Document gk1kG16kz924k1M63j7nBjo3

3M REPORT: BIOACCUMULATION STUDIES : gEH] 0800-373 "Marc8h, 1953 8 fifi (Bh re 2 0 rion oe rion mir -Fo . - i 000811807P : co{a0rd,i2ssThh.sttaoHtafSohostoemlwauetcrieootvnhpeverraosnmtedenocteurohwnlod,,edexsrpHetiernhndeicsimecae,anmtte1edsabcfcaiocsrtpeumtmdesoatnsaunrtcheemss)etnuatdnoydfismonlourb$e1il(fiartybeoqauvneedn,Kbemue.taasrsuirfneagimarelnystghoooronedyr(awn)doetxhspeohpsoounroe: ioFtohdntti his vPeryelrowpasorltuobl filfiyio,ermeavwneagneteowriftohmrotll hoewcuelloeawrdsmtseihazeusvufarTleo.rudewyo"h.fpi,scooe(unhxeebpeiwtclothyuelrdd=bheeah5xna0pdve,ipcoptrbh&ieaMmnloiddWneelraoargitweSfi7oFhe1a=wershy5p'.cLhPpuuurcsseur biiloaynerofthtehesMoelveecruallamremcobnrfaingcusra.tion may present some problems in crosiog through the pores of hbeoKU.g.. . : =`TrWhhaeerrteeapesoyrdotuaitssvteartreyacetohnoafftu0s0i3BngLu.wlh(e50n=0.50it0c0xupprpgebs."sseaedsotfapgaernta.co4n,ep.gI.n3"]wuhle"an.tnherseosliudbiuleistyns"u3ggs"gTlhe(50spypst)e.m =b-tTSahhatemeiesd)Iaanm.tdpail{vieIiatdwuiaTassalIbdsnli.teveTsirhde2seetd&i1tne0g3xitapnrrneohosvithiesdsoertweh2ngaafftirsodhfrfotou"orFrniwoshthsoheAlw"eeabrnoeddrye"aFminosdvheB2d, oaartstciiasfsctuhheestsvieinawe be(eraiernsege."qu6se,noptgiu.alp1as.n:aAyppsposaaormfeennhstye, hAheTasnSssDobEmetCeweQsecAineSntPOiCsPtBs,iarnel3stihbdououeogshkistnohemlyeawkbehoettrhreo.tuhtoFumogerhatstauhtnerheuapdmrbtobhebyerlyeCoamafgnriyaneedeanirdoastontnhhseatcrvhieieennswtgaiesmatrseprfipornrteoqoctucooeomcnlepttalf.yrorissTiSsogennssiafetircocstanhutordsseupeeasry {ltarihkeneiemrneofgvowoerefdrfPmeCtaBtthSewe,r(oi0aweplr2hr(o5irnemsso,nrrtmhdieei1df0U-rafiSeenrSsmee.o)ncvgbeeys. 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August 16, 19 "Environmental Laboratory (EE & PC) Fra Fate of Fluorochemicals "0352 G96r7n0612623 Evaluation of the Bioconcentration Potential of FM 3422 2 D. L. Bacon A. N. Welter eS Faia 09362 aT eo Co 25 secunmD Y O,contaminaRanorimton) | hEGTRY> Ta Ee. PSKeeivrweowroSms:grom he CURRENT OBIECTIVE: Progress Report 1. Evaluate Bioconcentration Potential of FM 3422. 2. wDheotleermifnieshupatnadk/eoratnidssculee.arance rates of FM 3422 in EE & PC - Div. | 3, Determine excretory pathway for FM 3422, F1u(oArqoucahteimci) cals aepohnyrABS5TCRoAmCTp.an(2R0A0D-.25t0+wCaormdpaTnsyscooenviacGteinntfaorrmaattaitonas viouted y he Tochicl Communications Cntr Toxicity (tBriaotcioonnc)en- . ce: DRLLBB MTE aR.wParokop-236ESKAWR ipnfeorsmaE tion Li W p-- 006251 3 -2- INTRODUCTION 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, FN3422, does bioconcentrate in organisms and if 50, 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 (Ietalaruspunctatus ) as the test species. The subject fluorochemical, FMG422 1s a white granular mater- i, ial, molecular weight-571, possessing physico-chemical properties which suggest that this material may bioconcemtrate. 