Document 3QdvYDEY9eVzmVkkDKdRx6mjD

AR226-0185 OES-24 Study Designs -- 3M Microbial Metabolism Program Goal: Tpoopoupltaitmiioznes ccoanpdaibtlieonosf tmheattabpoelrimziitnegnfrliucohrmoecnhtemoifcamlisc.robial A. Introduction The program and test systems described below offer a wide range of conditions designed to enrich for microbial populations that can metabolize fluorochemicals. Even though these systems are fairly complex and require significant maintenance, they are designed primary as "screening" systems to assess if microbial metabolism of individual fluorochemicals can occur. At the screening phase, all possible control systems, multiple replicates, etc. can not (and should not) be included because of the number of analytical samples generated and the. effort required to service each test system. Because of the complexity of many fluorochemicals, metabolism may be limited or not occur in most of the systems. If metabolism is seen in screening systems, they will serve to guide more detailed metabolism studies under a wider range of test conditions. The key to a successful microbial selection or enrichment program (i.e. one that maximizes the chance for enrichment of those organisms capable of metabolizing fluorachemicals) is often based on providing the following: - Use of natural ecosystems as the basis for enrichment - Use of natural nutrients from those ecosystems with supplemental trace minerals, co-factors and vitamins - Continual introduction of new microbes from different natural sources - Periodic replenishment of natural nutrients without diluting-out the species being enriched. - Providing a realistic time frame to permit enrichment and acclimation - Protection of microorganisms from toxic products or metabolites by use of low substrate concentrations, replenishment of nutrients, balanced medium (C:N:P, etc.), proper pH and provision ofa protective surface for growth (vermiculate, sand, soil, activated C, diatomaceous earth, etc.) Systems. - Enrichment in more concentrated (higher biomass & test material conc.) systems and examination of biodegradation in more dilute - Separation of systems selective for fast growing (zymogenous) and slow growing (autochthonous) species. Note that throughout the scheme outlined below, we have attempted to incorporate the principles outlined above for enrichment. Most of what is described has been gained from our background in microbial ecology and practical experience with isolation and culture of unique microorganisms. 3M Biodeg. Program 1 04469 B. Aerobic Enrichment Systems -- General Discussion Three select aerobic for faster systems growing are proposed: a sewage treatment based system species (Zymogenous), a sail based system to select to for slower growing species (Autothonous)and a pure culture system for examining specific metabolic capabilities (Cytochrome P-450 monooxygenase). 1._Sewage treatment based system `Secondary aerobic sewage treatment systems include primarily activated sludge (AS), rotating biological contacters (RBC) and trickling filter (TF) wastewater treatment plants. We also include as part of sewage treatment systems the immediate receiving water and top-most floc (sediment) on the banks of the receiving stream or pond (tertiary will be used, Wareham and New treatment). Bedford, and Two one sources of RBC plant, activated sludge Bridgewater, will be sampled. Wareham and Bridgewater are primarily domestic wastewater treatment plants (WTP) of about 1MGPD flow. New Bedford is a new 30 MGPD WTP which receives light industry and domestic waste (approximately 20:80). The New Bedford plant has been shown to have greater metabolic capabilities for certain classes of chemicals. The receiving water system, site for sediment collection, will be downstream from the Wareham STP