Document zQZRnxzE3vjdMQGqD3NvvOwym
BIODEGRADATION
TEST SUBSTANCE
Identity: N-ethylperfluorooctane sulfonamidoethanol; may also be referred to as 1N,-1E,t2.F2O3S,3E.4A,l4c.o5h,o5l,6o,r6F,M7,-73,482,28.,8-(h1e-ptOacdteacnaefsluulofroon-Na-m(i2d-eh,ydNr-oextyheytlh-yl)-, CAS #1691-99-2)
Remarks: Material is an off-white, waxy solid of uncharacterized purity. The following summary includes a history of studies.
METHOD:
MMoedtihfioeddsSfColAlSowTeeds:t;MoWdiafribcuartigonDeotfeOrmEiCnaDti3o0n2(ASt--aInndhaerrdenPtroBcieoddeugrreadianbcillutdye:d
with study); in study)
COD
analysis;
Shake-Flask Die Away
Study
(Procedure described
Test Type: Aerobic
GLP: No
Years studies were performed: 1976 - 1978
CInoonctualcutmt:ime:Acti7vadtaeydsstlou6dgmeocnotlhlescted from an aeration basin at the municipal iwnaosctuelatrteaaktemnenftropmlatnhte iDn eScta.tuParu,lA, lMaibnanmesaomtaanwufaasctpurirmianrgilfyacuisletdy .weArleteamlsaoteutilized wnaittuhratlhleyuenndreircshteadndciunltgutrheastctahpeasbelemoefdidaegwreardeintgheflmuoosrtoclhiekmeilycatlosh.ave developed
RTheimsatrekssti:ngTrheesuilntietidalinstouxdyygeinn1u9p7t6akweaseqauiWvaalrebnutrtgo oaxpyprgoexnimuapttealkye3ex%peorfitmheent.
theoretical oxygen demand for the hydrocarbon degradation products were detected.
moiety.
However,
no
Further experiments included testing on non-stabilized and surfactant-stabilized NN--EEttFFOOSSEE Aallccoohhooll. wIansthseimipnlityaladsdemeidctoonttihneumoeudsiaactiinvaantedetshlaundolgeso(lSuCtiAoSn.)TshtuedyN,EtFOSE Alcohol rapidly congealed and separated from the water phase. It was cpoousltudlnaotted"atthtaatckb"iotdheegcroaadgautiloantemdaNy-nEottFhOaSvEeAbleceohnoolbmsoelrevceudlebse.caSuisneceeenmzuylmseisons oafttwaactker(-Diincskoslounb,leL.mSo.leLicuu,leMs.Shca.,vePhb.eDe.)n,sahsoewcnotnodbSeCmAoSredesgursacdeapttiibolnesttoudeynzoyfmNe-.
EtFOSE Alcohol was conducted in 1978 using emulsified N-EtFOSE Alcohol.
RESULTS
006322
Noneofthe testing conducted could definitely prove loss of N-EtFOSE alcohol through biodegradation process. Analytical capability at the time could not verify the presence of degradation productsorgive explanations for why the test substance became non-extractable during some of the experiments.
IN SIONS
It appears that N-EFOSE Alcohol does not degrade, at least to detectable levels or by verifiable processes, under the conditions of these tests including with high cell densities and extended contact periods. These studies cannot rule out the possibility that conditions could be found that would allow for biodegradation, nor could they excude the possibility that a microorganism could eventually be enriched that could metabolically alter this chemical. Testing did indicate that N-
EtFOSE alcohol was not inhibitory to bacteria.
DATA QUALITY Reliability: Kiimisch ranking = 3. The analytical methodology is questionable. Sample purity was not sufficiently characterized. Procedural problems were present in several of the studies. Analytical methodology did not exist at this time
to detect possible degradation products.
REFERENCES
3M Technical Report Summary, "Biodegradation Studiesof Fluorocarbons", E. A. Reiner, 8/12/76 Request for Laboratory Work, Lab Request No. 3844, COD, Dale Bacon, July 1977. 3M Interoffice Correspondence, `Chronological Review of Biodegradation Studies on FM 3422", E. A. Reiner, August 12, 1977 3M Technical Report Summary, "Biodegradation Studiesof Fluorocarbon~s II",
Report number 4, E. A. Reiner, 1/9/78
OTHER SMuibnmnietsottear,: 5531M33Company, Environmental Laboratory, P.O. Box 33331, St. Paul,
Last changed: 5/18/00
006323
ke
Technical Communications
Ceti BEE
3m TECHNICAL REPORT SUMMARY
are. August 12, 1976
Environmental LABORATORY, DEPT. NUMBER __0222
Jicrororm COPIES: FroBFieaietoredeogfradFaltuioornochSteusdiseail ceaslsofi4 nFlthueoroEcnavribroonnssental
E Faremie e 1 lig
weR._L. Bohon
|_E. A. Reiner Wiecimer
--
--_-- securTy
To deternine the biodegradability of
= CsoocmipaelrAvChoonrtidneamtiisanlCOlpoesne)d)
selected 34 fluorocarbon compounds.
Toviores Wonka: 47816
Warb"ee0s67Re1turner Pgs. 29-37, 41-50
Fox IiFnfaSrUmeMtMioAnRiYn RthEiPs OpReTrt
Wo
eTreaes eding 12
coven
etn ont et pz her eoerts (TBSTRACT ond Conclusions. (System can accommodate 200.250 wards)
Ii he
Biodegradation studies using a Warburg respirometer were conducted
Conplorely
on FC-95, FM 3422, FC-128, and hydrogen analogs of FC-95 and
Prloetaisneckleudyewdorindotihnefroromapteinons | md sive pose bers fnew|
FaMppr34o2x2i.natNeohybdiroodgeegnradaanbailloigtyofwaFsC-9o5bswearsvedreaondilFyC-9d5e,graadlatbhloeu.gh an Fi 3422 and FC-128 both were demonstrated to undergo some
m"o=te=l--------------__| bFiMod3e4g2r2,adaftrioomn.theAtWtaermbputrsg tstoudiiseosl,ateanddegfrraodmataiosnubpsreoqduuecnttsaocftivated
HIyeMcChHemEiMcIalCsALreRpEGoI?STRY| sludge Study were unsuccessful.
Five ver
Select Kgmeerovl,wosprecoisfic, SM prods terms rem
-
Ttoeumssouinrvp.arEanmceltoesre,supgested
r ~2
EE PC - Div. Envron - Assess BaBicotleorgiya FBliuoosrcorceheenniincgal Biodegradable
.
.
re SPECIFIC PROBLEMS
remaining
to
reach
SA sbiective
Cboinotdiengureaddataitotnempptrsodwuicltls boef mFaide342t2o iansdolaotteherand3Midfelnutoirfoycartbhoens. Work with radioactivity labeled FM 3422 is being considered.
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006324
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BBIOIDEGORADDATIEONSGTSTURUDDIIAESODOFFFAFLLUTUOORROIOCCAARORBBOONNNSS
SUMMARY AND RECOMMENDATION
OOfYfoFtCbhii9so5decgoirsmapdoiaumtnpidroonbtaowbablsieodobebegscreaarudvsaeetdiiotinnibsWyarcboanumrpaglcectsletilumydaitefeslduoonrmiincFarCtoeb9di5..al
TBhieodergersiasdtaatnicoen culture, however,
has not yet been demonstrated.
oWscaecrtubiruvrraegtde.dstusdlTihueedsgeproosndtuuFcdMtise3s4o2o2fntahnFidMs F1bC4i22o1d2ed8girbdaontdohattiiocnnodnifcairaertmendootrthkdantioswpsnro.omveebSietohmediecWgoarntraibdnuaurtogiuosn indings
wfFoouurtludrFCebei95n.vgreesatWtialgryabtuirfogancsitolufidtitaehtseedbubisyoidngetghperuarddieafvbieielldoiptmyFeCnotf12o8tfheshanofullaudnoarbloeyctamircabadolen
pcroomcpeoduunrdes to confiim
the present findings.
sSotluwdbiielsitoyn. theThibsiopdreogbraldeambicloiutlyd boef oFvMer3c4o22meweurseinghiFnMder3e42d2byraditisoacltoiwvwiattyer alcatbievlietdyon(55itsncihy/dnromcolaer)bonanpdoprutriiotny.provBiidoeddegrtahdisatimoanteroifa3l hsaadtaurahtiegdhsoslpuetciiofnic of the labeled compound could be measured by detecting 14c0, evolution. y INTRODUCTION
sThteudysuoscfeptthiebielnivtiyrontmoenmtiaclrobfiaatle moofdiafniycactliaosns iosf canompiomupnodrst.antItparisamettheermoisntthe
important form of degradation for organic compounds.
~
SAovavsatstarirnayfacotf tohragtaniict wcaosmpoounncdesbeclainevbeedcboymplseotmeelythadtegrgaivdeend beynoumgihcrotoifmgeanainsdas. = Tthhiesprdoopcetrrincoendoiftimoincsr,obimailcroionrfgaalnliisbmislitcyoulisd dsteiglrladea caonmymoonrgmainsicconmcaeteprtiiaoln.(1).
APletrhfoluugohrincoamtpeoduncdosmpowuintdhssianregleextfrleumoerilnyesrehsiasvteanbteentoshboiwondegtroadraetlieoanse (2f)l.uoride
nieovnserabseeanresshuolwtn of the perfluoro
tcooofmbpuionondedenertggsroadonaafttiucoronam,lpoupdneedrgsfrlaudioantriitonhnai.tsedstFucoodrmyptowhuianssdsranehtaaiscvoienp,artnaeorde.lmyodiHoofrwiecvaetri,on
modification of its hydrocarbon components seemed possible.
Aennviurnodnemresnttandwiilnlg boef etxhepopsaerdtitaoltdheegsreadpartoidounctsproidnucatddsitiison'imptoorttahentusnidnecgeradtheed materials.
METHODS AND MATERIALS
Chemicals
\
The chemicals used in these experiments are shown in Table I.
006325
2
Ny
TABLE I. CHEMICALS USED IN BIODEGRADATION EXPERIMENTS
Fu 3022 HydrFoMge3n42A2nalog of
Cais
CgF17S0NCoHyOH
Calls
Cay7505NC HOH
FC 95
CgF17805K
Sipex-ols
Cafty7050Na
Cas
FC 128
CgFS0NCHZC00K
y
TSheepytewmeberre,ob1t97a5i.nedFfMro3m422Don(NR-eitckeFroseofaltchoehoClo)mmwearsciaildeCnhteimfiiceadlasDiv7i8s8ioCnC i7n45-2.
The FC 95 used was from lot 583. Lot numbers were not given for the FM 3422
hydrogen analog or the sipex-ols (RM 26442).