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 bloconcentrate. METHODS The hydrophobicity of FM3422 precluded a simplistic determination of its water solubility. Use of the Veith-Comstock technique 006252 a (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 Fujitaet.al. (1964). Channel catfish (Ictalarus punctatus) were acclimated to the following test conditions for a minimum of 14 days: temperature, 21+1C, 16-hour light and 8-hour dark photoperiod with a 30 minute transition period and were fed daily (Tetra Min). F342, previously impregnated on 3.5 mm glass beads was placed in a 114 * 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/e. Based on a previous study (1977) which indicated rapid uptake 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 FU3422 uptake analysis were obtained at the following time periods: control, 006253 : prior to introduction, 15 mates, 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 ive 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 electron capture detector (i%). Specifications and operating conditions were as follows: stainless steel column-length-6 feet. x 1/8"; column packing and support-10% Carbowax 20 Mon Chromosorb- W acid washed 60/80 mesh; operating temperature-isothermal 180C, detector-300C; carrier gas-5% methane in argon. Recovery rate was above 90%. Statistical treatment of the data utilized the 3 Trac system, MINITAR 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 bioconcentration studies with fish (1977). 006254 . a RESULTS . The water solubility of FH3422 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. (1077) 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 mature of the test substance. In consideration of the slightly volatile nature of FU3422, the test organisms were introduced into the newly filled experimen- tal aquarium and sampling of both channel catfish and exposure water began fifteen minutes later. Table 1a illustrates the mean concentrations of FM3422 in various layers of the exposure water. The test substance was quite evenly distributed throughout the aquarium as indicatbeyd concens trations of 0.49, 0.54, and 0.52us/2 of FM3422 in the top, middle i, 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/FH3422 layer would contrinpte to the variability of mean concentration values as indicated by the magnitude of the standard error (Table 1a). Upon analysis of the exposure water samples for FN3422, evidence was obtained that this material was being rapidly released into solution (Table 1b). These data suggest that concentrations approaching equilibrium values were attained within 1 hour of exposure. 006255 lm Table 1a -- MPeearniodConcentration of FM3422 Exposure Water: Experimental Sampling Site n Top layer 15 Middle layer 15 Lower layer 15 concentrationut/2 .49+.06 .54+.06 .52+.04 Mean concentration of exposure period + S.E. Table 1b -- Concentration (ppm) during Initial 4 Hours of test Sampling Site Top layer Middle layer Lover layer 15 min. 19 .31 EX 1 hour .a5 a9 | sa 2 hour 43 47 a7 4 hour .35 Laz .55 "Expressed as uz/t . WHOLE FISH UPTAKE AND CLEARANCE Uptake of the fluorochemical 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 FN3422 across the finteumencJ sen subsequent binding to lipophilic tissues. A slower rate of FW3422 uptake was noted for both organisms which was possibly accounted for by a decreased water/organism concentration gradient. No uptake plateau indicative of a steadv-state was obtained following analysis 006256 . r= Table 2 - FM3422 Time 11 5 nomuinr. 