Document 6y55dqBXE0RBaZzZnGL2q8B1

* b lQ X l+ fT Chemosphere, Vol.15, No.5, pp 537-548, 1986 Printed in Great Britain 0045-6535/86 S3.00 .CO Peraamon Journals Ltd. n 2 ,3 ,7 ,8-TETRACHLORODIBENZO-P-DIOXIN AND RELATED DIOXINS AND FURANS IN SNAPPINC TURTLE (CHELYDRA SERPENTINA) TISSUES FROM THE UPPER ST. LAWRENCE RIVER John J. Ryan*, Ben j a m i n P.-Y. Liu , and J a c q u e s A. Hardy, Food R e s e a r c h D i v i s i o n , H e a l c h P r o t e c t i o n B r a n c h , T u n n e y 's P a s t u r e , O t t a w a , X l A 0 L 2, C a n a d a W a r d B. S t o n e , N ew Y o r k State, Dept. E n v i r o n m e n t a l C o n s e r v a t i o n , D e l m a r , New York 12054, U.S.A. and P a t r i c k O 'K e e f e and J o h n F. C i e r t h y , W a d s w o r t h C e n t e r for L a b o r a t o r i e s and Research, New York State Dept. Health, Albany, New York 12201, U.S.A. Abstract Fat and liver samples from three snapping turtles (Chelydra s er pe nti na ) from the u p per St. Law r e n c e Riv e r were a n a l y s e d for t e t r a - up to oct a c h l o r i n a t e d d i b e n z o - p - d i o x i n s (PCDDs) and c h l o r i n a t e d d i b e n z o f u r a n s (PCDFs) by two gas ch r o m a t o g r a p h y - mass spectrometry t e chniques and by a bioassay. The t i s s u e s c o n t a i n e d o n l y 2 ,3 ,7 ,8 - ch 1o r i n e s u b s t i t u t e d P C D D s and P C D F s w i t h h i g h l e v e l s o c c u r r i n g for 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o - p - d i o x i n (2 ,3 ,7 ,8 - T C D D ) . On a wet w e i g h t basis, fat levels of 2 , 3 , 7 , 8 - T C D D w e r e 4 to 7 times h i g h e r than liver le v e l s w i t h up to 500 pg/g p r e s e n t in t u r t l e fat. One fat sample also c o n t a i n e d o v e r 3 n g / g of 2 ,3 ,4 , 7 , 8 - p e n t a c h 1 o r o d i b e n z o f u r a n ( P n C D F ) s u g g e s t i v e of a PCB s o u r c e of c o n t a m i n a t i o n . The T C D D le v e l s in the t u r t l e s are c o n sistent with the c o n s u m p t i o n of a fish diet c o n t a i n i n g about 20 pg/g 2 , 3 , 7 , 8 TCDD, a level c u r r e n t l y found in cel s a mples f r o m the same r e g i o n . C o m p a r i s o n of this limited snapping turtle data with that of other aquatic species from the same and d i f f e r e n t bodies of w a t e r s u g g e s t that the N i a g a r a River via Lake On t a r i o could be a pos s i b l e source of c o n t a m i n a t i o n of 2 , 3 , 7 , 8 -TCDD for the St. Lawrence River. The results also show that turtles are excellent monitors for PCDD and PCD F c o n t a m i n a t i o n . Human c o n s u m p t i o n of turtle tissue samples c o n t a i n i n g t h e h i g h e s t l e v e l s of 2 , 3 , 7 , 8 - T C D D a n d P C D F s so far d e t e c t e d in wildlife samples may constitute a hazard. INTRODUCTION 2 , 3 , 7 , 8 - t e t r a c h 1o r o d i b e n z o - p - d i o x i n (2 , 3 , 7 , 8 - T C D D ) o c c u r s as a c o n t a m i n a n t 537 783347 in orne f i s h s p e c i e * f r o m the C r e s t L a k e s , p a r t i c u l a r l y L a k e O n t a r i o and the Saginaw Bay area of Lake Huron (1-3). T y p i c a l l y posit i v e values range between 5- and 20 p a r t s p e r t r i l l i o n (ppt; p g / g ) w i t h the o c c a s i o n a l h i g h e r v a l u e . Other chlorinated d ibe n z o - p - d ioxins (PCDDs) and chlorinated d ibc n :ofurans (PCDFs) have been r e p o r t e d less f r e q u e n t l y and in s m a l l e r c o n c e n t r a t i o n s (4,5). There has been no d o c u m e n t a t i o n of the o c c u r r e n c e of 2 , 3 , 7 , 8 - T C D D or v a r i o u s P C D D s in o t h e r a q u a t i c s p e c i e s f r o m the C r e a t L a k e s . H o w e v e r , t i s s u e s of the s napping t u r t l e (Ch elydr a serpent i n a ) from New Yo r k Sta t e w a t e r s are known to accumulate and tolerate remarkedly high fat and liver levels of PCBs (6,7). In some c a s e s the c o n c e n t r a t i o n of PCB in a d i p o s e t i s s u e was a l m o s t II m a k i n g the tu r t l e an e x c e l l e n t b i o l o g i c a l m o n i t o r for the p r e s e n c e of this type of c h e m i c a l c o n t a m i n a t i o n . In a d d i t i o n , high levels of P CDFs