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Arch. Environm. Contam. Toxicol.

7, 237-244 (1978)

Archivesof

Environmental Contamination and Toxicology

The Accumulation of Low Molecular Weight Aromatic Hydrocarbons of Crude Off by Coho Salmon (Oncorhynchus kisutch) and Starry Flounder (Platichthys stellatus)
William T. Roubal, Susan I. Stranahan, and Donald C. Malins
Environmental Conservation Division, Northwest and Alaska Fisheries Center, National Marine Fisheries Service, NOAA, 2727 Montlake Boulevard East, Seattle, Washington 98112

Abstract. Coho salmon (Oncorhynchus kisutch) and starry flounder (Platichthys stellatus) were exposed to 0.9 _+ 0.1 ppm of a water-soluble fraction
(WSF) of Prudhoe Bay crude oil in flowing sea water. Both species accumulated a complex spectrum of low molecular weight aromatic hydrocarbons. Bioconcentration factors (ppm hydrocarbon based on dry weight of tissue/ ppm hydrocarbon in flow-through water) for most hydrocarbons in starry flounder muscle were substantially higher than for coho salmon muscle. After two weeks of exposure, for example, there were 17 ppm (dry weight of tissue) of C,- and Cs-substituted benzenes in muscle of starry flounder (bioconcentration factor of 1,700) but only 1.5 ppm of these compounds occurred in muscle of coho salmon (bioconcentration factor of 150). Generally, alkylated aromatic hydrocarbons accumulated in tissues to a greater degree than unsubstituted derivatives. In both species, accumulations of substituted benzenes and naphthalenes in muscle increased in relation to the degree of alkylation. Complex mixtures of aromatic hydrocarbons were found in gills and liver of starry flounder. Accumulated hydrocarbons in coho salmon exposed for six weeks fell below limits of detection within a week when fish were transferred to clean water. Starry flounder exposed for two weeks retained substantial concentrations (7 to 26 ppm) of C2-C3-substituted naphthalene and C4-Cs-substituted benzenes in muscle two weeks after the termination of exposure. Substantial variations were found in bioconcentration factors for individual hydrocarbons in both species. Thus the data reflect difficulties in relating specific sources of petroleum pollution to hydrocarbon profiles in tissues. An understanding of the accumulation and metabolic fate of aromatic hydrocarbons in marine organisms is of interest in relation to the impact of increased petroleum transport and drilling operations on marine environments (Varanasi and Malins 1977). A number of studies on the metabolism of petroleum hydrocarbons were carried out with a variety of marine organisms (Malins 1977a, Varanasi and Malins 1977); however, only a few studies have employed fish as experimental animals. These studies were largely concerned with the metabolic
0090-4341/78/0007-0237 $1.60 9 1978 Springer-Verlag New York Inc.

238

w . T . Roubal et al.

fate o f i n d i v i d u a l isotopicaUy labeled h y d r o c a r b o n s . R o u b a l et al. (1977a) s t u d i e d the a c c u m u l a t i o n a n d b i o t r a n s f o r m a t i o n of 14C-labeled b e n z e n e , n a p h t h a l e n e , a n d a n t h r a c e n e b y coho s a l m o n (Oncorhynchus kisutch). Lee et al. (1972) i n v e s t i g a t e d the u p t a k e a n d discharge of isotopically labeled b e n z o ( a ) p y r e n e a n d n a p h t h a l e n e b y m u d s u c k e r (GiUichthys mirabilis), s c u l p i n (Oligocottus maculosus), a n d s a n d d a b (Citharichthys stigmaeus). M e y e r h o f f (1975) s t u d i e d the a c c u m u l a t i o n o f benzene-l-14C in striped bass (Morone saxatilis) in relation to b e h a v i o r a l alterations. A n d e r s o n et al. (1974) investigated the a c c u m u l a t i o n o f n a p h t h a l e n e a n d 1 - m e t h y l n a p h t h a l e n e in s h e e p s h e a d m i n n o w (Cyprinodon variegatus). W h i t t l e et al. (1974a, 1974b) d e s c r i b e d the a c c u m u l a t i o n of 14C-labeled b e n z o ( a ) p y r e n e a n d b e n z a n t h r a c e n e b y j u v e n i l e herring (Clupea harengus) a n d investigated the u p t a k e of b e n z o ( a ) p y r e n e b y j u v e n i l e Atlantic cod (Gadus morhua). I n each o f the a b o v e studies it was s h o w n that fish readily a c c u m u l a t e a r o m a t i c h y d r o c a r b o n s w h e n e x p o s e d to individual c o m p o u n d s ; h o w e v e r , substantial d e c r e a s e s in tissue c o n c e n t r a t i o n s o c c u r w h e n the a n i m a l s are p l a c e d in clean sea water. I n a n a t t e m p t to u n d e r s t a n d the p r o p e n s i t y of fish to a c q u i r e a n d store h y d r o c a r b o n s f r o m c o m p l e x m i x t u r e s of p e t r o l e u m in the m a r i n e e n v i r o n m e n t , w e e x p o s e d c o h o s a l m o n a n d starry f l o u n d e r to a w a t e r - s o l u b l e fraction ( W S F ) of P r u d h o e B a y c r u d e oil in flowing sea water.

