t o -/ d ' /% O . / 0 .6 UX /> ho o .$ 6. H o .a o ./ -o . / -O . 1 ccb l 2- --d i --+ d* (Cblo^ UH 0 o - H A 3 . (o - 0 . / | * \-4 -0 .1 o -H l- t O.(o Flansok. Degradatonlk- %t>0C2re1moviil a2f7ter ;[da^s Incubation See attached se ctio n 1.6.3.2 fo r the formula used to calculate degradation. 1 S I. s' 1l.3 13.0 R e su lts o f blank n o .1 and no. 2 were averaged fo r each day x. The average 2 2o. \ t G . ) 17.0 1M> t l . v J mean 13 <?3 <)3 va lu es were used In the c a lc u la tio n s. cbl| + ^bl2 (from above) Cb,ave " -- l---- 1. The te s t m ate rial I s com pletely s o lu b le in w ater. T h e re fo re , the TOC DOC. 2. DOC a n a ly se s performed more than tw ice on the same sample were run f o r q u a lit y assu ran ce purposes. 002773 3M Environmental Laboratory Oata Sheet -results of the Modified OECO Screening Test- Lab Request'N o.: 55k 1 S Date o f S t a r t of T e st: 4- - 11- Bd -A n alyst: Wade A. S c h e ll____________ _ )# st M a te ria l : AMTuA-HR f . m - W i . ' n r ? '**n. i / - n S tandard M a te ria l; hydroqulnone, 98,5%. Lot m e o re tlc a l DOC o f T est M a te ria l: i HOT C At-Cm .XT&P) Measured TOC* of T est M a te ria l: 9 3 . 9 0 0 mg TOC/kg Inoculum: s o il and non-chlorlnated secondary e fflu e n t fron a m unicipal wastewater treatment p lan t Carbon A n a lyz e r: Doh'rmann DC-S^A Total Organic Carbon A n a lyze r .............. ....... 1001k7. A ld ric h A. Determ ination of TOC* o f T est M a te ria l; A sto c k s o lu t io n of the te st m aterial was prepared by d il u t in g / j O & O wq to a f in a l volume o f 5~0Q ml with deionized water cone, te st m aterial TOC In stock solution stock solution mg/l mg/l TOC test material mg/kg T h is re su lt was used to ca lcu la te the necessary amount o f te st m aterial to prepare a c u ltu re medium o f kO mg/l DOC' Z ,tZ O 2 0. Carbon Determ inations: BEST COPY AVAILABLE i C u ltu re medium Flask no. Mineral nutrient solu tion wlth test material and w lth 1n o c u 1um i 2 Mineral nutrient solution wlthout te st material but with 1nocuTurn Blank 1 Blank 2 E v a lu a tio n of Raw Data: Cone, of te st C alcu la te d OOC material In of te st m aterial f la s k , mg/l In f la s k , mg/l ^ 2 .6 V o .o 1 + a2 n ct,- n V 2 6> io .o b , + b. ... b Ct,2- J ------ n2--------- OOC A n a l yses ai ... a3 .. *1 .. aF hi b2 bbS3 b5P.. DOC - cone* af t e r x day s Incubiit Ion, im}/1 O' (Co) 7 lk 21 27 28 ^ ,0 *//. 7 li i . a fe. 1 1.Q> - 3 . V 3.ST 3.*/ 3.0 V.53.0 h.'i V/.3 ^ 1 ,0 30,9 30.7 f '7 -M -T. 3 3 Z .to 3,o */./ 3 V 3'Z, 3 .5 " 2.C 3. S V A * 30 ` % 3 - 4 3 .6 3 >Cs 3 , i CK1 - -c--l--+---c- 2-- b'l n .................. 1 C l . / ..C . ........ (Cb l0) / S " d, /- do /ZL O .t 'V - 0,/ o. / o .z Of if O. 9 O '? o .t o,<i 0,1 0 , o, A2. o . r i.L 0,9 . t ' O ... - 0 .1 o. / 0,1 - 0. ' -0. / c- Cb'2 "d<T+Td-- * <<W 0 . 9 /3 0 , & - o. ^~^Ave. ~ /V - o . t 0 .9 A2 o * c O Flask no. Degradaitlon - % >0C removiil a f t e r t da^| In cu b ation 7 1k 21 27 See attached se c tio n 1 .6 .3 .2 f o r the12 formula used to calcu late degradation. 1 2 3 3 W / 93,; 9 /. 