have been i d e n t i f i e d in the f a t t y tis s u e of a s n a p p i n g t u r t l e t a k e n in Che H u d s o n river, New Y o r k S t a t e , in the v i c i n i t y of a f o r m e r c a p a c i t o r m a n u f a c t u r i n g p lant ( 8 ) . (site 10 in Fig. 1). Since we were surveying fish populations from the Great Lakes for PCDDs and PC D F a and b e c a u s e t u rtles a p p e a r e d to be such good a c c u m u l a t o r s of chlorinated aromatic compounds, we analysed some turtle tissues for the PCDDs and PCDFs. This report gives the result of the analyses of liver and fat samples from three s n a p p i n g turtles taken in the upp e r St. L a w r e n c e River near Lake O n t a r i o . E x t r a c t s of two of the t u r t l e fat s a mples we r e also a n a l y s e d by a second g a s - c h r o m a t o g r a p h y (GC)-mass s pe ct r o m e t r y (MS) method and by a newly d e v e l o p e d i_n v i t r o b i o a s s a y for d i o x i n l i k * a c t i v i t y (9). Th e r e s u l t s s h o w some of the h i ghest 2 , 3 , 7 , 8 - T C D D and PCDF levels for any b i o l o g i c a l sample from Che Great Lakes area. EXPERIMENTAL Tissue Collection Abdominal fat and liver tissues were taken from three snapping turtles, two c o l l e c t e d in J u n e 1984 in the u p p e r St. L a w r e n c e R i ver ne a r La k e O n t a r i o , sites 1 and 2 in Fig. 1, and a third t a k e n in J u n e 1985 in the same r i v e r ne a r East M a s i e n a , sit* 3. Li v e r t issue fr o m a f o u r t h tu r t l e but a d i f f e r e n t c o l l e c t e d in the Ot t a w a River ne a r O t t a w a in July 1980 (site 4). M o r e d e t a i l e d i n f o r m a t i o n on ch* t i s s u e s a m p l e s is g i v e n in T a b l e 1. S a m p l e s of **1 (A n g u i l l a r o s t r a t a ) war* o b t a i n e d in 1 982 and 1 9 83 by F i s h e r i e s and O c e a n s C a n a d a f r o m two areas in ch* m i d d l e ( s i t e 5) and lower (site 6) St. L a w r e n c e R i v e r in Q u e b e c , one of its t r i b u t a r i e s the R i c h e l i e u Ri v e r (site 7), and the eastern part of Lake Ontario (site 8). Analysis A n a l y s i s of t i s s u e s was carried out to effect m e a s u r e m e n t of all tetra- through o c t a -PC DDs and PCDFs (up to 136 analytes). Crude tissue was first h o m o g e n i s e d to a u n i f o r m c o n s i s t e n c y and an a l i q u o t (2 to 3 g for fat and 5 g for liver and fish) t a k e n for analysis. The sample was then h o m o g e n i z e d or 783348 iff 539 Fig. 1 - Hap of irei from whi ch Che tu rt l e and f ish samp les we re collected, Hu mber* refer to spe cific sites mentioned, in text. Ta b l e 1. I n f o r m a t i o n on turtle c is m e * from tl ,e St. L a w re n c e and Ottawa R ivers . Turtle Date N um b e r Col 1eet ed* Location Species Ti ssues June 1984 June 1984 Jun e 1985 Ju^y 1980 S t e e l e 's Bey Clayton, St. Lawrence River, site Ia Goose Bay East, Alexandria, St. Lawrence River, site 2 East Masseoa, Sc L a w re n c e River, site 3 Ottawa River, Ottawa, site 4 a - r e f e r s 'to Figure""!"." snapping, Chelydra serpentina snapping, Chelydra serpentina I snapping, ' Chelydra serpentina Bland ings , Emydo idea blending i Fat iver Liver Fat Liver i L iver L iv e Height Kg 6.4 3.6 7.1 2.1 Age Sex Years 16 + 8+ 14 + shaken with 120 mL of acetone-hexane (2:1) and spiked with about 100 pg each GEfrfp 010538 i 783349 540 of a m i x t u r e of stable i s o t o p i cs l l y e n r ic h e d PCDDs and P C D Fs in o rd er to cal cul at e loaset io sample p r e p a r a t i o n . The sample e x t ra c t vaa then part i- t.iontd against w a t e r and the a q u e ou s and solid phases e x t r a c t e d a second time with hexane (10). After washing the combined hexane phases with water, a 101 ] p o r ti o n was taken, e v a p o r at e d to d r y ne s s in a rota ry e v a p o r a t o r u n d e r v a c u u m and w eighed* This wei g h t r e p r e s e n t e d the lipid con'tent of the tissue, The remaining hexane extracts were then defatted(with sulfuric acid and chromato- graphed on disposable glass columns containing Fiorisi! as adsorbant This latter step re moved the less p o l ar c on t a m i n a n t s sue h as PCBs