Materials and Methods


Coho salmon smolts (14.4 -+ 4.0 g) and starry flounder (12 months old; 54 -+ 20.g) of mixed sex were acclimated for several weeks in six 20-gal all-glass flow-through aquaria described by Roubal et al. (1977b). Fish were fed a daily ration of hydrocarbon-free Oregon moist pellet (Moore-Clark Co., LaConner, Washington)a equal to approximately 1% of their body weight. The WSF was prepared from Prudhoe Bay crude oil in filtered Puget Sound sea water at 10~ The sea water flow-rate through each aquarium was 900 ml/min. Coho salmon and starry flounder were exposed to 0.9 - 0.1 ppm of the WSF for 6- and 2-week periods, respectively. After the exposure periods, both species were transferred to clean sea water for two weeks to study the deputation of tissue of accumulated hydrocarbons. Hydrocarbon concentrations in exposure water were determined by extracting 500- to 1,000-ml samples with several 25-ml portions of carbon disulfide. Extracts were concentrated to about 1.0 ml using a small scale evaporative concentrator (Kontes Glass Co., Vineland, New Jersey). Gas-liquid chromatographic analyses were performed on the extracts. The limit of detection for individual hydrocarbons was 1 ppb as shown by analysis of standard hydrocarbon solutions. Hydrocarbons in organs from single fish were below limits of analytical detection, thus it was necessary to combine individual samples from several fish to obtain sufficient material for analysis; limits of detection for individual hydrocarbons varied according to the weight of tissue taken, the final sample volume and instrument characteristics for each analysis. These factors have been taken into account. A lower limit of detection of 0.05 ppm was obtained for 10- to 15-g samples; however, a lower limit of 0.5 ppm was obtained for a 1-g sample. Duplicate analyses were performed on muscle from both species. Muscle composites (10- to 15-gsamples) were obtained from each of two groups of coho salmon (two fish per group) and from two groups of starry flounder (five fish per group). With starry flounder it was not feasible, however, to conduct duplicate determinations on gills and liver

a Mention of commercial products is for information only and does not constitute endorsement by the U.S. Department of Commerce.

