Document omMgZVRgwRZD7B5zmDkOqq1Vo

BIOCONCENTRATION TEST SUBSTANCE Identity:N-ethylperfluorooctasnuelfonamidoethanolm;ay alsobe referredto as N-ETFOSE Alcoholor FM-3422. (1-OctanesulfonamideN,-ethyl1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-heptadecafluoro-N-(2-hydroxyethyl)-, CAS # 1691-99-2) Remarks: Materialisan off-whitwea,xy solidofuncharacterizepdurity. METHOD: Method/guidelinefollowed:3M derivedmethod outlinedinthe testprocedure fordeterminingtheaquaticBloconcentratioFnactorof N-ETFOSE alcohol. Type: Staticexposurewitha flow-througchlearancephase. GLP (Y/N): No Year: 1978 Species: Channelcatfis(hktalunipsunctatus) Supplier: Not notedinthereport. Analyticalmonitoring:ConcentratioonfN-ETFOSE alcoholinwaterand fishby GC withelectroncapture;temperature Length and weight:Not noted inthereport. Exposure period: 7 days Clearance period: 5 days Statisticamlethods: 3M Trac system,MINITAB 11program Test fishage: Not noted inreport. Loading: Not notedinreport. Pretreatment: Not noted. Test conditions: Dilutionwater:Carbon-filterweedllwater Dilutionwater chemistry:Not includedinreport. Stock and testsolutionpreparation:The primarystocksolutionwas preparedby dissolvin5g7 grams ofN-ETFOSE alcoholin50 ml of acetone,applyingthesolutionto 10 pounds of3.5mm glassbeads, evaporatingofftheacetone,and circulaticnagrbonfilterewdellwater throughtheglassbeads. The system was designedto resulitna final loadingratiof0.5grams perliterA.erobicconditionwsere maintained throughoutand thesystem was assumed to go tosaturationL.engthof timeallowedto circulatweas notnoted. Exposure vessels:114 litearquarium. Volume utilizewdas notnoted. Number of replicatesT:wo Number of fishper replicateN:ot notedinthe report. Number of concentrations:One plusnega'tivecontrol Water chemistry during thestudy: Dissolved oxygen: Not notedinthe report. Testtemperatur2e1:+1OC pH: Not noted inthe report. Photoperiod: 16 hours lightand 8 hours dark witha 30 minute transition period. Remarks field:Followingthe introductioonfthe channel catfishintothe experimentaland controlaquariasamples of4 channel catfishand threewater samples, top,middleand bottom layer,forN-ETFOSE alcoholuptake analysis were obtainedatthefollowingtime pedods: Control,priorto introduction1,5 minutes,one hour,two hours,fourhours,eighthours,twelve hours,sixte' en hours,twenty hours,twenty-fourhours,and attwenty-fourhour intervalfsora periodof7 days. The channel catfishwere then removed toclearancetanks for two hours and finalltyransferredtoa flow-throughsystem havingflowrates throughthe testchambers offourwater volumes per 24 hours. A sampling regimen identicatlothatofthe uptake phase ofthe studywas immediately initiateadnd terminatedafterfivedays. RESULTS N-ETFOSE AlcoholUptake by Channel Catfish(ictalarupsunctatus)- Whole fishsamples Time 15 min. I hour 2 hours 4 hours 8 hours 12 hours 16 hours 20 hours 24 hours 48 hours 72 hours 96 hours 120 hours 144 hours 168 hours Fish Group A (ug/g) 1 4 9 16 58 62 51 61 104 132 222 190 211 320 552 Fish Group B (ugig) 1 4 10 29 42 60 56 74 110 213 159 257 201 279 158 N-ETFOSEAlcohoCllearanceofChannelCatfis(hictalarpusnctatus-)Wholefishsamples Time 15 min. 1 hour 2 hours 4 hours 8 hours 12 hours 16 hours 20 hours 24 hours 48 hours 72 hours 96 hours 120 hours u Sample lost. Fish Group A (ugig) 306 269 252 260 260 253 235 238 237 -0 200 136 106 Fish Group B (ug,g) 319 216 309 242 242 206 177 255 201 190 227 226 78 -N-ETFOSE Alcohol Uptake of Channel Catfish(ictalarupsunctatus)Muscle Time Control 15 min. 1 hour 2 hours 4 hours 8 hours 12 hours 16 hours 20 hours 24 hours 48 hours 72 hours 96 hours 120 hours 144 hours 168 hours Muscle Concentration (ugig) <0.1 <0.1 3 5 10 22 31 38 49 61 118 127 135 153 197 147 N-ETFOSEAlcohoCllearanceofChannelCaffis(hIctalarpuusnctatusM)uscle Time 15 min. 1 hour 2 hours 4 hours 8 hours 12 hours 16 hours 20 hours 24 hours 48 hours 72 hours 96 hours 120 hours MuscleConcentration (ugig) 118 127 108 128 147 148 181 117 75 102 77 69 72 N-ETFOSE AlcoholUptake by BrainTissue and GastrointestinTarlactofChannel Catrish (ktalaruspunctatus) Time Control 15 min. I hour 2 hours 4 hours 8 hours 12 hours 16 hours 20 hours 24 hours 48 hours 72 hours 96 hours 120 hours 144 hours 168 hours Brain Concentration (ugig) <0. 1 2 8 28 50 100 115 149 240 225 352 328 606 588 695 905 GastrointestinalTract Concentration (ug/g) <0.1 <0. 1 6 12 41 54 110 119 206 201 230 510 533 504 688 533_ N-ETFOSE AlcoholClearance ofBrainTissue and GastrointestinaTlractofChannel Catfish (Ictalarupsunctatus) Time 15 min. 1 hour 2 hours 4 hours 8 hours 12 hours 16 hours 20 hours 24 hours 48 hours 72 hours 96 hours 120 hours Brain Concentration (ugig) 1152 761 712 602 511 582 618 559 523 586 450 423 417- GastrointestinalTract Concentration (ug/g) 281 630 325 507 302 263 1205 805 564 264 353 390 712 .N-ETFOSE AlcoholUptake by Liver,Kidney,and Gall Bladder of Channel Catfish(ictalarus punctatus) Time 15 min. 1 hour 2 hours 4 hours 8 hours 12 hours 16 hours 20 hours 24 hours 48 hours 72 hours 96 hours 120 hours 144 hours 168 hours LiverConcentration (ug/g) <0. 