Tfhleusoeroccahrebmoincalcsonswetrietuesnetleocftead
floarrgea
mnummbbeerr
ooff
r3eMasfolnuso.rocaFrCbo9n5
is essentially compounds.
the,
FaMfi3n4i2s2hisflaunoroicnatrebromnedpiraotdeucitn. theSippreoxd-oulcstioanndotfhe3MHyfldurooTgoecnarAbnoanlso,ganodf FFCM 1342282is. '
were selected for comparison to the fluorocarbon. Sipex-ols is an approximate
hydrogen analog of FC 95. These hydrogen analogs were tested because biologically
blyabielnezymfelsuowrhoicacrhbonnosrmhaalvley frermeoqvueentalyhydbreoegnenf.ounTdhutso,beitgrsaeteumietdoupsrloybabdleefluthoartinated
mciocurlodbimaolregcroowktphletoenlhyyddreoggreandeanfalluoogrsocacrobuolnds`.select populations of organisms which
WARBURG DETERMINATION
W(aArtbtuarcghmesnttu)dieMsicwreoroegracnoinsdmusctweedreacccoorldliencgtedto frthoem
attached standard procedures. the mixed liquor of the Pigs
Eayteatrceoantcmeennttratsiyosntemo,fwa2s0h0e0d,mganodf bsiuoslpoegnidceadlinsolaibdassaplersallittser.medium and used
WtaheterWianrsbuorlgubflleasskusb.strEamtuelssiwoenrse ewemruelsmiafdieedusininwgataerBlparcikosrtonteo maodddeiltioEnP-2to
ultrasonic probe, base 1/2 inch, at 100% power. Logarithmic dilutions
Bl
sinidweataerrm woefrethemadWearobfurgtheflatseksst. subCsotnrtartoelss, caonndtai1n/2edml1/w2asmlploafce1d0 gin/1 thgelucfoirsset
eSoiltuhteironglourcodseeioornizdeediownaitzeerd wiantetrh.is side am. The second side arm contained
2
006326
.
+
wOixtyhgenreaudpitnagkse waats 1f0-i1r5stmionb,seirnvteedrvianlseactho eflsatsahblifsohr athepeernidoodgeunpoutso a1.c5tihvrist.y.
y
Tmhoinsitwoarsingfolfloorweadppbryoxiamdaditteiloyn2ohfrst.he Afdidristtiosnideofatrhmeansdecocnodntsiindueedaromxygen
ceovnatlauiantiinogn golfuctohsee,toxaicrietaydiloyf dtheegrpardeavbiloeusmlayteraidadle,d maaltleorwieadl.a further
Semicontinuous Activated Sludge Studies
A week=long on FM 3422.
semicontinuous activated The microorganisms used
wselruedgeobt(aSiCnAeSd), stasudbyefwoarse,confdruocmtPeidgs
Eye
T3reSaCtAmSenrteacPtloanrts. anOdnetaHpuwnadtreerd
aFsiftay cmolntroofl atcotiavatfoeudrtshl.udgeFMwa3s422adwdaesd
to added
tadodi3tiroenactionrcsrebaesleodw thtehe FwMat3e4r22sucrofnacceentrianti1o/n2
ml by
of 33
absolute alcohol. Each mg/l. Pure ethanol was
saedmdiecdonttoinounoeussluredagcet--ocrosntaiisnisnhgownreaicntoFriguasrea1.control. The operation of the
Tthhee ScCoAnStenrtesactoofrseawcehrereaaecrtaotrewderfeors2t3irhrresd.wwitihtha50m0agmnle/tmiicn.stoifrraeirrtwohiplreevent
settling. one liter
ofAfstueprertnheataanetratwiaosnrepeprliaocde,d
wtihtehsplurdigmearywaseffsleutetnltedfrfoomr
atnhehoPuigrs and
Eye Plant.
wFeMre342t2akweansataddtehde asttarttheabnedgiennndionfg eoafchtheteTsetstcycClyecslesand1,f2r,omatnhde4.supeSarmnpalteasnt after settling.
)
The 13"
haiegrhatwiiotnh caha4s"berisnteursneadl idniatmheeteSrC.AS
Astusdiidees awremrealplloevxeidgldarsasinacgyelinodfertshe
supernatant leaving the 500 ml with the settled sludge undisturbed.
Analytical
yr
Swaemrpeleesvaltuaaketnedabtythtehinterlmayienraticohnroomfattohgerapfhiyrst(CWeanrtbrualrgRessteuadrychonanFaMly3t4i2c2al work ;
rteoq.a Nsom.allAS9v4o1l2u)m.e, Tahned ssaemppalreastewderoen eWoxetlrmactesdiliinctaopldaitcehsl.oromTehtehadneev,eldorpiiendg
solvent system was were visualized by
t1h0e:90iodeitnheanoslt,arcchhltoercohfnoirqmue(Va:nVd). coTmhpeardedevetloopkendownplates
standards with a detection limit of one ug of FM 3422.
SgaasmplcehsromfaotrogtrhaephSyCAwSitshtudayn weelreectreoxntraccatpetdureintdeotenc-toocrt.anolExtarnadctsieopnasrawteerdeby pbyerfcoernmterdifuignincgapapted2560,7m0l04polfyoprro1p0ylmeinneutecse.ntrifuge tubes and phases separated
RESULTS AND DISCUSSION
Warburg - FM 3422
eRexspuelrtisnenftrowmastheperWfaorrbmuerdg bsytudfyireotn sFoMni3c4a22tinargeFMsum3m42a2rizaendd 1in3sFmigaurTee2if.n This
J
wmga/tLeroftotmheakeFMem3u4l22sioannsaloogf. appSrionxc.e F2M4,0304022mga/n1d oiftsthhyedrFoMge3n422anaalnodg1a1r,e00n0ot
Voefrythessoelucbolmepouinndswatienr,conittacwtas wiftehlt thtehatmicfrooromrignagniasnmsemuinlsitohne
would put more Warburg study.
006327
* BEST
|
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STEP 1: Ad test compound, mreedsira,gaannidsns || :
p _ "STEP 2: Aperatee asnd mix :
JE i
SIT LEN
I
J)
2
i
A
7>
Ee. STEP 5: Re-add /
test compound,'
and media. / Repeat /
magnetic
stirring bar
air sparger
oy
:
--
&
.
=
of
supernatant
men
==| Fuge drain
STEP 4: Drain supematant.
.
s!tudge ".
TC
STEP 5:
;
3
i
i
LLdH
="
Setotpeaerrastioenean.d mixing.
A
FIGURE 1: Test cycle for semicontinuous activated sludge reactor.
006328
.
15.
=
FM 3422
`-i
BEST COPY AVAILABLE
s-
3 2 z
& ya
rJ yl-
i E $ge0 0 BO~I~
2
TT
Ja
7 PL
mTial 3p422gAdidjtoi n,
5
A
Hon E
dN Analog
3g . "
/
~,
. y
30mu00si22ng
(u 1mo6 l
Fu 3422 Ge~300 mg/1
)
w!
.
o
.3
J
P, ort+o 2s sas
time hours
FIGURE 2: Warburg study of
No
Lo =~ glNguacddoistieon
10 aos Te igs
FM 3422 and its hydrogen analog)
.
20 006329
Hydrogen Analog
~1860 mg/1
6
Wehmiullesiftiheed,FMth3e422FMan3a42l2ogwawsasnorte.latAivpeplryoxiemaastiellyyemonuelsihfoiuerdWaasndresqtuaiblreedontoceput
3
c75o%meobfactkheouFtM o3f422emuilnstioone.mulsIinona,bouatndtwtohistomattherreiealhouprrso,ceeedxecedsstoFMslo3w42l2y
emsuollisdiogne,lw.hich had not been used in the experiment, turned into a semi-
Caotmptlheetehigchheesmticaclonocxeindtartaitoinonof(vt16heihmyodlreosc)arwboonuldcomrpeoqnueinrte 8o7f ythmeolFeMs34o2f2 0, based on the following equation:
CaFy SON (CHICO + 5.50, + CFSON,H, + 4C0, 4,0
eMxipcerroibmieanlt,oxiodnaltyio2n-3rmaircelryo emoxlceesedsof60o%xyogfenthueptackheemiwcaaslcbosxeirdvaetdi.on.HowIenvetrh,is goxlyugceonseupttoaktehewaFsM 3c4o2n2tincuuilntgureataltshoe pernoddoucfedthiisncerxepaesreidmeonxty.genAdudpittaikoen, of ccuolntfuirrem.ing that the FM 3422 emulsion was not inhibitory to the microbial
OUnpontheaddoitthieornhaonfd,thethemohsytdrcoognecnentarnaatleodg oefmulFsMio3n422ofshtohweedansalioggn,ifiecnadnotgentoouxsicity.
o$(xF2yigg.eunr8eupS2t)yalktieusrscd,ieeaseTtdhoeatnnhedegaweatnsidvonegoetnsoluorspeestcooorrfertdehceteivheoynndraaonfdgteenrhottshneoaxloyaggd'edsnitioeoxvnyogleuontfioUgnpl.tuackoesSeimcuirlvaer
hTyedsruolgtesnwearnealoogb.tained in a second Warburg ex3periment with FM 3422 and its
3
Aonbasleyrsviesd woafsFMgre3a4t2e2rhatshanshwoowunlditbetoebxepeqcutietdefpruorne.impuTrhietioexsygeinn uthpetakceompound.
bIitodiesgrcaodnacbeliev,abbluettnhoatt dseotneicctaatbiloen bpyrodtuhcinedldaeyegrradcahtrioomantopgrroadpuhcyt.s thIattiwseraelso
pdeogsrsaidbelde. thHatowesvoemre,oufsitnheg htyhdinroclaarybeorncchormopmoanteongtrsaphoyf wFeM w3e4r2e2 munoalbelceu.letowedreetect .
kannyownmatiefritahleshyfdormoegdenasanaalorgesoufltFMof3t4h2e2 biitosdeelgfraidsattiooxinc.of TFhMin342l2a.yerIatndisgnaost 3
tchheromaantaolgorgapthoybesh9o0w%edputrheiswimtahtertiwaolmtaojobrecoinmtpaumrie.nantGsa.s have been the cause of the observed toxicity.
cThhreomactoongtraampihnantsshomwaeyd
SCAS - FM 3422
.
Tihseolasteemictohentihnyupooutshesaiczteidvatdeegrslauddagteion(SpCArSo)ducsttsudyofwaFsM a342s2e.conTdhiasttesmtputdytwoas ceonnddoufcteedachove2r4.-har.perciyoclde,of T1heweeFkM w3i4t2h2 ssaammpplleess taadkdeend aint tanheetihnaintoilatisoonlutainodn rsmaaplildlya ssuerpfaarcaetedarefaromtothaellolwiqsuiigdniphfaiscea,ntamnidcraosbiaalredseuglrtadamtaiyonh.ave had too
Nn-oocnteawnolleaekxstwrearctesoffortmheed sasampalerseswuelrteofaneaxlpyozseudrebyofgasthechrFoMma3t4o2g7ratpohyt.he
3.
maiccidr,oorigtanwiosunlsd.notIfhasvoemebeoefntdheeteFcMte3d4.22 hTahde bseuelnfondiecgraadceidd itos ntohte ssuuflffiocniiecntly volatile to pass through the gas chromatography colum.
