Document 5kLGDoZaZzzL2QqgBOyeyY26J

3M REPORT: BIOACCUMUIATION STUDIES -O200- b i t March 8, 1993 UA6-0370 Ca> -p . CIj-> ^ epa -otb 000811807P oooaiifl07P a Report No. 2 (8116178) "Evaluation o f the Bioconcentration Potential o f FM 3422.* This report covers new experiments based on the study in 1 (above), but using a shorter (!) exposure (to a sat'd, solution and under the same ciffcumstances) and more frequent measurements only with channel catfish. However, the methods indicated for measurement of solubility and K,,, are fairly good and the basis for state-of-the-art protocols. Hence, I accept the value o f solubility = 50 ppb and log Ko. = 5. Further, with this very low solubility, even with the low measured v.p., one would expect a moderate Henry's Law con stant leading to volatility from water or a wetted surface. ' On the other hand, the MW is 571, which puts it toward the upper part o f the range o f molecular sizes'for which expected behavior is linear with xespect to K,*. That is, the molecular configuration may present some problems in crossing through the pores o f the lipid bilayer of the several membranes. a. The report is very confusing when it expresses test agent cone, in "ul/1* and residues in 'ug/jg.* The system was loaded at a rate o f 0.5 g/L (500,000 ug/L)[end o f para. 4, pg. 3] when the solubility is 50 ug/L (50 ppb), whereas you state that *0.5 ul/1 = 500 ppb.* b. The data in Tables 2 & 3 are shown for *Fish A* and *F!sh B,* as if these were sequential analyses o f the same individuals. The text notes that four fish were removed at each time [para. 6, pg. 3]. Apparently, . .the:sample was divided to provide 2 fish for whole body and 2 for tissues via being *tissuemized* (whatever that is). {It is. interesting in this regard to note the problem in not having a protocol for dissection other . than som e QA/SOP in a book somewhere. For a number of years there were frequently significant differ ences between PCB residues in lake trout measured by Canadian scientists in comparison to those by NYS DEC scientists, although they both thought that they agreed on the same protocol It turns out that there were two errors or differences. The Canadians only used the portion of the fillet above the median line for PCBs, whereas the U.S. group used the entire fillet minus the mid-line. Second, the Canadians removed material (fins, mid-line) by a rigid protocol, Le., cutting a 6-mm notch to remove the mid-line and taking off the top 25 mm to remove the dorsal fin and associated fat. The DEC lab varied the fat removal so as to take off all fat by cutting in 1-5 mm more or less. These differences are veiy small, but reflect 006249 3M REPORT: BIOACCUMULATION STUDIES March 8, 1993 the advisories given the public by separate agencies. Anyway, the U.S. values are much lower than those o f the Canadians, even though the fish come from the same water.} c. The reporting and plotting o f arbitrarily designated data ("Fish A* - Fig. 1 and "Fish B" - Fig. 2) is not very scientific At least they should be on the same plot, as a range or recalculated as an average at time t. As an average value, the whole body residues were still climbing when the investigators terminated exposure. The avg. values for the times from 24 hrs on are 107,172.5,190-5, 223.5, 206, 299.5, and 355, implying a rate o f "equilibrium* - approaching +20%/day! That is far from equilibrium. Further, although the general protocol resembles state-of-the-art, the small number o f test organisms per tim e interval (should be > 6) means that what the investigators see as an "outlier" may really be a typical point from a wide range o f responses. Curiously, although the authors were concerned about the increasing uptake with weight in Report No. 1, they do not report body weight, lipid com position, or length (determinant o f gill size) for the test subjects. The rate constants should be calculated and divided (kt/k2 = BCF), but the loss rate appears to accelerate with time. This may be a feature o f the incomplete equilibrium, tissue distribution, or enterohepatic cycling via bile. d. By the time one gets around to the specific tissue distributions (which are expressed as up to 5 significant digits!), the authors are confusing individual and temporal variation with "plateaus" and "erratic" patterns. It is unlikely that 2 specimens at each time can yield comparable data unless there are criteria for age and size class. Reporting BCF by specific tissue, without attention