MARINE BIOLOGY

Marine Biology 46, 35-40 (1978)

9 by Springer-Verlag 1978

Persistence of Polychlorinated Biphenyls in Marine Bivalves W.J. Langston Zoology Department, West'field College, London University; London, England

Abstract

B i v a l v e s (Cerastoderma edule and Macoma balthica) w h i c h had p r e v i o u s l y b e e n e x p o s e d to A r o c l o r s | 1242, 1254 and 1260 w e r e able to r e d u c e their t i s s u e b u r d e n s of c h l o r o b i p h e n y l s w i t h 2 to 5 c h l o r i n e atoms in s h o r t - t e r m static assay systems. E l i m i n a tion rates d e c r e a s e d w i t h i n c r e a s i n g c h l o r i n a t i o n and r e m o v a l of isomers w i t h m o r e than 5 c h l o r i n e atoms was not recorded. P o s i t i o n , in a d d i t i o n to t~e number, of c h l o r i n e atoms i n f l u e n c e d the p e r s i s t e n c e of c h l o r o b i p h e n y l s . Isomers w i t h m o s t " o r t h o " - s u b s t i t u t e d c h l o r i n e atoms w e r e least p e r s i s t e n t . E x p e r i m e n t s w i t h s i n g l e isomers i n d i c a t e d v a r i a t i o n in the e l i m i n a t i o n of low c h l o r i n a t e d isomers b e t w e e n b i v a l v e species. T h e s e b i o l o g i c a l and c h e m i c a l i n f l u e n c e s on t i s s u e r e s i d u e s , t o g e t h e r w i t h e n v i r o n m e n t a l p a r a m e t e r s such as t e m p e r a t u r e and s u s p e n d e d solids, are c o n s i d e r e d in r e l a t i o n to the use of b i v a l v e s as b i o - i n d i c a t o r s of m a r i n e pollution.

IntroduNon

B i v a l v e s , such as the m u s s e l Mytilus edulis (Linnaeus), have b e e n i n c l u d e d in surveys of c h l o r i n a t e d h y d r o c a r b o n c o n t a m i n a t i o n of the m a r i n e e n v i r o n m e n t (Holden, 1973; M a r c h a n d et al., 1976). Their s u i t a b i l i t y as i n d i c a t o r species i n c l u d e s f e a t u r e s such as a r e l a t i v e l y s e d e n t a r y habit, w i d e s p r e a d a v a i l a b i l i t y in large numbers, and an a b i l i t y to conc e n t r a t e p o l l u t a n t s to a d e t e c t a b l e level from the s u r r o u n d i n g sea water. Biom a g n i f i c a t i o n of r e s i d u e s has not b e e n e v a l u a t e d fully in m a r i n e o r g a n i s m s , and b i v a l v e m o l l u s c s , b e c a u s e they b e l o n g to a low t r o p h i c level in m a r i n e food chains, may r e f l e c t c o n t a m i n a t i o n of the e n v i r o n m e n t by PCB. Previously, s e l e c t i v e a c c u m u l a t i o n of p e n t a c h l o r o b i p h e n y l s has b e e n demons t r a t e d in Cerastoderma edule (Linnaeus) and Macoma balthica (Linnaeus) e x p o s e d exp e r i m e n t a l l y to c o m m e r c i a l p o l y c h l o r i n a t e d b i p h e n y l m i x t u r e s (Langston, 1978). Some lower c h l o r i n a t e d isomers w e r e a c c u m u l a t e d v e r y slowly, p a r t i c u l a r ly in c. edule, s u g g e s t i n g a r a p i d turno v e r - r a t e of these isomers. The p r e s e n t study a s s e s s e s the p e r s i s t e n c e of PCB in these b i v a l v e s , t o g e t h e r w i t h the influence of some e n v i r o n m e n t a l p a r a m e t e r s on

tissue r e s i d u e s , in an a t t e m p t to increase their v a l u e as i n d i c a t o r s of environmental contamination.