* performed by Commercial Chemicals Division
006330
7.
h bTehceanus-eoctoafnotlheexltorwacvtoslactioluiltdynootf btehissespoalrvaetnetd.byFrtohiznenlnaoynerextcrharcomtaetdogsraampphlyes sftoirlltheinxisltayaetr athniaslysdiast.e and could be extracted into a more volatile solvent Tehxrpeeerimaedndti.tionTsheoFfMFM34232422setitnle3d3 wpiptmhinthceremseonltisdswearned mfaodrethdeurmionsgttphaertSCArSemained i(naltthheougrheancottoriwnhesnolutthieons)upwearsnataapnptrowxaismawtietlhydr1a0w0n.mg/lT.he Tfhiinsalmactoenrcieanltrwaatsionnot homogeneously distributed and accumulated on the sides of the reactors. WARBURG FC-95 The results of Warburg studies with FC-95 are graphed in Figure 3. Nmaoteorxiyaglenaulpstoakceauwsaesd noobsetrovxeidc eafsfeactrse.sultSiopefl-tohle,adanditaipopnroxoifmaFtCe-95h.ydrTohgiesn aefnfaelcotgs.of FC-95, was shown to be readily biodegradable and to have no toxic TFhCe-95Siwpaesl-oilnscowmapsletseollyublseoluabtleallatco40n0c0entmgr/alt,ionbsuttweasstedcom(paslehtieglhy aisn s1o70l0utmigo/n1)a.t 4c0o0mpmogu/nLd.s aTrheechlaacrkacotferdiesgtriacdaaltliyonnownbiitohdeFgCr-a9d5abwlaes.expected since perfluorinate
; WARBURG Fc-128 OThxeysgeenruepstulatkse ciunrdviecsatefrothmatWarFbCu-r1g28stiusdireesadoinlyFbCi-o1d2e8graasdasbhloew.n iAnssFuimguirneg 4. bmiaoxdiemgurmadoaxtyigoenn oucpctuarkse aoscciusrrsehdowwinthbielnowt,heap7-phrro.ximexapteerliyme7n0t%alopfetrhieodth.eoTrheitsical * oexnydgeonf utphteakeexpeirsimgerneta.terThtehsaen erxepseulcttsedaraendsoampepwehaartedintoqbueestcioonntinsiunicneg atth:isthe FC-128 is known to be an impure chemical.
C1750NSClHyCO0K + 4.250, + HY + CF SOMH + 4C0, + 31,0 + K
\
.
006331
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BEST COPY AVAILABLE
.
172 mgt
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1
1?
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1
|
3
tine hours
Blucose
FIGURE 3: Warburg study of FC 95 and Sipex-ols.
606332
3
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. } 60
500 mg/1
L5
g %-
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34.
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22
.
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100 mg/1
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5
6
006333
-10- . REFERENCES: (1) AMliecxraonbdiearl, FMa.l;libBiiloidteyg.radaAtdivo.n:ApplP.robMliecmrsobioaflMo7:lec$u5l-8a0r, 1965. Recalcitrance and
(2) Chapman, P. J.; Department of Biochemistry, University of Minnesota,
St. Paul, Minnesota, Personal Communications 2/24/76.
D
2
006334
:
E7./10A./75Reiner
ATTAGRENT IT
3
STANDARD PROCEDURE FOR WARBURG DETERMINATIONS
J. Design experiment and calculate concentrations of materials to add. 2. Fill water bath (OI water if left in bath). 5. Adjust temp. of bath (several hrs. or overnight).
4. Place manometers in desired order.
5. Prepare thermobarometer. Add aboutSulof 1,0 to 1 flask. 6. Stehteyouwtereglcaaslsiwbarraeteidn).desired order (to match the manometer with which
7. wLiitghhtlsyolvgernotu.se Acdednt0e.r2-wemlll 1t0o%pKOwHi.th stopcock grease that can be removed 8. PKreeepparreefrsaimgpelreastedinunDItilwatuesred.(or according to request) to add to side ams. 9. Prepare cells (keep cells cold at all times but avoid freezing).
9
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C. D.
Resuspend Determine
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E. Take sample of final adjusted sludge for standard MLSS analysis.
10. A2ddsidseampalremss).to side ave (usually 1 ml if one side arm, % ml to each side arm.if |
11. Add 2 ml of washed cells to Flasks. 12. Add filter paper strip to alkali in center cup.
15. Attach flasks to the correct manometer. 14. Retighten flasks after about 5 min. shaking in bath.
15. Leave stopcock open to utmosphere, and let temp. adjust for an additional 10 min.
16. Adjust level in manometer to 150 with stopcock open (close stopcock). 17. begin readings (always adjust closed arm of manometer to 150 mm before reading). IS. AJ contents of side urns sccording to emperinent design requirements.
006335
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19. Take readings periodically (on open arms) throughout course of experiment. 20. Disconnect and clean flasks.
AB.. WRaisnhseowfifthgrweaatseer.with acetone. CU.L. ARciindsewawsiht.h DI Water.
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--------------------------------------------
Subject: CBhiroodneoglroagdiactailonRSetvuideiwesof 5 on FM 3422 1
August 12, 1977
TO: D. L. BacoN FROM: E. A. REINER
TOhfeFIfMol3l42o2winovgerrevtiheewscoutrhseeproofgrtehsesFattheatofhasFlbueoernochmeamdiecailnstPherobjeicotd.egradation study
Teehxmepuelrsfiiimorensntti.nstweaTptheeirnWpaorruobrduurbcgieodwdoerbgkyrawdsaaostniicocanotnidsoutncu.tdyedWohufesniFnMgFM3a4n2324u22nwasstwaasbailWeiaxzrpebodusrePgdHotoSwg5e2n uptake
oacftitvheatetdheosrleutdigcealas0,thedemsaonlde fcoarrbotnhesohuyrdcreo,car0,Bonupptaokretioenquiwavsaloenbtsertovedabwoiutthi3n%
a(TfLCo)u.houHrosw.everN,o tdheegsleadraetsiuolntsprwoedruectsconwefriermeddetbeycteadsbeycontdhiWna-rlbauyregr scthurdoym.atography
nsTohunifsefmiuwcloirsekinftiwaefsdorFfiModlel3no4t2w2iefdiwcabsayticaotontn.edmupctAtsedsetomfioccroonlatlieocnntueo-uwdseeegkraacdptaeitrviiaotoneddwpirstolhdudugcdetasilsytinudrqyeuaaudnsdtiiitntigioens of
F(fGLC3)422i.n added in
n-Nooctdaengorladaexttiroanctpsrodourctbsy wTeLrCeindetetehcytlablaecebtyatgeasextlriaqcutisd.chrThoomatFofgr5a4p3h2y ethanol rapidly congealed and separated from squeous suspension. As
daegrreasdualtt,iont.oo little surface area may have been available for detectable microbial ;_
ttAhoimseitnhcaorpdeparwsoeaaschthtehtoenlbiekdeeevlfeiflheoopcoetddivoeftomwiimtachkreopboinalolyntcohmxloiodcir,fiincasattteaidbolnbeipoFhfienF3yf4l2s2342e(2mP.uCBl)siLoi(nu1s).haisn osrhdoewrn
Stabilized PCB emulsions aro biodegradable; | nomemulsified PCB's are mot.
Htyoiiwoeenlvdeprre,otdhuytclhtesacioenftcauFtbeMati3eo4xn2t2raobcfytaebmeluietlhseimrfaitTeeLdrCiaFloMr3tGh4La2Ct3. cionuItlSdCAabSlesoreiwadacestnotriisfmipeaodgsasaiisnblbediiotddoengortada-
hqoumaongteinfyeoutshelylodsisstorifbuFtMed34t22hrboyughtohuits athpeprosaycshtems.ince FM 3422 did not remain
abDycitebi-ivaoaultaoeygdiecsxalpluedrgidemeegnritansdaEtrwielorenen.mecyoeMnredauscfutlraesedkdst.qouaqnFutlaiansttkiisetsawteiorvfeelFysMacm3re4ia2fs2iucwreeerdedaetsphlae0,kteino1,nwi2o,tfh4F,M
5422 and
r8edduacyst,ionaciidnifGiLeCd,peaekxthreaicgthetd waansd aonbasleyrzveedd Peak was observed that was not present in
ifnorthFiMs 3842-2dabyyexGLpCe.rimeNnot.conAsinsetyentGLC control cultures
7
006358
D. L. Bacon
-2-
August 15, 1977
Stthhueecncefsimsndaielvecionsnteocveennntu-rtdaratiyieonantdapmotefidvieeathyctlornatanacsiefnteiarntsgeofeexmFtuirlasci3tf4ai2be2ldeacFcPMlMi3m432a42t2.2edwacUsnuelxtfpuoerucentsdedwtloeyr,e wtdahesactrepartseheseemn(tasteeberoitaahtltaicncahueisdniinbtgaiarlthgirasanpdhpsef)ai.knawlaTsFhMefon3re4m2w2eldyccoohnbtesameiirncviaenldglyunsai(mdnpeoltnetsbi.ifoileoTdghiiGcsLaClslupyge)gaeksftreodm wPiaMdsen3t4fi2o2fuyndasthoianslrymeasitunelrtsiaamolpflbetsyheTaLceCixdtirifanicetoduirontolaabnpdH/ano1drpbaryniaolrCyetntitocraaellxtrRtaeecsctehinaoirnqc.uhe.fAatiltTeehdmi.pstsmaTthfeeorial acidification step in the extraction procedure was found unnecessary and dropped. MtoedcihfniicqauteiornesduciendthtehemevtahroidasbiloiftymakiinngresFuMlt3s4.22 Oeumrulasbiionlsityandtoinobttahienexrterpaecattiaobnle ATlestuelmtasteoveinroctuhlea,samien aidndciutbiaotniontopPeirgi'osd Eiysedselmuodngset,ratweedreinalstohetraitetdachaesdsboaurrcegsraphs. oFMf m3i4c2r2oodrugrainnigsmtsh.e fAirystears-evoelnd-dDaeycatgurrowtshoilpersiaomdp.le Hgoawveevenro, lolsosssowfasextorbascetravbelde agrfatpehr t3)h.e sNeecwondDepceartiuordsoainld ainndcrDeeacsaetdurinacstuibvsaetqeudentsluaddgaeptwievreetrfaonusnfdercsapa(bsleee of pdeerciroedasiasngwetlhle aasmouinntsoubfseeqxuternatctagbrloewthFMpe3r4i2o2dsd.urinHgowetvheer,firisttmussetvenb-edanyotegdrowtthhat sthtaetrilaelltoefchntihqeusee cwualstunroetsubseecdamien caollntaomfintahteesde beyxpetrhiemeanltrse.adyThduesv,eloiptedisacpcolsismiabtleed culture present in the laboratory.
iNodenmteitfaybolaitpeosssihbalvee y(ebtutbeIenfeeildenitmipfrioebda.ble)AnmaettatbeomlpitteissneopwarbaetiendgbymadTeLCt.o Very
dcuilsttuirnecst, spboutts a osfpotthisofmastiemriilaalr
wienrteenspirteysewnatsinalstowoob1s35ervpepdm
FM in
a34c2o2ntcroonltaicnuilntugre.