1 11 11 31 67 89 99 161 149 329 261 481 469 384 561 Kidney Concentration (ugg) <0.1 <0.1 <0.1 37 59 77 93 149 99 189 200 359 315 349 152 Gall Bladder Concentration (ugig) <0.1 <0. 1 <0. 1 <0.1 <0. 1 158 245 271 242 1383 1585 1573 2875 3261 13805 N-ETFOSEAlcohoCllearancoefLiverK,idneya,ndGallBladderofChannelCaffis(hktalarus punctatus) Time 15 min. 1 hour 2 hours 4 hours 8 hours 12 hours 16 hours 20 hours 24 hours 48 hours 72 hours 96 hours 120 hours LiverConcentration (ug/g) 504 650 758 779 507 446 728 420 443 575 316 76 507 Kidney Concentration (ug/g) 274 567 439 447 390 559 515 365 259 351 203 182 285 GallBladder Concentration (ug/9) 1543 4908 1286 5058 3710 4321 4247 2495 3912 2810 1221 1411 6497 CONCLUSIONS No reliable conclusions can be defived from this study. General observations can be made. N-ETFOSE exposure. alcohol concentrations increased in the fish tissues over the course of Lipophilic organs and organs possessing a large surface area at the water/organ interface possess the highest uptake rate constants. The brain and gastrointestinal tract have potentially high bloconcentration factors due to the lipophilicnature of N-ETFOSE alcohol. The muscle filleatppeared to exhibit the least potential to bioaccumulate NETFOSE alcohol. The gall bladder showed the highest concentration of N-ETFOSE alcohol There is a potential for excretion of N-ETFOSE fecal pathways. alcohol through both udnary and No signs of toxicitywere apparent at the exposure concentration. While concentration decreased in the whole fish samples clearance phase, the time required for complete removal days used in the test. at the end of the is longer than the 5 Submitter:3M Company,EnvironmentLaalboratorPy.,O.Box 33331,St.Paul, Minnesota,55133 DATA QUALITY ReliabilityK:limischranking3. There isa lackof descriptiofnorthe study's methodology and characterizatioonfthe testorganisms. There isno attemptat correlatingn,or isthereany recordof,the size,age, and body fatcontentofthe fishas itrelatestothe concentrationof N-ETFOSE alcoholfound inthe various samples. This isespeciallyimportantdue tothe factthatolder,largerfishare likeltyo have more fatcontentthan younger,smallerones. The vigorousmethod used to solubiliztehe testsubstance appears to have resultedina supersaturatedsolution.The solubilitiyscharacterizedas being 50 ppb, butthe concentrationinthe aquarium was givento be 500 ppb, and itwas noted thatthe N-ETFOSE alcoholwas presentas a suspension. Moreover, itwas alsonoted thatexcitabiliotfythetestorganisms would cause disturbanceofthe bottom layer and re-suspensionof N-ETFOSE alcoholintothewater. Test substance purity was notcharacterized. Thirdpartyreviewby Dr.James W. Gilletotf CornellUniversit(yobservations includedwith report)alsoindicatea lackof reliability. REFERENCES 3M TechnicalReport "Evaluationofthe BioconcentrationPotentialof FM 3422" A. N. Welter,Project9970612623, Fate ofFluorochemicals,Report Number 2, August 16,1978 3M requested expertoverview,"BioaccumulationStudies",Dr.James Gilleft, CornellUniversityM,arch 8,1993 OTHER Last changed: 5/18/00 3M RE:PORT: BIOACCUMULANON STUDEES March 8, 1"3 ReportNo. 2 (8116178')Evaluadonof dteBioconcentratiPoontmijalofFM 342?-- lliisreportcoversnew experimentsbasedon thestudyin l (above),but usinga shorter exposure (to a safd. solutionand under the same circumstancesa)nd more frequentmeasurements only with channel catfish.However, the methods indicatedfor measurement of solubilitaynd X. are fairlgyood and the basis forstate-of-the-parottocols.Hence, I acceptthevalue of solubilit=y 50 ppb and log K.. = S. Further, with thisvery low solubiliteyv,en with the low measured v.p.,one would cqxxt a moderate Henry'sLaw constantleadingto voLatUitjyrom water or a wettedsurface.On the other hand, the MW is571, which puts it toward the upper partof the range of molecularsizes'fowrhich expectedbehaviorislinearwith.,xespetcotK, That b, the molecular configuratiomnay presentsome problems in crossingthrough the pores-of the lipid bilayerof the severalmembranes. a. The report isvery confusing when itexpresses testagent conc. in "UW and residues in *ug(gw vMe sptem was loaded at a rate of 0.5 g/L (500,000 ug/L)[end of parl 4,pg. 31 when the solubilityis50 ug/L (50 ppb), whereas you statethat'0.5ulA - 500 ppb.0 b. The data in Tables 2 & 3 are shown, for OFLshA7 and O]FishB," as U these-weresequentialanab= of the same individual&7le textnotes thatfour fishwere removed