and c h l or i na te d di ph eny l ethers as out lined p r e v i o u s l y (11). Furth er c le anup of some sample extracts was carried out by reversed phase high pressure liquid chromatography (HFLC) (12) with methanol as eluent and by collection of two fractions. Some sample extracts w e re p u r i f i ed a f t er F lo r i s il b1y the rIe c e n t l y d e v e l o p e d c h r o m a tog rap hi c p r o c ed u re (13) of a c t i v at ed carbon jdispersjed on silica gel. In the latter ease, a mixture of eyelahexane-dieh1orooecbane eluted the non-planar aromatics fo ll owe d by the e l u t io n of the pl an a r aro ma ti cs such as PCDDs and PCDFs with toluene. M e as u re m e n t of the PCDDs and PC D F s in sample e x t ra c t s was e f f ec te d by GCI M S . The GC was a P a r i a n 3700 f i t t e d with a 25 m DB-5 (J & V, 0.25 m m l.D.) bonded phase fused silica c a p i l l a r y column. The column wae heat ed in steps r a pidly from 100 to 180"C (c_ 1 min) and then m o r e sIlowly to 240 *C at 5*C per min such that 2 , 3 , 7 , 8 - T C D D and oct acb lorod ibenzo-pj-d iox in (OCDD) eluted at about 11 and 22 min, r e s p e c t i v e l y . The GC was c o upled d i r e c t l y to a VG- Analytieal ZAB-2F MS operating at low resolution (2,000) for liver and fish e x tracts and at high r e s o l u t i o n (10,000) for jfat ext r a c t s. C o m p o u nd s el ut i ng from the GC were ionized by electron impact and detected by -selected ion m o n i t o r i n g of ions from eseh c o n g e n e r group as d es c r i b e d (14,15). Q u a n t i t a tion of the an al ytes in ext racts was ac hi e ve d by c o m p a ri ng the r e sp o ns e of e xt e r na l sta nd a rd s i n jected e i t h e r b e f o re orj a fter sample e x t r a c t s . Values reported were corrected for manipulative and purif ieation losses using the isotopically labelled internal standard recoveries, Detection limits were about 2 ppt for mos t -analytes except for OCDD which, due to hi g he r re age nt blanks and lower r e c ov e r i es , was about 10 ppt Method performance was m o n i t o r e d by the s i m u lt a ne o u i p r o c e s s i n g of l a b o r a t or y r e a ge n t b l a n k s in each sat of f iva samples, by the use of seven carbon-13 labelled PCDD snd five c h lo r i n e- 3 7 l a b e ll e d PCDF i n t er n a l s t a nd a r d s and by the p a r t i c i p a t i o n in interlaboratory studies. C b n f i rm s t i on of R e s i d u e s in the tur t l e fat S ep a r at e 4 g a l iquot s a mples of erude turtle fiat f r o m a n i m al s 1 and 2 taken at the same time as above t i ssues w e r e spiked w i t h in tern al isotopic standards, e x tr ac t e d w i t h d i c h l o r o m e t h a n e anId p a r t iIt i o n e d a g a in s t s ul fu r ic acid. After neutralization and passing through a column of KOB treated Jemsilica, the ex tract was p r o c e ss ed though a i - a u t o m a ted sy st e m conta in ing 783350 541 a c i d i c a l u m i n a , e l u t e d w i t h d i c h 1o r o m e t h a n e - h e x a n e (1: 1) o n t o c h a r c o a l in -Celite, and reverse flow eluted with xylene onto a neutral alumina column. Final el u t i o n of the neutral alumina col u m n was carried out w i t h 1:1 d i c h l o r o methane hexane. The GC-HS was equipped with a 60 m SP-2330 polar stationary p h a s e c o l u m n and MS r e s o l u t i o n w as 1 0 , 0 0 0 . T h i s t e c h n i q u e (16) is s p e c i f i c for 2 , 3 , 7 , 8 - T C D D due to the c o m p l e t e s e p a r a t i o n of this isomer from the o t her 21 T C D D i s o m e r s on the p o l a r GC c o l u m n . In v i t r o b i o a s s a y p r o c e d u r e The sample was e x t r a c t e d with d i c h 1o r o m e t h a n e , tr e a t e d w i t h s u l f u r i c acid and n e u t r a l i z e d as d e s c r i b e d a b o v e , and t he e x t r a c t was s o l v e n t e x c h a n g e d to be n z e n e to a final v o l u m e of 1 mL. D i m e t h y 1 s u 1foxi d e (DMSO; 50 u L ) was added and the benzene evaporated under a flow of dry nitro g e n . The DMSO extract was d i l u t e d 1 : 1 0 0 0 in c u l t u r e m e d i u m and a s e r i e s of t e n - f o l d d i l u t i o n s w e r e a d d e d to X B F . e p i t h e 1 ia l c