2 2 3 ,r ^ 2 .6 J mean 3 1o 7 V 93 h.! ?2- R e su lts o f blank no.1 and no. 2 were averaged fo r each day x . The average values were used In the c a lc u la tio n s. - **bl | + ^b 12 (from above) blave 2 1. The te st m ate rial Is com pletely so lu b le In w ater. T h ere fore , the TOC DOC. 2. DOC a n a ly se s performed more than twice on the same sample were run f o r q u a lit y assurance purposes. 002774 3M Environmental laboratory Data Sheet -results of the Modified OECD Screening Test- lab Request No.; SSbl S Date of S ta rt of Test; F-H -ftO 'A n a ly s t ; Wade A. Sch e ll f a s t M a t e r ia l; AMTwtfcP. F-c-g^'T . uPT-s.-bo'S Standard M a te ria l: hydrogulnone, 98.5%, lo t T h e o re tica l DOC of T est M a te ria l: M O T C f l u im . A T D ) Measured TOC* of T est M a te ria l: 1 9 7 .0 0 0 mg TOC/kg Inoculum; s o l i and n on -chlorln ated secondary e f flu e n t ffo n a m unicip al wastewater treatment p lan t Carbon A n a lyze r; Dohrmann 0C-52A T otal O rganic Carbon A n alyze r .......... 1001b7. A ld ric h A. Determ ination of TOC* of Test M a te ria l: A stock so lu tio n of the te st m aterial was prepared by d ilu t in g with deionized water. mg to a f in a l volume of 2 -- m* cone, te st m aterial TOC In stock solution stock solution mg/l mg/l TOC test material mg/kg T h is re s u lt was used to calcu la te the necessary amount o f te s t m ate rial to prepare a c u ltu re medium o f b0 mg/l DOC' 1 ,4 4 0 B2 Carbon D e te rm in ation s; rt2 jO O O - b e s t c o p y a va ila ble C u ltu re medium F la sk no. Mineral nutrient solu tion wlth test material and w lth Inoculum 1 2 Mineral nutrient solu tion without test material but with t nocu turn Blank 1 Blank 2 . E v a lu a tio n of Raw Oata: Cone, of te st material In f la s k , mg/1 10% C alcu la te d DOC1 of test m aterial In f la s k , mg/l Jo, 0 DOC Anal yses *i ai ooc - cone* af t e r x day s Incub,it lo n , m<)/! o (Co) 7 lb 21 27 28 4M 3/. 2 O o .k 30.3 a .y .r ?.? z.% 1 -4 i-2 . z-s- 2 .o / o a 1 + a2 ... an ct," n lo Z lt>, 0 b, + b, ... b ut2 n _C. , " -C--1--+---c- 2-- n 1 Ccbi 2- _d l +_ ^ hi - hy-- bj bl b5r ci c . ^lol a..* - V t- f SOS v / . x ~~PT9~ 4 /. t.f 0. } -o. w . s * l.t. ` 7 -0 .1 O. 1 1-4 0 1,4 - 0 ,1 2A 3.53, H 3.0 . 3.9 /.9 3 .6 0.7 O -T 2 .9 /.S" _ 0.9 0 -4 o .1 a . <o /. 2. /. i 1,0 0 -4 0 ,4 1- -3 /x ZS J- if 1-7 .2 , 2.7. 2. V 2 .0 0 ,9 0 .1 1 -X o .t o ,/ o ,r .4 o - ft 0 ,t -0 . : - 0. l 0 ,(o - o . 0 ` > o Flask no. Degrada Cion - % >0C rernov,>1 a f t e r : da^s In cu b ation 7 lb 21 27 See attached se ctio n 1.6.3.2 fo r the formula used to calcu late degradation. I JO .X 2 2 ,2 . `t i! 1S -Z % . iT J mean 3 7 1 4 U 1 4 -i 1C R e su lts o f b lan k n o .l and no. 2 were averaged f o r each day x . The average v a lu e s were used In the c a lc u la t io n s . . m Ctl | + Cb l j (from above) .1. The te st m ateriel I s com pletely so lu b le In w ater. T here fore , the TOC DOC. 2 DOC a n a ly se s performed more than twice on the same sample were run f o r q u a lit y assurance purp0S^ * Q 2 ^ ^ r - " 3M Environmental Laboratory Oata Sheet -results of the Modified OECD Screening Test- BEST COPY AVAILABLE Lab Request Mo,; S Oate o f S t a r t o f T e st; A - - 11 Ana1y s t ; Wade A , S c h e ll >e st M a t e r ia l; S tandard M a te ria l; hydroqulnone, 98.5%. Lot 100147. A ld rich .theoretical DOC o f T est M a te ria l; 6 5 . 