at each time finm 6,pg. 31. Apparently, -.thsea-mple was dividedto provide 2 fishf.orwhole body and -2.fortissuesviabeing Itissuemized('whatever thatis).fItis.interestinign thisregardto note the problem in not havinga protocolfordissectioonther ..thasnome QA/SOP in a book somewhere. For a number ofyearstherewere frequentlysignificandtifferences between PCB residuesin lake troutmeasured by Canadian scientistisn comparison to those by NYS DEC scientistasl,thoughthey both thoughtthattheyagreedon thesame protocol Itturnsout that therewere two errorsor differences'.Me Canadians only used the portionof the fluetabove the median lineforPCBS, whereas the U.S. group used the entirefillemtinus the mid-line.Second,the Canadians removed material(fm, mid-line)by a rigidprotocolL,e.,cuttinga 6-mm notch to remove themid-lineand takingoffthetop 25 mm to remove thedorsalflnand associatefdaL Ile DEC labvariedthefatremoval so as to take offallfatby cuttingin 1-5 mm more or less.These differenceasre verysmall but reflect 2 3M REPORT.-BIOACCUMUIATION STUDEES March 8,1"3 theadvisorigeisventhepublicby separataegenciesA.nyway,theU.S.valuesaremuch lowerthanthose of the Canadians,even though the fishcome from the same water.) c. 'Me reportingand plottingof arbitraridleysignateddata (*FishA7 - Fig.1 and 'FishBm - Fig.2) isnot verysmentifiq At leasttheyshould be on the same plot,as a range or recalailateads an averageat time t. As an averagevalue,the whole body residueswere stillclimbingwhen the investiptorsterminated exposure. Ile avg.valuesfor the times from 24 hrs on are 107, 172.5,190.5,223.5,206,299.5,and 355, implyinga rateof 'equilibrium-'-approaching+20%/dayl That isfarfrom equilibrium.Further,although the generalprotocolresemblesstate-of-the-atrhte,smallnumber of testorganisms per time interval(should be Z6) means thatwhat theinvestiptorseeas an 'outlierm'ay reallybe a typicalpointfrom a wide range of responses.Curiously,althoughthe authorswere concerned about the increasinguptake with weight in Report No. 1,theydo not reportbody weight,Upid composition,or length(determinantof gillsize)for the testsubjects.Ile rateconstantsshouldbe calculateadnd divided(k)k = BCF), but the lossrate appears to acceleratweith time. 71iismay be a featureof the incompleteequilibrium,tissuedistribution, or,enterohepaticcycling-viabfle. d. By the time one getsaround to the specifitcissuedistribution(swhich areexpressedas up to 5 signifimnt digits!t)h,e authorsare confusingindividuaalnd temporalvariationwith "plateausaond lerratidplatterns. Itisunlikelythat2 specimens ateach time can yieldcomparable data unlessthereare criterifaorage and sizeclass.Reporting BCF by specifictissuew,ithoutattentionto tissuelipidlevelisdisingenuousor at leastpoleadmg. e. Itisnot clearhow theauthorshave documented excretiovniaurineper se,sinceurineconcentrations, conjuptes,etc,were never measured. Presenceof materialin kidney merely reflectvsascularperfusion. On the otherhand, the tentati,v@ieew of the importance of biliaryexcretion(alreadywell establishefdor lipophflesi)snot understandable,Indeed,one might note thatpll bladderdistentiocnould mean positive acceleratioonf bfliareyxcretion(influencebdy hepaticlevels). L 7he beliefthatthe absenceof toxicsymptoms forthe exposureregime used constitutedsefinitioonf this fluorocarbonas anon-toxieisan abuse of the term,since(as Paracelsussaid)'allthingsare toxicand it isonly a matter of dose which determineswhether a chemicalisa poison.'Ifthe investigatorwsere able to determine the N-Et FOSE alcoholas such,then itwas not conjugated(statementsabout.t,hisroutenot withstanding). 3 "6,m 6747-1 I-A TECHNICAL REPORT SUMMARY Date August 16, 197E TO: TECHNICAL COMMUNICATIONS CENTER - 201-2CN (Important- Ifreportisprintedon both sidesofpaper,sendtwocopiesto TCC.) Oi%fision Environmental Laboratory (EE & PC) Project Fate of Fluorochemicals Report Title Evaluation of the Bioconcentration To D. L. Bacon Author(s) A. N. Welter Notebook Reference Potential SECURITY lO- 0 Open (Company Confidential) M Closed. (Special Authorization) 3M CHEMICAL REGISTRY of FM 3422 Dept. Number 0222 Proi&ct Number 9970612623 'Report Number 2 Employ@ Number(s) No. of 09362 a Including Covershoot 25 Now Chemicals Reported 0 Yes No KEYWORDS: lselect terms from Thesaurus. Suggest applicable terms.) 3M other EE & PC'- Div. CURRENT OBJECTIVE: Progress Report 1. Evaluate Bioconcentration Potential of FM 3422. 2. Determine uptake and clearance rates of FM 3422 in whole fish and/or tissue. 