e l l s w h i c h w e r e i n i t i a t e d o n e day p r e v i o u s l y w i t h i r r a d i ated 3T3 m o u s e f i b r o b l a s t c e lls. T r e a t e d as w e l l as p o s i t i v e and n e g a t i v e control cultures were incubated for 14 days with r e f e e d i n g every 3 to 4 days with freshly made sample dilutions. The cultures were then evaluated for a l t e r e d g r o w t h and m o r p h o l o g y as d i s c u s s e d e l s e w h e r e (9,17). RESULTS T h e P C D D s and P C D F s f o u n d in the s e v e n t i s s u e s f r o m the f o u r t u r t l e s are l i s t e d by i n c r e a s i n g d e g r e e of c h l o r i n a t i o n in T a b l e 2. O n l y 2 , 3 , 7 , 8 - c h 1o r i n e Table 2.. PCDD and PCDF levels (pg/g) in four turtle fat and liver camples. Turtle No.1 Tissue 2378-Fb 2378-Dc 12378--F 23478-F 12378-D Total HxF* Total HxD* 1234678-F 1234678-D 12346789-D Lipid Z . 1 Fat Liver 12 474 ND(2) 152 63 ND (2 ) 23 ND(2) 2.3 34 93 ND(2)d 107 ND(2) 29 12 ND( 2 ) 3.0 ND(2) 4.5 27 11 2 Fat Liver 6.0 232 ND( 2 ) 95 33 ND( 2 ) 16 ND(2) 17 36 71 ND( 2) 32 ND(2) 13 4.6 ND( 2 ) 6.4 ND( 2 ) 4.0 ND(10) 9.2 3 Fat Liver CO 330 370 600 3020 104 890 102 69 3.7 17 74 74 100 480 22 160 18 13 3.7 ND(10) 16 4 Liver ND( 2) ND (2) ND (2) ND(2) ND (2) ND( 2) ND( 2) ND (2) ND (2) 16 - s - c f . Table 1; b - chlorinated dibenzofuran; e - chlorinated dibenzo-p-dioxin; d - not detected followed by detection limit in pg/g in brackets; e - hexachlorodibenzofuran including the 123478-, 123678-, and 234678-isomers; f - hexachlorodibenzo-p-dioxics including the 123678-, 123478-, and 123789-isomers. g a #> 054q 783351 0 a. 542 substituted FCDDs snd PCDFs were detected is these m a p l e s (Figure 2V with the tetra- and penta-chlorinated congeners predominati ng in conee ntration over fuM -- TIT* MMSifflfl un-# KM weiM - TfTM turns uecf/itHSFRoOmNxtimo i ISSM I HO lJUriU *//* UT1-0 ii i TMi - o |: Ilr`i --u.-t-. JL|.u..i i ^ ,,y,n , ,, r* I13STI- *> T1WC (HIM.) errA oct* "//- > (CMAT* OCT* Iiii**m*o !)w Fig. 2 - GC/HS profile of fat sanple from turtle 1 shoving presence of 2,3,7,8-chlorine substituted FCDDs and PCDFs; peak responses are r epor te d at dif fe re nt a tt en u a t i o n s for eac h .congener group the hi gher chlorinated groups (hcxi-, hepta- and octaj-). The pres en ce of only 2 , 3 , 7 , 8 - c h l o r i n e s u b s t i t ut e d F C D Ds and F C D F s ia s i m i l a r to that f o un d in h uman t i ssues and in fish from the Great Lakes fl -- 4 JlS3 . H o w e v e r , the d i s t r i b ut i on of F C DD s and PCDFs among the v a r io u a c o n g e n e r g ro u p s in Che t u r t l e tissues is c o m p a r a b l e Co that found ia fish (te tra- and pe ot a - h i g h e s t and h ig her c h l o r i na t ed PCDDs and PCDFs lover) and d i f f e r e n t to that fou nd in h uman tissues (for PCDDs, tetra- loveat and oeta- highest; for PCDFs, penes-, bexa-, h e pt a - a i m il a r c o nc e nt r a t io n ) (13). A l t h o u g h |the n u m b e r of s a m p le s ia small, a distinction in tissue level exists be tvean the snap pi ng turtle tissues from the St. Lawrence Kiver and the Standings turtle liver sample from the Ottawa liver. Tba latter sample shoved only a small amount of OCDD near the detec tion limit of 10 ppt, a d i f f e r e n c e w h i c h no d o u b t r e f l e c t s tha l o c al it ie s from which the animall were obtained. The tissues from turtle 3 contain the same types and about tha same c o n c e n t r a t i o n of P C D D s as t h o s e f r o m t u r t l es 1 snd 2. Howev er , t ur t l e 3 shows a d i f f e r en t P C D F p r o f i l e tfcian do t u r t l e s 1 snd 2. Firs t ly , a d di.t i.o n a l PCDF (1,2 , 3 , 7, 8 - F nC D F , h e|x a d h l o rJo d i b e n z o f u r i o i (HxCDF), I( and h e p t a e h l o r o d i b e n z o f u r a n (HpCDF)) are p r e s e n t in .turtle 3 and, secondly, the levels of some of the PCD? (p ar ticularly 2,3,4' ,7,8-PnCDF and 2,3,7,S- GENR 010541 783352 543 t e t r a c h l o r o d i b e o c o f u r a n (TCDF) ) ire au eh h i g h e r in this a n i m i . On vet wei ght basis, the 2 , 3 , 7 , 8-TC DD content of the three fat s amp le s vis 4 to 7 tiaes higher than the liver aamp lea* However, on