Environ. Sci. Technol. lg85, 19, 836-841
Bioavailability and Biotransformation of Aromatic Hydrocarbons in Benthic Organisms Exposed to Sediment from an Urban Estuary Usha Varanasl," Wllllam L. Relchert, John E. Stein, Donald W. Brown, and Herbert R. Sanborn
Environmental Conservation Division, Northwest and Alaska Fisheries Center, National Marine Fisheries Service, Seattle, Washington 981 12 Phylogenetically diverse benthic organisms [amphipods (Rhepoxynius abronius and Eohaustqrius washingtonianus); clams (Macoma nasuta); shrimp (Pandalus platyceros); fish (Parophrys vetulus)] were exposed to an urban estuarine sediment [ 16 ppm of two to six benzenoid ring aromatic hydrocarbons (AHs)] to which trace amounts of [3H]benzo[a]pyrene(BaP) were added. The techniques used to assess uptake and metabolism of AHs were gas chromatography/mass spectrometry (GC/MS) for AHs, high-pressure liquid chromatography/fluorescence spectrometry for AH metabolites in fish bile, and radiometric analyses for biotransformation of [3H]BaP. Generally, the extent of metabolism of [3H]BaP( M . nasuta < E. washingtonianus < R. abronius IP. platyceros N P. vetulus) was negatively correlated to tissue concentrations of AHs (three to six ring), except that amphipod species accumulated higher concentrations of AHs than did clams, indicating that other factors (e.g., feeding strategy and rate of excretion) also influenced accumulation of AHs. Radiometric and GC analyses for BaP in both sediment and tissues suggested that not all of the BaP (and presumably other AHs) extracted chemically from sediment was bioavailable.
Introduction Information on bioavailability (e.g., uptake and bioaccumulation) and disposition (e.g., metabolism, tissue distribution and excretion) of contaminants in marine biota is essential to delineate cause-and-effect relationships between chemicals in the marine environment and observed biological abnormalities. Generally, two types of studies are conducted to assess bioavailability and disposition of chemical contaminants: (a) field studies to determine concentrations of chemicals in tissues of organisms sampled from contaminated areas (1-4) and (b) laboratory studies to determine bioaccumulation and metabolism of radiolabeled xenobiotics added to sediment, water, or food (5-8). The field studies yield useful information about xenobiotics that are not appreciably biotransformed or about organisms that lack the ability to metabolize xenobiotics significantly. For compounds such as aromatic hydrocarbons (AHs), which primarily exert their toxic effects after metabolic activation, field studies provide only partial information because metabolites in tissues are not routinely measured. However, AH metabolites have recently been detected in bile of fish from contaminated areas, showing that AHs are taken up by fish from their environment (9). Nevertheless, our lack of knowledge of migratory patterns of most marine animals or of the exact route of uptake of contaminants makes it difficult to correlate accumulation of chemicals in organisms to the presence of chemicals in their immediate environment. Laboratory studies, on the other hand, provide detailed information on uptake and metabolism of one or two conthminants per experiment. This information cannot be easily extrapolated to field situations in which an organism can be exposed simultaneously to a myriad of chemicals. Moreover, the bioavailability of added chem836 Environ. Sci. Technol., Vol. 19, No. 9, 1985
icals may not accurately reflect the bioavailability of the chemicals already present, for example, as in sediment from contaminated areas. Accordingly, we conducted a study that combined the methodologies used in field and laboratory studies to obtain a better understanding of the bioavailability and disposition of sediment-associated chemical contaminants in phylogenetically diverse benthic species. Sediment from a contaminated estuary in Puget Sound, WA (Duwamish River delta), was used to examine the uptake of selected AHs (two to six benzenoid rings) already present in the sediment. In addition, radiolabeled benzo[a]pyrene (BaP) was added to the sediment to serve as an indicator hydrocarbon to obtain information on the metabolism and disposition of sediment-associated AHs by these organisms.