3. Determine excretory pathway for FM 3422. Fluorochemicals (Aquatic) REPORT ABSTRACT: (200-250 words) This abstract information isdistributed by the Technical communications alert 3M'ers to Company R&D. Itis Company confidential material. Center to Toxicity (Bioconcentration) cc: DLB RLB MTE Xft.lk R.Prokop-236-OB EAR SKW ,i"nfiotrimaaltsi@o.n L;7k tlj 3M CON Fl DE NTIA L -2- INM@RODUCTION Living organisms possess the ability to concentrate and accumulate high concentrations of lipophilic organic compounds either directly from their environment or from their food source, a phenomenon which is well documented (Burnett, 1971; Gustafson, 1970). This ability to bioconcentrate seemingly non-toxic substances might- well pose a hazard for the ultimate predator species. This investigation was conducted to determine whether a fluorochemical, FM3422, does bioconcentrate in organisms and if so, does the material depurate rapidly. Selective organ systems were analyzed for the subject fluorochemical as potential uptake sites. Since the aquatic environment serves as a primary mode of entry for FM3422 into the environment, this study was conducted using the channel catfish (Ictalarus:.@punctat)usas the test species. The subject fluorochemical, FM3422 is a white granular material, molecular weight-571, possessing physico-chemical properties which suggest that this material may bioconcentrate. Thus, the material was found to be relatively water insoluble and possessed a high distribution coefficient, data which is indicative of a highly lipophilic molecule... This profile is generally accepted as that of a substance which will tend to bioconcentrate. METHODS The hydrophobicity of FM3422 precluded a simplistic ation of its water solubility. Use of the Veith-Comstock determintechnique -3- (1975) wherein water in a constant-level reservoir was continuously saturated with FM3422 by circulating the water through a bed of inert substrate impregnated with FM3422. Samples for analytical analysis were obtained via a sampling port. Replicate studies were performed. The distribution coefficient of FM3422 in n-octanol/water was determined using the methods described by Chiou et. al. (1977) and Fujita et. al. (1964). Channel catfish (IctaZarus punctatus) were acclimated to the following test conditions for a minimum of 14 days: temperature, 21+1 0 C, 16-hour light and 8-hour dark photoperiod with a 30 minute transition period and were fed daily (Tetra Min). FM3422, previously impregnated on 3.5 mm glass beads was placed in a 114 t aquarium, forming a layer approximately 2 cm above the gravel filter which was then covered by sand. The test tank was then filled with carbon-filtered well water. This system under continuous aerobic conditions generated a saturated water solution of FM3422, having a final loading ratio of 0.5g/c. Based on a previous study (1977) which indicated rapid uptal-.e and depuration rates for FM3422 in this species, the bioconcentration test was modified so that the uptake and clearance periods were seven and five days respectively. Following the introduction of the channel catfish into the experimental and control aquaria samples of 4 channel catfish and three water samples, top, middle and bottom layer, for FM3422 uptake analysis were obtained at the following time periods: control, prior to introduction, -415 minutes, one hour, two hours, four hours, eight hours., twelve hours, sixteen hours, twenty hours, twenty- four hours, and at twenty-four hour intervals for a period of seven days. The channel catfish were then removed to clearance tanks for two hours and finally transferred to a flow-through system having flow rates through the test chambers of four water volumes per 24 hours. A sampling regimen identical to that of the uptake phase of this study was immediately initiated and terminated after -five days. Individual (whole fish, n=2) and pooled tissue samples (n=2) were weighed, tissuemized, refrigerated and stored in ethyl acetate prior to the analytical determination for FM3422. Whole fish were blotted before the weighing procedure. The pooled samples consisted of the following tissue: brain, gills, liver, gall bladder, kidney, gastrointestinal tract, skin, muscle and skeleton. Tissue and water samples were analyzed for FM3422 using a Hewlett-Packard model 5713 gas chromatograph equipped with an :r3) electron capture detector (@Ti Specifications and pperatin g conditions were as follows: stainless steel column-length-6 feet. x 1/811; column packing and support-10% Carbowax--20 Mon ChromosorbW acid washed 60/80 mesh; operating temperature-isothermal 1800 C, detector-300OC; carrier gas-5% methane in argon. Recovery rate was above 90%. Statistical treatment of the data utilized the 3M Trac system, MINITAB II program. Bioconcentration factors and uptake rate con- stants were calculated based on formulae proposed by an ASTM commit- tee developing a standard method for conducting bioconcent ration studies with fish (1977). -5- RESULTS The water solubility of FM3422 was 50 ppb (Veith-Comstock technique) coupled with the added observation that this chemical was slightly volatile (Mendel, 1977). Using the method described by Chiou, et. al. (1977) the ,distribution coefficient for FM3422 was determined to be in excess of 105, in an n-octanol/water system. Values of this order of magnitude are indicative of the highly lipophilic nature of the test substance. In consideration of the slightly volatile nature of FM34.