to. tissue lipid level is disingenuous or at leastjnisleading. e. It is not clear how the authors have documented excretion via urine per sey since urine concentrations, conjugates, e tc , were never measured. Presence o f material in kidney merely reflects vascular perfusion. On the other hand, the tentative view of the importance o f biliary excretion (already w ell established for lipophiles) is not understandable, Indeed, one might note that gall bladder distention could mean positive acceleration o f biliary excretion (influenced by hepatic levels). f. The belief that the absence o f toxic symptoms for the exposure regime used constitutes definition o f this fluorocarbon as "non-toxic" is an abuse o f the term, since (as Paracelsus said) `all things are toxic and it is only a matter of dose which determines whether a chemical is a poison.' If the investigators were able to determine the N-Et FOSE alcohol as such, then it was not conjugated (statements about, this route not withstanding). 3 006250 /form 6 7 4 7 -1 1-A TECHNICAL REPORT SUMMARY D ate August 16, 19 TO: TECH N ICA L COMMUNICATIONS CEN TER - 201-2CN (Important -- If report is printed on both sides o f paper, send two copies to TCC.) O lvifio n E n v i r o n m e n t a l L a b o r a t o r y (EE & PC) Project Fate of Fluorochemicals Report Title Evaluation of the Bioconcentration To D. L. Bacon P o te n tia l of A u th o r! s) A. N. W e l t e r Notebook Reference S E C U R IT Y >> D Open (Company Confidential) E9 Closed (Special Authorization) 3M C H EM IC A L ^ REGISTRY w FM 3422 Dept* Num ber 0222 Project Number 9970612623 Report Number 2 Em ploye Number(s) 09362 No. of Pages Including Coversheet 25 New Chemicals Reported Q Yes g ] No KEYW ORDS: (Select terms from 3M Thesaurus. Suggest other applicable terms.) CURRENT OBJECTIVE: Progress Report 1. E v a l u a t e B i o c o n c e n t r a t i o n P o t e n t i a l o f FM 3 4 2 2 . EE & PC - D i v . 2 . D e t e r m i n e u p t a k e a n d c l e a r a n c e r a t e s o f FM 3422 i n whole fis h and/or t i s s u e . 3. D e t e r m i n e e x c r e t o r y p a t h w a y f o r FM 3 4 2 2 . F l u o r o c h e m i c a l s R EP O R T A B ST R A C T: (200-250 words) This abstract information is distributed by the Technical Communications Center to (A q u atic) alert 3M'ers to Company R&O. It is Company confidential material. T o x ic ity (Bioconcen tra tio n ) V- c c : DLB RLB MTE m .% R.Prokop-236-cB EAR SKW Information Liaiso] Initials: Z to 3M CONFIDENTIAL 006251 -2- INTRODUCTION Living organisms possess the a b il i ty to c o n cen trate and accumulate high c o n c e n tra tio n s of l i p o p h i l i c o rg a n ic compounds e ith e r d ire c tly from t h e i r environment or from t h e i r food source, a phenomenon which i s w ell documented (B u rn e tt, 1971; G u stafso n , 1970). This a b ility to bioconcentrate seemingly nop-toxic sub stances might w ell pose a hazard fo r the u ltim a te p re d a to r sp e c ie s. This i n v e s t i g a t i o n was conducted t o d eterm in e w h e th er a f l u o r o c h e m i c a l , FM3422, d o e s b i o c o n c e n t r a t e i n o r g a n is m s and i f so, does the m aterial depurate rap id ly . S electiv e organ systems were analyzed for the subject fluorochem ical as p o te n tia l uptake sites. S in ce t h e a q u a t i c e n v iro n m en t s e r v e s as a p r im a r y mode o f e n t r y f o r FM3422 i n t o t h e e n v i r o n m e n t , t h i s s t u d y was c o n d u c t e d u s i n g t h e c h a n n e l c a t f i s h {la ta la n ^ \ punatatns ) as the t e s t species. * The s u b j e c t f l u o r o c h e m i c a l , FM3422 i s a w h i t e g r a n u l a r m a t e r - *, i a l , molecular weight-571, possessing physico-chem ical p ro p e rtie s w h i c h s u g g e s t t h a t t h i s m a t e r i a l may b i o c o n c e n t r a t e . T h u s , t h e m a t e r i a l was found t o be r e l a t i v e l y w a t e r i n s o l u b l e and p o s s e s s e d a high d is trib u tio n c o e ffic ie n t, data which is in d ic a tiv e of a highly lip o p h ilic molecule... This p ro file is generally accepted as th a t of a substance which w ill tend to b io c o n c e n tra te . METHODS The hydrophobicity of FM3422 precluded a simplistic determin ation of its water solubility. Use of the Veith-Comstock technique 006252 (1975) w herein w a te r in a c o n s t a n t - l e v e l r e s e r v o i r was c o n ti n u o u s l y s a t u r a t e d w i t h FM3422 by c i r c u l a t i n g t h e w