a lipid basis the liver samples c o n t a i n e d 970, 350, and 462 pg/gj 2,3,7, 8-TC DD in t u r t le s 1, 2 and 3, r e s p e c ti v el y, levels w h i c h are 1 to 2 times high' er than the fat sample levels. To c o n f ir m the 2 , 3 , 7 , 8 - T C O D c o n c e n t r a t i o n s in some of the p o s i t i v e turtle fat samples, a n al y si s of turt le fats 1 and 2 was also c a r r i ed out by two separate l a bo r at or ie s using, in one case a d i f f er en t c h r o m a t o g r a p h i c cleanup and CC-K S i ns t r u m e n t a t i o n and, in the o t h e r case, a c o m p l e t e l y u n r e l a t e d in vi_tr^ bioassay vhieh measures 2,3,7,8-TCOD like sctivity. The results from the three di ff er en t p ro ce d ur es are giv en in T a b le 3. The ag re em en t b e t w e e n the two c h e m i c a l - p h y s i c a l t e c h n iq u e s is q u i t e a c c e p t a b l e g iven the level being measured and the use of different fat from the same animals. The results of tha vitro bioaaaay indicate^ that both samples of turtle fat have 2 ,3 ,7,8 - T C D D - 1 ike activity. P r e s e n t a t i o n of ,the b i o a s s a y data as a range ric he r than a d i s c r et e n u m b er ia a c o n s e q u e n c e of u s i ng a t e n -f ol d d il ution. However, Che semi-quantitative values are consistent with the results gener ated by chemical analysis. In order to com pare the 2 , 3 , 7 , 8-TCDD v a l u e s found in the turt le samples wi th other biota, res ults of the analysis of eel samples from Lake O ntario and Che St. L a w r e n c e H i v er area are givenj in T a b l e 4. Eel is a fatty fi sh (a verage lipid content of the ele v en samples i J T a b le 4 was 2421 and cont ai ns i relatively high levels of 2,3,7,8-TCDD (1). Thj`Ie a v e r ag e v a l u e s found here on a vet tissue b s a ia of about 20 ppt 2 ,3,7 j,8-T C D D v i t h s m a ll e r a m ou nt s of o cher PCDDs and PCDFs are at least an or d e r of m a g n i t u d e lover than chose found in the turtle fat samples. A l t h o u g h the s a m p l i ng is limited, the two eels from the R i c h e l i e u R i v e r (sice 7) v h i e h does pot r e c e i v e an input f r om Lake O nt a ri o contained no measureable 2,3,7,8-TCDD. T a b l e 3. C o m p a r i s o n of 2 , 3 , 7 , 8 - T C D D lavels in pg/ g in turt le fata 1 and 2 u s i n g two CC-HS methods and a bioassay (2,3,7, IS-TCDD equival e n t a ). Turtle Fat Ho. 1 GC-KS HPH* 474 NTSb 586 B ioasaay HYS C 200-2000 2 232 203 100-1000 a - Health Protection Branch,! results from T a b le 2; b - Hev Y o r k S t a t e Dept. Health, fat from the same turt]le but us in g different sample preparation and GC-KS c o n d it i o n s; e - the [same sam p l e as in (b) but after extraction andi lipid removal vith aulfurie acid G E b lp 0 lO542 783353 544 Table 4. 2 , 3 , 7 , 8 - T C D D level* is A m e r i c a n eel s a m p l e s 1 c o l l e c t e d in 1982 and 1983 from tbe Great Lakes region. Origin Lake St. Pierre, St. L s v r e n e e Riv er , site 5^ X a a o u r a s k a ,' St. Lawrence River, site 6 Richelieu River, site 7 (tr ibut sry of St. Lawrence) Eastern part Lake Ontario, site 8 Humber Samp lea 2 2 2 5 Humber Positive 2 2 0 4 Concent ration ppt 16,29 Average positives in ppt 23 15,22 19 N D C ,HD - 19 1 5 , 1 0 , 3 . 7 , HD 12 a - average length and veigbt of river eel sample* vas 960 mm and 2.1 kg; b - refer to Fig. 1; c not detected DISCUSSION This ii the first rep o r t to d o cument tbe pre'sen e e of 2 , 3 , 7 , 8 - T C D D in turtle t issues. The c o n c e n t r at i o n s f o u nd in1 t u r tl e s f r o m the St. L a w r e nc e R i ve r are some of the h i g he s t k n o wn for w i lId l i f e Jbiota. R a p p e et al. (8) r e po rt e d h i g h levels of PCDFs (up to 3,000 ppIt tota1l) in the fat of a turtle taken f r om the H u d s o n river. 