Tihneocuclounnt.rolBcouthltuTrLeC acnodntGaLiCnecdonfoinrlmyed2.7thipspmloowf FFMM 33442222 tlheavtelwaisn ptrheesecnotntrionl.its
wArintutmebnerwiotfhextpheeriombejnetcstivaereonfowdeutnedremriwnianygiwnhatthecaluasbeosratFoMry342a2ndtootbheecrosmearenobne.ing : peoxltyrmaecrtiazbelde,. oArnmoadtitfeimepdt itso baeifnogrmmatdhaettocandetbeermcionneveritfedFMba3c4k22thoasunmbeoednificoendjuFgMat3e4d2,3 btyheapcoiidntorobfasfeluhoyrdirdoelyisoins.relAelastehoutgohthiet miesdiuan,liktehlisy ptohsatsibFiMli3t4y22isisbedienggraidendvetsoti gaactceldi.matAend cautltteumrpetswialrle bneecemsasdaeryusitongefsfteecrtilethetecchhnainqgueeitnoFMcla34r2i2fywwhihcehthmearkevsiabilte ncoonnbdiuocdteegdratdoabdleet.erniAnematiefritahlesnboanldaentceectaebxlpeeripmoernttionwitohr p1rcodulcatbeolfedFFMM33424222iwsill be located in the ethyl acetate phase, the water phase, or in the microbial solids.
oOcoftmhpaeoruhnyfddurtouixsryelcohsbeujmbeisccttaiiltvlueytsevdearrycearstbioomnil,loaorakndtfoorFCF-Mb1i2o38d42e2gi.sradaaFptCo-is1o2sn8ibplrhaoesdudcaetgscraarodbfaotxiFyoCln-12ps8ra.oldtucTitnhipsolface FM 3422. FC-128 was found to be readily degradable in Warburg studies.
* iIsnfararceodm,pleGxLCedanfdorTnLCofanPaMly3s4i2s2 o(f8/t17h/e77e)x.tract now suggest that this material
00635
D. L. Bacon
-3-
August 15, 1977
peIoldsueisntitivioefnigcoianatliotnhtehaonffloutroheriogbciianoradbleolgnyraeldxiapneet,citoenidt,priosdHouiwcmetpvoerortf,anFtbMect3ah4ua2st2evheaosfdFpertMoerv5me4in2n2'etsowhcbaoenttariasm)ore hhaavpepepnlianngnetdo wtihlils gmiovleecuulse.theItansiswerlsikweelynetehda.t the noncostly experiments we
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EN
TECHNICAL REPORT SUMMARY
70: TECHNICAL COMMUNICATIONS CENTER -- 201208
Im=preoport irnprinttos osh sofs pps,mdtocop0TiCCe.
FAR
Vers
Environmental Laboratory (EE PC)
Fate of Fluorochencals in the Bnvironnent
0222
biodegradation Studies of Fluorocarbons - 11
`
b. 1. bacon
E. A. einer
ass
a
"+ 40671, p. 54-56; 45727, p. 1-30; 46269, p. 7, 14, 15, [Neof Paws Trading Coreraiost
18, 21, 25, 29; 41947, p. 50, 51, 55; 44191, p. 27-32
20
eM
Ham rnd
RBESEmo,
soplicable tm.)
Tdoecoemvpaolsuiattieont.he susceptibility of FM 3422 to biological
FEE(nElBvuiioorrd.oPecgeharbeacindevias,ctsai,lon)
(Degradation) EoATASTAIA,oRRS TI at Th aSsaraion Sond Ten SomSre
tsBiiuoosndceegptrteasidtbaitpliriootncyedosuftruedFsiiosu$s42ea2dreatvdoeessemcrorodibibifecidcawmthiiicocrnhosbiaalollfoxdceogmthrmeaodnaetbviiaoldnus,agtriaodnBeitooidfsogntehaedss
test methods. They include semicontinuous activated sludge md shake
iflmansokcudlaiev-earwea'yststauidnieeds wFirtohn sseevqeureanltiaelnviardoanpmteinvtealtrasnosufrecress.. MSiocmreobsisaelrces *
were selected for their likelihood of containing microbial enriched in the capability of degrading FM 3422 or similar
populations luoro-
ccohuenmitcianlgs.andAnaanlayltyisceasl fporrocreedleuarseesd.usflevdoriindce,l.udedInGtLeCr,prTeLtCa,tio1n4Co-fscbiinotsillation
dFmodaeeHfttgeerT3rraH4igd2aea23lnt4.ti2Eo2x,nos`ntTairhtbetissishuellbivtibzosnieldsdalitfonicrgglmoiiumctlmaystdl,iheoenssaseonildtvisdetdisrtuissdFifoeifsascfeutfdwlhiatenstiotttoeyhscitiqnfrduoceernuurtmlseitvudtuesarnpbteteyisnv.pdterehlodeybNjoolenerontxgwsoeaxrnsiaicoccclatusbeidlity
by Tou acer solubility.
ev
006368
.
2.
SUMMARY AND CONCLUSIONS
ocFpoMtnid3im4tu2i2monwfsaosrdbefisoouclnrodigbietcdoalbinedetcghorimaspdlaretetipeoolnry.t.reFsMTihse3ts4ae2n2tcwotanosdibtaiiloosdnoesgrfaoaduranetdicotoonnhwsanivddeeerreadthsettroonbeg affinity for organic solids.
neInmeuultasrlialolne,xppHetro(i7mh.ee1nt-t7es.r5o)gd.eenseCorouinsbceedmnithcreraroetbiiinoa,nls FcMuolft3u4Fr2M2es3,w4a2s2unerdaxenprgoesadeedrofabrsiocma c1os0nu0dr-if5ta0ic0otnmasgn,/tl.asntdaMbniicelriaozr-ed tmoirocgarfnolibusiomarslochwseeonruireccaeolsbst.aweirneeItdsweoalrsecdtefeevdletlboeptcehaadtusferthooemsfesotihsleoiurrocrepsrweavwsietoreuestrleiokaretlmcyeonntttoinshulaouuvdsgeesde.exvpeolsSouoprmeeed entaxrtpanuosrsfaeeldrlsytoeobfnortithchehheidlgohwcucl(et1lu2lr0e0sd0encmgsa/pi1at)bylecaunlodtfulrdoeewsgr(aavdl1il00nogwmegdf/l1au)cocrlocciehlmelamtidiceoamnlssit.toieFsFM.M343W242e2e2ktwloays proceed for six months.
wTohuisldsatluldoywcbainondoetgrraudlaetioount that a microorganism could
othfetphiosssicboimlpioutnyd,thnaotrcocnadnitiitonesxclcuodueldtbhee pfoosusnidbitlhiatty eventually be enriched that could metabolically alter
pTteherisssulitscsthemioinfcattlhheisuneqdnueviritreothneemxetncetonnsdfiiovtreioenxssttuedndydeesdsctrrpioebrneigdoldyisnsuutnghagilestsetrreeptodhratbt.yFmMeNte3av4be2or2ltihiceslealstistk,aeclkyt.hteo
smIeetsdnitmoebinsstesrlviekodeflyaaqfuftaiotniisctoyercfboosrytsootregomarsng.iacnsicTsasbulgsegucehs1tssasumtmhtaahtroisezFeMspr3et4sh2e2entrexeplieneraissmeoeidlnttsoorctohinenduetchnteveidron-
in the study of FM 3422 biodegradation.
006369
.
"5.
THBLE 1 SUMMAFRMY3O42F2EXBPIEORDIEMGERNATDSATIPOENRFOSRTMUEDDY IN
Experinent 1) 1a0c-tdiavyatseedmicsolnutdigneuboiusodegrada-
stitoanbilsitzueddy Foif 3s4u2r2f.actant 2) 6s-tnuodnythwisthhakweeefkllaysktrdaines-faewrasy,
3)exMpaetreriimaelnst.balance
4) Fluoride release
5) Aatcteetamtpetsexttorarcetcaobvleer nFoMne3t4h2y2l fron cell cultures: A) Hydrolysis B) Binding to Containers ) Volatilization from Cells D) Hot ethyl acetate extracts E) Reflux with Dioxane
Findings Nion eFtMhy3l422acbeitoadteegreaxdtartacitosn pbryodTuLCctsorweCrLGe, detected
Asegotmahyeiltnimnaeocsetdae>t7ge5r%aedxaottfriaotcnhtesp,riondiutHcoitwaselvleywre,reeth>ySdl0ebteaccanetdte.adtein ferxotmrac7t-adbalye-olFdM c3u4l2t2urweass. missing from extracts
FaAnind3a4st2ot2leimpdpsrtesptehonatsmeesaisnuorftehe7-twhdaeatye-fror,ladcetctiuholynltsuraoecfsetMafcta.ei,leds.
dTchueilrslisn,gapFpsaMorlei3dn4st22lydbrewycianasgm.eduneoWnhteeoxntFrmHaicx3te4a2d2bwlievtohltaotsgirlorwianzpaitdiloyn
to be' explained by a metabolic phenomenon,
Fdliueo-raiwdaey Fi 3422.
cstoundcieenstrawtitihoncudlitdurneost iancccrleiamsaeteddurfiong This suggests no modifications occur
to the perfluoro portion of FM 3422,
. ;
NeexittrhaectrabalceidFior3b42a2s.e hydrolysis freed non-
nIortreevxeprsliabilnenobinnedxitnrga.cttaoblceultFuMre342c2o.ntainer did tNiolniezxitnrgacattabl1e00PCM. 34f2r2omwacsult1u/r5erseocloivdesredanbdy volacondensing. ErtehcyolveraceatdadtietioenxatlracFtMs3a42t2.60C. did not rReecfolvuexriendg aclulltnuorneexstorlaicdstabwliethFf1,434-2d2ioxfarnoem the OSofliFdMs3o4f22 sh7o-wdsayt-hoaltd ncoulbtiuordeesg,rCaodmaptlieotne recovery occurred.