a t e r t h r o u g h a bed of i n e r t s u b s t r a t e im pregnated w ith FM3422. Samples f o r a n a ly tic a l a n a ly sis were ob tain ed via a sampling p o rt. R e p lica te stu d ie s were performed. The d i s t r i b u t i o n c o e f f i c i e n t o f FM3422 i n n - o c t a n o l / w a t e r was determ ined using the methods d e scrib e d by Chiou e t . a l . (1977) and F u jita e t . a l . (1964). Channel c a tfis h (Iatalarus punctatus) were acclim ated to th e fo llo w in g t e s t c o n d itio n s f o r a minimum of 14 days: tem p erature, 21+1C, 16-hour l i g h t and 8-hour dark pho to p erio d w ith a 30 m inute t r a n s i t i o n p e r i o d and w ere f e d d a i l y ( T e t r a M in). FM3422, p r e v i o u s l y i m p r e g n a t e d o n 3 . 5 mm g l a s s b e a d s was p l a c e d i n a 114 a q u a r i u m , f o r m i n g a l a y e r a p p r o x i m a t e l y 2 cm above th e g r a v e l f i l t e r which was th e n c o v ere d by s a n d . The t e s t t a n k was t h e n f i l l e d w i t h c a r b o'n - f i l t e r e d w e l l w a t e r . T h i s system under continuous aerobic conditions generated a satu rated V w a t e r s o l u t i o n o f FM3422, h a v in g a f i n a l l o a d i n g r a t i o o f 0 .5 g / A . Based on a p re v io u s stu d y (1977) which in d ic a t e d ra p id uptake a n d d e p u r a t i o n r a t e s f o r FM3422 i n t h i s s p e c i e s , t h e b i o c o n c e n t r a t i o n t e s t was m odified so th a t th e uptake and c le a ra n c e p e rio d s were seven and five days re sp e c tiv e ly . Following the introduction of the channel c a tfish into the experim ental and co n tro l aq u aria samples of 4 channel c a tf is h and t h r e e w a t e r s a m p l e s , t o p , m i d d l e a n d b o t t o m l a y e r , f o r FM3422 uptake a n a ly sis were obtained at the follow ing time periods: c o n tro l, 006253 -4p r i o r t o i n t r o d u c t i o n , 15 m in u te s , one h o u r, two h o u r s , fo u r h o u r s , e ig h t hours, twelve hours, six te e n hours, twenty hours, twenty- four hours, and at tw enty-four hour in te rv a ls for a period of seven d ays. The channel c a t f i s h were th en removed to c le a ra n c e ta n k s f o r two h o u rs and f i n a l l y t r a n s f e r r e d to a flow -thro u g h system having flow r a te s through the t e s t chambers of four w ater volumes p e r 24 h o u rs. A sam pling regimen i d e n t i c a l to th a t o f the uptake phase of th is study was immediately i n i t i a t e d and term inated a f t e r five days. In d iv id u a l (whole fis h , n=2) and pooled tis s u e samples (n=2) were weighed, tissu em ized , r e f r ig e r a te d and s to re d in e th y l a c e t a t e p r i o r t o t h e a n a l y t i c a l d e t e r m i n a t i o n f o r FM3422. Whole f i s h were b l o t t e d b e fo re th e w eighing p ro ce d u re. The pooled samples c o n sisted of the follow ing tissu e : b rain , g i l l s , liv e r, g a ll b la d d e r, kidney, g a s t r o in t e s ti n a l t r a c t , sk in , muscle and skeleton. T i s s u e a n d w a t e r s a m p l e s w e re a n a l y z e d f o r FM3422 u s i n g a H e w le tt- P a c k a r d model 5713 gas chrom atograph e q u ip p e d w ith an p3 e le c tro n capture d e te cto r (N i00). Specifications and op eratin g v conditions were as follow s: s ta in le s s s te e l column-length-6 feet, x 1 / 8 " ; co lu m n p a c k i n g a n d s u p p o r t - 1 0 % Ca rbowax 20 Mon C h r o m o s o r b - W a c id washed 60/80 mesh; o p e ra tin g te m p e ra tu re -iso th e rm a l 180C, detector-300C ; c a r r i e r gas-5% methane in argon. Recovery ra te was above 90%. S t a t i s t i c a l t r e a t m e n t o f t h e d a t a u t i l i z e d t h e 3M T r a c s y s t e m , MINITAB I I p r o g r a m . B i o c o n c e n t r a t i o n f a c t o r s a n d u p t a k e r a t e c o n s t a n t s w e r e c a l c u l a t e d b a s e d on f o r m u l a e p r o p o s e d by an ASTM c o m m it tee developing a standard method for conducting bioconcentration studies with fish (1977). 