*12, the test organisms were introduced into the newly filled experimental aquarium and sampling of both channel catfish and exposure water began fifteen minutes later. . Table la illustrates the mean concentrations of FM3422 in various layers of the exposure water. the test substance was quite evenly distributed throughout the aquarium as indicatea by concentrations of 0.49, 0.54, and 0.52pi/9 of FM3422 in the top, middle and lower water layer of the aquarium. Since FM3422 was present as a suspension, excitability of the test organisms resulting in unusual disturbance of the bottom sand/FM3422 layer would contribute to the variability of mean concentration values as indicated by the magnitude of the standard error (Table la). Upon analysis of the exposure water samples for FM3422, evidence was obtained that this material was being rapidly released into solution (Table lb). These data suggest that concentrations approaching equilibrium values were attained within 1 hour of exposure. -6- Table la Mean Concentration of FM3422 Exposure Water: Experimental Period Sampling Site Top layer Middle layer Lower layer n concentration a iii/t 15 .49+.06 15 .54+.06 15 .52+.04 aMean concentration of exposure period + S.E. Table lb -- Concentration (ppm) during Initial 4 Hours of testa Sampling Site Top layer Middle layer Lower layer 15 min. .19 .31 .23 1 hour .45 .49 .54 2 hour .43 .47 .47 4 hour .35 .42 .55 aexpressed as u.EI9. WHOLE FISH UPTAKE AND CLEARANCE Uptake of the fluorocliemical FM3422 by channel catfish has been tabulated (Table 2) and illustrated (Figs. 1, 2). During the first 24 hour exposure period, both Fish A and Fish B showed a rapid uptake of the test substance. This initial uptake rate may be attributable to movement of FM3422 across the'integument With subsequent binding to lipophilic tissues. A slower rate of FM3422 uptake was noted for both organisms which was possibly accounted for by a decreased water/organism concentration gradient. No uptake plateau indicative of a steady-state was obtained following analysis -7- Table 2 FM3422 Uptake by Channel Catfish (IctaZaruspunctatus)a Time Fish A Fish B 15 min. 1 1 1 hour 4 4 2 hours 9 10 4 hours 16 29 8 hours 58 42 12 hours 62 60 16 hours 51 56 20 hours 61 74 24 hours 104 110 48 hours 132 213 72 hours 222 159 96 hours 190 257 120 hours 211 201 144 hours 320 279 168 hours 552 158 a Expressed as pg/g. of Fm 3422 content of those fish comprising the composite organism A (Figure 1). The 168-hour value for the test fluoroch;ainicalmay be an outlier as these values encompassing the period from 72 120 hours suggest a plateau (steady state) at FM 3422 concentra- tions of n,200 ug/g. A plateau effect was observed to occur with sample B, having an uptake value of approximately 220 Ug/g FM 3422 (Figure 2). Concentrations of FM 3422 bioaccumulated by these test organisms were observed to vary independent of whole fish weight. Clearance of FM 3422 by the channel catfish required in excess of 5 days (Table 3). -8- Table 3 FM3422 Clearance of IctaZarus punctatuma Time 15 min. 1 hour 2 hours 4 hours 8 hours 12 hours 16 hours 20 hours 24 hours 48 hours 72 hours 96 hours 120 hours a Expressed as ug/g. b Sample lost Whole Fish A 306 269 252 260 260 253 235 238 237 -b 200 136 106 Whole Fish B 319 216 309 242 242 206 177 255 201 190 227 226 78 TISSUE ANALYSIS: MUSCLE Of the tissue analyzed for FM3422, the muscle layer of the channel catfish was found to possess the'least propensity to bioaccumulate this fluorochemical (Table 4, Figure 3). A plateau effect was observed to occur in the concentration range of 100-200 pg/g (Table 4). Tissue weight was an independent variable, not affecting the bioaccumulation level of FM3422. Depuration of the sub ject fluorochemical, FM3422, was delayed, approximately 50% of the material having cleared within four days (Figure 3 Table 5). TISSUE ANALYSIS - BRAIN, GASTROINTESTINAL TRACT Uptake of FM3422 by brain and the gastrointestinal tract was elevated due to the lipid nature of these materials (Table 6). Brain uptake of FM3422 continued throughout the experiment (Figure 4) -9- Table 4 FM3422 Uptake by Ictalarus Punctatus Muscle Time Control 15 min. 1 hour 2 hours 4 hours 8 hours 12 hours 16 hours 20 hours 24 hours 48 hours 72 hours 96 hours 120 hours 144 hours 168 hours Muscle <0.1 <0.1 3 5 10 22 31 38 49 61 118 127 135 153 197 147 a Expressed as og/g. Table 5 -- FM3422 Clearance of Ictalarus puntatus muscle Time Muscle 15 min. 118 1 hour 127 2 hours 108 4 hours 128 8 hours 147 12 hours 148 16 hours 181 20 hours 117 24 hours 75 48 hours 102 72'hours 77 96 hours 69 120 hours 72 a Expressed as ug/g. -10- Table 6 FM3422 Uptake by Ictalams punctatus brain tissue and the gastrointestinal tract of IctaZrus punctatusa Time Control 15 min. 1 hour 2 hours 4 hours 8 hours 12 hours 16 hours 20 hours 24 hours 48 hours 72 hours 96 hours 120 hours 144 hours 168-hours a Expressed as ug/g. Brain <0. 