2 , 3 , 7 , 8 - T C D F and 2 ,3 ,-4,7 ,8 - P n CD F were m e a s u r e d at 43 and 620 ppt, r e s p e c t i v e l y , but the p r e s e n c e jof PCD D s vas not a s s a y e d than the H u d s o n r i v e r turtle. It is of i nt e r e st tjo note that turtle 3, in a ddit io n to c o n t a i n i n g a hig h PCDD load s i m i l a r to jthat of t u r t l es 1 and 2, also had PCDF levels (especially 2 , 3 , 4 , 7 , 8 - P n C D F ) even higher than those of tbe Hudson River turtle. Recently, Ko rfmac her et al. (18) reported qualitacive results of over 300 ppt 2,3,7,8-TCDD in the fat and eggs of a snake, wh ic h vas taken f r o m a c o nt am i na te d lake in I r k a m a s . All these reports on tissue levels in reptiles for 2,3,7,8-TCDD are samevJst lover than the 2.7 and 16 p arts per b i l l i o n (ng/g) f ound in the liver and adlipose,, r e s p e c t i v e l y , of a snake from the Seveso area of Italy (19) after the accidental release of 2,3,7,8-TCDD from a triehlerophenol explosion. Comparison of these results with that reported in other wildlife shows that fish c o n t ai n lover amounts of 2 , 3 , 7 , 8 - T C D D w i t h one of the mor e c o nt a m i n ated speeies, eels, from Lake Ontario showing levels of about 20 ppt. Eggs of the h e r r i n g gull (Larus_ ar gent atu s ) . a fish e a t i n g bird, c o n t ai n h i g h e r levels of 2 , 3 , 7 , 8 - T C D D ( p r es e n tl y about 60 to 100 ppt in Lalte O nt a r i o) than fish do, but n ei the r s pe cies a p pears to a c c u m u l at e and s t o re 2 , 3 , 7 ,8-TCDD as m uc h as I1 the s n ap pi n g t u r t l e does. As far as ve are aware, P^CDF levels of tbe m a g n i tude found in the t issues of turtle 3 have not bee n seen in w i l d l i f e samples. 783354 GENP 010543 I ft* 545 Indeed this ani mal species appears to be uni q ue in its a b i l i t y to tole ra te these contaminants. Because of their non-mobile habits, turtles would appear -to be an e x ce l le n t biological screen for P C D Ds and PCDFs. This a bi lity of t u rtles to a c c u m u l a t e toxic c h e mi c a l s has b e e n n o t ed by S t o n e ec al. (6) in working with the related contaminant PC3. | They found very high fat levels (over 1000 pg/g) of PCBs in c er tain c a s e s | f r o m a p pa re n tl y h e a l t h y s na pp i ng turt l e s p e ci m en s. T a v i t h at al. (20) also nIo t ed Chat a n o t he r a q u a t i c turtle, the C a s p ia n t e r r a p i n (Cl emmy i e ^ e ^ ^ a ) , c o n t a in ed PCB levels over 20 times h i g h e r than that found in two bir d species tak en from Che same area. The detection of dioxin-like activity determined by the bioassay and the samiquantitative correspondence with the chemieal procedure suggests that the b i o a s s a y could be used as an initial ind ic at o r foj the p r e s e n c e of PCDDs and PC D F s upon w h i c h a p ri o r i ty r a n ki n g for s u b se q u e nt c hemical a n a l ys i s could b,* bas ed . This b i o a s a a y is based on the 2 , 3 , 7 , 8 - T C D D induced r e v e r s i b l e inhibi- tion of cell growth and a ltera tions in m o r p h o l o g i e s 1 c h a r a c t e r i s t i c s of a line of mouse .epithelial cells co-cultured with irradiated mouse fibroblast cells (17). This a s sa y is p o t e n t i a l l y useful as a raIpid and i n e x p e n si v e semi- q u a n t i t a t i v e b r o a d sereen for a g g re g a t e jbiologi'eal a c t i v i t y even in the p r e s e n c e of a large excess of PCBs ad. eia be us ed as a s u p p l e m e n t to mor e specific chemical analysis. j The live r/adi pos e ratios for 2,3,7,8- TCDD in these three turtles varies b e t w e e n 0. 14 and 0.22 on a wet weight basics. This rstio is s om e wh at higher than that found by Stone at al. (6) for PC1B (0.04( to 0.07} and DDE (0.03 to 0.09) in the same species from the same acate andj by P e a r s o n et al. (21) for d i e l d r i n (0.03 to 0.09) in the b o x turtle (T e r r a p e n e C a r o l i n a ) 70 days after a sing l e i n je ct e d dose. The h i g h e r r e l a t i ve amount of 2 , 3 , 7 , 8 - T C D D in the t u r tl e liver than in the fat w h e n c ompared |to the Jother c h l o r i n a t e d c o mp o un d s is in a g re em e n t w i t h e x p er i m e n t al data in rata (22) and m a y r e f le ct a recent e x p os u r e to the f o r m e r c o n ta m i n an t or an e x pIo s u r e 'cIlose to one c a us in g ad ve r se effects* C h e l y d r a se rpent ins are o m ni vo r ou s and fee d on s i g n i f i c a n t am ounts of both ani ma l and p l an t food. The TCDD levels in tI,he t u rtles are c o n s i s t e n t w it h