outfall, since New Bedford discharges far out into the Bay. There are no TF plants in the nearby area, but microbial growth from the Bridgewater RBC plant should mimic attached growth from a TF plant. Fresh raw sewage from the Wareham plant will be collected on a weekly basis and used as the primary growth medium. DOC from each of these plants is typically below 40 mg/L, 50 Supplementation with activated sludge extract to achieve levels of ~100 mg C/L wil be done. AS extract is prepared from dried Bridgewater AS by mixing one part dried sludge with 4 parts water and autoclaving for 30 min. After cooling the mixture is settled and fitered through a fiberglass filter. TOC of this extract is in the range of 3500 mg/L. Test vessels for acclimation will be baffled 300-mL Erlenmeyer (DeLong neck) flasks containing ~100 mL of enrichment medium and sealed with a polyurethane plug. Natural sewage will be supplemented with OECD mineral salts at the concentrations specified for the OECD 301B test, trace minerals (Trace Minerals Corp. 0.01 mL/100 mL) and yeast extract (5 mg/100 mL). A sandy soil wil also be added (I g/100 mL) to provide a surface for microbial growth as well as an additional source of microorganisms. This soil was collected by American Ag Services, Inc. on 10/28/98 and is classified as a loamy sand with 77% sand, 21% silt and 2% clay. It contains 2.5% organic matter and has a pH of 4.9. Activated sludge, RBC sludge and receiving water sediment (1 g/L each d. wt. basis) will be the primary source of microorganisms. Test material will be added at a concentration of 20 mg a.i.lL. A blank control flask containing inoculum but no test substance will also be included. Once per week, media from acclimation systems will be centrifuged and two-thirds of the supernatant and 10% of the biomass will be removed. Fresh medium and test material will be added. Additional fresh biomass (activated sludge, RBC sludge and sediment at a 3M Biodeg. Program 2 04470 `concentration of adjusted to ~7.0, 10% of the original concentration) if necessary. Weekly addition of will be added ~100 mg CIL and from the pH natural sewage and weekly basis AS extract should (perhaps by 50 mg allow the microbial population cells/L based on a 50% growth to increase on yield from C). a On a weekly extracted and basis, test analyzed medium for DOC, and10% parent moaftetrhiealceanntdrifpuogteedntibailommaestasbowliiltesb.e aDneapleynzeddi.ng Tohnetahcecltiemsattisounbsptraonccees,s fwoilalmcosnttoipnpueersuntmilayevaildseoncbeeofexttersatctmeadterainadl degradation metabolism has occurred is seen, a more or for a definitive maximum of 8-10 weeks. If microbial assay will be conducted. Assay for rates of degradation and metabolite formation will be conducted in sealed 20 or 40-mL serum bottles containing two-thirds of the volume as nutrient medium, inoculum and test material (biomass concentrations in the 10-50 mg/L range). Bottle headspace will periodically be assayed for carbon dioxide and volatile organics with a Thermoglas 1200 TOC analyzer. Medium will also be assayed for parent material and metabolites via appropriate LC-MS technology. Prior to toxicity. initiation of The assay testing, system each fluorocarbon will be examined for will consist of 125-mL Erlenmeyer flasks its to microbial which 50- mL OECD mineral salts medium containing 20 mg/L test substance and 10 mg/L c"oCn-tlaaibnelallel disngordeiduiemntbsenezxocaetpet wtielsltbesuabdsdtaendc.e.BlaTnhkecfolnatsrkoslswiwlilllhbaeveinaclruedseedrvaonidr above the test medium that contains 750 uL 0.1N KOH. The assay wil be initiated by adding 1.0 mL of medium from the blank control flask, described above, and conducted sealing the vessel at 4-6 hours and with a silicone stopper. for several days (e.g. Assay at days for 1, CO, 2 and will 4) be by removing 100 uL from the KOH reservoir and counting on the LSC. Toxicity will be assessed by comparing the rate of "CO; evolved from the test substance and control systems. 