Materials and Methods [G-3H]BaP (70 Ci/mmol) was purchased from Amersham (Arlington Heights, IL) and purified before use (10). For liquid scintillation spectroscopy (LSS), Insta-gel, Dimilume-30, and Soluene-350 were purchased from Packard Instrument Co., Downers Grove, IL. All other chemicals used were reagent grade or high-pressure liquid chromatography (HPLC) grade. Sediment was obtained by Van Veen grab from the West Waterway of the Duwamish River delta, Seattle, WA/ Puget Sound, WA (47"35' N; 122"21' W). The top 2 cm of sediment was collected, mixed in a large cement mixer, and then stored at -20 "C until used. This allowed all experiments to be done with the same sediment. Before the exposure studies were initiated, sediment was mixed for 15 min in a cement mixer after addition of [3H]BaP dissolved in acetone, and then the sediment was placed in the exposure aquaria (see Table I). The trace amount of [3H]BaP added to the sediment did not measurably change the concentration of BaP in the sediment. Test Organisms and Exposure Procedures. Exposure conditions and methods of collection of the test organisms are given in Table I. The exposure systems for clams (Macona nasuta), shrimp (Pandalus platyceros), and fish (Parophrys vetulus) consisted of glass aquaria (125 X 74 X 20 cm) containing a hard plastic grid (1cm square openings) covered with 300-pm nylon mesh (Nitex from Tobler, Ernst and Traber Co., Elmsford, NY) to support the sediment 4 cm above the floor of the aquaria. The grids were rinsed in flowing seawater for 1week before Duwamish River delta sediment (test sediment) containing added [3H]BaPwas placed in the aquaria. Seawater was introduced by way of PVC pipe into the aquaria beneath the grid and allowed to flow upward through the sediment. The sediment was allowed to stand for 1day with flowing seawater before addition of test animals. The seawater depth in the aquaria was kept at approximately 10 cm above the sediment. In the aquaria containing fish and shrimp, additional seawater was added from above the seawater surface. Amphipods (Rhepoxynius abronius or Eohaustorius washingtonianus) and test sediments were held in PVC cylinders (15 cm diameter X 20 cm high) with side and bottom openings covered with 500-pm nylon mesh
Not subject to US. Copyright. Published 1985 by the American Chemical Society
Table I. Information on Sites of Collection, Test Organisms, and Exposure Conditions species clams, Macoma nasuta no. of animals length/weight volume, L, of sediment mCi of [3H]BaPadded to sediment volume, mL, of acetone to dissolve [3H]BaP flow rate, L/min, of seawater up through sediment flow rate, L/min, of seawater added to water column water temperature, "C photoperiod light:dark, h period, weeks; animals held before start of experiment source of animalsC
amphipods@ Rhepoxynius Eohaustorius a bronius washingtonianus
shrimp, Pandalus platyceros
fish, Parophrys uetulus
210 30-50 mm/ 8f3g 40 5.8 10
900
3-8 mm/ 3 mg 3.6 0.6 0.75
2400 3-8 mm/ 5 mg 3.6 0.6 0.75
85 26 f 3 mmb/ 12f6g 45 18 10
162 12 f 1 cm/ 15f4g 45 18 10
1
1
1
1
1
0
0
0
4
4
12-15 12:12
15 12:12
15 12:12
10-15 0:24
10-15 12:12
1
1
1
1
1
Discovery Bay, WA
Deception Pass, WA
Deception Pass, WA
Port Susan, WA
Useless Bay, WA
150 R. abronius or 400 E. washingtonianus were placed in each exposure cylinder which contained 600 mL of sediment. length. Shrimp and fish were caught by otter trawl, amphipods by sieving of substrate, and clams by digging.