2 hours 4 hours 128 hhoouurrss 16 hours 2204 hhoouurrss 4782 hhoouurrss 96 hours 120 hours 144 hours 168 hours Uptake by Channel Fish A 41 92 16 6528 51 16014 212322 190 21 320 852 Catfish (Iotalarus punctatus) * Fish B 14 10 29 6402 se 170 31539 257 201 279 158 2 Expressed as ug/g. of FU 3422 content of those fish comprising the composite organism A (Figure 1). The 168-hour value for the test fluorochehicalmay . be an outlier as these values encompassing the period from 72 - ' 120 hours suggest a plateau (steady state) at FM 3422 concentra tions of "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). 006257 . ae Table 3 -- FM3422 Clearance of Ictalarus punotatus Time 115homiunr. 42 nhoouurrss 128 hhoouurrss 2106 hhoouurrss 4284 hhoouurrss 9762 hhoouurrss 120 hours Whole Fish A 236096 225620 225630 223385 23-7, 123060 106 Whole Fish B 231169 234029 220462 215757 129001 222267 78 2 Expressed as ug/g. sample lost 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 ug/g (Table 4). Tissue weight was an independent variable, not affecting the bioaccumulation level of FM3422. Depuration of the subject 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 1ipid nature of these materials (Table 6). Brain uptake of FM3422 continued throughout the experiment (Figure 4) 006258 . o- Table 4 -T-etFaMla3r4u2s2PuUnpcttaaktues bMyuscle CToinmterol 115homiunr. 24 hhoouurrss . 128 hhoouurrss 1260 hhoouurrss 4284 hhoouurrss 9752 hhoouurrss 112404 hhoouurrss 168 hours Mu<s0c.l1e 301 105 2321 3489 16118 112375 115037 147 a Expressed as ug/g. Table 5 -- FM3422 Clearance of Ictalarus puntatus muscle Time Muscle 115homiunr. 42 hhoouurrss 128 hhoouurrss 2106 hhoouurrss 4264 ohouurrss 72 hours 19260 hhoouurrss 111287 110288 x v 114487 := 181 . 1107735 . 1| 77 | 6792 Expressed as ug/e. 006259 -10- Table 6 -- tFhMe342g2asUtprtoaiknetesbytinIcatlaltarruasctpunocftaItcutsalbrruasinputniscstuaetusand Time C1o5ntmrion.l 21 hnoouurrs 48 hhoouurrss 1162 hhoouurrss 2240 hhoouurrss 4782 hnoouurrss 96 hours 114240 hhoouurrss 168 hours Expressed as ug/g. Brain 02.1 288 10500 111159 224205 335228 606 659858 905 Gastrointestinal Tract <00..11 126 5a4 111190 . 220016 253100 533 658084 533 Table 7 -- FofM3I4c2t2alCarluesarpaunnccieatoufs Brain and the Gastrointestinal Tract Time 115homuinr.s 24 hhoouurrss 128 hhoouurrss 2106 hhoouurrss 2448 hhoouurrss 9762 hhoouurrss 120 hours Brain 1176512 670122 551812 851589 558263 445203 417 Gastrointestinal Tract 623801 ; 352057 > 236032 1820055 256544 339503 - 712 a Expressed as ug/g. 006260 S11 Table 8 -- Ftfhe34C2h2annUepltakCeatfbyishLiver, Kidney, and Gall Bladder of Time Liver 151 mhionu,r <110.1 24 hhoouurrss 1311 i128 hhoouurrss 687 1260 hhoouurrss 16219 2448 hhoouurrss 312499 9762 hhoouurrss 428611 112404 hhoouurrss 436894 168 hours 561 ? Expressed as ug/g. Kidney <<00..11 <370.1 7597 12439 1829 230509 331459 452 Gall Bladder 0<0..11 00..11 1508.1 224715 1324823 11558735 23827651 13805 Table 9 --- FofM I3c4t2a2laCrluesarapnucnectabtyusLiver, Kidney, and Gall Bladder Time Liver Kidney Gall Bladder 151 mhionu.r 560540 526774 41950483 = 24 hhoouurrss 777598 44379 51028568 128 hhoouurrss 451067 