the consumpt ion of a fish diet containing about 20 ppt 2 , 3 , 7 , 8 - T C D D , a level currently found in eel samples from the same region and suggest that turtles b i o s e e u m u l a t e 2 , 3 , 7 , 8 - T C D D to tba asm extent as jdo l a b o r a t o r y rats (23) and beef ca t tl e (24). In the form er ease E o c i b a at al. (23) fed l a bo r at or y rats c o n t i n u o u s l y for 2 y e a rs a diet c o n t a i n in g 22 ppt 2 , 3 , 7 , 8 - T C D D (about 1 ng/kg b o dy w e i g h t per day) wh i c h r es u l t ed in a t i s s u e r e s i du e level of 540 ppt in . I I both fat and liver. In the oth er ease, J e n s e n et al. (24) fed be ef cattle for 28 days a diet containing 24 ppt 2,3,7,8-TCDD (about 0.8 ng/kg body weight per day) and this r e s u l t e d in a fat level of about 80 ppt w h i ch was i n cr eas in g and e s t i m a t e d to pea k at about 600 ppt. It U s also n o t e w o r t h y that the older t u rtles in this report (turtles 1 and 3 )| h a d the h i g h e r fat 2,3,7 ,8-TCDD residues suggesting that the levels from specimens from the same location may GENP 010544 II \ 783355 6 546 i n cr ea s e v i ch age aod / o r size - a r e l a t i o n s h i p w h i c h is also s u g g e s t i v e for PCS (6). |J It appears that ouch of the aquatic biota from'jLake Ontario and the St. L a w r e n c e Bi v e r c o nt a in s a p p re c i a bl e a m o un t s of 2,3 j[7,8 - T C D D (1-3,23). More limited data from two tributaries of the St. Lawr ence Biver, the Ottawa and Biehelieu Bivers, appear to show that they do not 1 contribute TCDD to this watershed. It is p o s s i b l e that 2 , 3 , 7 , 8 - T C D Di is e\n\ t e r i n g the St. L a w r e n c e B i ve r f r om L a k e O n t a r i o w h i ch in turn could h a v e o r i g i n a t e d in the N i ag a ra Bi v e r (site 9) c u r r e n t l y the only c o n f i r m e d sour ee of| 2 , 3 , 7 , 8 - T C D D in the area (26). Sources of the other PCDDs (excluding 2 , 3 , 7 , 8--TCDD) and Che PCDFs have not been identified. Turtle 3 contained much h i gher,l evels of PCDFs than did turtles 1 and 2 (in a dditi on to the PCDD r e s i du e levels). This result 'I suggests a second component as a souree for the levels in turtle 3 possibly from PCB. In s u pport of this, the PCBs in the fat q|f tur t l e 3 w e re over 800 Ug/g, m o re than 20x the levels .found in tfhe fatj of o t h er two animals. Certainly the low levels of the higher chlorinated PCDDs and PCDFs in turtle and fish sa mples w o u ld not indicate p e n t a c h l o r a p h e n o 11 as the source. The turtles from which these tissue levels of1 2,3,7,8-TCDD and ocher [ P C D D s / P C D F s were found sppeared to be no rm a l in both |a pp e a r a n c e and b e ha vi or, Wh ether they have been subjected to any long term adverse effects such as r e p r o d u c t i v e a b n o r m a l i t i e s or c h r onic d i s e a s e is p r e s e n t l y not known. C u rr en t r e g u l a t i o n s for 2 , 3 , 7 , 8 - T C D D in fish are b a s e dI on a r|[ e s i d u e level b e t w e e n 10 and 20 ppt. Thus the habitual consumption of these turtle tissues by humans may constitute a health risk. CONCLUSION Analysi s of turtle fat samples by both ch'emieal' and bioassay techniques has shown the presence of up to 500 ppt levels of 2,3, 7,8-TCDD in samples from the St. L a w r e n c e r i v e r along with les a s r a m o un t ! of| ot h e r PC D D s and PCDFs. One s a m pl e also showed a very high PCDF r e s i d ue level !s u g g e s t i v e of PC8 s as an additional source. The 2,3,7,8-TCDD concentratio ns in turtles are higher than those foun d in eel s am ples from the same area [and s ug gest tu rt l e t i ss ues ire an axe el lent m o n i t o r for thia type of c o n t a m i n a t i o n . The s o u r c e of the 2 , 3 , 7 , 8 - T C D D levels could be the N i a g a r a r i v e r vti a Lak''Ia O n t ar i o . ACKNOWLEDGEMENTS The a n t bo r a are gra teful to B ay m o n d e L i z o c t e for sample p u r i f i c a t i o n and to Wing F. Sun for help with the mats spectral measurements. Adrien Gervais and C l a u d D e s j a r d i n s of F i s be r i e a and Oceans C a n a d a'I are t h a n k e d for their collection of f i^h samples. These studies were supported in part by NXEHS grant ES03561. BEFEBENCES 1. J.J. Byan, P.