Hydrocarbons in Salmon and Flounder

239

because of the limited number of these fish available in the weight range employed. Accordingly, analyses were conducted on a single composite sample of liver and gills from the fish used in the analyses of hydrocarbons in muscle. Composites of gills and liver from coho salmon weighed 1 g or less. Thus there was insufficient tissue for gas chromatographic analysis because the hydrocarbons were below limits of detection. Both species were analyzed at weekly intervals, starting one week from the beginning of exposure, and continuing two weeks after the termination of the exposure (depuration). Excised tissued were thoroughly rinsed in isotonic saline solution, blotted and weighed, and then digested at room temperature in 10-ml portions of 4N NaOH in 40-ml glass centrifuge tubes with tight fitting, Teflon-lined screw caps. Extracts of digests were analyzed for hydrocarbons using capillary column gas-liquid chromatography with mass spectral confirmation (MacLeod et al. 1976); a Hewlett-Packard model 5840 gas chromatograph with flame ionization detector and either OV-101 or SE-30 glass capillary columns (30 meter glass capillaries WCOT, J and W Scientific Co., Orangevale, California) were employed. Muscle lipids of coho salmon and starry flounder were extracted by the method of Hanson and Olley (1963) and determined gravimetrically. Hydrocarbon losses occurring during workup of samples were determined by spiking controls (water and tissues) with reference compounds and quantitating recoveries. With the exception of the more volatile hydrocarbons (benzene, toluene, and xylenes) recoveries ranged from 81 to 86%. The recoveries for benzene, toluene, and xylenes were 61 to 63%. No hydrocarbons of the WSF were detected in control coho salmon. Accordingly, corrections for background levels in exposed salmon were not required. Some tissues of starry flounder controls contained small amounts of alkylated naphthalenes (generally below 20 ppb, dry weight of tissue). In reporting the hydrocarbon concentrations in organs of exposed starry flounder, appropriate corrections were made.

Results

Composition o f the WSF


T h e m a j o r i t y o f the w a t e r - s o l u b l e c o m p o u n d s in t h e W S F c o m p r i s e d l o w m o l e c ular w e i g h t volatile a r o m a t i c c o m p o u n d s , i.e., b e n z e n e , t o l u e n e , x y l e n e s , o t h e r alkylated benzenes and naphthalene, and alkylated naphthalenes. The composition o f t h e W S F is given in T a b l e 1. S e v e n a n a l y s e s o f W S F w e r e p e r f o r m e d during t h e c o u r s e o f the e x p e r i m e n t .

Analysis of Hydrocarbons in Coho Salmon


I n T a b l e 2 d a t a are given o n the a c c u m u l a t i o n o f h y d r o c a r b o n s in m u s c l e (12.5% lipid) o f c o h o s a l m o n . N o h y d r o c a r b o n s r e p r e s e n t a t i v e o f the W S F c o u l d be d e t e c t e d in m u s c l e after o n e w e e k o f e x p o s u r e . H o w e v e r , after t w o to six w e e k s significant a m o u n t s of h y d r o c a r b o n s w e r e f o u n d . A f t e r five w e e k s (the t i m e o f m a x i m u m h y d r o c a r b o n a c c u m u l a t i o n ) , b i o c o n c e n t r a t i o n f a c t o r s f o r C3-substit u t e d b e n z e n e s , n a p h t h a l e n e , the c o m b i n e d 1- a n d 2 - m e t h y l n a p h t h a l e n e s , and C2-substituted n a p h t h a l e n e s a n d C a - s u b s t i t u t e d n a p h t h a l e n e s w e r e 50, 80, 160, 85, a n d 140, r e s p e c t i v e l y . T h e c o m b i n e d C4-/Cs-substituted b e n z e n e fraction, p r e s e n t at = 0.01 p p m in f l o w - t h r o u g h w a t e r , c o m p r i s e d the m a j o r p o r t i o n o f the h y d r o c a r b o n s in the c o h o salmon. M u s c l e c o n t a i n e d 5.5 p p m o f this f r a c t i o n after five w e e k s , e q u i v a l e n t to a b i o c o n c e n t r a t i o n f a c t o r o f 550. A f t e r six w e e k s o f e x p o s u r e a significant decline in all h y d r o c a r b o n c o n c e n t r a t i o n s w e r e o b t a i n e d in c o h o s a l m o n muscle. W h e n e x p o s e d fish w e r e p l a c e d in c l e a n w a t e r , no e v i d e n c e w a s f o u n d f o r h y d r o c a r b o n s o f the W S F in m u s c l e tissue after a p e r i o d o f a w e e k .