355990 43372110 2106 hhoouurrss 472208 356155 24429457 2448 hhoouurrss 454735 235591 23891102 9762 hhoouurrss 31766 210832 11242111 120 hours 507 285 6497 2 Expressed as ug/g. 006261 : -12- with no apparent plateau effect. At 168 h the concentration of FM3422 in this organ was equivalent to 905ug/g tissue. The brain tissue levels of FM3422 remained elevated upon completion of the clearance phase of this test; 417ug/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 de- puration. TISSUE ANALYSIS - Gallbladder This organ demonstrated a delayed "uptake" of Fi3422, detectabl levels 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 clear- ance 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 FU3422 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 phen. omenon associated with depuration data obtained for the gastro- intestinal tract and the gall bladder. 006262 ' -13- . Table 10 -- cCoemnptirlaattiioonnFaocftoWrhsoleDurFiisnhg EaxnpdosOurrgaentoSyFsMt3e4m22Bi'ogon- Sample Whole Fish #1 Whole Fish #2 Skeleton Skin Gills Liver Kidney Gastrointestinal Gall Bladder Brain Muscle Weigght 10.8:4.5 11.542.4 11.842.3 2.9+.9 641.2 51.1 .36+.1 1.4+.4 .03+.02 .19+.03 7.0%2.0 BioconFcaecnttorration 575 406 501 534 542 a4 700 1064 26548% 1344 ` 281 BCF - calculated using plateau method average water concentration of FM3422, 0.52 ppm ' " "Data expressed as mean + S.D. 5 dseven day BOF. value, absense of plateau effect. 006263 . -1a- Table 11 -- (UCphtaankneel sample Whole Fish A Whole Fish B Brain Gills GastroiTrnatcetstinal Liver Gall Bladder Kidney Muscle Skin Skeleton RCaattteisCh)onstant of FM322 in Ucpotnaskteantratceomposite Rapid 3.241.3 5.7 6.43.1 8.6 7.702.4 9.3 5.4+3.3 7.3 6.0+2.4 7.3 4.542.3 82.1% 4.01.4 1.810.6 6.743.2 3.141.0 6.3 co5.4 8.9 as Iotalarus Phase pwictatus Slow Phase 2.7 3.0 5.1 2.7 4.4 3.6 3.0 3.6 2.4 2 Uptake rate constant at 168 hours Values similar at all time periods ES `Lh 006264 = -15- A tabulation of the bioconcentration factors (BCF) determined at an approximate steady state, using the plateau method,provided evidence demonstrating the lipophilicity of FU3422 (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 bloconcentrated FM3422. A tabulation of uptake rate constants for whole fish and organ systems provides further evidence that the lipophilic nature of Fu3422 does influence uptake rate (Table 11). Both brain and the gastrointestinal tract had 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 data variability. DISCUSSION . In these studies the principal mechanisms for initial uptake of FU3422 were the gills and integument. Substantiating evidence comsist- ing of uptake rate constants which exceeded all other organ systems ex- cepting brain, gastrointestinal tract and the gall bladder. The gill ci 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 006265 -16- subject of recent papers by Granmo and Kollberg (1975) and Bass and Heath (1977). 