006370
whe
INTRODUCTION
QhWaausrrbbueeraegrnlyredwseopsrickrroimebevetadrliuicantiaenxgppreertvihimeoeunsstusscrweehppitocrihtb,il(ic1)to.nyvionTfchiiFnsMglw3yo4,r2k2shtioonwcmeliducdirenodcbriteawalosedsdeegmorsxaeydagacetenion uuFtMnislt3ia4z2b2altiizaosendtbheeymulussonilaeocncselxioomgfaetneuodnupbsuiroiclaforigbeoidncaFlsMou3sr4lc2ue2d,.geoxiIynmgmeetndhiesuaepttealtkyoefoeablxoplveoerwiimnteghnetasde,dnidwsoiigtsehenouosf cOlanerevbeoplnosaspciocbroituliniottneydofifsotrhteh3%amtoolbfeicotudhleeeg,roaxdyagTbehlnee rreefarqsauoginmreendftosrtotohcfiosmFpMlree3mt4ae2i2lnysmaoyxwnihedaxipvzleeaibtneheeedn.hyfdorromred as a result of the emulsion formation by sonication.
sAtethmteeimcopobtnssteirnvtuoeoduissoxoylagacettneivuaFptMtead3k4e2s2lwudedgreeegrau(ndSsaCutASci)coensbsipforudoled,gucrtaasdsatwfieroronem
tthheosWearfbruormgaflsausbksseqfuoelnltowing study. In the SCAS study
ApshdoadlsueetdioianncsctuhomiufslaFmtMainn3ng4e2r2o,n intthheeetrFhMaenaoc3lt42o2rwewrraealplaisdddleyadndcdowinirgteehcatlletydhe tabonidotlshoeegpbiaciraoalltoesdgoilcfiardlosm.retahTcethoiwrsast,er
sdeepgarraadtaitoinvefernoznymtehse.water phase may have made FM 3422 unavailable to react with
FInM t3h4e22prteosebnitodergeproardta,tifounrthareer adetstcermipbtesd.to characterize the susceptibility of
METHODS AND MATERIALS
Mmoestthodmsethaonddsmaarteeridaelsscrwiebreed iunsedthereepxepaetreidmleynttahlrousgehctoiuotn.theTheexpefroilmleonwti.ng standard Extraction Procedure
a11n00d..00innmllversoatfmepedlthe5ys0lwteiamrceeest.aetxet.PrhaacstTeheedseispnaamprpolaletsyipornwoepwryaesletnheeennsucrecenadptprbeiydfuwgcieetnhttrupbieofsluygbpirynogpaydfldoeirnneg10caps ;
mvionluutmeesofatthaeboustolv1e2n,t00p0haxsegraavnidtyt.he Ainccroerarseecdtiovnolhuamse extraction. After shaking the voluse of the aqueous
otfo btehemwadaeterfoprhatshee fdoelclroewaisnegd phase increases rom 10.0 to
91.10.0mln.l present
aTnhdertehfeorveo,luamedeotfertnhienaettihoynl in the water phase which is
oafcettahteenusmoblveerntofpphpasmeofdecFrMea3s4e2s2 extracted into ethyl acetate is
ofrriogmin1a0l.l0yto made by
multiplying the concentration found in the ethyl acetate phase by 0.90.
Media
Tchoensbiastseadl sofa:lts media used in the semicontinuous activated sludge experiment
1.0 g/1 NH,C1 2.0 8/1 KPO, 0.25 g/1 MgSO, 7H,0 Adjusted to pH 7.2 with strong acid
006371
so
The standard media used in die-away and subsequent studies contained: 133 ppm of FM 3422 (emulsified) 2 8/1 Kyip0, 2.4 /1 YM Broth 830 mg/1 Alconox * 6 8/1 Ethyl acetate pH A7.2
eItthywlasapcreetpaatreeidnby100emumllsioffyi1n2g.50.g2/1gAolfconFoMx34s2o2ludtiiosnsoltvheadt hiandwbaeteenr-psraetpuararteedd fuosring30etsheyclondascewtiatthe-asamtoudrealteSdUTwatTeirs.sumiEzmeurl.sifTihceatiroensuwlatsingacecmoumlpsliiosnhewdabsytmhienxing diluted in a KPO, solution which in turn was diluted in a weak YM broth media. bTyheinsoucruflaacttianngt cthoensteaimneidnigameadnidacwoemrpearidnegmotnhsetradtiesdsotlovebdeonxoyngteonxiccontcoenmtriactriooonrganisms Tdheepsleeticoonmparraitseosnsfrwoemrethmesaedecuilntiutriealslyw,ithafttheorsoeneofhonuorn,surafnadcatfatnetr-coonnetaidnayingofcultures, exposure to the surfactant-containing media.
Chemical Analyses
TLC and GLC analyses are described in the Progress Report by A. Mendel (2). Chemicals
The following chemicals were used in this study:
1) Sublimated FM 3422 (see report of Art Mendel- (2)
2) Radioactive FM 3422 (see report of Art Mendel -(2))
3) Ethyl acetate - Mallenckrodt,.A.R.
4) YM Broth - DBIaFcCtOo -YeaBsatctEoxtDreahcytdr-ate1d4.3%
BaDcItFoC-OPeMpatlatneExtract -- 2143.38%%,
Bacto-Dextrose - 4T6%
5) Ether - Anhydrous,J. T. Baker,"Baker Analyzed."
6) Siponate DS-10 - A purified alkylaryl - sulfonate supplied by Alcolac, Incorporated.
7) Alconox - Aphomsipxhtautrees,ofcaarlbkoynlaatreyslsualnfdonsaytneesr,gisltaiucrylageanltcsoho-lssuuplpflaiteeds, by Alconox, Incorporated.
8) 1,4-Dioxane - Reagent grade - repackaged, supplier unknown. 9) Aquasol - NXuyclleenaer-.based scintillation solution - supplied by New England
006372
te EXPERIMENTAL
Semicontinuous Activated Sludge Study
soIinrmpaGlnLyCeiaandrdlaeidesretomsituctdohyne,timnenudooiuadsegasraacdtaanitviaeottnheadnporsloldudusgcoetlsut(ioSoCfnA,SF)Masn3td4u2d2FyMwei3r4n2e2whdirecathpeicdFtlMeyd3b4c2yo2nTgwLeaCasled amanyd nsoetpahraavteedbefernomobthseerwvaetdebrecpahuassee e(n1)z.ybesItcwoausldpnoosttul"aattteadckt"hatthbeiocdoeaggrualdaatteidon FshMow3n422tomobleecmuolrees.suscSienpcteibelmeultsoioennszymoef waatttearck-in(s3o),lubalesemcootnedcuSlCeAsS hdaevgreabdeaetnion study of FM 3422 was conducted using emulsified Fi 3422.
PabriiloirtytotothsetaibniiltiizaetioFnM o3f422thiesmusltsuidoyn,s. seTvheeraltwosurmfoascttasnattsiswfearcetotreyswteerdefAolrcotnhoexir and Siponate DS-10. These were both used in the SCAS study.
eTthheylemualcsetiaotnes. foPrortthiiosnsstoufdythwiesresmoalduetiobny Alconox or Sipanate DS-10, and emulsified
wdeirsesoltvhienngad3dgedotfoFM 3g/4l22soilnut3iomnlsooff by mixing for 30 seconds with a tissunizer.
aDTnShd-e10co,onenc2oe.7nftrgta/h1etieottnwhsoylsoufracfchaeectmtaiatcen,atslsawnedirne3tugh/se1edemFaMusls3c4io2o2nn.tsrotlhEsum.sulsfioornmsedwiwterheonSlyg/1ethAyllcomaocextaotfe
Sfirxeshsemaicctoinvtaitneudoussludagcetivtoateeadchslwudigte. uniTthsiswesrleudgseetwausp bcyollaedcdtiendg 2frloimteanrsaoefration
wbaassinsetattletdheamndmitchiepablotwtaosnte500trnela,tmceonnttapilnainntg inallPigo'fs tEhyee,SeMtitnlneedsocteal.ls, Thweas sludge
rreeatcationre,d. anOdneidelnittiecralofmmeeddiiuummrweaasddtedhenatadtdheedbetgointnheingseotftleedachslutdegset ciyncleea,ch
Tinhetsheemebadsiaalwesraeltsmadmeedibyumddielsuctriinbgedporitniotnhse moefthtohdesabaonvde-mdaetesrcirailbsedseecmtuilosni.ons
v.
TFaMbl3e4222 psrheoswesnttheinco1n.5ceLntrcaotnitoennstsofofetehayclh arceeatcattoer,atAlctohneoxb,egiSninpionngateofDSt-h1e0,Firasntd
test cycle.
TABLE 2
-
SEMICOCNOTNICNEUNOTURSATIAOCNTIOVFATOERDGASNLIUCDGECOMRPEOAUCNTDOSRSINAT THE BEGINNING OF THE FIRST TEST CYCLE
Rsecaacstor 21 34 65
AceEtthaytle 556600 446600 9%0
Initial
Concentration
Alconox 8300 8300 1700
(ppm)
5-10 8300 8300 1700
BM 3422 00 551100 110000
006373
-
T(h1e), saenmdicoisntibnruioeufslyaocuttilvianteedd sinludFgieguprreoc1.edure was described in a previous report
ceTehnindstroifefxutpgheeerdi.mfeinrtsTthweaasnsdolcilodanssdtucanttdeesdtcefncoytrcrli1ef0.ugdaatyAesftwweeirrtehtheesxatmsrpalamecpslteesdtawskeeernpearaatttaektlehyne,, betTghhieenyniwnegreand
acgeanitnrifaufgtaetreawdajsusteixntgracttoepditw1owittihmesc,oncfeinrsttrataetdthseulfpuHriocf extracted only once at pH 7. The ethyl acetate extracts
atchied,samTphlee c(evlplH7s)wearned vere then evaporated,
resuspended in methanol and separated by TLC.
PLThriiotsbtlleefmosoarmwininotghscoafluoisademsdintwgheerdeelvolesolssotpoefdfrsiononmearlelsaucstrepoaercntd5,oerdsasnoddluimrdoissntgiontfhyeetahfceitrossrutssptee1n,sdte2,dcycs3l,oel.iadnsd 4.
wweerree This
claolossntotrofilrnlocemrdearbseyeadcbtlotohrewi6.nragteaInosftalrleevaasnpuoobrfsaetqaiuioernn,tonatetnshdterwecaayctctleoerrss,suwrfeforaaecmeibnrogofuagenhadtch1b0arscekacottfoors.olids
their original volume with deionized water prior to draining or sampling.