240
Table 1. Hydrocarbon content of flow-throughexposure water

W. T. Roubal et al.

Hydrocarbons Cyclohexane Benzene Toluene Ethylbenzene m-Xylene o- and p-Xylenes Ca-Substituted benzenes C4-Substituted benzenes Ca-Substituted benzenes Naphthalene 1-Methylnaphthalene 2-Methylnaphthalene Ca-Substituted naphthalenes Ca-Substituted naphthalenes

Concentrationa (ppm) (Mean std. deviation)b 0.02 0.002 0.04 0.006 0.4 0.05 0.005 0.002 0.2 0.003 0.07 ---0.03 0.03 0.005 0.01 0.003 trace~ 0.003 _ 0.002 0.003 _ 0.001 0.003 _ 0.002 0.01 0.005

a Values corrected for losses during workup b Content of the hydrocarbons, cyclohexane through naphthalene was determined seven times during exposure. Values reported for the methylnaphthalenes are averages for two analyses. Single values are reported for Ca- and Ca-substituted naphthalenes. Hydrocarbon content of flow-through water (= 0.8 ppm at the beginning of solubilizer operation) was only slightlygreater ( - 1.0 ppm) after five weeks of continuous operation c __.0.001 ppm was the lower limit of hydrocarbon detection Biological samples were not analyzed for cyclohexane, benzene, or toluene of the W S F (Table 1) because the chromatographic procedure used for tissues did not effectively resolve these components.

Analysis of Hydrocarbons in Starry Flounder


Tables 2 and 3 provide data on levels of hydrocarbons in muscle (2% lipid), gills, and liver of exposed starry flounder. In contrast to coho salmon, where no petroleum hydrocarbons were detected after one week of exposure, exceptionally high levels of the hydrocarbons (e.g., 93 ppm of the combined C4-/Cssubstituted benzenes) were found in starry flounder muscle in this time period (Table 2). Hydrocarbon bioconcentrations in muscle were 500, 9,300, 700, 2,400, 2,400, and 3,400 for Ca-substituted benzenes, the combined C4-/Cs-substituted benzene fraction, naphthalene, the combined 1-methyl and 2-methylnaphthalenes, C2-substituted naphthalenes, and for Ca-substituted naphthalenes, respectively. Considerable bioconcentrations of hydrocarbons occurred in liver and gills during the exposures (Table 3). The combined C4-/Cs-substituted benzene fraction in starry flounder was 1 to 2 orders of magnitude greater than in coho salmon. After two weeks these hydrocarbons reached the highest concentration in liver (110 ppm), equivalent to a bioconcentration factor of I 1,000. Concentra-

H y d r o c a r b o n s in S a l m o n a n d F l o u n d e r

241

tions of hydrocarbons up to Ca-substituted naphthalenes increased in each tissue in proportion to the degree of alkyl substitution. Similar conclusions can be drawn from the data on exposed coho salmon. A significant decline in concentrations of all individual hydrocarbons occurred in the muscle of exposed starry flounder after two weeks. Over the same period the liver of starry flounder showed an increase in hydrocarbon accumulations; however, no comparable changes were observed in the gills. After maintaining exposed starry flounder for two weeks in clean sea water, levels for several of the hydrocarbons were still found to be elevated in muscle tissue: 26 ppm for the C4-/Cs-substituted benzene fraction, and 7.0 and 8.0 ppm for Ca- and Ca-substituted naphthalenes, respectively. However, levels for petroleum hydrocarbons in the gills and liver of starry flounder were either below limits of detection or at relatively low levels two weeks after termination of the exposure.
(0. kisutch) (P. stellatus)

T a b l e 2. H y d r o c a r b o n s

in muscle tissue of coho salmon

and starry flounder

e x p o s e d to the w a t e r - s o l u b l e fraction o f P r u d h o e B a y c r u d e oil u s i n g f l o w - t h r o u g h e x p o s u r e a Coho salmon Weeks of exposure

"~

- +

+
= ~'-

+
E g'-

~,-Hydrocarbons C.,-Substituted benzenes C:.-Substituted benzenes C+-C~-Substituted benzenes Naphthalene 2-Methylnaphthalene I-Methylnaphthalene Cz-Substituted naphthalenes C:,-Substituted naphthalenes I.I l0 150 20 30 30 30 50