Materials and Methods Persistence of PCB Mixtures

Cockles, Cerastoderma edule (Linnaeus), w e r e e x p o s e d for 10 days to 0.25 p p m A r o c l o r s 1242, 1254 and 1260, a d s o r b e d on a l u m i n a p a r t i c l e s and s u b s e q u e n t l y t r a n s f e r r e d to a q u a r i a c o n t a i n i n g 10 1 clean sea w a t e r for p e r i o d s of up to 21 days. C o c k l e s w e r e also u s e d to i n v e s t i g a t e the e f f e c t of i n c r e a s i n g the a m o u n t of s u s p e n d e d m a t e r i a l on the a c c u m u l a t i o n of A r o c l o r 1254 over 30 days. The c o n c e n t r a t i o n of s u s p e n d e d p a r t i c l e s was inc r e a s e d by r e m o v i n g the sand in w h i c h b i v a l v e s w e r e u s u a l l y held. The d e n s i t y of s u s p e n d e d a l u m i n a p a r t i c l e s , m e a s u r e d u s i n g a h a e m o c y t o m e t e r , was I x 106 parts m13 in a q u a r i a w i t h sand and 3.6 x 106 parts m13 in a q u a r i a w i t h o u t sand, a l t h o u g h i d e n t i c a l a m o u n t s of c o n t a m i n a t e d m a t e r i a l w e r e i n t r o d u c e d into b o t h sets of aquaria.

0025-3162/78/0046/0035/S 01.20

36

W.J. Langston: Persistence of PCBs in Marine Bivalves

Persistence of Individual Chlorobiphenyls Cerastoderma edule a n d Macoma balthica w e r e e x p o s e d t o 0 . 2 5 p p m 4-4' d i c h l o r o b i p h e nyl and 0.025 ppm 3,4,5-3',4',5' hexachlorobiphenyl o n a l u m i n a f o r 10 d a y s f o l l o w e d b y a 21 d a y p e r i o d in c l e a n s e a w a t e r at 8oc. To i n v e s t i g a t e the effect of t e m p e r a t u r e on the turnover of PCB, cockles were also treated with dichlorob i p h e n y l a t 15oc.

Chemical Analysis

P C B r e s i d u e s in b o t h s p e c i e s of b i v a l v e s w e r e e x t r a c t e d in r e d i s t i l l e d hexane using a Soxhlet apparatus and cleanup of samples achieved with alumina columns. For the calculation of PCB concentrations, extracts were analysed using a P y e s e r i e s 104 g a s c h r o m a t o g r a p h with e l e c t r o n c a p t u r e d e t e c t o r s a n d 83 x 0.3 cm (internal diameter) glass columns p a c k e d w i t h A p i e z o n L. C o l u m n a n d d e t e c t o r temperatures w e r e 190 ~ a n d 2 2 0 o c , r e spectively and the flow rate of oxygenfree nitrogen maintained a t 60 m l m i n - 1 . Quantification of PCB was determined by peak height comparison with standard

solutions. Isomers represented by indiv i d u a l p e a k s w e r e e s t i m a t e d in a d d i t i o n to t o t a l P C B c o n c e n t r a t i o n s . Reported P C B v a l u e s a r e i n p p m w e t w e i g h t of t i s sue and are mean concentrations for 5 individuals. Detailed qualitative c o m p a r i s o n s of PCB residues were performed by high resolution chromatography using support c o a t e d , o p e n t u b u l a r (SCOT) c o l u m n s . T h e s e c o l u m n s , 1 5 . 2 m x 0.51 m m ( i n t e r n a l d i a m e t e r ) w e r e c o a t e d w i t h A p i e z o n L. The gas flow rate through column and e l e c t r o n c a p t u r e d e t e c t o r w a s 2.5 a n d 60 m l m i n - 1 , r e s p e c t i v e l y . The column oven was operated at 200oc, the inject i o n p o r t at 2 1 0 o c a n d t h e d e t e c t o r a t 220oc. Under these conditions and with t h e i n j e c t i o n of s o m e i n d i v i d u a l P C B i s o m e r s , t h e i d e n t i t y of m o s t p e a k s w a s determined using the data of Sissons and W e l t i (1971). Results

Persistence of PCB Mixtures

T i s s u e r e s i d u e s in Cerastoderma edule exp o s e d to P C B m i x t u r e s f o r 10 d a y s f o l l o w e d b y a d e p u r a t i o n p e r i o d in c l e a n

Table i. Cerastoderma edule. Persistence of PCB mixtures during 21 day depuration period. Bivalves were previously exposed to 0.25 ppm Aroclor on particles of alumina Post-exposure Tissue concentrations (ppm) ~ standard deviations period (days) Aroclor 1242 Aroclor 1254 Aroclor 1260 O

6.83 • 1.58

20.54 ~

4.97

5.03 • 0.53

7

4.85 • 2.18

20.07 •

7.36

5.53 • 1.18

14

3.03 • 1.68"