-7. Lsu, J.C. Pilon, D. Levi s, B.A. M c L e o d and A. G er va i s, Env ir onmental S c i . T e c h n o l . 18 (1984) 719-^721. 2. P.0, Keef e , C. Meyer, D. Bilker, X. A l d o u a i B. J e l u s - T y r o r , X. D illon. R. I '1 D o n n e l l y , X. B o r n and K. Sloan, C h a m o s p h e r e 12 (1963) 3 2 5 -3 3 2 . 783356 GENP 010545 .o V 547 3 . R.L. Harless. E.O. Oswald, R,G. Levis, A.E. Dupuy , D.D. McDaniel and H. Tai, C h e a o s p h e r e 11 (1982) 193-98. 4. J.J. Ryan, P.-Y. Lau, J.C. P i l on and Dj. L e v i a i n * C h l o r i n a t e d D io xins and D i b e n z o f u r a n a in the Total E n v i r o n m e n t ; C . C b o u d h a r y , L.R. Ke it h and C. Rap pe, ads., B u tt er v or th , S t o n e ha a, Hass., Chp. 6, pp 87-99 (1983). 5. D.L. S t al li n g , L.M. Smith, J.D. P e t t y ,j J.V. H o g a n , J.L. J o h ns on , C. R appe and H.R. Buacr, in, Hunan and E n v i r o n a e n t a 1 iRiaks of C h l o r i n ated Dioxins and Related Compound. S.E. Tcher, | A .L . Toung and A.?. Gray, eds., Plenun (1983) pp. 221-240. i 6. V.B. Stone, E, Kiviat and 5 . A. I Butkas, Hew York State Fish Came J. 27 (1980) 39-50. 7 . F.G. Olafsson, A.M. Bryan, Bush a'nd V. Sto n e , Ch eaosph ere 12 (1983 ) 1525-1532. 8 . C. R a p p e , H.R Buser, D.L. Stalling, L.M. Smith and R.C. Dougherty, Mature 292 (1981 ) 524-526.' j 9. J.F. C i e r t h y and D. Crane, F u n d a m e n t a l App 1 T o x i c o l . 5 (1985) 754-759. .I 1 0 J.J. Ryan, D.T. W i l liams, B . P.-Y. Lau and T. Salcuoa, is Ch 1or in a ted D i o x i n s and D i b e n z o f u r a n a in the T o t al E nv, i ronaent II, L.H. Keith, C, Rappe and C. Cboudhary, eda., B u t t e r v o r t h , Stoaeham, Maas., Chp 17, pp. .11 12. 13 . 205-214 (1985). j J.J. Ryan, R. L i z o t t e and W.H. H e v s o me , J. C h r o m a t o g r . 303 (1984) 351- 360 . J.J. R y an and J.C. Pilon, J. Chromat^o^r. 197' (1980) 1 7 1 -180 . United States Environmental Protection . A g e n c y , Region VIZ Laboratory B r anch, D e t e r m i n a t i o n of 2 , 3 , 7 , 8 - T C D D in Soil l and S e d i m e n t , Kans as City, K a n a a i , Sep t . 1983. 14. B.P.-T. Lau, W.-F. Sun and J.J. Ryan, 32nd A n n . Conference Mass Spec- trometry, San Antonio, Texas, Hay (1984). 13. J.J. Ryan, R. Lizotte and B.P.-T. Lau[ Chem otphere 14 (1985) 697-706. 16. P.V. O 'Kee f e , R.M. Smith, D.R. H i l k n r , K . M . A l d o u a and V. G i l d a y in, Chl orinated Dioxina and Dibenzofurani in t^e Total Environment I I . L.B. Keith, C. Rappe and G. Cboudhary, eda. Chp. 10, pp. 111-124 (1985). j Buttervorth, Stoneham, Hass., 17. J.F. C i e r t h y and D. Crane, T o x i c o l . App 1 . P h a r m a c o l . 74 (1984) 91-98 . 18. V.A. Korfmacher, K.R. Rowland, R.K. Kit chum,| J.J. Daly, R.C. McDaniel and M.V. P l um me r , C h e m o a p h e r e 13 (1984) 1 2 2 9 - 1 2 3 3 . 19. R. Fannrtli, M.G. Castelli, G.P. M a r t e l l i , A. N o s e d a and S. C ar at t i n i, B u l l . Env i r o n . C o n t a n i n . T o x i c o l . 24 ^(1960) 4 6 0 -4 62 . 20 . A. T a v et z , I. Sidis and A. C a s i th , 1C o m p . B i o c h e m . P h y s i o l . 75C (1983) 377-382. | 2 1 . J.E. P e ar so n , K. T i n s l e y and T. H e r m a n d e z , Su_l^. Env iron * Cont s a i n . T o x i c o l . 10 (1973) 360-364. I 22. L.S. Bir nba um, Env i r o n . Health Per s p e c t . 61 (1985) 11-20. 23. R.J. K o e ib a , D.C. Kayes, J.E. Bey e r , R.M. C a r r e o n , C.E. Wade, D.A. GEKP 010546 783357 * r 548 Dittenber, R.P, Kalnina, L.E. Frauaon, C .H . Park, S.D. Barnard, R.A. Hunncl and C.C. Huaiaton, Toxicol . Ap p 1 . Pharmaco 1 . 46 (1 978 ) 27 9-303 . 24. D.J. J e nson, t.A. H u nncl, H.H. Mahlc, C.V. K o e h e r and H.S. B i g g i n a , J. A g r i c . Food C h e n . 29 (1981 ) 265-268 . 25. D.L. S t a l l i n g , R . J . H o r a t r o n , L.M. S a i c b and M. S i m o n , C h e a o a p h e r e 14 (6/7) 627-643 (1985). 26. l .M . S a i t h , P . V . O ' K e e f e , K . M . A l d o n a , D . R . B i l k e r and J . E . O 'B r i e n , Env i r o n . S c i . T e c h n o 1 . 17 ( 1 983 ) 6-10 . (Received in Germany 30 January 1986; accepted 17 February 1986) 783358 GENP 010547