4 * ^ C / A l l C A R S okJ A S i M i ) i p b C o l O R f t AW' C C A R I& o W } Measured TOC* o f T est M a t e r ia l; mg TOC/ko Inoculum; sol! and non-chlorinated secondary affluent frona municipal wastewater treatment plant__________ Carbon Analyzer; Dohrmann OC-S^A Total Organic Carbon Analyser A. Determination of TOC* of Test Material; A sto c k so lu tio n of the te st m aterial was prepared by d ilu t in g w ith deionized water mo to a f in a l volume o f ml cone, te st m aterial TOC In stock solution stock solution mg/1 mg/1 TOC test m aterial mg/kg T h is r e s u lt was used to c alcu late the necessary amount o f te st m aterial to prepare a cu ltu re medium o f <*0 mg/1 DOC* 0 Carbon Determ i n a t i o n s ; ^ C ulture medium Flask no. Mineral n utrient solution wlth test material and w lth Inoculum 1 z Mineral n u trien t solution wlthout test m aterial but wlth Inoculum Blank 1 Blank 2 E v a lu a tio n of Raw Data: e V A L U A T / o t J t>F c o m p o u n d - see s t a n d a r d MATERIAL. A H ovC . Cone, o f te s t m aterial In fla s k , mg/1 3 0.6 --------------------------I-- C alculated DOC* DOC o f tes t m aterial Anal In fla s k , mg/I yses ai a, _ Z O .O *3 1 + *2 an **t j" n 30. G Z O .o b1 + b 7 " b,, Vn ..b !_ . -fez-- b3 *>l. *v DOC - cone, af t e r x days Incubiitto n , m<J/I 0 (e,,) 7 14 21 27 28 \1\ 1 / .3 1 H .4 - IG .Z . J& .& 2 -3 1.7 /7*3 2 , o /7.8 /6 .2 2 >( O ' ST >6 2 .0 / . 2- /. /. s~ /<; 2.. V 2 -3 s-.s. / 3 /-<? z ,n OAS* 1I-* M 2, ) , (fi / JT A7 MO /.V O' 8 0 .0 A Sn . o A 2- 0 -6 hH 5 .8 r . C* + C2 Cb* f n ccb ,2. d * + d * C] J; 1 o . i 0 . 2, C~ -o .v o.< r I-S ' 1, / / 6 _dL / . A w - 0.2. 0 . * 4 o ,/ 0. / A / . <d 0 0 'V / S" /2 /.O /.3 0 .9 0, ! 0 ./ o. / o .r o .i 0 .9 . - o . / -O ' -O ' / .& ~0.J C. Ll S. ^ '**1 + b'o.ve ` o.-- - /V -0 - aV / % 0 (o Flask no. 1 2 J moan Degrada tlo n - % )0C removi>1 a f t e r c d a ^ | In cub atio n 14 21 27 ? 2 .r 9 0 .6 ? 3 .7 f/.z ? /,7 ^ .o / 0 2 W 3? 7 2- 7 7 7 7 ^3 See attached se c tio n 1.6.3.2 fo r the formula used to calcu late degradation. R e su lts o f blank n o .I and no. 2 were averaged f o r each day x . The average va lu e s were used In the c a lc u la t io n s _ ^b lj + ^ b l2 (from above) C*>*ave ------2--------- 1. The te st m a te ria l I s com pletely so lu b le In w ater. T here fore , the TOC DOC. .2 DOC a n a ly se s performed more than tw ice on the same sample were run fo r q u a lit y assurance purposes. 002776 17111.__l - J _ - l l T l l l ~ i r i ~ ~ T I T 1 1 H -- I I ---------------- J----------------- |-------- 1----- l ,----------------- :-------------------------------- 3M Environmental Laboratory OECD Modified Screening Test with DOC A n a ly sis A n a lyst: Wade A. Schel I oate of Start of Test: 4-11-1980 Lab Request No.: 5541 S H 7 1 1 1 1 1 1 H 1 Till -- !~TT~Z------`l - ! -----._^!1___111^l.LJT _ 1 .... ...... 1 ~ ~ " -- ;------------------------------ APPENDIX 3 "002779 3M Envlronrental Laboratory I OECO Modified Screening Test with DOC A n a ly sis i Analyst: Vade A. Schell Date of Start of Test: 11-1980 Lab Request No.: 55^1 S / -1 002780 3M Environrental Laboratory OECO Modified Screening Test with DOC A n a ly sis A nalyst: Wade A. Schell Date of Sta rt of Test; 4-11-1980 Lab Request No.: 5541 S ~j --4