20.92 •

6.55

5.66 • 0.94

21

2.49 • 0.89**

21.97 • 11.O9

8.77 • 1.85

*P <0.O1, **P
Tissue concentrations of chlorobiphenyls C12 C13 C14 (Peak i) (Peak 4) (Peak 6)

(ppm) • SD C15 (Peak iO)

C16 (Peak 19)

O

0.08 + O.O1

2.30 + 0.47

O.61 + O.13

0.90 + 0.25

O.15 + 0.05

7

0.04 + O.O1

1.28 + 0.68

0.43 + O.21

0.76 + 0.32

O.12 + 0.04

14

0.09 + 0.09

0.43 + 0.40***

O.14 + 0.08***

0.53 + 0.23*

O.14 + 0.05

21

0.05 + 0.06

0.33 + 0.23**

O.15 + O.iO***

0.48 + O.21"*

O.13 + 0.06

*P <0.05, **P <0.02, *** P
w.J. Langston: Persistence of PCBs in Marine Bivalves

sea w a t e r of up to 21 days are p r e s e n t e d in T a b l e I. T h e r e is a m a r k e d d i f f e r e n c e in the r a t e at w h i c h the 3 c o m m e r c i a l m i x t u r e s w e r e e l i m i n a t e d from c o c k l e s during depuration. Bivalves previously e x p o s e d to A r o c l o r 1242 lost 63% (P <0.OO1) of their PCB b u r d e n after 21 days, i n d i c a t i n g a r a p i d t u r n o v e r of this m i x t u r e , the h a l f - l i f e in c o c k l e t i s s u e s b e i n g b e t w e e n 7 and 14 days. Biv a l v e s p r e v i o u s l y e x p o s e d to the m o r e highly chlorinated mixtures, Aroclors 1254 and 1260, did not s i g n i f i c a n t l y reduce their PCB t i s s u e c o n c e n t r a t i o n s during d e p u t a t i o n (Table I).

37

p u r a t i o n or the o r i g i n a l dose m a t e r i a l . R e s i d u e s of A r o c l o r s 1254 and 1260 indicate similar, a l t h o u g h less p r o n o u n c e d , trends o w i n g to the a b s e n c e of lower c h l o r i n a t e d h o m o l o g u e s in these m i x t u r e s . C o n f i g u r a t i o n of PCB isomers, as w e l l as c h l o r i n a t i o n , i n f l u e n c e d their pers i s t e n c e in b i v a l v e tissues. It was app a r e n t that the e a r l i e s t e l u t i n g isomers in an i s o m e r i c series, those w i t h the l a r g e s t n u m b e r of " o r t h o " - s u b s t i t u t e d c h l o r i n e atoms (Sissons and Welti, 1971), were lost s e l e c t i v e l y (Fig. I).

Persistence of Individual Persistence of PCB Homologues

C o m p a r i s o n of SCOT c o l u m n c h r o m a t o g r a m s of c o c k l e s s a m p l e d after 21 days d e p u r a tion w i t h the s t a n d a r d A r o c l o r 1242 ind i c a t e s s e l e c t i v e loss of e a r l y eluting, lower c h l o r i n a t e d h o m o l o g u e s (Fig. I). Q u a n t i f i c a t i o n of c h l o r o b i p h e n y l s repr e s e n t e d by i n d i v i d u a l s h o r t - c o l u m n c h r o m a t o g r a p h i c peaks c o n f i r m s that h o m o l o g u e s c o n t a i n i n g 2 to 5 c h l o r i n e atoms per m o l e c u l e w e r e e l i m i n a t e d by c o c k l e s (Table 2). G e n e r a l l y , p e r s i s tence of c h l o r o b i p h e n y l s i n c r e a s e d w i t h c h l o r i n a t i o n . D i c h l o r o b i p h e n y l was present in small q u a n t i t i e s t h r o u g h o u t , ind i c a t i n g a r a p i d turnover, w h i l s t tric h l o r o b i p h e n y l d e c r e a s e d by 50% d u r i n g the first 7 days d e p u r a t i o n . T i s s u e conc e n t r a t i o n s of t e t r a c h l o r o b i p h e n y l w e r e s i m i l a r l y r e d u c e d after 21 days. E a r l y e l u t i n g p e n t a c h l o r o b i p h e n y l s (Peak 10) w e r e s i g n i f i c a n t l y r e d u c e d (P <0.02), but later e l u t i n g h o m o l o g u e s , the m a j o r c o m p o n e n t s of A r o c l o r s 1254 and 1260, w e r e not e l i m i n a t e d by cockles. The r e l a t i v e c h a n g e s in peak h e i g h t s of c h r o m a t o g r a m s are shown in Fig. 2. A r o c l o r 1242 r e s i d u e s in b i v a l v e s a f t e r d e p u r a t i o n c o n t a i n e d m u c h larger p r o p o r tions of p e n t a - and h e x a c h l o r o b i p h e n y l and less di-, tri- and t e t r a c h l o r o b i p h e n y l than those at the s t a r t of de-