006254 RESULTS The w a t e r s o l u b i l i t y o f FM3422 was 50 ppb ( V e i t h - C o m s t o c k technique) coupled with the added observation th a t th is chemical was s l i g h t l y v o l a t i l e (Mendel, 1977). U s in g t h e method d e s c r i b e d by C h io u , et^. a l . (1 9 7 7 ) t h e d i s t r i b u t i o n c o e f f i c i e n t f o r FM3422 was d e t e r m i n e d t o b e i n e x c e s s 5 o f 10 , i n an n - o c t a n o l / w a t e r s y s te m . V a lu e s o f t h i s o r d e r o f magnitude are in d ic a tiv e of the highly lip o p h ilic nature of the te s t substance. In c o n s i d e r a t i o n o f t h e s l i g h t l y v o l a t i l e n a t u r e o f FM3422, th e t e s t organisms were intro d u ced in to th e newly f i l l e d experimen t a l aquarium and sampling of both channel c a tfis h and exposure w ater began fifte e n minutes la te r . . T a b l e l a i l l u s t r a t e s t h e mean c o n c e n t r a t i o n s o f FM3422 i n v a rio u s la y e rs of th e exposure w a te r. The t e s t su b s ta n c e was q u ite e v e n l y d i s t r i b u t e d t h r o u g h o u t t h e a q u a r i u m a s i n d i c a t e d ,w by c o n c e n - 4 t r a t i o n s o f 0 . 4 9 , 0 . 5 4 , and 0 .5 2 p Jt/Jl o f FM3422 i n t h e t o p , m i d d l e and l o w e r w a t e r l a y e r o f t h e a q u a r i u m . S i n c e FM3422 was p r e s e n t as a suspension, e x c ita b ility of the te s t organisms re s u ltin g in unusual d istu rb an ce of the bottom sand/FM3422 la y e r would c o n trib u te t o t h e v a r i a b i l i t y o f mean c o n c e n t r a t i o n v a l u e s as i n d i c a t e d by t h e magnitude of the standard e rro r (Table l a ) . Upon a n a l y s i s o f t h e e x p o s u r e w a t e r s a m p le s f o r FM3422, evidence was o b ta in e d th a t t h i s m a te ria l was b e in g ra p id ly re le a s e d in to solution (Table lb ). These data suggest th a t concentrations approaching equilibrium values were a tta in e d w ithin 1 hour of exposure. 006255 -6- T a b l e l a -- Mean C o n c e n t r a t i o n o f FM3422 E x p o s u r e W a t e r : E x p e r i m e n t a l Period Sampling S ite Top l a y e r Middle lay er Lower l a y e r n c o n c e n t r a t i o n 3^ i f Z 15 .4 9 + .0 6 15 .5 4 + .0 6 15 .5 2 + .0 4 aMean c o n c e n t r a t i o n o f e x p o s u r e p e r i o d + S . E . T a b l e l b -- C o n c e n t r a t i o n (ppm) d u r i n g I n i t i a l 4 H o u rs o f t e s t 3- Sampling S ite Top l a y e r Middle lay er Lower la y e r 15 m in . .19 .31 .23 1 hour .45 .49 .54 2 hour .43 .47 .47 4 hour .35 .42 .55 E x p re sse d as v i / l WHOLE FISH UPTAKE AND CLEARANCE U p ta k e o f t h e f l u o r o c t i e m i c a l FM3422 by c h a n n e l c a t f i s h h a s been ta b u la te d (Table 2) and i l l u s t r a t e d (F igs. 1, 2 ). During the f i r s t 24 hour exposure p e rio d , both Fish A and F ish B showed a rapid uptake of the te s t substance. This i n i t i a l uptake rate may b e a t t r i b u t a b l e t o movement o f FM3422 a c r o s s t h e | i n t e g u m e n t % y i t h s u b s e q u e n t b i n d i n g t o l i p o p h i l i c t i s s u e s . A s l o w e r r a t e o f FM3422 u p tak e was n o te d f o r b o th o rg a n ism s which was p o s s i b l y a c c o u n te d f o r by a d e c r e a s e d w a t e r / o r g a n i s m c o n c e n t r a t i o n g r a d i e n t . No u p t a k e p la te a u i n d i c a t i v e o f a s t e a d v - s t a t e was o b ta in e d fo llo w in g a n a ly s is 006256 -7- T a b l e 2 - FM3422 U p t a k e by C h a n n e l C a t f i s h (Ic ta la ru s punatatus) Time 15 m in . 1 hour 2 hours 4 hours 8 hours 12 h o u r s 16 h o u rs 20 h ours 24 h o u rs 48 hours 72 h o u rs 96 hours 120 hours 144 hours 168 hours Fish A 1 4 9 16 58 62 51 61 104 132 222 190 211 320 552 Fish B 1 4 10 29 42 60 56 74 110 213 159 257 201 279 158 a Expressed as yg/g. o f FM 3422 c o n t e n t o f t h o s e f i s h c o m p r i s i n g t h e c o m p o s i t e o r g a n i s m V A ( F i g u r e 1 ) . The 1 6 8 - h o u r v a l u e f o r t h e t e s t f l u o r o c h e m i c a l may be an o u t l i e r as th e s e v a lu e s encom passing th e p e r i o d from 72 120 h o u r s s u g g e s t a p l a t e a u ( s t e a d y s t a t e ) a t FM 3422 c o n c e n t r a tio n s of ^200 yg/g. A p la te a u e f f e c t was observed to occur w ith s a m p l e B, h a v i n g an u p t a k e v a l u e o f a p p r o x i m a t e l y 2 2 0 y g / g FM 3422 ( F i g u r e 2 ) . C o n c e n t r a t i o n s o f FM 3422 b i o a c c u m u l a t e d b y t h e s e t e s t organisms were observed to vary independent of whole fis h w eight. C l e a r a n c e o f FM 3422 by t h e c h a n n e l c a t f i s h r e q u i r e d i n e x c e s s of 5 days (Table 3). 