1. 2 8 28 50 100 115 149 240 225 352 328 606 588 695 905 Gastrointestinal <0.1 <0.1 6 12 41 54 110 119 206 201 230 510 533 504 688 533 Tract Table 7 -- FM3422 Clearance of Brain and the Gastrointestinal Tract of Ictalame punctatusa Time 15 min. 1 hours 2 hours 4 hours 8 hours 12 hours 16 hours 20 hours 24 hours 48 hours 72 hours 96 hours 120 hours Brain 1152 761 712 602 511 582 618 559 523 586 450 423 417 Gastrointestina.1 Tract 281 630 325 507 302 263 1205 805 564 264 353 390 712 a Expressed as ug/g. Table 8 FM 3422 Uptake by Liver, Kidney, and Gall Bladder of the Channel Catfish Time Liver 15 min. 1 hour 2 hours 4 hours 8 hours .il2 hours 16 hours 20 hours 24 hours 48 hours 72 hours 96 hours 120 hours 144 hours 168 hours <0.1 11 11 31 67 89 99 161 149 329 261 481 469 384 561 a Expressed as pg/g. Kidney <0.1 <0.1 <0.1 37 59 77 93 149 99 189 200 359 315 349 -452 Gall Bladder <0.1 <0.1 <0.1 <0.1 <0.1 158 245 271 242 1383 1585 1573 2875 3261 13805 Table 9 -- FM 3422 Clearance by L iver, Kidney, and Gall Bladder of IctaZarus punctatus Time 15 min. 1 hour 2 hours 4 hours 8 hours 12 hours 16 hours 20 hours 24 hours 48 hours 72 hours 96 hours 120 hours Liver 504 650 758 779 507 446 728 420 443 575 316 76 507 a Expressed as pg/g. Kidney 274 567 439 447 390 559 515 365 259 351 203 182 285 Gall Bladder 1543 4908 1286 5058 3710 4321 4247 2495 3912 2810 1221 1411 6497 -12- with no apparent plateau effect. At 168 h the concentration of FM3422 in this organ was equivalent to 905pg/g tissue. The brain tissue levels of FM3422 remained elevated upon-completion of the clearance phase of this test; 417pg/g (Table 7, Figure 4). The gastrointestinal tract uptake of FM3422 identified three phases: rapid and slow uptake components with a demonstrable plateau ,(Table 6, Figure 5). The first two phases persisted for 24 and 48 hours respectively while the plateau effect was minimally of 96 hoursduration. As may be noted (Table 7 and Figure 5), this organ did not clear FM3422 readily, rather an inexplicable rise in organ content of FM3422 occurred during the latter stages of depuration. 'TISSUE ANALYSIS - Gallbladder This organ demonstrated a delayed "uptake" of FM3422, detectable leve ls being initially noted at 12 hours exposure (Table 8). The "uptake" pattern differed from those of other organ systems being analyzed in that the concentration of fluorochemical increased throughout the exposure period. This organ showed anerratic cle arance pattern, an initial depuration followed by increased levels of fluorochemical, a phenomenon which was observed throughout the remainder of the experiment (Table 9 Figure 6). The remaining tissues tested exhibited similar levels of FM3422 uptake. In all cases a steady-state concentration could be calculated using the plateau method. Depuration. data did not establish definitive evidence for the clearance of FM3422 by these tissues: gills, skeleton and skin. The liver did exhibit pronounced depuration of the test chemical followed by rapid uptake, a phenomenon associated with depuration data obtained for the gastrointestinal tract and the gall bladder. -13- Table 10 Compilation of Whole Fish and Organ Syst'e*mB*06oncentration Factors During Exposure to FM3422 Sample Whole Fish #1 Whole Fish #2 Skeleton Skin Gills Liver Kidney Gastrointestinal Gall Bladder Brain Muscle Weighte 9 10.8+4.5 11.5+2.4 11.8+2.3 2.9+.9 .64+.2 .51+.l .36+.l 1.4+.4 .03+.02 .19+.03 7.0+2.0 Bioconcentration Factor 575 406 501 534 542 474 709 1064 26548 d 1344 281 a BCF - calculated using plateau method b Average water concentration of FM3422, 0.52 PPM cData expressed as mean + S.D. d Seven day BCF.value, absense o f plateau effect. -14- Table 114 -- Uptake Rate Constant of FM322 in IctaZaruspunctatus (Channel Catfish) Sample Uptake rate constant composite Rapid Phase Slow Phase Whole Fish A 3.2+1.3 5.7 2.7 Whole Fish B 6.4+3.1 8.6 3.9 Brain 7.7+2.