Chlorobiphenyls

C h l o r o b i p h e n y l levels in Cerastoderma edule and Macoma balthica p r e v i o u s l y e x p o s e d to h e x a c h l o r o b i p h e n y l w e r e not s i g n i f i c a n t ly r e d u c e d d u r i n g d e p u t a t i o n . In contrast, e l i m i n a t i o n of d i c h l o r o b i p h e n y l f r o m c. edule was rapid, 90% of the tissue b u r d e n b e i n g lost after 21 days.

10 day dosage

A1242

A1254

A1260

400

~" 800 700 10 day dosage followed by 21 days in clean seawater

600 500. 400 300. 200

A

100

124679101iV189

568910!12117119J~1} 14 18 21

13 16 19 21 24 26

peak number B

240

180

120

retention

60

o

time (min)

Fig. i. Cerastoderma edule. SCOT column chromatograms. A: Aroclor 1242. B: residue in cockle 21 days after exposure to Aroclor 1242 was terminated

Fig. 2. Cerastoderma edule. Changes in ordinarycolumn gas chromatographic spectra of Aroclors 1242, 1254 and 1260 in bivalves during exposure and depuration. Percentage change in peak heights in tissue chromatograms relative to standard peak heights has been calculated from the mean of 5 individuals (each peak in the standard = iOO units)

38

W.J. Langston:

Persistence

of PCBs in Marine Bivalves

Table 3. Cerastoderma edule and Macoma balthica. Persistence of chlorobiphenyls in bivalves previously exposed to 0.025 ppm 3,4,5,-3',4',5' hexachlorobiphenyl and 0.25 ppm 4-4' dichlorobiphenyl. Values are tissue concentrations (ppm) • standard deviations, nd: no data Postexposure period (days)

Dichlorobiphenyl C. edule M. balthica

Hexachlorobiphenyl C. edule M. balthica

0

7.80 • 1.42

0.20 ~ O.ii

0.58 • 0.37

7

4.O2 s 3.48*

nd

O.19 • O.O8

nd

14

2.09 ~ 1.61"*

nd

O.19 ~ 0.06

nd

21

1.O7 • 0.67**

O.11 ~ 0.05

0.33 • O.21

20.13 •

3.69

14.52 ~ 10.59

*P
from those

15(]

D u r i n g the same period, 25% of the dic h l o r o b i p h e n y l r e s i d u e was lost f r o m M. balthica (Table 3).

high

~density

Temperature

Accumulation

of d i c h l o r o b i p h e n y l in Cerasf a s t e r (P <0.O01) at 15oc than at 8oc. E l i m i n a t i o n of this i s o m e r w a s also f a s t e r at the h i g h e r temtoderma edule w a s

E

20

0.

0

115~c E

low

U e-

density

0

U

2

lC ,m

P

r e,0 ~J

I.-

0 0 exposure 5 1.0 depura on ~-~ Days

14

21

F i g . 3. C e r a s t o d e r m a e d u l e . U p t a k e and e l i m i n a t i o n of 4-4' dichlorobiphenyl at 8 ~ and 15oc. Bars represent standard errors

Fig. 1254 (I x mina cles

I

I

15 30 Exposure period (days)

4. Cerastoderma edule. Uptake of Aroclor in a high (3.6 x 106 parts ml -I) and low 106 parts ml -I) density suspension of aluparticles. PCB concentration on the partiwas 500 ppm. Bars represent standard errors

w.J. Langston: Persistence of PCBs in Marine Bivalves

p e r a t u r e (Fig. 3). The h a l f - l i f e at 8oc was 7 days and r e s i d u e s w e r e still det e c t a b l e at 21 days, w h i l s t at 15oc the h a l f - l i f e was 5 days and no trace of dic h l o r o b i p h e n y l was d e t e c t a b l e after 14 days.