2. Soilisediment based system Soillsediment studies will be conducted in 50-mi test vessels containing 20 g of the soilisediment mixture. Individual test vessels will be sacrificed at each time interval. Three soils and two sediments, freshly collected, will be used as the source of microorganisms. Sails will be from several diverse sources (hardwood forest, pine forest and a river bank soilisediment from Bridgewater), mixed and screened through a 2-mm mesh screen. The two sediments will be collected from the Agawam River from areas below the Wareham WTP and at low tide from a brackish site behind the town of Wareham. These sediments will be combined in equal volumes based on dry weight. Equal amounts of each soil and the one sediment mixture will be combined (dry wt. basis) supplemented with trace minerals (1.0 mLikg, yeast extract (50 mg/kg) and soil extract (100 mg C/kg). Soil extract is prepared from a rich organic soil or humus by mixing one part with four parts water and autoclaving for 30 minutes. After cooling and filtration through a glass fiber filter, TOC is in the range of 500-800 mg C/L. Test 3M Biodeg. Program 3 004471 material will be added at 20 mg/kg. Blank control soils/sediment systems will also be set up and will contain all ingredients except test substance. On a weekly basis, individual test vessels will be sacrificed and analyzed for the particular fluorocarbon. Soil extract and yeast extract will be added as fresh nutrients and to adjust soil moisture. 3. Pure culture system Springborn maintains a bank demonstrated to metabolize of bacteria, actinomycetes complex molecules in a and fungi manner that very have been similar to mammalian (microsomes) and plant species. These species have been isolated from natural ecosystems (soils primarily) and contain the cytochrome P-450 monooxygenase enzyme system. Mammalian systems (liver hepatocytes) are known to rapidly metabolize Et-FOSE-OH and there is potential for these microorganisms to do the same. We have reviewed a portion of our data base consisting of nineteen species and ten complex molecules and have selected three fungi and one actinomycete based on their ability to metabolize the greatest range of molecules. These species are: Cunninghamella echinulata, Mucor circinelloides, Phanerochaete chrysosporium and Streptomyces griseus. These cultures will be grown in complex media in 250-mL shake flasks in the presence of the chemical of interest for seven days. Following growth, cells will be separated from broth and both will be extracted and examined for the parent material and metabolites. Also, since certain cultures may not readily metabolize difficult to degrade substances, resting cell suspensions (washed to remove complex organics growth nutrients) may be examined for their ability to transform the test substance. If certain cultures are observed to metabolize the chemical of interest, definitive studies will be conducted to establish kinetics of degradation and to identify metabolites. C. Aerobic Enrichment Systems -- Test System Summaries 1. Sewage Treatment Acclimation System (Zymogenous sp.) a. Test vessels -- 300-mL baffled Erlenmeyer flasks, polyurethane & s.s. stoppers. b. Test medium -- Natural sewage from one A.S. plant + OECD mineral salts + trace minerals & yeast extract (50 mg/L). Supplementation will be done with A.S. extract to achieve ~100 mg/L C. Also, an appropriate surface for microbial growth (sandy soil). Total volume 100 mLAask; incubation at 22C. c. Inoculum -- A.S from Wareham & New Bedford, attached microbial growth (periphyton) from Bridgewater RBC STP, receiving water sediment (floc) from Wareham STP (each @1000 mg/L d.wt. solids). 