006257 -8- T a b l e 3 -- FM3422 C l e a r a n c e o f I c t a l a r u s p u n a t a t u a sa Time 15 min. 1 hour 2 hours 4 hours 8 hours 12 hours 16 h o u r s 20 hours 24 hours 48 hours 72 h o u rs 96 h ours 120 hours Whole F ish A 306 269 252 260 260 253 235 238 237b 200 136 106 Whole F is h 319 216 309 242 242 206 177 255 201 190 227 226 78 Expressed as yg/g. Sample l o s t TISSUE ANALYSIS: MUSCLE Of t h e t i s s u e a n a ly z e d f o r FM3422, t h e muscle l a y e r o f th e channel c a t f i s h was found to p o s s e s s th e l e a s t p ro p e n s ity to bioaccum ulate t h i s fluorochem ical (Table 4, Figure 3) . A v p la te a u e f f e c t was observed to occur in th e c o n c e n tra tio n range * of 100-200 yg/g (T able 4 ) . T is s u e w eight was an independent v a r i a b l e , not a f f e c t i n g th e bioaccum ulation le v e l o f FM3422. D e p u ra tio n o f t h e s u b j e c t f l u o r o c h e m i c a l , FM3422, was d e la y ed , a p p r o x i m a t e l y 50% o f t h e m a t e r i a l h a v i n g c l e a r e d w i t h i n f o u r d a y s (Figure 3 Table 5). TISSUE ANALYSIS - BRAIN, GASTROINTESTINAL TRACT U p t a k e o f FM3422 by b r a i n an d t h e g a s t r o i n t e s t i n a l t r a c t was ele v ated due to the lip id nature of th ese m a te ria ls (Table 6 ). B r a i n u p t a k e o f FM3422 c o n t i n u e d t h r o u g h o u t t h e e x p e r i m e n t ( F i g u r e 4) 006258 i T a b l e 4 -- FM3422 U p t a k e by Io ta la ru s Punotatus Muscle Time Control 15 m in. 1 hour 2 hours 4 hours 8 hours 12 hours 16 h o u rs 20 h o u rs 24 hours 48 hours 72 h o u rs 96 h o u rs 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 Expressed as yg/g T a b l e 5 -- FM3422 C l e a r a n c e o f I o t a l a r u s p u n t a t u s m u s c l e ct Time Muscle 15 m in . 1 hour 2 hours 4 hours 8 hours 12 h o u rs 16 h o u rs 20 h o u r s 24 hours 48 hours 72 h o u rs 96 hours 120 hours 118 127 108 128 147 148 181 117 75 102 77 69 72 a Expressed as yg/g. 006259 -10- T a b l e 6 -- FM3422 U p t a k e by Ia ta la ru s punctatus b r a i n t i s s u e and the g astro in testin al tra c t of Ictalrus punctatusa Time Control 15 min. 1 hour 2 hours 4 hours 8 hours 12 h o u rs 16 h o u r s 20 hours 24 hours 48 hours 72 hours 96 hours 120 hours 144 hours 168 hours a Expressed as yg/g. Brain <0.1 2 8 28 50 100 115 149 240 225 352 328 606 588 695 905 G astrointestinal <0.1 <0.1 6 12 41 54 110 119 206 201 230 510 533 504 688 533 Tra< T a b l e 7 -- FM3422 C l e a r a n c e o f Q B r a i n a n d t h e G a s t r o i n t e s t i n a l T r a c t o f Ictalarus punotatus Tune 15 m in . 1 hours 2 hours 4 hours 8 hours 12 h o u rs 16 h o u r s 20 h o u rs 24 hours 48 hours 72 h o u rs 96 h ours 120 hours Brain 1152 761 712 602 511 582 618 559 523 586 450 423 417 G astrointestinal Tract 281 630 325 507 302 263 1205 805 564 264 353 390 712 a Expressed as yg/g. 006260 -11- T a b l e 8 -- FM 3422 U p t a k e b y L i v e r , K i d n e y , an d G a l l B l a d d e r o f the Channel C atfish Time Liver 15 min. 1 hour 2 hours 4 hours 8 hours i 12 hours 16 hours 20 hours 24 hours 48 hours 72 h o u rs 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 yg/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 T a b l e 9 -- FM 3422 C l e a r a n c e b y L i v e r , K i d n e y , a n d G a l l B l a d d e r of Ictalarus punetatus* Time 15 min. 1 hour 2 hours 4 hours 8 hours 12 hours 16 hours 20 hours 24 hours 48 hours 72 h o u rs 96 hours 120 hours Liver 504 650 758 779 507 446 728 420 443 575 316 76 507 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 Expressed as yg/g. 006261 -12- w ith no a p p a re n t p l a t e a u e f f e c t . At 168 h th e c o n c e n t r a t i o n o f FM3422 i n t h i s o r g a n was e q u i v a l e n t t o 9 0 5 y g / g t i s s u e . The b r a i n t i s s u e l e v e l s o f FM3422 r e m a i n e d e l e v a t e d upon c o m p l e t i o n o f t h e clearance phase of th is t e s t ; 417yg/g (Table 7, Figure 4 ). The g a s t r o i n t e s t i n a l t r a c t u p t a k e o f FM3422 i d e n t i f i e d t h r e e phases: ra p id and slow uptake components with a demonstrable p lateau (Table 6, F ig u re 5 ) . The f i r s t two p h a se s p e r s i s t e d f o r 24 and 48 hours r e s p e c t iv e ly w hile th e p l a t e a u e f f e c t was m inim ally o f 96 h o u r s d u r a t i o n . As may b e n o t e d ( T a b l e 7 a n d F i g u r e 5 ) , t h i s o r g a n d i d n o t c l e a r FM3422 r e a d i l y , r a t h e r an i n e x p l i c a b l e r i s e i n o r g a n c o n t e n t o f FM3422 o c c u r r e d d u r i n g t h e l a t t e r s t a g e s o f d e puration. TISSUE ANALYSIS - G a l l b l a d