39

known, but seems m o s t likely to be b a s e d upon the i n c r e a s e d v o l a t i l i t y and sOlub i l i t y of low c h l o r i n a t e d i s o m e r s (Zitko et el., 1971), since b i v a l v e s do not possess r e c o g n i s e d d e t o x i f y i n g e n z y m e systems (Lee et al., 1972). V a r i a t i o n in the p e r s i s t e n c e of PCB's in b i v a l v e s may, t h e r e f o r e , be the r e s u l t of i n t e r s p e c i f Density of Suspended Solids ic d i f f e r e n c e s in the c h e m i c a l s t r u c t u r e and p e r c e n t a g e c o m p o s i t i o n of their lipA c c u m u l a t i o n of A r e c l o r 1254 in Cerastoid pools. derma edule was faster (P
40

W.J. Langston:

Literature Cited Crump-Weisner, H.J., H.R. Feltz and M.L. Yates: A study of the distribution of polychlorihated biphenyls in the aquatic environment. J. Res. U.S. Geol. Surv. 1, 603-607 (1973) Cunningham, P.A. and M.R. Tripp: Factors affecting the accumulation and removal of mercury from tissues of the American oyster Crassostrea virginica. Mar. Biol. 31, 311-329 (1975) Denton, G.R.W.: The uptake and elimination of polychlorinated biphenyls (PCBs) in the hardshell clam Mercenaria mercenaria, 194 pp. Ph.D. thesis, London 1974 Holden, A.V.: International cooperative study of organochlorine and mercury residues in wildlife 1969/1971. Pestic. Monit. J. 7, 37-52 (1973) Hughes, E.D.: Steric hindrance. Q. Rev. chem. Soc. 11, 107-131 (1948) Jensen, S. and G. Sundstr~m: Structures and levels of most chlorobiphenyls in two technical PCB products and in human adipose tissue. Ambio 3, 70-76 (1974) Koeman, J.H., M.C. Ten Noever de Brauw and R.H. de Vos: Chlorinated biphenyls in fish, mussels and birds from the River Rhine and Netherlands coastal area. Nature, Lond. 221, 1126-1128 (1969) Langston, W.J.: Accumulation of polychlorinated biphenyls in the cockle Cerastoderma edule and the tellin Macoma balthica. Mar. Biol. 45, 265-272 (1978) Lee, R.F., R. Sauerherber and A.A. Benson: Petroleum hydrocarbons: uptake and discharge by the marine mussel Mytilus edulis. Science, N.Y. 177, 344-346 (1972) Lowe, J.I., P.R. Parrish, J.M. Patrick, Jr. and J. Forester: Effects of the polychlorinated biphenyl Aroclor | 1254 on the American oyster Crassostrea virginica. Mar. Biol. 17, 209-214 (1972)

Date of final manuscript acceptance:

November

25,

Persistence

of PCBs in Marine Bivalves

Marchand, M., D. Vas and E.K. Duursma: Levels of PCBs and DDTs in mussels from the N.W. Mediterranean. Mar. Pollut. Bull. 7, 65-69 (1976) Prestt, I., D.J. Jeffries and N.W. Moore: Polychlorinated biphenyls in wild birds in Britain and their avian toxicity. Envir. Pollut. 1, 3-25 (1970) Risebrough, R.W., G.L. Florant and D.D. Berger: Organochlorine pollutants in peregrines and merlins migrating through Wisconsin. Can. Fld Nat. 84, 247-253 (1970) Schoor, W.P.: Problems associated with lowsolubility compounds in aquatic toxicity tests: theoretical model and solubility characteristics of Aroclor 1254 in water. Wat. Res. 9, 937-944 (1975) Sissons, D. and D. Welti: Structural identification of polychlorinated biphenyls in commercial mixtures by gas chromatography, nuclear magnetic resonance and mass spectroscopy. J. Chromat. 60, 15-32 (1971) Vreeland, V.: Uptake of chlorobiphenyls by oysters. Envir. Pollut. 6, 135-140 (1974) Widdows, J.: Effect of temperature and food on the heart beat, ventilation rate and oxygen uptake of Mytilus edulis. Mar. Biol. 20, 269-273 (1973) Zitko, V., O. Hutzinger and S. Safe: Retention times and electron capture detector responses of some individual chlorobiphenyls. Bull. Envir. Contam. Toxicol. 6, 160-163 (1971)

Dr. W.J. Langston Marine Biological Association The Laboratory Citadel Hill Plymouth PLI 2PB, Devon England

1977. Communicated by J. Mauchline,

Oban