Research Toxic Effects of Unresolved Complex Mixtures of Aromatic Hydrocarbons Accumulated by Mussels, Mytilus edulis, from Contaminated Field Sites PETER DONKIN, EMMA L. SMITH, AND STEVEN J. ROWLAND* Petroleum and Environmental Geochemistry Group, Department of Environmental Sciences, Plymouth Environmental Research Centre, University of Plymouth, Drake Circus, Plymouth PL4 8AA, United Kingdom
Exposure of marine mussels (Mytilus edulis) to an unresolved complex mixture (UCM) of aromatic hydrocarbons isolated from a crude oil has been shown to reduce their feeding rate by 40%. The present study was undertaken to determine whether UCMs bioaccumulated by mussels in the field are also toxic. The feeding rate of mussels derived from polluted sites increased when they were placed in clean water, pointing to a loss of toxic agents from the tissues. At the end of the depuration period, water in which mussels from an oil-polluted site had been held contained a UCM. Steam-distillation extracts of the tissues of mussels taken from several polluted sites were shown to be highly toxic to the feeding activity of juvenile mussels. The tissues of mussels from these sites contained UCMs. Nontoxic steam distillates from clean mussels did not. Steam-distillation extracts of mussels from an oil-polluted site were fractionated by normal-phase high-performance liquid chromatography. A fraction, largely comprising a “monoaromatic” UCM, reduced the feeding rate of juvenile mussels by 70%. Two later-eluting fractions containing aromatic UCMs also produced smaller depressions in feeding rate. These results support our contention that some aromatic UCM hydrocarbons constitute a forgotten pollutant burden in the marine environment.
Introduction The Blue Mussel, Mytilus edulis, is an important sentinel organism used in global marine monitoring of water quality (1-3). Mussels in polluted environments accumulate a wide variety of pollutants by filtration of water and exhibit a variety of health effects (1-5). Among the hydrophobic organic pollutants accumulated by mussels, a feature known as the unresolved complex mixture (UCM) of hydrocarbons is commonly observed (6). Such UCM hydrocarbons comprise both aromatic and nonaromatic compounds (7). The toxicity of UCM hydrocarbons has rarely been studied (8, 9). Such evidence as it exists suggests that nonaromatic UCM hydrocarbons are largely nontoxic to mussels, although toxicity can be introduced by oxidation of nonaromatic UCMs * Corresponding author phone: +44 1752 233013; fax: +44 1752 233035; e-mail:
[email protected]. 10.1021/es021053e CCC: $25.00 Published on Web 10/01/2003
2003 American Chemical Society
to oxygenated, but still mainly unresolved, compounds (8). Recently, however, we were able to demonstrate that an aromatic UCM isolated from a North Sea crude oil by highperformance liquid chromatography (HPLC) was able to elicit a nonspecific narcotic toxic response in mussels exposed in the laboratory (viz., a >40% reduction in feeding rate in 24 h (9)). Furthermore, a number of populations of U.K. mussels exhibiting impaired health, as measured by a well-accepted assay known as scope for growth (SfG′; 10) contained high concentrations of UCM hydrocarbons (9). In the present study we sought to establish whether UCMs bioaccumulated by mussels in contaminated field sites are toxic, and which if any of the UCM subfractions are most toxic. Three experimental approaches were used to provide evidence for the toxicity of bioaccumulated UCMs: (1) depuration/recovery experiments in clean water to establish whether the loss of UCMs from the tissues of polluted mussels resulted in an improvement in their health; (2) toxicity testing of steam-distillation extracts of mussel tissues, comparing clean and UCM-polluted sites; (3) fractionation of these extracts and toxicity testing of the fractions.