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 1pid layer underlying the skin which theoretically based on the lipophilicity of Fi3422 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 ome views the 1ipid layer as a binding site for FU3422, 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 5 substantiated by the present study. Transport of FU3422 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/integument interface, 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 Iotalarus punctat: 006266 TL -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 maye+then be either excreted within fecal matte: 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. 006267 Ln -18- The edible portion of the channel catfish, the muscle fillet, did not bioaccumulate FM3422 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 (Zotalarus punctatus) to Bloconcentrate the fluorochemical FM3422 has led to the following conclusions: i 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% 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 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. 6. Clearance of FU3422 by components of the digestive system contributes to the erratic nature of the depuration curves. 7. FM3422 at the exposure concentration did mot elicit signs of toxicity, therefore, under the conditions of this experiment, this fluorochemical can be considered non-toxic. Ahi dls ANW/ vp 006268 Co -19- BIBLIOGRAPHY 1. B4a9s7s-,502M,ich1a97e7l. L. and Alan G. Heath. Water Research E11s: 2. Burnett, R. Science 174:606, 1971. 3. KCohbinoeur,t,C. R.T.,L. FreEendv,. VS.ci.H.,TecShc.hm1e1d:d4i7n5g,, D1.977.., and 4. Eaton, John - Chairman, ASTM Draft Guidelines, 1977. 5. Elnabarawy, M. T. Technical Report, 1977, 6. 1F9u5j4i.ta, T., Iwasa, J., Hansch, C. J. . Am. Chem. Soc. 86:5175, 7. Grammo, A. and S, Kollberg. Water Research 10:189, 1976. 8. Gustafson, C. G. Eav. Sei. Tech. 4:814, 1970. 9. Mendel, A. Technical Report, 1977. 10. 3V2e:i1th8,49,G. 19D.75.and Comstock, V. M. J. Fish Res. Board Can. 11. Welter, A. N. Technical Report, 1977. 4 > 006269 Co TLTolo BESTCOPY AVAILABLE COdWaEn LL E S E e a ap TL pe er Re fl Lom o i3 Hi Ha. [ z ig ie LE ig 5i3a JTTH T P RT F TFFRrF ER Fre pTEH To PEER HR 8 hf TT SEENEEN EE HTT em spe [b 2 e b EE EN Z& HFER o Hoa HR EE rETeFeTE ee E re FE rere a EeRreeere rr Ee ee Co Ee TT rer rt by BREE RE FL EM LE En EEpr 5 HEHE eee 0 5 S i] Fr EH E R E Pe ee ig3 SH CT a TT E E EaTEE iz [Ee Fi E Hi$3Ee3E0 pr 7iFssEsE sTsE riaesdaiTo E rmr ay E sSHFi rE eePr ET n PLEye Fre Meee rer =) TT fe JR F ee eT ereer eT 9 iiEE PT eT i Trree ITor to TT sal greemptite LLLome LL SEE SER r Ae A PH Cr H EEFre IT TE i EEE ee RH Te J E H E Ebrh ee TEHTT Sa CERT cE Tere rd Lgl SLE nL fb Ping Slime he Feber lil gL Tine, hours 24 48 72 96 120 144 168 24 48 72 26 120 Figure 1 - Composite of designated Whole Fish A, one organism per data point, concentration of FM 3422 in water ) equals 0.52 ppm. Slope Intercept y equals 1.511% 006270 . ? so CUTER RE cn fol TE #1000 i: Lode J ! o $l ds se +i:BESTCOPY AVAILABLE AE ee H me | :z Nn ! gC nR EH I IFAT A TA rr 3; PL | Bo TRA H 5 3 CHE TH ERE TE ITT RTT LETT : e ing F G2 E nnTS ili HfhE = HTT Gi Gf Dd ee EL HL ee u5 H F HH r ile i eE e EEHeEeREeH Re FE ePr rerE ereeEpeEHH vozs i HJ f Ee I Hr Te T oda LTTE sit dHrE iia bfrE i22d oHem EE RH 1 5 Fer re Ee s: HERR ih bop Ee Er] E Jeet e e l |] SLE eT plies LL SE RE EE Le Ee fi: T BaaE dese EEa e ite brid] HT eeT e H 7 ik LESAE EE il ean Ali id nl Locipin | TE # 24 48 72 96 120Tim1e4,4 ho1u6r8s 24 48 72 96 120 Figure 2 - 65 Composite of designated Whole Fish data point, Cy = 0.52 ppm. Slope B, one organism per Intercept y equals 006271 >. .