(11). These cylinders were placed in a glass aquarium and held 4 cm above the aquarium bottom by a hard plastic grid. This allowed seawater to flow under, around, and through the cylinders. At each sampling time, amphipods and clams were placed in clean seawater for 4 and 24 h, respectively, to allow elimination of any associated particulates. Amphipods were sampled at 1 week; clams, shrimp, and fish were sampled at both 1 and 4 weeks. All samples were stored at -20 'C. Analytical Procedures. Concentrations of selected AHs in sediment and tissues were determined by using solvent extraction, column chromatography and capillary column gas chromatography (GC) with a flame ionization detector and using GC/mass spectrometry (GC/MS) for confirmation of peak assignments (12-13). Quality assurance steps included analyses of (a) replicate samples (aliquota of a single sample), (b) samples of contaminated sediment and tissue used as standards, (c) laboratory blanks and laboratory blanks with added standards of the one to six ring AHs shown in Table 11, and (d) internal standards added to each sample; deuterated naphthalene, acenaphthene, and perylene were added at the start of the extraction procedure, and hexamethylbenzene was added just before GC analysis. Concentration of BaP-derived radioactivity (Le., [3H]BaP and its metabolites) in sediment and tissues was determined by methods described earlier (6,B). A chloroform-methanol extraction procedure was used to remove [3H]BaP and its metabolites from tissues (14). The hydrocarbon was separated from organic solvent-soluble metabolites by thin-layer chromatography (TLC) on silica gel plates with hexane-acetone (6:l v/v) in a paper-lined tank or hexane-toluene (4:l v/v) in an unlined tank. Specific activity of [3H]BaPin tissues and sediment was calculated by dividing the total radioactivity in the final GC extract, which contained only parent hydrocarbons, by the total BaF' in the final extract as determined by GC. Measurement of BaP-like fluorescence in bile of sole was done according to the method of Krahn et al. (9) using HPLC/ fluorescence spectrometry. Bile samples were analyzed from fish exposed to (a) test sediment (Table 11) having no added [3H]BaP,(b) test sediment having added [3H]BaP, (c) a reference sediment (Dosewallips River delta) from a nonindustrialized site in Puget Sound,
Thoracic
Table 11. Concentrations of Aromatic Hydrocarbons (AHs) in Sediment from the Duwamish River Delta" ng of AH/g of sediment. wet wt
no. of benzenoid rings
XAH
1 1 1 1
isopropylbenzene n-propylbenzene indan 1,2,3,4-tetramethylbenzene
NDb ND ND ND
2 2 2 2 2 2 2
naphthalene 2-methylnaphthalene 1-methylnaphthalene biphenyl 2,6-dimethylnaphthalene acenaphthene 2,3,5-trimethylnaphthalene
200 f 20 90 f 20 70 f 20 20 f 0 40 f 10 160 f 30 30 f 10
3 3
3 3 3
210 f 40 fluorene phenanthrene 1200 f 200 anthracene 320 f 60 1-methylphenanthrene 130 f 20 3,6-dimethylphenanthrene 50 f 10 2000 f 330
4 4 4 4
fluoranthene pyrene benz [a]anthracene chrysene
2000 f 300 2400 f 440 1010 f 160 1500 f 230 6900 f 1100
5 5 5 5 5
benzo[ e]pyrene benzo [a]pyrene perylene dibenz[a,h]anthracene benzofluoranthenes
1100 f 240 1160 f 110 320 f 20 230 f 30 2440 k 270 5300 f 600
6 6
indenopyrene benzo[ghi]perylene
860 f 90 620 f 70
600 f 100
1400 f 400
(x
"Concentrations of AHs f SD, n = 6) were determined at the start of exposure, and these values were not significantly different from those determined after 4 weeks. Concentration of [3H]BaPin sediment was measured frequently, and no significant change was observed over the 4-week exposure. bDetection limits varied from 5 to 23 ppb. Percent recovery of internal extraction standards: naphthalene-d8, 56 f 12; acenaphthene-dlo, 68 f 14; perylene-d,,, 61 f 6.
WA/Hood Canal, WA (47'41' N; 122'54' W), having no detectable levels of BaP, as measured by GC, and no added Environ. Sci. Technol., Vol. 19, No. 9, 1985
837
Table 111. Benzo[a Ipyrene and Metabolites in Benthic Organisms Exposed to Duwamish River Delta Sediment"
hydrocarbon and metabolites pmol of BaP equiv/g of tissue (wet weight) % BaP % BaP metabolites
length of exposure, weeks 1 4
amphipods, whole body E.w.~ R.u.
clams (M. nasuta) hepatobody' pancreas
shrimp (P. platyceros) hepatobodyC pancreas
bod9
fish (P. uetulus) liver bile
4900 f 50 2400 f 110 430 f 120 2600 f 1200 13 f 4 600 f 310 36 f 6 890 f 170 950 f 270 4800 f 1700 41 f 13 400 f 95 35 f 5 960 f 270 78 f 3' 22 f 38
26 f 4 74 f 4
>95
95