-4 9.3 5.1 Gills 5.4+3.3 7.3 2.7 Gastrointestinal Tract 6.0+2.4 7.3 4.4 Liver Gall Bladder 4.5+2.3 82.1 a 6.3 3.6 - - Kidney, Muscle 4.0+1.4 1.8+0.6 b 5.4 3.0 - - Skin 6.7+3.2 8.9 3.6 Skeleton 3.1+1.0 3.5 2.4 a Uptake b Values rate constant at 168 hours simitlar at all time periods -15- A tabulation of the bioconcentration factors (BCF) determined at an approximate steady state, using the plateau method,provided evidence demonstrating the lipophilicity of FM3422 (Table 10). Although the gall bladder.possessed the highest BCF for comparative purposes these data can be ignored for reasons to be discussed. Of the remaining organ systems the propensity to bioaccumulate FM3422 was most prominent in the brain and gastrointestinal tract. The edible portion, muscle, minimally bioconcentrated FM3422. A tabulation of uptake rate constants for whole fish and organ systems provides further evidence that the lipophilic nature of FM3422 does influence uptake rate (Table 11). Both brain and the gastrointestinal tract bad high uptake rate constants. A second premise may be established that being the greater the surface area exposed to the material the greater the uptake rate, hence elevated uptake rate constants for both skin and gills. The former instance provides a physiological basis for uptake while in the latter case both mechanical and physiological mechanisms were operative. Clearance rate constants could not be calculated due to da:ta variability. DISCUSSION In these studies the principal mechanisms for initial uptake of FM3422 were the gills and integument. Substantiating evidence consisting of uptake rate constants which exceeded all other organ systems excepting brain, gastrointestinal tract and the gall bladder. The gill cii culation functions as a transport system dispersing the fluorochemical to various sites in the body. The importance of this system in terms of both uptake and transport of foreign chemicals has been the -16- subject of recent papers by Granmo and Kollberg and Heath (1977). (1976) and Bass Movement of FM3422 across the integument also contributes to the body burden of FM3422 based on uptake rate constants. Definition of the extent of this contribution cannot be determined as the exact transport pathway has not been established. Our previous study (1977) did demonstrate the existence of a highly lipid layer underlying the skin which theoretically based on the lipophilicity of FM3422 should serve as a storage (binding) site for this chemical. Vascular perfusion of this area could serve to transport the fluorochemical to other sites in the body, however, the magnitude of perfusion is an unknown. Similarly, if one views the lipid layer as a binding site for FM3422, the nature of the binding becomes of relevance in determining the ease with which material may be transported elsewhere. It was demonstrated previously in our laboratory that the more highly lipid-containing organs tended to bioconcentrate the subject fluorochemical more readily. This finding has been substantiated by the present study. Transport of FM3422 to the brain and gastrointestinal tract by the circulatory system of the fish seems self-evident as these organs are highly vascular. Evidence obtained in this study suggests that the subject chemical is bound tightly as it does not depurate appreciably. By implication a similar situation may \ exist at the oil layer/integumefit inter face, thereby reducing the overall contribution of the integument as a source of fluorochemical for other organs in the body. Organs contributing to the digestive process of IctaZarus punctatz -17- presented an interesting profile. The gall bladder, serving as a reservoir for bile manufactured by the liver, possessed the greatest concentration of fluorochemical observed in these studies. When samples were obtained for FM3422 analysis, distention of the gall bladder was observed, indicating that at our sampling time we were in reality analyzing a concentrated sample of bile. It is also probable that this organ participates in the excretion of FM3422 from the body. The composition of bile is such that given the lipophilicity of FM 3422,, binding sites, complexation and/or conjugation of this fluorochemical to biliary components is a distinct possibility. Contraction of the gall bladder with the resultant emptying of its contents into the gastrointestinal tract may account for the sporadic nature of the clearance curve for this organ. One may view the sequence of events occuring in the depuration phase of this study as it relates to the digestive system in the following manner: FM3422 is cleared at a measurable rate by various organs, being transported to the liver by the circulatory system. In the liver, FM3422 either binds, complexes and/or becomes a conjugate of newly manufactured bile. The bile is then stored in the gall bladder being expressed into the duodenal area of the intestine. This material maytrt@hen be either excreted within.fecal matter and/or some FM3422 may