d e r T his organ dem o n strated a d e la y ed "uptake" of FM3422, d e te ctab l l e v e l s b e i n g i n i t i a l l y n o t e d a t 12 h o u r s e x p o s u r e ( T a b le 8 ) . The "uptake" p a tte rn d iffe re d from those of o th er organ systems being v analyzed in th a t the concentration of fluorochemical increased th ro u g h o u t th e exposure p e r io d . T h is organ showed an e r r a t i c c l e a r anee p a t t e r n , an i n i t i a l d e p u ra tio n fo llow ed by in c re a s e d l e v e l s of flu o ro c h e m ic a l, a phenomenon which was observed throughout th e remainder of the experiment (Table 9 Figure 6). The rem aining t i s s u e s t e s t e d e x h ib ite d s i m i l a r le v e ls of FM3422 u p t a k e . I n a l l c a s e s a s t e a d y - s t a t e c o n c e n t r a t i o n c o u l d be c a lc u la te d using the p latea u method. Depuration' data did not e s t a b l i s h d e f i n i t i v e e v i d e n c e f o r t h e c l e a r a n c e o f FM3422 b y t h e s e t i s s u e s : g i l l s , s k e le to n and s k in . The l i v e r did e x h ib it pronounced d ep u ratio n o f th e t e s t chem ical follow ed by ra p id uptake, a phen omenon a s s o c i a t e d w ith d e p u r a t io n d a t a o b t a i n e d f o r t h e g a s t r o i n t e s t i n a l tr a c t and the g a ll blad d er. 006262 -13 T a b le 10 -- C o m p ila tio n o f Whole F i s h and Organ System B io c o n c e n t r a t i o n F a c t o r s D u rin g Exposure t o FM3422a,C Sample Whole F ish #1 Whole F is h #2 Skeleton Skin G ills Liver Kidney G astrointestinal Gall Bladder Brain Muscle Weight0 g 10.8+4.5 11.5+2.4 11.8+2.3 2.9+.9 .64+.2 .51+.1 . 36+. 1 1.4+.4 .03+.02 .19+.03 7.0+2.0 Bioconcentration Factor 575 406 501 534 542 474 709 1064 26548d 1344 281 uLBCF - c a l c u l a t e d u s i n g p l a t e a u m e t h o d DA v e r a g e w a t e r c o n c e n t r a t i o n o f FM3422, 0 . 5 2 ppm cD a ta e x p r e s s e d as mean + S.D. dSeven day BCF,value, absense of p la te a u e f f e c t. 006263 -14- T a b l e 11^ -- U p t a k e R a t e C o n s t a n t o f FM322 i n la ta la r u s punctatus (Channel Catfish) Sample Uptake ra te Rapid Phase constant composite Slow : Whole F i s h A 3.2+1.3 5.7 2.7 Yihole F i s h B 6.4+3.1 8.6 3.9 Brain 7.7+2 .4 9.3 5.1 G ills 5.4+3.3 7.3 2.7 G astrointest inal Tract 6.0+2.4 7.3 4.4 Liver 4.5+2.3 6.3 3.6 Gall Bladder 82. l a -- Kidney 4.0+1.4 5.4 3.0 Muscle 1 .8 + 0 .6b -- Skin 6.7+3.2 8.9 3.6 Skeleton 3.1+1.0 3.5 2.4 a Uptake r a te c o n stan t at 168 hours k Values simi'.lar at a ll time p erio d s 006264 -15- A t a b u l a t i o n o f t h e b i o c o n c e n t r a t i o n f a c t o r s (BCF) d e t e r m i n e d at an approxim ate ste a d y s t a t e , u sin g th e p l a t e a u m eth o d ,p ro v id ed e v i d e n c e d e m o n s t r a t i n g t h e l i p o p h i l i c i t y o f FM3422 ( T a b l e 1 0 ) . A l t h o u g h t h e g a l l b l a d d e r , p o s s e s s e d t h e h i g h e s t BCF f o r c o m p a r a t i v e p u r p o s e s t h e s e d a t a can be i g n o r e d f o r r e a s o n s t o be d i s c u s s e d . Of t h e r e m a i n i n g o r g a n s y s t e m s t h e p r o p e n s i t y t o b i o a c c u m u l a t e FM3422 was most prom inent in th e b ra in and g a s t r o i n t e s t i n a l t r a c t . The e d ib le p o r tio n , m uscle, m inim ally b io c o n c e n tra te d FM3422. A ta b u la tio n of uptake ra te constants fo r whole f is h and organ systems provides fu rth e r evidence th a t the lip o p h ilic nature of FM3422 d o e s i n f l u e n c e u p t a k e r a t e ( T a b l e 1 1 ) . B o th b r a i n a n d t h e g a stro in te stin a l tra c t had high uptake rate constants. A second p r e m i s e may b e e s t a b l i s h e d t h a t b e i n g t h e g r e a t e r t h e s u r f a c e a r e a exposed to the m aterial the g reater the uptake ra te , hence elevated u p tak e r a t e c o n s ta n ts f o r both s k in and g i l l s . The form er in s t a n c e provides a physiological basis for uptake while in the l a t t e r case both mechanical and p h y sio lo g ic al mechanisms were o p e ra tiv e . C learance r a te c o n sta n ts could not be c a lc u la te d due to d a ta v a ria b ility . DISCUSSION In these s tu d ie s the p rin c ip a l mechanisms fo r i n i t i a l uptake of FM3422 w e re t h e g i l l s an d i n t e g u m e n t . S u b s t a n t i a t i n g e v i d e n c e c o n s i s t ing of uptake ra te co nstants which exceeded a ll o th e r organ system s ex c e