~
0 27 SS 0.40 0.18 2.0 1.5 0.12-+ 0.06 0.20 0.10 0.16 0.08 0.44 0.30 0.40 0.40 NF' NF NF NF NF NF NF NF

0.31 SS~' 0.30 0.12 1.5 0.07 003 0 I O - 0.05 0.10 0.03 0.31 0.30 0.23 +- 0.09

2.4 30 170 50 100 70 40 30

0.66 SS 0.90 0.12 1.7 0.14 0.07 0.31 0.01 0.22 0.00 0.36 - 0.00 0.15 0.02

2 50 550 80 190 130 85 140

0.55 SS 1.5 0.09 5.5 1.0 0.24 0.06 0.56 0.14 0.40 0.08 0.90 0.24 0.70 0.22

I l0 200 40 70 50 40 80

Starry flounder Weeks of deputation (2 weeks exposure)


1 2 I 2

C2-Substituted benzenes C:~-Substituted benzenes C+-C~-Substituted benzenes Naphthalene 2-Methytnaphthalene l-Methylnaphtha[ene C_~__-Substituted naphthalenes C:rSubstituted naphthalenes

20 500 9300 700 2800 2000 2400 3400

5.5 15 93 2.1 8.3 6. l 24 17 SS

2.0 5.7 34 1.5 3.6 4.3 9.7

4 70 1700 240 470 330 540 1000

1.0 -.- 0.30 2.2 1.2 17 - 6.2 0.72 0.30 1.4 - 0.60 1.1 O.SO 5.4 2.3 5.0 2.0

I 6 980 lOO 110 113 270 420

0.27 -. 0 18 9.8 0.30 0,33 0.34 ~ 2.7 2.1

0.06 0.03 0.40 0.02 0.03 0.O! 0.80 0.00

l0 2600 270 20t) 270 700 1600

NF 0.30 0.02 26 1.6 0.80 0.04 0.60 0.02 0.82 m 0.04 7.0 1.6 8.0 O.10

" 0.9 - 0.1 ppm (total hydrocarbons) in flow-through water. See Table 1 h Bioconcentration = ppm hydrocarbon in dry weight tissue/ppm hydrocarbon in water Mean -'- Standard error of mean for two 10- to 15-g composite samples each prepared from separate groups of coho salmon (2 fish/ group) a Mean - Standard error of mean for two I0- to 15-g composite samples each prepared from separate groups of starry flounder (5 fish/ group) " SS = Single sample value t NF = Not found: below limits of detection (60.05 ppm)

Fable 3. Hydrocarbons in liver and gills of starry flounder (P. exposurea Gills

stellatus) exposed to the water-soluble fraction of Prudhoe Bay crude oil using flow-through

Liver

Weeks of exposure Weeks of exposure


1 2 1 2 2 1 2

Weeks of depuration (2weeks exposure)

Weeks of deputation (2-weeks exposure)

Hydrocarbons

o o

o o

e ~

8 o

o ~

oo o

o o~

o g

6 6.0 36 11,000 110 2,900 29 ND ~ 3,000 30 320 9.6 10 0.38 6 0.19 170 5.0 100 2,100

1.6

10

2.6

0.23

0.2

1.8

1.1 3.1 21

1.1 110 2,300

1.1 3.4 23 100

Nl ~ NF ND

200

3,600

500 1,000 800 1,000 25 NF 5.3 4,400 22 2,000 i0 1,500

1.5 3.0 2.4 l0

1,100 2,000 1,600 2,800

3.3 6.2 5.0 28

300 300 300 900

0.90 0.90 1.0 9.0

ND NF NF 0.25

150 770 600 800

0.44 2.3 1.8 8.0 7.6

250 400 310 320 700

0.75 1.2 0.94 3.2 3.5

160 170 170 400 860

0.47 0.50 0.50 3.9 4.3

20

0.31 NF NF 0.22

Ca-Substituted benzenes Ca-Substituted benzenes C4-C~-Substituted benzenes Naphthalene 2-Methylnaphthalene l-Methylnaphthalene C2-Substituted naphthalenes Ca-Substituted naphthalenes