undergo re-uptake for bodily distribution. Excretion of FM3422 is not solely via fecal matter as a similar scenario can be described for renal clearance wherein elevated levels of FM3422 are maintained through the first 72 hours of depuration. The edible portion of the channel catfish, the.musele fillet, did not bioaccumulate FM34-22 to an appreciable extent substantiating our previous observation for this tissue. In general, no adverse signs were observed during the duration of the experiment attesting to the non-toxic nature of this chemical under the conditions employed in this test. CONCLUSIONS This study of the ability of the channel catfish (Iotalazw punctatus)to bioconcentrate the f luorochemical FM3422 has led to the following conclusions: 1. Both lipophilic organs and organs possessing a large surface area at the water/organ interface possess the highest uptake rate constants. 2. The brain and gastrointestinal tract have BCF's in excess of 10 3 due to the lipophilic nature of the subject fluorochemical. 3. The gall bladder due to its'storage function showed the highest concentration of FM3422 on a ug/g basis. 4. Whole fish or organ weight was an independent variable, not a determinant in the bioconcentration of FM3422. 5. Excretion of FM3422 consists of both urinary and fecal pathways. 1 6. Clearance of FM3422 by components of the digestive system contributes to the erratic nature of the depuration curves. 7. FM3422 at the exposure concentration did not elicit signs of toxicity, therefore, under the conditions of this experiment, this fluorochemical can be considered non-toxic. ANW/vmp -19- BIBLIOGRAPHY 1. Bass, Michael L. and Alan G. Heath. 497-502) 1977. Water Research 11: 2. Burnett, R. Science 174:606, 1971. Chiout C. T., Freed, V. H., Schmedding, D. W., and Kohnert, R. L. Env. Sci. Tech. 11:475, 1977. 4. Eaton,, John - Chairman, ASTM Draft Guidelines, 1977. 5. Elnabarawy, M. T. Technical Report, 1977. 6. Fujita, T., Iwasa, J., Hansch, 1964. J. Am. Chem. Soc. 86:5175, 7. Granmo, A. and S. Kollberg.- Water Research 10:189, 1976. 8.* Gustafson, C. G. Env. Sci. Tech. 4:814, 1970. 9. Mendel, A. Technical Report, 1977. 10. Veith, G. D. and Comstock, V. M. J. Fish Res. Board Can. 32: 1849, 1975. 11. Welter, A. N. Technical Report, 1977. 1000 IF bO t. -T 7 4.4 .7r4 6+ + +-+1--14 -T 1L cq 4-1 4 3 > 4) IL lz IL L)CL- 3. z -H 2Pk 20 X.- U) Lq Ei Li ri in . .. -7t.- t7 T- T 1 ... + -71 ... --4 4 7-:- r@T- -7- 7 -@ll .7k ti'pa'it' T-TT . .......... Clearan'C'B, I--@-77 7- T-T", 77! 2 7 i -7 --7 24 48 Figure I 72 96 120 144 168 -24 Time, hours 48 72 96 '-12--0-- Composite of designated Whole Fish A, one organism per data point, concentration of FM 3422 in water (CW) equals 0.52 ppm. 'Slope Intercept y equals 1.511. 1000 21+ be c,@ cq co 'Lit -1.@ :7L T T 77 zLa Ll _j 0 LnUl T@T T T7 T T@. LAI, .1 4 i L-L- -LL17 F-.1'. 7 -T@F k L-: 174 7' -T- -T T L I* I Ir 71 7T-7 -77, -7T -r @7 L 24 48 72 96 120 144 168 24 48 72 96 120 Time, hours Figure 2 - Composite of designated Whole Fish B, one organism per data point. Cw 0.52 ppm. Slope Intercept y equals 1.699. 4 @-7- r'T-7',t L: 4 7 T= 7-] Tr tic bo C4 ci cr3 7 rs4 4 f 1 I i t -F.T- T- F-I @.4--I tr -r4 I-f-- 7-7' 1 4 t+ 1. T:- I- -.L tz- I 1 - -1@;I -'t -A*: '@t 4.:i. 7-- 1 2 z :3 u (L-r4 z (D t2 C03 5 L4 Ul Ei 7-@ -7 !:F f7@ rf -T 7-1 :F-F q-T t ..ec..... .... . . . t j. 5 7'-:- '-7 "7 --7---l 24 48 Figure 3 72 96 120 144 168 24 48 72 96 120 Time, hours Muscle Composite Data C w 0.52 ppm. Slope 2 tissue analyses per point. Intercept y 1.225. 10 0. --7 cC@4 Ce) 4 -r23- 4 +-:K T @f+LL 7 :T --r-r-r- --T---7- .7 -7 I L ff". -T V f,.L: . 7 -7 :"77 i: T I I.. ....... JA 13 r4 rj 7. "-'-7 7 ... .... .... . T, 7T 7-. 7r-- r I ask r'an'.-in 7- -1-7-!- T'! 48 Figure 4 .. ... . .. .. ... 72 -ii@--120 14-4-16.;'-8-42-. 48 72 Time, hours 96 120 Composite Data of Brain Tissue - 2 tissue analyses per point. Cw 0.52-ppm. Slope Intercept y 2.357. 4.daoaaluj adols 'Wdd Zg*o = fiko 'JUTod aad S9sATTUV anssiz Z - :@OvJl TvuT4S94UTOJISVD ;o e4m(I 9;Tsodtuoo sanoq 'AWTJ, OZT 96 ZL St, tZ 89T 'DtT OZT 96 ZL 7--7- .7-77-[ -7 "-7 'g ains -ra ,4- -7 7':-7* ++4 j . .. . .t lp 1 1 -4 44 1 4;- LLL 4i i4 tI j f7 4i-t"r -t t-',-7++-t-,- ... . .. ul u,' Ll P4 OT M t t@ .. .. 7 1 :3 44- 't 4 L- L iuIDOTI; oZ 1 DOOT TAM P!5 r 1000 -r 'r-7--rT' 4 -TT-! ba ba 0 :31 IL m 0 C,4 u cq Ir w cr) @n ;Ll 0 7= 7- >4 (D IC.) El $4 ZE4 :toci CL Z.,.4 LE 4J L3 r. 0 U) us ti I21 :7@ J',T-T" =7-1 IT t f . . . . ... . . .-T 'r r -JE Lt 77--7 --t :7-77--777--' 7 T. r -L--4 t f L7- ?7 1.1kte1. r 71 r7.- 4 t 7r--- Figure 6 77-7-----7--'7 :--7 7-*-*-- . . . .. ... . . 96 12 14t 168 @ime, ours 24 48 72 96 120 Composite Data of two gall bladder analyses per point. cw = 0.52 ppm. Slope Intercept y 1.364.