- CTT ng i Ligh RT poopy Fa2-IT L. LBEESTCOPY AVAILABLE ; HEE ii . s dpm m nsabre umshenssi efusnsnat roped nle n be ritsngwdage erred E SET EhE ee B Ee ea ae eh ba pEzSassesatasssestsaazassaantsasansaad naaiannss MELE ARAR ENSARENTRG DEER RRTHEE ER TR RE iE i Th DFOD3aJ S s=momElcA dhieE rELeE LH m TL e I 0 e MBeeAe B MA T MEME maema Ba eA rE Eerer r(ETEEEE FT H EP E EEE FETreeeEA eeEEErTrekT THTHTH A BE EH EE EFEH HE AA EprRe E EE PRe E RE HHH A s2 HA OT e ASHEn S HE SHEIEINe NENTES e SHRI EEA NEASRNIRAe RES ty HE ae HEE ii [fit nl rE TRARR ETD 1] tig {f3f8f AgfE i {TTT LE TENT e PR ee E H TH E EE | (Efe 1333 E S E H EIH EHE ee E RE TET TT {3d EE ETFE Ee EE TT E A E mEE RE m RE ! hi ii Si Pi i LPL F SH F H TEEP FEA ArEHCA E PE A PERE er ort E i FE Fe ed RR E : PEFPPT RH EHTH FERRER E FEE E TT tpi 1 I a Hi a Tr a1 T i i di omed d| ed Sl iLSTE d i l CT | : 24 48 72 96 120 144 168 24 48 72 96 120 Time, hours Figure 3 - CM,usc=le0.5C2ompppoms.iteSDlaotpae I-nt2ertciespstuey a=nal1y.s22e5s. per point. 006272 T:ET pen eiCne DRi IEBETSHTC;IOPIY AVArILAr BLE so 080mi i 1 thee ed 1 Ssiri H ET ey reTeA aT NLErg | i . po a HTH A TR AHA HH HEH EET ET RE nT do ee Lr 3 ELE WY Ps da silt pretend ( Emme Bio EE Tr HHEePA E RR e ITT EERE 342 ,r FE ETe T EE e e E ERe LE Tr i is fdH iE HEE) } Ahm en ir EEE 35% FR rd er Te eer Toon: i SRHE EH ETT ioc Te emt mb fT TTI TTT ERNE] toe CTT if EET E SSR EER eee eE erC Alea errreer y gHHeeEePr EE HEH HH Jo RH PH PR RT Pe PR HF ed ee ee apominp bi Freed I De Hf quid i yah a "mesmo Time, hours Figure 4 - Composite Data of Brain Tissue - 2 tissue analyses per point. Cy = 0.52 ppm. Slope Intercept y = 2.357. 006273 ; pe WET eis a sed bebe Hada : CT BESTCOPY AVAILABLE f , l Cid Heo HI i @ 2000. bee ini FH be #1AFr L e L EH = L pe E :r rE E e A E reI rT eeHe E eTpEn 2 i SEE EE EsTI E A TH TH 8: ST [i] FET r IN Pb 81m 00 E A r probe e e eer ; 2hid S SF R 5 HE Te Ee TO er ERE re TeCrre AE HE s i JE HE JAH EE FET EEr ER HH id EH TE ETLL[E eH LHEE E is lnm lc a: I i 5 ER $He5l E rE eRE e op r Th ER [Eobi TEEEER1 Lr er rere EET EE eert rr Ete Time: hoses Epi 006274 Figure 5 - Composite Data of Gastrointestinal Tract - 2 tissue analyses per point. Cy = 0.52 ppm. Slope Intercept y = 2.153. - Cr [SER EERE en FO mete fica Prim : i Lob oe rr re LLpLT NS J i L 0o RT R r RERerr eeeH ee E Tree e HE rAT 4 T+e+ Not Hi Ader a fo THeEE HH gE S175, T i By TT i ERp rT S3PiEoEaa9 lS : E EE CTaE E EEe R RPTET Ra Pt EL 1 S ET FE LEETe er T E ET E TEE E ETE E FTrEPEEFE HeeH Hr LEK) EH 8. Ee Fhe dPEe3 J Lo E m E T E I E HHETETIE ii] LES SHHE he 5 i FOCI TITER: TE Tie] in 2 FH HHH He Pee ee rr i or HHGi EEE ee Er Cg UTI PE A Re TITI: P po rrees EE T hE E TT ET eT | EEE ee S E e E oz T I Lr E L e e rr re rTeEE ART I I ii: CON STTLIee He RIdF HEanaersPitincunsspees TriTal Fre iree aan ! geo 98 12q df Mew 20 46 72 86 120 Figure 6 - Composite Data of two gall bladder analyses per point. - Cy = 0.52 ppm. Slope Intercept y = 1.364. 006275