p tin g b r a in , g a s t r o i n t e s t i n a l t r a c t and th e g a l l b la d d e r. The g i l l c i cu lation functions as a tran sp o rt system d isp ersin g the fluorochem ical to v a rio u s s i t e s in th e body. The im portance of t h i s system in term s of both uptake and tra n s p o rt of fo reig n chem icals has been the 006265 -16 s u b j e c t o f r e c e n t p a p e r s by Granmo and K o llb e r g (1976) and Bass and Heath (1977). Movement o f FM3422 a c r o s s t h e i n t e g u m e n t a l s o c o n t r i b u t e s t o t h e body b u r d e n o f FM3422 b a s e d on u p t a k e r a t e c o n s t a n t s . D e f i n i t i o n of the extent of th is co n trib u tio n cannot be determined as the exact tra n s p o rt pathway has not been e s ta b lis h e d . Our p revious study (1977) d id dem onstrate th e e x is te n c e of a h i g h ly l i p i d l a y e r u n d e rly in g th e s k in which t h e o r e t i c a l l y b a s e d on t h e l i p o p h i l i c i t y o f FM3422 s h o u l d s e r v e a s a s t o r a g e ( b i n d i n g ) s i t e fo r th is chemical. Vascular perfusion of th is area could serve to tran sp o rt the fluorochem ical to other s i t e s in the body, however, th e magnitude of p e rfu s io n is an unknown. S im ila rly , i f one views t h e l i p i d l a y e r as a b in d in g s i t e f o r FM3422, t h e n a t u r e o f t h e binding becomes o f relevance in determ ining the ease w ith which m a te ria l may be t r a n s p o r t e d e l s e w h e r e . I t was dem onstrated p re v io u sly in our la b o ra to ry th a t the more h ig h ly lip id - c o n ta in in g organs tended to bioconcentrn/te th e v s u b je c t flu o ro c h e m ic a l more r e a d i l y . This f in d in g has been s u b s t a n t i a t e d by t h e p r e s e n t s t u d y . T r a n s p o r t o f FM3422 t o t h e b r a i n and g a s t r o i n t e s t i n a l t r a c t by th e c ir c u la to r y system o f th e f i s h seems se lf-e v id e n t as these organs are highly v ascu lar. Evidence obtained in t h is study suggests th a t the su b je ct chemical is bound tig h tly as i t d o e s n o t d e p u r a t e a p p r e c i a b l y . By i m p l i c a t i o n a s i m i l a r s i t u a t i o n may e x ist at the o il layer/integum&rit in te rfa c e , thereby reducing the o v e ra ll co n trib u tio n of the integument as a source of fluorochem ical fo r other organs in the body. Organs c o n t r i b u t i n g t o t h e d i g e s t i v e p r o c e s s o f Iotalarus punetati 006266 -17- p r e s e n te d an i n t e r e s t i n g p r o f i l e . The g a l l b la d d e r, s e rv in g as a r e s e r v o i r fo r b i l e m anufactured by th e l i v e r , possessed th e g r e a te s t c o n c e n t r a t i o n o f f l u o r o c h e m i c a l o b s e r v e d i n t h e s e s t u d i e s . When s a m p l e s w e re o b t a i n e d f o r FM3422 a n a l y s i s , d i s t e n t i o n o f t h e g a l l b l a d d e r was o b s e r v e d , i n d i c a t i n g t h a t a t o u r s a m p l i n g t i m e we w e r e in r e a lity analyzing a concentrated sample of b ile . It is also p r o b a b l e t h a t t h i s o r g a n p a r t i c i p a t e s i n t h e e x c r e t i o n o f FM3422 f r o m th e body. The co m p o sitio n of b i l e i s such t h a t given th e l i p o p h i l i c i t y o f FM 3 4 2 2 , b i n d i n g s i t e s , c o m p l e x a t i o n a n d / o r c o n j u g a t i o n o f t h i s flu o ro c h e m ic a l to b i l i a r y components i s a d i s t i n c t p o s s i b i l i t y . Con tra c tio n of the g a ll bladder w ith the re s u lta n t emptying of i t s c o n t e n t s i n t o t h e g a s t r o i n t e s t i n a l t r a c t may a c c o u n t f o r t h e s p o r a d i c nature of the clearance curve for th is organ. One may v i e w t h e s e q u e n c e o f e v e n t s o c c u r i n g i n t h e d e p u r a t i o n phase of th is study as i t re la te s to the d ig estiv e system in the f o l l o w i n g m an n e r: FM3422 i s c l e a r e d a t a m e a s u r a b l e r a t e by various organs, being tra n s p o rte d to the l i v e r by the c irc u la to ry ' s y s t e m . I n t h e l i v e r , FM3422 e i t h e r b i n d s , c o m p le x e s a n d / o r becom es a co n ju g ate of newly m anufactured b i l e . The b i l e i s then s to r e d in the gall bladder being expressed in to the duodenal area of the i n t e s t i n e . T h i s m a t e r i a l may -nt-hen be e i t h e r e x c r e t e d w i t h i n , f e c a l m a t t e i a n d / o r some FM3422 may u n d e r g o r e - u p t a k e f o r b o d i l y d i s t r i b u t i o n . E x c r e t i o n o f FM3422 i s n o t s o l e l y v i a f e c a l m a t t e r as a sim ila r scenario can be described for renal clearance wherein elevated l e v e l s o f FM3422 a r e m a i n t a i n e d t h r o u g h t h e f i r s t 72 h o u r s o f d e p u r ation . 