1,100

NF

.~

a b c d e

0.9 --+ 0.1 ppm (total hydrocarbons) in flow-through water. See Table 1 Bioconcentration = ppm hydrocarbon in dry weight tissue/ppm hydrocarbon in water Single values for 5-g composite samples prepared from 10 fish NF = Not found; below limits of detection (~<0.10 ppm) ND = Not determined

P-

Hydrocarbons in Salmon and Flounder

243

Discussion

Water-soluble aromatic hydrocarbons were readily accumulated in muscle, liver, and gills of starry flounder and muscle of coho salmon exposed to about 1 ppm of a WSF of crude petroleum. Gas chromatographic analyses revealed complex hydrocarbon profiles. Species differences found in the tendency to bioconcentrate the aromatic hydrocarbons suggest that demersal fish may be more susceptible than pelagic fish to petroleum contamination; however, additional work is necessary to verify this hypothesis. As with other marine organisms (Neff et al. 1976, Anderson 1975, Varanasi and Malins 1977) a strong tendency exists for both coho salmon and starry flounder to accumulate alkyl-substituted aromatic hydrocarbons in preference to the unsubstituted structures. In addition, the present findings imply that the accumulations of the water-soluble hydrocarbons in fish increase in proportion to the extent of ring substitution. It was shown in a previous study (Roubal et al. 1977a) with benzene, naphthalene, and anthracene that tissue levels of unsubstituted aromatic hydrocarbons in exposed coho salmon increase in relation to the number of benzenoid rings. Accordingly, at least two structural properties of the aromatic hydrocarbons have been shown to be related to their biological fate in marine fish. Insufficient data exists on the metabolism and excretion of the alkylated aromatic hydrocarbons in marine organisms (Varanas'i and Malins 1977, Malins 1977b) to know whether the tendency to accumulate these molecules in fish is related to slower rates of enzymatic oxidation and conjugation of the alkylated compounds in comparison to rates for the unsubstituted analogs. The decreases in hydrocarbon values observed in both coho salmon and starry flounder are of interest. Previous findings have indicated that the aryl hydrocarbon hydroxylases (AHH) of certain salmonids (Gruger et al. 1977) and starry flounder (Gruger, personal communication) are induced by exposure of the fish to aromatic hydrocarbons. Although no firm evidence is available, it seems likely that the changes in hydrocarbon concentrations observed in the present work were related to the induction of enzyme systems responsible for the metabolism of aromatic hydrocarbons. Wide variations in AHH activities were observed in marine fish previously (Malins 1977b). These individual differences in enzyme activities may account, at least in part, for the wide range of accumulated hydrocarbons found in the tissues of both species of exposed fish. It was suggested that the degree of hydrocarbon contamination in marine fish exposed to petroleum may increase in relation to the lipid content (Whittle et al. 1974a). The muscle of coho salmon was richer in lipid that starry flounder muscle. Thus, it appears that other factors were more influential than lipid content in the observed species differences in hydrocarbon accumulation and retention. Less than 15 ppb of the alkylated aromatic hydrocarbons (e.g., Ca- through Cs-substituted benzenes and C1- through C3-substituted naphthalenes) were present in the WSF. Nevertheless, these compounds became major petroleum components of tissues in both exposed species, notably the starry flounder. Great differences in the bioconcentration factors were observed for individual aromatic hydrocarbons in both coho salmon and starry flounder. We feel that these differences may complicate attempts to relate tissue hydrocarbon profiles to hydrocarbon profiles of specific sources of petroleum pollution.

244

W . T . Roubal et al.

Acknowledgments. This study was supported by the Bureau of Land Management through interagency agreement with the National Oceanic and Atmospheric Administration, under which a multiyear program responding to needs of petroleum development of the Alaskan continental shelf is managed by the Outer Continental Shelf Environmental Assessment Program (OCSEAP) Office. We also thank Donald W. Brown and John S. Finley for assistance in gas-liquid chromatographic analyses and Joanne Parker for maintenance of fish stocks.