. dTehxetegrrraeacdtawsteiroeonfnpotrhnoeeduwscutssppeootnfsdeFdMthas3to4l22iddewsvaesalnodapcecdiunmountlhaeTtLieCnxgtp.rlaactteTsshisotfhwaattshweoturcluedentbrioinftduhigcaaittneest.htehat a
TDchSoe-ur1re0sewweeorrfeetpihrneedsifecinanttailoinntsestthtehacetyxcltberi.aocdtesgSproaotdfsattiwhohenicihnoiftcitoahuleldssbauemrpflaaetcstt.rainbtuTthteeodsoektpsoploAatclsecowndeourxreinogF the groenpelac(eedxcbeyptneiwn srpeoatcstorfr4o)m altate24r hsoaumrpsl.es and these new spots were completely
tIThtheesasepepecaaorrnesdicttoihnoasntisdFewMrietd3h42th2oihgbhaedconepolttlimddemegnrscaiodtneiddei,stioaatnnsdalefaos1rt0-bditaooydeagardcaecdtlaeitcmitaoatnbi,loenpl.eervieold,, under v
Shake Flask Die-Away Studies with Adaptive Transfers
3
FiGtMroww3at4s2h2 ovwefirtyahhdesitefeqfruioecgnuetlniteaoltuosaqduacaeplntltiivpteoapttuirlvaaentlsiyfoenrmseaiwnsausnruetirtnihieetnitarteemmdoevdaiflaorocfotnwtoFaMirne3ai4sn2og2nsre.moumlsFSiiCrfsAitSe,d
sftourdiceesl.lulTahrismatwearsiadlu.e toOnFtMhe34o2t2'hservhearnyd,lowthweateentrirseolucboinltietnytsaonfd idtise-aawfafyinictuyltures
cThoeuldseecaosnidlyrebaesonextfroarctsetdaretliinmginshaatkiengflthaissk psrtoudbileesm oisf ntohnahtomtohgiseneaoduasptisvaempltirnagn.sfer
pcraopcaebldeureofisgroanwthaccoenptxeednobmieotthiocdsoforenrraircehliyn-gococrugrarniinsgmsorgfarnoimcnamtautrereiatlhsa.t are
ofIrnbotmaainnMea'dtstefmrDpeotcmatatuorn,uombbtAaeliranboamfoargspaolnuarincsteu,ss. tcaaTkpheaenbsleefrsooofmurgacrreeosawsthintochnaltudFweMedr3e4t2h2rl,eiekeilsnyooiclutlosaahmwapevlreees sbleuedngeexfproosnedthteo MfeiturooropcohleintiacnalHsa,st`ebiTorleoagtimcealntslPluadngte fatromPigt'hse EDyeec,atMuirnnpelsaontt,a. and wSeerpearaetveentsuhaalkleyfcloamsbkinceudltuirnetso waerseingslteartmeadstefrromcultthuersee, various inocula, but they
006374
teetattt eee.fe 3 21 3 -
0 1: at cos. comms
meedrieas,rpaanndisns
STEP 2:
CF=vo%%8ndl
AAT oes
St
aa]
ro
STEP 5: Ro-atd
test compoun
aTnodepkmeeed.ia.
megotic iz spargor
stirring bar
E'S Aerate moun and mix
]
--]
CT]
B=
Jp
drain
C=)
STEP 4: Drain supernatant.
-
se
-- CE
EL
Bs 3wd
STEP 3: STtRopeserraetioan ean,d mixing.
FIGURE 1 Test cycle for semicontinuous activated sludge reactor.
006375
-s-
acIconinidttiiafailinleiydn,gwit5t0hhemlsaudlaofpfutricivuceltautcrrieadnsmfetedoirupsmH.we1,reThseucpoptnlodetuamcletnetdceodnintweingttlhsasasofsEtarhtleuernaamteseydekrNawfCellrasekssolution,
wTeeoxrttearlaccptohemadbsientewdosepptraiirmeaostr,iotnoanwGdaLsCtheaancaiclnoysmsipidlsei,shoefAdctbihydeifFcilecanasttkriiofwnuagsahateniddonws.iatlhtEaxSdtodrliavtceitnostn tawwneodrwetaismlheasit.negrs
beelniemfiintateadndfsruolmfutrhiec exactirdactaidodnitpiornocceaduusreed.'thSealtfoardmdaittiioonn opfraovendewtounbiedeontfinfoied
peexatkracintabtihleitGyLCoafnaFlMys3i4s2.2. AcTihidsandedwiltyi-onfowramsedaplesaokofapnpoarevnatlluye modification of FM 3422, and it was converted back to FM 3422
wians ima pcrhoevmiincgalthe on thin-layer
Ptlhaetegsr.owtShinpceerioidt,waist pwarseseonbtvibooutshlyinnotextarmaecttsabotlaikcenpriondiutcita.lly and at the end of
lTohnaeltyeprirneipmaptrrhaaottviewodnataeonrdf-sstatheatnumdraeardtdieadpreuotscheyedlduirnaeceetdaaretlseyc,risbhaaenkddeeitnfhlytalhsekamcceuetlthtaoutdrese-ssaadntidufrmfaaettreeedrdiwaafltrseormswetechreteion
anlnoydtenumoustlesdiinficinleudidtseidnpritenhpiastrhaemteidcoiunal..turTeAhsmeeadniorane.esmuultlT,hsuisfh,ioweedtvheeFrM,in3i4t2th2iealwFaMsFM34i23n24t2e2wnatscioonnnocatelnltyarsastceiopomanprlawetatesed-
wvaasriatbhleecoanncdenittsractoinocnentthraattwioonuldknohawvneobnleyentporebseenstomheatdhinagll lteshse sified. As with the standard media, this initially-used media
FtMhan3421233bopopmn, ewmhuliech was demonstrated fo
bine niotsntporxeiscentcoe.microorganisms by the fact that cell respiration occurred normally
eIvneirtyial8lyd,aysc.ultTurraenssfweerretismheaskewneraet lraotoemrtcehmapnegreadturteo 7(2d0a-y2s5Cf.o)r caonndvetnriaennscfee.rs made pCeurlitoudr.es were extracted both at the beginning and end of each culture growth
FThiegurreesu2.lts Iwn emroestquictaseesv,aritahbeleamofurnotm
otfraFnMsf3e4r22tothtartancsfoeurl.d
beThiesxtrcaacntebde
seen with
in
ethyl acetate at the end of the incubation period was less than that which was
v
ethxetraicntiatbilaellyi-memxetdriaactteeldy mfaotlelroiwailngwacsulntoutrereicnoocvuelraatbiloen.froUmsutahlelyw,oek5-0o%ldorgmroorwen of
;
csutlhtyulreasc.etaNtoe dcuelgtruardeatieoxntrapcrtosd.uctsAtwetrhiesdpiosicnotv,ereidt bwayseniottherknToLwCn wohretGhLeCrofthethe
umantreercioavlesrewdhiFcMh 3c42o2ulwdenreotbebiengextcroamcptleedt,elyor dwehgeratdheedr, to the biomass of the culture container.
itmeotra.biotlsicmaeltlaybomloidtiefsiedwerteo
bound
AponrtuimobneroofftheexpeFrMim3e4n2t2stwoebreecosmuebsneoqnueexnttrlyactcaobnldeuctbyedettohyldetaecretmaitnee.what caused a
006376
"10-
1.0 0.9
os
ggor
M5 X)
z 0s
goa
0.3
zg 02
or JM.
v
25 4
s
12
16
20
Weekly Transfer #
*wIeneiktliyaladcaopntcievnetrattriaonnssfewrepreerinoodts.measured in the second and third
FIGURE 2. eaRtadhtayiplotivoaefcettfairtnaeanlsefxettroraeicxntipsteiraoilfenwFteM.ek3-4o22ldcoshnackeentrfaltasikoncuilntuerqeusalfrvoolmume
006377
ue
TLC and GLC Analysis of Extracts from Shake Flask Studies
aInmaotneeriTaLlC wehxipcehrimpernotd,uceedthyvleryacedtiastteincetxtrsapcottss owifth3 caulstmuarlelserweRrgethfaonundFMto342c2o.ntain TTwhoe ToLfCthveisucaullitzuriensg pprroocdeudcuirneg stuhgesgeestsepdotsthactonttahiennedew13s5potpspmcoonftaFiMne3d422flpurorioocrartboons. extraction,
TFhMe34t2h2irdacccluilmtaurteedcoinntoacuilnae)d. lesDsesptihtaen t3hipspmgroefatFMdi3f4f2e2re(nicnetrionducienditwiialthctohnecentra
atpiopnr,oxitmhaet"enleyw"equsaplotsintfernosmittyh,e
beuxttraFcMts342o2f
the was
tdherteeectecdulbtyurTesLCpornolvyedintotbhee
of extract
of the two 135 ppm cultures.
nTehwe sepxottracitssoloatfedthbeyseprtehpraereactuilvteurTeLsC waenrdeagcoamibninreedp.uriTfhieedmabtyertihailn-lcaayuseirngchtrhoimsatog
irnaphoyu.r lAabtteomnpltysyiweelrdeemdadaemattoercihaalracwtietrhizGeLCthcihsarmaactteerriiaslt.icsGLiCdenotfictahlistomatFeMri3a8l22,
mMaeatseurrieamlensthsowweedreitnotto mhaavdee oafcatrhbeonaymlounatbsoofrpmtaitoenriaatl"eiunjmecatnedd.an IaRbsosrppetcitorna offairtlhiys
FcMhar34a2c2t,erbiustticnotofovaerfllaupoprioncgarwbiotnhcoFmMpo34u2n2d,bweetrweeenal8soapnrdesSeynm.t. BTahnidss ssiumgigleasrtedtoa
ctoomnpleoxf'wtihteh uankmnaotweriianlto ltihkee
muraesas.
spMeacstsropshpeocttormastoebrtasihnoewdedfaomlmloonwiian,g
direct inserC0,, glycerin,
daentderFmMin3e4d2,2. ThTeheprreesleantcieveocfonucreenatwraastinoontonfotFeMd.3422FMto34o22thearppmeaatreerdiailns'tchoeumladssnot be .
hsapveectbreaewnordkuetotovoitlsatbieliinzge actompalehxiegdh.er GtCe-mmpaesrsatusrpeecthoafntpheureunkFnMow3n422m;atetrhiiaslmyaiyelded
miadiennltyifigeldycbeyrinmasass ispdeecntiasfiegdlybceyritnh.e) masUsnliskpeec.the (wIonrkfacitn,omuorrelabt,hanFMon3e422pewaakswas
ntoemtpedreatteucrtee."d, Thailsthporuogdhuctthehassamneotlibqeueind pfhuarsteh,erCcahrabroawcatxer2i0zMe,d.wasItusdeodesatnoathiagphpeerar
to be a major degradation product.
Fluoride Release Determinations
Mnoeascuhraenmgeentisn oeifthF~ercocnocnetnrtorlatciuolntsu,restakoerncuilntiutrieasllythaatndsahfotweedr v7SOd%aysd,ecrheaavseesshoiwnn ethyl acetate extractable FM 3422 concentration.
oInf athihseteerxopegreinmeeonuts, cnuilnteurecultthautrehsawderbeeenprempaairnetdainaendd fionroc6u-l8atterdanwsifthersa sonpalFlM a3m4o2u2n-t
caonndtaeixntriancgtemde,diat.hreeThwreereeogfrotwhneseincuthletuprresesewnecree ogfrowenmulinsiftiheedabFsMen3c4e22ofandFMex3t4r2a2cted,
aadnddittihorne.e weTrhee sixty-one, and
eetxhtyrlactaecdetparteioprhatsoecwoalsl grreomwotvhedafnrdomimamleldicautletluyreasf.terFiFfMty3-4o2n2e, fifty-six percent of the initially extractable FM 3422 could not
bLeessextthraancte0.d1fprponm tofheFthwraese pcruelsteunrtesinthaatnyhaodf bteheenngirnoewnculitnurtehse. prHeasdencaellooffFtMhe3422.
nF"oneixontrarcetlaebalsee FtoM 3t4h2e2mbeediean, d1e4g0rapdpemd winoultdhehacvueltbuereengrfoowunnd.withTheFsMe34r2e2sulwtisthincdoimcpalteete
mtehatta,bolasicaexlpleyctaetdt,acktehde.perfiuorinated portion of the FM 3422 molecules is not
006378
12
Attempts to Free Nonextractable FM 3422 by Hydrolysis
ieAnxpctoursbascaittbailboelneweixFtpMhla3na4ac2tc2iloifnmraetfqeoudrenmttihlceyroocbabinsanelortvactbuielotnuerxettshraatciste5dt0%haitonrtoitmoehrtahesylobfeaectnheetactoiennjiuatigfaatlteleryd-
mOFenptowlaysmertiozeddetefromrimniengifa an extractable form.
naocniedxtorracbtaasbelehydmraotleryisails,
The could
coobnjveecrttivFeMof342t2hibsacekxpetroi-
FInM t3h4i2s2 tion of
oenxpetrhiemesntta,ndacrudltmureedsiawwheirceh heaxtdrabceteendgwriotwhn ionthytlheapcreteasteen,ce FM 3422 in the ethyl acetate extract was determined. The
Tohfeemcuonlcseinftireadcultures were
NtLahedeningtwheasxstrouafscettidemdet.oagaiCinoncnrecaaefsnteterraththyeeddrpHoH:lPyOs,ifsowlalasotwiu8ns0geCd.hyfdaornrodlaycasitidspi,fHica1alltoirosnapmHpaln1ed3s 2fW0oerrmeovlaarirous
neutralized and adjusted to equal volunes before extraction at room temperature.