3M Biodeg Progfrown ` 04472 d. no Test test Material -- Test system will be material wil also be included dosed at 20 a.i. and flasks will mg/L. Blank be somewhat controls with larger (500- f1l0a0sk0s,mLid)enttoicaaclcionmemvoedraytweaaynaelyxtciecpatl needs for that they five test substances. contain no biomass Additional will also be included as analytical controls. established via respirometry (34 Potential day study in toxicity of test a miniaturized material will be test system using "C-benzoate). Sterile controls may be added based on analytical requirements. e. Enrichment Conditions -- Once per week, test medium will be centrifuged and 2/3" removed owifllthbeesurpeeprlnaacteadntwiathndfr1e0sh% mofedthieumbiaonmdastseswtillmabteeriraelm.oveAdn. adMdietdiionuaml amount amount of will A.S., also RBC microbes and sediment be added weekly. Medium pH equivalent to 10% will be adjusted to of the original ~7.0 with 0.1N HCl or NaOH if necessary. Enrichment will continue for at least 8-10 wks. bf.iomAansaslywsililsbe-- eOxtnraactewdee(ikflayppbraospirsi,attee)stanmdeadniaulmyzaedndf1or0p%aroefntth&e mecteanbtorliiftuegse.d cSoanmdpulcetsednootnassuspaeyrneadtainmtmse.diaAtneallyyswiislofbeforaefmripgleuragtsedm.ayCabreboinncalnuadleydsibsasweildl be on the test material. indicated below. Analytical results will trigger need for definitive testing as ag.ctiTveesitngMraetdeirenital(a.Ri.eq[u2i.r0emmgendtasy--0,En1.r5icmhgmewnetekslyystfeomrs9wwilelekrseq,uiarned116..00 mmggfoorf toxicity assay]. mL medium]. Definitive assays will require about 10 mg [set up 16 vials with 20 h. Definitive biodegradation, Assays -- For enrichment systems showing metabolism profiles will be developed in 20 positive signs of or 40-mL serum cvioanlsc.en~trVaitailsonwiflrlomcotnhteaiancc2l/im3atveodleunmreicahsmennuttrmieendtiummed(i1u0m-5a0nmdginsoolciudls/uLm) at a plus test material. CO, Volatile Headspace organics and will be assayed on days 0, 1, perhaps O;. Medium will be 3, 7, 14, assayed 21 for and 28 for parent and metabolites.on days 0, 7, 14, 21 & 28. 2. Soil Based System (Autochthonous sp.) a. Test vessels -- 50-mL culture tubes with polyurethane stoppers. b. Inoculum ~ Three soils and two sediments collected from various sites in the Cape Cod area will be used. Soils will be sieved through a 2 mm screen and mixed. Sediments will be collected at low tide and mixed on an equal d.wt. basis with the soils. Ten grams of the soil mixture will be added to each culture tube. . Test Material --Test material will be added at 20 mg/kg. Blank controls with no test material wil also be included. Need for abiotic controls will be dependant 3M Biodeg. Program 5 04473 on test material. Potential established via respirometry toxicity of test material would not need since it will be done in the sewage system to be re- d. Enrichment moisture holding Conditions -- capacity (MHC) Soils with will soil be adjusted to extract prepared approximately 75% froma rich organic psoaislsoirngpeawta.terMtHhCrouisghdettheermcionleudmnonundtriiledbrseoaikl-stharmopulgehs. placed in a ~ Additional column and soil extract, trace minerals and yeast extract will will continue for at least 8-10 wks. be added on a weekly basis. The enrichment e. will Analysis -- On be sacrificed, a weekly extracted basis, blank tubes and analyzed for and tubes parent & containing metabolites. test material Based on danoasleytdicwailthrteessulttms,atefrrieaslh asnadmpklineesticosf otfhedesgraamdeatisoonilwiislledbiememnotnimtixotruerdeovwielrl tbieme.re- f. for Test Material Requirements -- Enrichment systems each tube times enough tubes for 10 weeks, and will require 6 mg 2 mg for toxicity [0.4 mg assay]. Definitive assays will require about anothe6r mg. 