References
Anderson, J. W., J. M. Neff, B. A. Cox, H. E. Tatem, and G. M. Hightower: The effect of oil on estuarine animals: Toxicity, uptake and depuration, respiration, In F. J. and W. B. Vernberg (eds.): Pollution and Physiology of Marine Organisms, pp. 285-310. New York: Academic Press (1974). Anderson, J. W.: Laboratory studies on the effects of oil on marine organisms: An overview. Am. Petrol. Inst. Publ. No. 4249 (1975). Gruger, E. H., Jr., M. M. Wekell, P. T. Numoto, and D. R. Craddock: Induction of hepatic aryl hydrocarbon hydroxylase in salmon exposed to petroleum dissolved in sea water and to petroleum and polychlorinated biphenyls, separate and together, in food. Bull. Environ. Contam. Toxicol. 17, 512 (1977). Hanson, S. W. F., and J. Oley: Application of the Bligh and Dyer method of lipid extraction of tissue homogenates. Biochem. J. 89, 101P (1963). Lee, R. F., D. Sauerheber, and G. H. Dobbs: Uptake, metabolism and discharge of polycyclic aromatic hydrocarbons by marine fish. Mar. Biol. 17, 201 (1972). MacLeod, W. D., D. W. Brown, R. G. Jenkins, L. S. Ramos, and V. D. Henry: A pilot study on the design of a petroleum hydrocarbon baseline investigation for Northern Puget Sound and Strait of Juan de Fuca. NOAA Tech. Memo. ERL MESA-8, Marine Ecosystems Analysis Program Office, Boulder, Colorado (Nov. 1976). Malins, D. C.: Biotransformation of petroleum hydrocarbons in marine organisms indigenous to the arctic and subarctic. In D. Wolfe (ed.): Proceedings of Symposium on Fate and Effects of Petroleum Hydrocarbons in Marine Ecosystems and Organisms. New York: Pergamon Press (1977a). Malins, D. C.: Metabolism of aromatic hydrocarbons in marine organisms. Ann. N.Y. Acad. Sci. 298, 482 (1977b). Meyerhoff, R. D.: Acute toxicology of benzene, a component of crude oil, to juvenile striped bass (Morone saxatilis). J. Fish. Res. Board Can. 32, 1864 (1975). Neff, J. M., D. Dixit, B. A. Cox, and J. W. Anderson: Accumulation and release of petroleum derived aromatic hydrocarbons by four species of marine animals. Mar. Biol. 38, 279 (1976). Roubal, W. T., T. K. Collier, and D. C. Malins: Accumulation and metabolism of carbon-14 labeled benzene, naphthalene and anthracene by young coho salmon (Oncorhynchus kisutch). Arch. Environ. Contam. Toxicol. 5, 513 (1977a). Roubal, W. T., D. H. Bovee, T. K. Collier, and S. I. Stranahan: Flow-through system for chronic exposure of aquatic organisms to sea water-soluble hydrocarbons from crude oil. In: Proceedings of 1977 Oil Spill Conference (Prevention, Behavior, Control, Cleanup), p. 551. American Petroleum Institute, Washington, D.C. (1977b). Varanasi, U., and D. C. Malins: Metabolism of petroleum hydrocarbons: Accumulation and biotransformations in marine organisms. In D. C. Malins (ed.): Effects of Petroleum on Arctic and Subarctic Marine Environments and Organisms, Vol. II, p. 175. New York: Academic Press (1977). Whittle, K. J., J. Murray, P. R. Mackie, R. Hardy, and J. Farmer: Fate of hydrocarbons in fish. Int. Counc. Explor. Sea (1974a). Whittle, K. J., P. R. Mackie, and R. Hardy: Hydrocarbons in the marine ecosystem. S. Aft'. J. Sci. 70, 141 (1974b).

Manuscript received July 14, 1977; accepted October 3, 1977.

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