006267 -18- The e d ib le p o r tio n of th e channel c a t f i s h , the. muscle f i l l e t , d i d n o t b i o a c c u m u l a t e FM3422 t o a n a p p r e c i a b l e e x t e n t s u b s t a n t i a t i n g our previous observation for th is tissu e . In g e n e ra l, no adverse s ig n s were observed during th e d u ra tio n of the experiment a tte s tin g to the non-toxic nature of th is chemical under the co n d itio n s employed in th is t e s t . CONCLUSIONS T h is s t u d y o f t h e a b i l i t y o f t h e c h a n n e l c a t f i s h (.Iatalarus punotatus) t o b i o c o n c e n t r a t e t h e f l u o r o c h e m i c a l FM3422 h a s l e d t o t h e following conclusions: 1. Both lip o p h ilic organs and organs p o ssessin g a larg e surface area at the w ater/organ in te rfac e possess the highest uptake rate constants. 2. The b r a in and g a s t r o i n t e s t i n a l t r a c t have BCF's in excess q o f 10 due t o t h e l i p o p h i l i c n a t u r e o f t h e s u b j e c t f l u o r o c h e m i c a l . 1 V' 3. The g a l l b la d d e r due t o i t s s to ra g e fu n c tio n showed the h i g h e s t c o n c e n t r a t i o n o f FM3422 on a y g / g b a s i s . 4. Whole f i s h o r organ w eight was an independent v a r i a b l e , not a d eterm inant in th e b io c o n c e n tra tio n of FM3422. 5 . E x c r e t i o n o f FM3422 c o n s i s t s o f b o t h u r i n a r y and f e c a l pathw ays. 6 . C l e a r a n c e o f FM3422 by c o m p o n e n ts o f t h e d i g e s t i v e s y s t e m contributes to the e rra tic nature of the depuration curves. 7. FM3422 a t t h e e x p o s u r e c o n c e n t r a t i o n d i d n o t e l i c i t s i g n s of to x ic ity , th ere fo re , under the conditions of th is experiment, th is fluorochem ical can be considered non-toxic. /) A ANW/ vmp 006268 -19- BIBLIOGRAPHY 1 . B a s s , M i c h a e l L. a n d A l a n G. H e a t h . W a t e r R e s e a r c h 11: 497-502, 1977. 2. B u r n e t t , R. S c ie n c e 1 7 4 :6 0 6 , 1971. 3. C h i o u , C . T . , F r e e d , V. H . , S c h m e d d in g , D. W ., a n d K o h n e r t, R. L. Env. S e i . T e c h . 1 1 : 4 7 5 , 1977. 4 . E a t o n , J o h n - C h a i r m a n , ASTM D r a f t G u i d e l i n e s , 1 9 7 7 . 5 . E l n a b a r a w y , M. T. T e c h n i c a l R e p o r t , 1 9 7 7 . 6 . F u j i t a , T . , I was a , J . , H a n s c h , C. J . Am. Chem. S o c . 8 6 : 5 1 7 5 , 1964. 7 . Granmo, A. a n d S. K o l l b e r g . W a t e r R e s e a r c h 1 0 : 1 8 9 , 1 9 7 6 . 8. G u s t a f s o n , C. G. Env. S e i . T e c h . 4 : 8 1 4 , 1 9 7 0 . 9 . M endel, A. T e c h n i c a l R e p o r t , 19 7 7 . 1 0 . V e i t h , G. D. a n d C o m s t o c k , V. M. J . F i s h R e s . B o a r d C a n . 32:1849, 1975. 1 1 . W e l t e r , A. N. T e c h n i c a l R e p o r t , 1 9 7 7 . 006269 I ; F i g u r e 1 - C o m p o s i t e o f d e s i g n a t e d Whole F i s h A, o n e o r g a n i s m p e r d a t a p o i n t , c o n c e n t r a t i o n o f FM 3422 i n w a t e r (C ) e q u a l s 0 .5 2 ppm. S lo p e I n t e r c e p t y e q u a l s 1 .5 1 1 . 006270 S CYCLES X IQ D IVISIONS PER INCH F i g u r e 2 - C o m p o site o i d e s i g n a t e d Whole F i s h B, one o rg a n is m p e r d a t a p o i n t . C = 0 . 5 2 ppm. S lo p e I n t e r c e p t y e q u a l s 1.699. W 006271 w* -22- .3^1P:lBESTrrCO'PFY;' AVAIL-ABLE S E M i - L O G A R I T H M 1C S CYCLES X IQ DIVISIONS PER INCH Figure 3 Muscle Composite Data - 2 tissue analyses per point. Cw = 0.52 ppm. Slope Intercept y = 1.225. 006272 Z it.5 X ID DIVISIONS PER INCH BIST COPY AVAILABLE Figure 4 - Composite Data of Brain Tissue - 2 tissue analyses per point. Cw = 0.52 ppm. Slope Intercept y = 2.357. 006273 Figure 5 - Composite Data of Gastrointestinal Traot - 2 tissue analyses per point. C = 0.52 ppm. Slope Intercept y = 2.153. 006274 O IETZG EN C O R PO R A T IO N bfi bD 3. CM CM CO S H O <i> 0>)J -p O . ci fa-ii 1--i III A G "iJi0) >4) Q+J aC 1*0 tn ui ^ "-P ) W 5- ; i F ig u re 6 - Composite D ata o f two g a l l b la d d e r a n a ly s e s p e r p o i n t . C = 0 . 5 2 ppm. S lo p e I n t e r c e p t y = 1 .3 6 4 . w 006275