TFhMe34r2e2suwlatss oexftrtahcistabelxepebreifmoernet careellushloawrngrionwtTha,bleVe3.ry FM 3422 could be extracted even after hydrolysis. In
Alnitatlveeraofgetohef r2e8m.a4i%nionfgthe fact, it appears that
tlhoantgecrouelxdpobseurerectoovetrheedseihnydarosleycsoinsd ecxotnrdaicttiioonns. decreased the amount of FM 3422
TABLE 3
FOLRLEOCNOIVNEGRYACIODF O"RNONBEASXETRHAYCDTRAOBLLYES"ISFMAT342820,C
pH
Tim8e0aCt
B4Mo3f42"2NonReexctorvaecrteadble"
7
0
1
0
1
0.25 hr,
2.7
"
12
.
3.9
1
2.5 hr,
Ls
1
25 hr.
.
"0.8
13
0 hr.
8.2
13
2.5 hr.
EX
13
25 hr.
0.4
006379
15
Materials Balance Experiments
BOiFodtehgesreadaetxipoenrimsetnutdsieswasworteo in the aqueous, solvent, and
scdooe}ntidedurscntipenhdeasuteshseingorfe1lea%xtCti-vrmeaMcpt4oe2rd2t.icounlstTuhreoefsprFaiMfmta5er43ry2gopbojooessceteirve
in the presence of emulsified 1'C-FM 3422.
shFIatmMdant3bdh4eai2ers2nde(xmF1paM0e0irn3i4tpm2pa2emin)nt.me,e'ddifTtahoh.ires samtpeapdnridoaaxridwzsaastmeleiydnioa2c0uwlatasrtaepndsrwfeioptrahsredaathbweoetteehkrlowygietnhiegogauensrdvawwluisitphuoocnuetthtMehco.r
aeelxxsttorraagccrttoeewddnaiffmotmreerdoinaoenteewloWyeekea.kftOTeAfhrtreigerntohowicotsufhl.patothiienoTtnhn,,reewga,nccduulltttuhurrreeeses cindooentnttaicicnoainlntgaicnuilvnLiagGr-FeFisM3w34e493c55ewwaevree
schruaolktneurntehsef,orabqruaeinonugasiddnigatnidtohneeatilhrylfciofantcceeetenanttremaitPnihuoatnseestsoawn1ed/0rC0-eeFxpMpttmra3.ak4ce2tn2eTdhe.emulcTsuhilrotenusrwesaesnew-eaardledesdsahsespneletsre
TehxetrSaocltieddscuflrtounretsh,e An omnsferred to
gadrdoemd ctuoltAquuraseosl"'()esseinntisaclilnytniollsaotlifiordnosmvwiceaarlcsgh,porafensdtehncetonmiitnnoigdt.ially) combustion cups, ried in an oven at 1030 C. gor
ccaoonumdnbtucesodtm.ib|ounsStwaeadmsluetssrianvpgeprAeegdricincohuenant'essdciwPniattcihklalarandtiocinnotmebfrulnsuatilidosnctoenaqtnuadiiapnrmidengngute.annchoTrhgoani1c40.dtiwtoifnhgroouanrnsd
their added
driardeicotalcytivtoitAyqucaosmolp.a,red
to
known
volune
samples
of
IC-FM 3c4o3r3reecutuiiooni,onand
ATghnsiuosl,siegxypCevr-aiFsmen3it4n2a2wdavseermtucleosnnitdolunyctwneaodsttnwsoottabtiiamldeidsze,dedtwoIintthhteheAlccufolintrousxrt,esatatntedhamtpLtt,habdrtohbkeeeeMnracpbirpdalryy3:422 bbreofkoerne. Fi 3422 addition because the week-01d wnstabilized emulsion had slzeady
The results of the first attempt at this experiment are shown in Tables 4 and 5. .
ExCturlatcutreed
ECxutlrtaucrteed
;
Initially
After 1 Week
Ethyl acetate
10382118
1028268
Water
0.04820,008% 0.02%20.02%
Solids
-
0.38.48
Total Recovered
10382118
102326.4
Table 4. Pinercfeinrtst#of1 S2tMaantdearridalDseviBaatliaonnceofexpienriitmieanltlsy. added Mc-FM 3422 recovered
006380
1a
ECxutlrtaucrteed Initially -- CCuullttuurree
Extracted
Aer,
SEMleetchetordonoif CDnaeptteurrtneinGiLinCg tFM 4T22aContcentiraCto ioonmney
ECxotnrcaecnttration Foo Aled
9)iggpypntd.2ropm ----------L1S11R5mtplpasZ12pnppm
AAMcacotcweorumintatleddfForr.. =
103
BREE e 83 e pputse .0 pon
113 put? pom
foentration
WTR
MaAtcecoruinatled For. 75%
102%
Table 5. bdCyeovnicsaectniitnortnaitllifaortnoioonftheFcMoufni3t4r2is2ntgMiaantnedertibhayyllselaBeccaetltraaontnceeceeaxxptptreaucrrtiesmeGnL+Ct.1asstadnedtaerrdmsined
TinheTxaobslueslts6oafndth7e. second rum of the Materials Balance experiment are shom
ECxutlrtacutreed Initially
ECxutlratcutreed After 1 Week
GErxotwrnactCeuldtuArfetser Fifteen Minutes
Ethyl acetate 89.743.6
3.92.8
33.623.0
Water
0.3
0.40.3
1020.4
Solids
-
3.94.6
0.120.05
Total Recovered 93.7:4.9
a0.2416.7
0.73.0
:
Table 6. TPeecrcoevnetred+ oinnesesctaonnddarodf dtweoviMaattieorniaolfsiBnailtainaclleyexapdedreidmeMncts..F 3422
MEeltohcotdroonfCaDpettuerreminGiLnCg FSMci4n2t2ilClaotnicoenntrCaotuinotning Culture Extracted Initially 99.8 ppmi0.7 ppm 99.6 ppasd.0 ppm Culture Extracted After 1 We. 44.0 ppatS.7 ppa 44,3 ppat10.8 ppm AGfrtoemr C1u5lMtiunrueteEsxtracted 3L1ppmi2.7 ppm 37.3 puts.3 ppm Table 7. MaCaontndecrebinyatlresalteicBotanrloannocfecaFpeMtxup3re4er22imGLeiCnn.tetashyldetaecremtianteedebxytrascctisntiflrloamtitohne sceocmoenidng
006381
-15-
wrTaehsecofvroeeursniudelststhoaoftfFwMthhee3ns42e2FMewx3ep4re2er2inmwoeatnstsoabdtwdaeeirdneedav.saluanaIbnwlnetshtedaebsfipiliritszetedtahteetmeumflpastci,tonti,thatthecoambpilleittey
tithtoaatteixowtnrnasoctftabcitolhaiisgzueldmaatteeemrudilasclilounmwspassbnrooeftakFMlwois3t4t2h2ia.nftaerThfie7swdhpaorysuerssuomfaobnslhysakihitnatgpi.pnegnWwesidthihnavtehtehisspbroeecviepd.
weixtpheriamevnitablaes the uptake df
bweiloll,ogiacnadl incultthuirse,formI,t iist proesmsaiibnlseextthraatctsaubrlfeactdaenstpitfeaciclointtaatcets Fi 3422. However, it is more likely that emulsification increases
tmhiecrnobuimablersoolfidFsM. 3422 molecules contacting and, subsequently, binding with
Abaosttnreometpcteovdeatraebodtvheii,sniteailxalplelroyif,metnbhtie.t"iCAt-s7wMaiss 3ns4oe2te2nwraeisnconvToeatrbeldeac6ca,ofwtmenortsetsdigofnfoirftihicinsanttmhaetbeisroeilcaoolgnidccaoluld ascotliidvse hFaMd34a2c2cumiuslattheadt, itTwhaesmoasbtsoprrboebdabblyetheexplcaenlaltuiloanr mfaotrertihails laonsdsbooufndrasdoio wsatsronnogtlysothsattrointglcyoublodwnnodtgbheatexittraccotueldd bnyotebtehylvolacaettialtiez.ed Hforwoemvetrh,e ictelaluplpaarrently meaxtpelrainaaltiobny hfeoartithneg tloow10r3ecovC.erieLso.ss to volatilization is the most probable
wTahsisaelsxopenroitmenetxtraalcstoabslueggefsrtosm tthheatpraebgsroorwpntiocnultisureasphtyhsaitcawlerpehesnhoavkeennonw.ithFM 3422 etomublesiefxipeldaiFnMed34b2y2 faomreotnalbyoli1c pmhiennutoemse.non,Thipsartriatceulaorflyresmiovnacle itshetooorgarnaipsimds had wnhoettbheeernorancocltitmhateedFito34F22Mwh34i2c2h. isItprceasnu'mtabbelydbeotuenrdminteodthferocneltlhsisisexpeevreinmteunatlly FmMeta3b4o22lizaendd ctoonfainromtahteironchbeymicaalmetfhoromd.sucThhisaswGouLlCdthraetquitrhee rreeccoovveerryedomfattehreiablounisd still FM 3422.
Adsorption on Culture Containers
:
Aispotshsaitblteheexmpaltaenraitailonwasfobreitnhge dsetcrroenagsley bionunedxttroactthaeblepolFMypr3o4p22yleinnegtreowsnt ccuullttuurrees
wceornteaionbetrasi.nedThiinsboptohssgilbaislsityanwdaspolcyopnrsoipdyelreenderceomnottaeinseirnsc.e sNiemvielratrherleessusl,tsit was
investigated in the following procedures:
3
Aonlmyediina wtahsatprietpadrieddnotthatcondtiafifneretdhefnruotmritehnetsYtManbdraortdh1n3e3cmegs/sLaryFMas342a2nietmruolgseinon media Tshoruerecewefroer ceexltlracgtreowdthi.mmedTieantemlly,ofththriese wmeerdeiacwaapspedadwdietdhtpooleyapchropoyflneinneecatpusb,es. waenrdetshhraekeewnerfeorpolnueggweedewkitahndfeoxatmrascttopepderasn.d anThaelyczaepdpbeyd aGnLdC sustionpgpertehde tsutbaensdard procedures.