3. Pure Culture Systems (Cytochrome P-450 Enzyme System) a. Test vessels -- 250-mL Erlenmeyer flasks, polyurethane & ss stoppers. (b.soyTbeesatnmgeridtis-ugmluc--osGer)ofwotrhalwlilslpebceiecs.onTdoutcatlevdoilnucmoem~p6l0exmLmicfrloabsiko;loigniccuablatmieodniaat 26C cc.ircineMlilcoirdoeso,rgPahnainsemrsoch--aetFeoucrhryspseocsipeosriu[mCuannndinSgthraempetlolmayceecshignruilsaetuas,] Mucor will be. evaluated for their ability to metabolize test materials. d. Test Material -- Test material will be added at 20 mg/L. Blank controls with no test material will also be included. . Growth Conditions slants to fresh medium --_ Actively (Stage 1). growing Stage 1 cultures will be transferred cultures will be grown for from 3-6 agar days. and 6.0 mL will be transferred to 60-mL of fresh medium containing test material (Stage 2). Stage 2 cultures will be grown for seven days until the early to mid- stationary phase of growth. Cells will be separated from broth and each examined may also analytically. Based on be grown in complex results from growing medium without test cell suspensions, material to the cells early rsteastuisonpaernydepdhaisne,minceernatlrisfaulgtesd maenddiumwaschoendt.aininWgatsrhaceed micneellrsalsw,ouvlidtamtihnesn anbde test material (resting cell suspensions). separated for analysis. After 72 hr, cells and broth will be 3M Biodeg. Program 6 04474 f. Analysis -- and analyzed Cells and for parent broth are extracted (at & metabolites. Foam sttiompepserzsermoaayndbeafatneralsyezveedn days) based on potential volatiltyoftest materials or degradation products. g. Test Material Requirements and an additional 9.0 mg if resting -- Assays will require cells are studied. 9.0 mg for growing cells h. Definitive identification Assays - Establish kinetics of degradation and metabolite D. Test matrix tThaeblseasmp1laensdu2ndseurmsmtaurdiyzeantdhethporsoepotosebdetaesstsasyaemdploinngamawtereikx.lyTbaabsilse. 1 Fdiivsepltaeysst substances (Group 1) will 2) as well as test systems be will examined be added first. over Additional test substances (Group time. Table 2 presents more detail of the "0" in actual Table 2 numbers ofsamples indicates the day of for each initiation test substance on a weekly for that particular test matrix basis. A and that no sample was taken. Pure culture studies are sampled on day "0". E. Additional test matrices Asodidlitainodnasletweastgmeastoruicrecseswilplroivnicdleuddebayn3aMe.robTihcessleudwiglelsbaenaddsdeedditmoetnhtiss taesxtweinllthaes near future. Also, the need to evaluated as analytical results ianrceorrpecoeriavteed.sterile control matrices will be Anaerobic Enrichment Systems A. Sewage Treatment Acclimation and Assay Systems sTteosptpevress.seAlsss--ay16b0as-emdL osnerOuPmPbTotStl8e3s5w-i3th40c0riamnpdedsubbustyelquruebnbtemroodrifTiecfaltoionns by Birch et al. Atleast three bottles will be set up for each test material, Test medium -- resazurin will be OECD added mineral to A.S. salts + extract. trace Total minerals & vitamins and 0.0002% volume ~100 mLbottle after inoculum addition SITnPo'csulwuilml b--eMmixitxuerdeeoqfuaalnlayeruonbdiecrdciognedsitteirosnslutdogmeafirnotmaiWnaarneaehraobmic&itNyeawndBeaddfdoerdd to the test medium (10% viv). Incubation wil be at 35C. wTietshtnMoatteerstiamlat--eArdidailtailosnoiinnctlhuede3d0.