Materials and Methods Sample Sites. Whitsand Bay is home to an open coast population of mussels and has a generally high water quality. For the greater part of the year this can be used as a “clean” reference population. The mussel population at Port Quin resides in a small rocky inlet and has been used previously as a clean reference site in an Irish sea study (10). At Sutton Harbour mussels transplanted from Port Quin were caged and placed at a 2 m depth hanging from a jetty on a marina used largely for recreational boating activity. Mussels at West Hoe were transplanted from Port Quin without caging (i.e., direct to the intertidal rocks), close to the outfall of the main sewer for Plymouth. Samples were also taken from natural mussel populations at Instow, where commercial harvesting for human consumption is banned because of sewage pollution, and Cattedown, due to the close proximity of a marina and harbor. All of these sites are located in and around Plymouth in the southwest part of England. Mussels were also collected from New Brighton and Kirkcolm by Dr. T. Crowe during ecological investigations (11). New Brighton is a site with severely depressed mussel scope for growth (10) and a mussel bed ecosystem indicative of pollution impact (11), while Kirkcolm in comparison is a clean site (10, 11). All the mussel populations were native populations except for those at Sutton Harbour and West Hoe, which were transplanted from Port Quin. The depuration/recovery experiments were performed with mussels from Whitsand, Port Quin, and Sutton Harbour due to the proximity of these sites to the Plymouth laboratory. The testing of steam distillates was performed on all the sites ranging from clean to highly polluted. Fractionation experiments were performed on a clean (Whitsand) and a polluted (Sutton Harbour) site for comparison. Depuration/Feeding Rate Recovery Experiments. Mussels (35-40 mm shell length) were collected from the field, cleaned of superficial debris and encrusting organisms, and used for laboratory study the same day, or if collected when low tide occurred in the late afternoon, they were cleaned, held overnight in moist air at a temperature close to that of the ambient water temperature, and then used for experiments the next morning. The maximum air exposure time VOL. 37, NO. 21, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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that mussels were subjected to was 20 h, but most experiments were carried out with shorter air exposures. The mussels were allowed to recover for 2.25 h in 2 L beakers (one mussel per beaker) containing 2 L of filtered offshore (clean) seawater. Previous studies have shown that mussels recover rapidly after 24 h of air exposure, most of the recovery occurring within the first 5 h (10). The mussels handled using the reduced time scale procedure described above (50%). Aromatic hydrocarbons in such mixtures are known to be toxic to mussels (9). We do not infer, of course, that all of the toxic components are represented by the UCM. Indeed it is to be expected that depurated compounds will include and perhaps be dominated by more hydrophilic substances not amenable to GCMS analysis. Nonetheless, UCM compounds are clearly among the depurated toxicants. Further support for this interpretation comes from GC-MS analysis of the mussel tissue from the Sutton Harbour transplants, which show a bioaccumulation of UCM compounds as compared to their native Port Quin counterparts (Figure 4). Toxicity Testing of Steam-Distillation Extracts of Mussel Tissues. To establish whether mussel tissues (in addition to the depurated water) also contained toxic chemicals, tissues from mussels were extracted by cyclic steam distillation and the toxicity of the extracts was tested by measuring their VOL. 37, NO. 21, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. GC-MS total ion current chromatogram of a solid-phase extract (diethyl ether eluate) of water depurated for 24 h from mussels collected in April from Sutton Harbour (minus the procedural blank). Both resolved (including some biogenic, marked with an asterisk) and a large proportion of unresolved (UCM) chemicals are present.
FIGURE 3. Feeding rates of juvenile mussels exposed to steam-distillation extracts of tissues of mussels from various U.K. sites. The feeding rate is expressed as a percentage of the control feeding rate (n ) 8 ( standard deviation). impact on the feeding rate of juvenile mussels (Figure 3). Mussels were taken from sites at various points along the U.K. coast known to range from clean to highly polluted. Cyclic steam-distillation extraction as carried out in these studies recovers only those chemicals which are steam volatile and significantly soluble in hexane. Many UCM hydrocarbons, in addition to hydrocarbons that are usually resolved by capillary gas chromatography, have these properties (15, 16). Steam-distillation extracts of mussels from Port Quin, Kirkcolm, and Whitsand had little or no detectable toxicity (i.e., no depression in feeding rate compared to controls) using the methods described. Mussel tissues from these sites have been shown previously to contain low or very low levels of aromatic hydrocarbon pollution on the basis of a steamdistillation extraction normal-phase HPLC analytical method which is sensitive to aromatic UCM hydrocarbons (10, 13) and on the basis of solvent extraction/GC-MS methods (9). Using a semiquantitative GC-MS approach (e.g., Figure 4), no UCM contamination could be detected in the tissues of Port Quin, Kirkcolm, or Whitsand mussels in the present 4828
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study under conditions where UCMs were clearly apparent in more polluted mussels. Distillates from Instow and West Hoe mussels caused mean depressions of feeding rate of 15% and 25%, respectively, indicating some toxicity. Instow is remote from significant sources of oil pollution, though exposed to sporadic pollution by raw sewage derived from a small town. West Hoe mussels are exposed to some urban and harborderived pollution and at certain states of the tide, to highly treated sewage effluent, though the tidal influx of open seawater at this site may result in substantial periodic dilution of pollutants. GC-MS analysis of steam distillates of the tissues of mussels from Instow and West Hoe failed to reveal the presence of significant UCMs (Figure 4). In contrast, steam distillates of tissues of mussels from Sutton Harbour, New Brighton, and Cattedown were highly toxic, depressing the feeding rate of juvenile mussels by more than 60% (Figure 3). Interestingly, steam distillates of mussels from each of these sites also contained substantial UCMs (55-76% relative to resolved components) when examined by GC-MS under the same conditions (identical proportions
FIGURE 5. Feeding rates of juvenile mussels exposed to HPLC fractions 1-5 of combined steam distillates from a clean (Whitsand) and a polluted (Sutton) site (n ) 4 ( standard deviation). Mussel feeding rates upon exposure to Whitsand (dark gray bars) and Sutton (light gray bars) distillates for 24 h are expressed as a percent of the control feeding rate when the same mussels have previously been exposed to seawater to 24 h.