006382
.
-16-
Sample hn
1a 1s LC Pe Pes Fa Fa Fas
Cap Polypropylene Polypropylene Polypropylene Polypropylene Polypropylene Polypropylene Foan Foan Foam
TimePriinorPotloypErxotpryalcetnieonTube 15 min, 15 min, 15 min, 7 Days 7 Days 7 Days 7 Days 7 Days 7 Days
%FMIn34i2t2ialRleycoAvdedreedd 7 i i 7s 78 8 92 93 92
Table 8. Percent of initially added FM 3422 extracted from polypropylene tubes.
The Tesults of this experiment are shown in Table 8,
kTnhoewnr.easoTnhsereforalsthoe islonwo FeMxp3l4a22natrieocnovefroiresthoebtgarienaetder irnectohviesrieexsperoibmteanitnedarweitnhot
ctehdeurteu,besthethactontweenrtesshoafketnhewittuhbesfonaenvesrtopcpaemres,in cInonttahcetwweietkh-loenigthesrhaktihnegpoplryo--
bperfoopryelenien acnayp eoxrpetrhiemefnotasn. stopper. Also, the foan stoppers had not been used
:
pDheesnpoimteenotnhereipnecaotmepdlleytesereencovienryearolfieFrM e34x2p2e,rimtehnetsse. resIunltesarldyo neoxtpereixmpelnatisn, thheigh
ddreeocvnoeevleoarpfietedesraontdfhelFoMcwul3r4te2u2croevweehrraeidesogbrtooafwinnFeMfdo3r4i2ns2ietvwieeanrleldyayosbb.etfaoirnIeendtthihenisecetelhxlyplecrauiclmetetunarttewehiatedhxtracts
annpoetpgelaisrgisginbtilhfeaitcmaintcthrleoybiiandlcirfefpaeosrpieunnlgtatilfoornsossm, otfhtehaebrieilcnioitvtyeiraitleosFeMxatf3rt4ea2rc2t 7reFdMcaoyv3se4.2r2iesIftr,owmetrhteeherefore,
culture with time requires the presence of biological solids.
Recovery of FM 3422 Fron Cells By Volatilization and Condensation AdtshueripnrMgeavtietorhiueaslltsywonBohatoleuadrn,sce dthreexypienmorgsitmpeernpitroodbi.as bltehFaotrexptihtliasvnoaltriaeotansiolnif,zoerditthfweraosmlobsteshleioefsvoelFdiMdst3h4ap2th2asiietn imtighftrobmeceplolsssibalned ctoollqeucatnitnigtatiitveblyycornedceonvseartisoonrbeond FaM"c3o4l22d bfyingveorl.a"tilizing
006383
o
-17-
A a
LX
2
>
CWojaltode]r Tube
.
;I
i
0
Aspirator Tube
i
:
oy |
Test Tue
!$dE" 1 Cold Fi. nger ol
:
i
.
;
FM 34S2a2-mCpolnetaining * ;
:
i
4a
:
Stean Bath
Figure 3. aApnpdarcaotnudsensuasteidont.o recover FM 3422 from cells by volatilization
006384
.
-18-
Twhheichfewaassibimulnityinodfuptlhiicsataep.proaFcMh 3w4a2s2 e(1vamlgu)atweadsbyplatcheed fionlltohweinbgaseexpoerfimaenltarge
test tube. effectively
A second smaller made into a cold
test tube finger by
was placed inside the first, and was continuously running water into its
bPralesamceoevdeadndinanadsapitshrteaetainsnubgbaltiihmtatf(erFdoimguFrtMehe3432)t.2op,waAsfThtheeedr bf1a5rsoemminioutfstetshs,iusrftahacepepacroialntdtuosfaiwnabgseorwtswheesnvoluse
oafddeetdhyFlM a34c2e2tawtea.s sGuLbCliannaatleydsiasndicnodlilceactteeddthinattw3o2%trainadls4.2% of the originally
T1h0ealabocvelelscuublltiumraetsioninprtohceedsutraendwaarsd mtehdeina.repTehaetesdmailnldiuhpolciuclautme fuosrintghewseeekc-uolltdures
came from
dereexsmtocrvraeicdbt.eedd
wTaaihphtepheatrueanertdquorusgaileendaenvobdouilsouemmxaepFssoMssoe3wdf4a2s2etothattycrhlcaelnisamfcsaeettrteeraadetmdebcaut(tlo1ht0utrmhfeleo.)r,ou45Ttahnemedrisnettuhutebceeusll.toiubqrueTtishhdeewpheseatrhsheoeyvs:e
aocfettahteenwoansehxitnrgasctefdromFMth3e422cowladsfriencgoevrerweedr.e aTnhaelyczeeldlsbyweGrLCe, dbruitedvuenrdyer1intietlsrogen
and again exposed in the steam bath for an additional twohours,
TeqhxuetarnatrciettsaaulbttliseveloiynftortehiecstohvyeelxrpeedarcibemyteantsteublaairnmedaptsirhoeonswnunasnibndlyTcoanbbdloeeunnsd9a.ttiooTnht.eheFPMceerl3lh4sa2,p2swtahcsaotmpnolwteatsenot
Tecovery would have been possible had the cells been exposed to the stoam bath
fthoartasolnoengoefr ptheeriobdo.undAlFtMho3u4g2h2 mtahyishaevxeperbeiemnentchedmiidcanloltyrmuoledifoiutedtbhye ppeotsasbiobliilsint,y
it did indicate that a large fraction of the bound FM 3422 was unchanged,
Culture #1
Culture #2
Extracted into Ethyl Acetate
15.6%
21.08
~
Volatilized from "Wet" Cells
0.5%
0.3%
Volatilized from N, Dried Cells 25.68
2.8
:
Total FM 3422 Recovered
4.7%
43.1%
Table 9. Percent of initially added emulsified FM 3422 recovered from cells
by ethyl acetate extraction followed by volatilization and
condensation of remaining FM 3422,
HotEthylAcetateExtracts
bIsindodmiaantsisoa,ntteeomxfpttr2a.5cttoimolinnsocfrweeaFrsiee34td2ho2eneerumesucilonsvgieorhnyototofewttehheyekl-FoMalcde3,t4a2t21e0.smolrTbheciduslttuoprreotscheedtuhcaruteltuhiarndevobleveend
wwgearrtoewenrshosnaakmepnnluetsrf.ioerntT1h5ememdirineaus.tuelstiFnoMng 3a41222.r5oetmanlurlyssaismohpnalkewesar,s twoahlisacolhloawdcdoensdtoariptntoeiodtnwo1,o3f31p0tphemml FFdMMei53o442n222i,zbeyd
tohreabtio40ngssC,. oarnd60egtrC.actePdhawsiethsepeqauraaltiovonluwmaessionfsuertehdylbyacceetnattreifautgatriooonm tatemp1e2r,a0t0u0rerpm
acnhdromtahteogcgroanrpcheyyn.tration
of
FM
3422
in
the
ethyl
acetate
phase
was
determined by gas
006385
ER
-19-
The results of this extraction procedure are shown in Table 10.
sample AqCuoemopuossitPihoanse
TeEmxpterraacttuiroen (C) pApcmetFaMte34E22xtrianctEthyl
1 Deionized Water
2
1s
2 Deionized Mater
2
133
3 Microbial Culture
2
52
4
"
"
2
ss
5
"
0
3
6
we
"0
5
,
0
8
-
"
60
56
Table 10. H1otmientuhtyelsacoeftastheakienxgtrwaictths eomfuwlastiefrieodrFbMio3l4o2g2ic(a1l33cpuplmt)u.res after Taehtxets6r0eagCra.ebsiuillsittsnyoihonifdgihFceaMrte34t2ht2ahnatftrhhooamtttheeetxhgybrliaocmtaacesedst.aatte2T2heexCt.armaocuTtnhiteonorfedoaesFsoMnn3o4f2to2rietmhxpetrroavlceotweetdrhe wcciootnnhfciienrnmtr1Satthemiionpnurteoevfsi.oFuMsT3h4oi2bs2serirnvaaptitdihoenlo4s0tshCa,tfreeoxmtuarlasnciotfnieaisdccnlFoiMtma3tK4ne2o2dwnci.sultaubTrhseeorbsreuedgsgubelstytssceallalsso
physical/chemical absorption process rather than biological degradation,
Dioxane Extraction of FM 3422 from Culture Solids
Ta
v
AbeItylnteeceamttpzhptoyrsnlelictamoacipetnrtuaearprteeyeatdecxeopttuehelircdsitmboeeornbtsor,efercvooiauvttrewiraogesnadswbfeycorhuerrnoedmfialnttuhioxatgtiirnaagalpllhlyt.hhoeifncTdhtueehlesrteeudrFpeMbryos3b4olp2ler2imodssbnlowewtenisstehoxwt1iar,tpa4hpc-atdretiehdonextalnye, caused by the presence of dioxane. Central Research overcame this anaiyticel ccpuorlnotcbullrueesmsivaentldhyatthtehhaatveexpFbeMerei3nm42e2mnatiinswtaansiontreedpmeeawttiaetbdho.lwieceaTklhlleyyretssrluatlnetsrsfeedr(siTnaobnsleeFveM1n1)34d2ad5ye.smcoobnnsyttarsiahntaieknegquFiltaesk
media for over six months.
006386
REN
"20.
CwuEixlctthruarEcettheydl Acetate at: 15 min. 15 nin. 15 min. 7 days
7 days
7 days
7 days 7 days
BE%yxtoErfatcFtMhea3422
Acetate
09.3 9.3
101.3
25.8
27.1
su
0.4
2.6
PCrfaootrcteuEdxruterreacting
Solids
No solids present No solids present
No solids present
$ReFcFroMovne3r4e2d2
Solids
---
-
(Refluxed in Dioxane for 2 hrs.)
77.9
"
80.1
(Nr, ie&dresoflliudxsedwiinth 75.0
Dfoxane for 2 hrs.)
(fRoerfu1semdin.i)n Dioxane 75.5
(DEixotxraancetedatwiRtohom a
Temperature)
RTeoctoavlery%
of FM 3422
99.3 09.3
101.3
103.7
107.2
106.1
103.9
7.6
SERS Table 11. (RietchovDeiroysaonfe,FM 3422 from culture solids by extracting or "refluxing
v
ReFERBNGES
(1) Reiner, E. A., Biodegradation Studies of Fluorocarbons, 3H Technical Report,
August 12, 1976.
=~
(2) NMoenvdeeslb,erA.1,8, An1a9l77y,tical Methodology on FM 3422, 3M Technical Report,
(3) CEDoinncvskiesrroovnna,nteinoLt.na,lS. VSooLlli.uu,ti3,oMn.SNoct..o,S2,oPmh.eSDu.Tmoex(riBcr1iO0t7ri6gs.ahniCcoluCmobmipao)u,nds",B"ioEdnevgirzaodnasteinotne,lAn
fr
oEwAR/cen
006387