-50 mg/L (carbon basis) range. Blank controls 5M Biodeg. Program 7 004475 eExntrriaccthtmhernotugChontdhietsieopntsu--m Owhnilaewmeaeikntlayibnaisnigs,anaadedroabdidcitcioonndailtiaocntsi.vatCeodnstliundugee enrichment for at least 8 wks. Analysis -- On a weekly basis, remove 5 mL of mixed liquor from the test vMeestshelasn,ecaenntdricfaurgbeotnesdtiomxeiddieuwmil,l eaxltsroabcte afnodllaonweadlyiznetfhoer hpeaarednstpa&cemevtiaaboGlCit.es. Tmegs/tL MCat-eraisasluRmeequ7i5rmegm/eLnttesst--mEatnerriicahlm,e3n0t0smysLtemmesdwiiulmr=eq7u.i5rem2g2.p5ermgves[s5e0l]. Definitive Assays -- Definitive test systems will be used to evaluate metabolic rates in more detail and will be designed based on results from the initial test bottles. Definitive assays will probably contain less biomass to facilitate analytical measurements. B. SoiliSediment Acclimation and Assay Systems Tsteosptpevress.seAlsss--ay16ba0s-emdL osnerOuPmPbTotStl8e3s5w-i3th40c0riamnpdedwobrutkyolfrSuubfbietraoertTael.flAotnleast three bottles will be set up for each test material. Test medium -- OECD mineral salts + trace minerals & vitamins and 0.0002% rmeUsiabzoutrtlien waifltlerbeinaodcdueludmtoadadintiaoqnu.ifer sediment extract. Total volume ~100 Inoculum -- Inoculum will be obtained from an anoxic receiving water or aquifer. Water and sediment will be collected under conditions to maintain anaerobicity and added to the test medium (~5 g d.wt. basis). Incubation will be at 22C. Test Material --Addition in the 30-50 mg/L. (carbon basis) range. Blank controls. with no test material also included. Enrichment Conditions -- On a weekly basis, add additional aquifer extract (5 mL) through the septum while maintaining anaerobic conditions. Continue enrichment for at least 8 wks. A(annaaleyrsobiisca-llOyn),acewneterkilfuygebatseisst, mreedmiouvme,5exmtLraocft amnidxeadnalliqyuzoer fforropmatrheentte&st vessels metabolites. Methane and carbon dioxide wil also be followed in the headspace via GC. Tmegs/tL MCat-eraisasluRmeequ7i5rmegm/eLnttesst-- mEanterriicahlm,e3n0t0smysLtemmesdiwiullmr=eq7u.i5rem2g2.p5ermvges[s5e0l]. Definitive Assays -- Definitive test systems will be used to evaluate metabolic rates in more detail and will be designed based on results from the initial test 3M Biodeg. Program 8 04476 bottles. Definitive assays analytical measurements. will probably contain less biomass to facilitate 3M Biodeg. Program 9 04477 as we PEPc FErEEElHHEnREEREETIHR= RHTBNEREEIRERDEANNS BEEH ESREhmREHiEUab mREaEEEneRMnE E R ERFRhEERi p ERRLAH SARE eE PEtEpbLEhR HEtCRlRHeE ERlRE]E lBEFElRERtERREhEER fE BBefeeeuE eisthmataEananta rm E SEEEEHE ELHS)] T SLHHHE EEH E C (Ch i ET |=a E bPSaEsA lEaRsE ysL]siE H eEhasteslE y eslaulsniB Bs[obseetsaE eteatbyal 2$ "a A" evRgmaSm E sgn aRrievneorinne 33 E m e en m EEE memes! Ff tPtERim t PEEE FtFREtEEm t ES FFt REEEEeff P E tE Et t E E EEE FEES m e a men1n 7n 2t t 3 Jft ftt tt t tft et t tf f cr f 3mf f fef ff 2 | m 1 n ef emmfe ef f teft|| Ee mEm t a = t m rE m2n i F1 J t REt ER FJ REm ER Fn f REfEft rFtf RmEf tEft t EEeRtE E m n ee 1 n t m n 31fee 3 e 3 tfe 2 m et m nffe3 fx f3 ffll 3 3 2 | em eee m e m m mm f mf fe m m t mnn ees t n ett ettft t m tt r tf tf rtefi3 xf mm vm t t f eem .emlffn 2 e m e m p r 3 ne tt n m t BH n A 3 e Ff 3emefBt R5mEffE BEER em e m nmn tt tt t J t tf te eff f n ete ft e E ETmEr) |BCIIET) S EisEen [Gsren h C (Thhe LGRTEEE CCeIeEs ==5 on CTael cont) weetsang iea t E Ema wH t r H te t FOEm m TEt EEmEEr mRE Cf fBteEEt eEE1 f f BAEf f eR BFEeEe Em C e et m e mf ee f te e e mfm e n mt e tet r t2t l t 3 fffll n mt e e mt f e mt e e lf teffl 3 E e m 7 s e H t mHt t HHmeHffe m r HHt eHmf t e i FEeE f 4 f BRrEJffll BEreE e 7 m eemtm em t ef m mf tm ee mt o t t f e t f e t t e 4ffffllll || n nt t eH BEe Efm e EHt eHtm BEeE f Bf e | BEE EETemn eme eO e ttn O e eetm O e mt D tt t Ot e eA l FR3 aEE| t mtt e tett en ete m etemt t f fffl Ee = m m t H t m m HEe eH e HmHt tH B e t H o t Befl BEE xoS28 mmr artwonnAhE el,(CETrLesSTETLS,teere))E(CTeLSTeereerresl)s) ((PTSelhe[oelee] (CIhSIEE[D6T[0Y) m EEm TTsms STABILITY IN WATER TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC-95. (1-Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6.7.7,8,8,8heptadecafluoro-, potassium salt, CAS # 2795-39-3) Remarks: Testing is in progress. 04481