FIGURE 4. GC-MS total ion current chromatograms of steam distillates from tissues of mussels from various U.K. sites. The same proportions of extracts were examined in each determination, and the data are represented on the same vertical and horizontal scales. Chromatograms of nontoxic distillates (Port Quin, Whitsand) or relatively low toxicity distillates (Instow, West Hoe) comprise resolved chemicals. Chromatograms of toxic distillates (Sutton, Cattedown, New Brighton) comprise resolved and significant proportions (55-76%) of unresolved (UCM) chemicals. of extracts injected) as the less polluted mussels from Whitsand and Port Quin (Figure 4). Mussels from New Brighton are known to be heavily polluted with steam-volatile aromatic hydrocarbons (13). Indeed, using a direct solvent extraction procedure followed by cleanup and GC-MS analysis (9), tissues of mussels from New Brighton were shown in the present study to contain 708-764 µg/g dry weight of nonaromatic UCM hydrocarbons and 235265 µg/g dry weight of aromatic UCM hydrocarbons. We have no quantitative data for Cattedown mussels, but this site is visibly oil polluted due to its proximity to a marina and commercial harbor in Plymouth. Thus, distillates of the tissues of mussels shown herein to be highly toxic to clean mussels (Sutton, New Brighton, Cattedown) also contained >50% UCMs of organic chemicals,
whereas distillates of mussels which were nontoxic or exhibited relatively low toxicity contained no detectable UCMs. Although this provides circumstantial evidence that some bioaccumulated UCM chemicals may be toxic, as shown in laboratory experiments (9), the data by no means prove the relationship. This required a more detailed examination of the toxicity of the tissue extracts. HPLC Fractionation/Toxicity Testing of Steam Distillates. Clearly there are a number of possible steam-volatile components (cf. refs 10, 12, 13, and 16) which may be toxic to mussels. In an attempt to establish the relative importance of such compounds to the total toxicity, we fractionated distillates derived from Sutton Harbour (polluted) mussels by normal-phase HPLC (cf. ref 9) and measured the toxicity of the resulting fractions in replicate (n ) 4) using the juvenille mussel feeding rate bioassay. For comparative purposes, distillates of mussels from Whitsand (clean) were treated in the same way. The weight of material recovered in each fraction was measured. In the earlier HPLC fractions (F1F6), a detectable weight (ca. 0.2-0.8 mg from a 16 g tissue) was only observed when Sutton Harbour mussel distillates were analyzed. Later fractions were dominated by the presence of biogenic materials that occurred in both Sutton and Whitsand animals. Only one of the HPLC fractions (F3 from Sutton mussels) exhibited a pronounced narcotic effect, reducing the feeding rate by about 70% compared with controls (Figure 5). This fraction comprised mainly aromatic UCM hydrocarbons with about four to six double bond equivalents (DBEs; i.e., mainly no more than one aromatic ring) plus a natural resolved hexaunsaturated hydrocarbon, squalene (Figure 6). This approximate composition of the UCM fraction (viz., about four to six DBEs) was assigned by HPLC analyses of authentic aromatic hydrocarbons including benzene (four DBEs), tetralin (five DBEs), dialkyltetralins (six DBEs; ref 17), indene (six DBEs), and naphthalene (seven DBEs) and by the cooccurrence in this fraction of squalene, which has six double bonds. The same fraction in Whitsand mussels contained squalene as the only hydrocarbon detected (Figure 6). Importantly this fraction produced no detectable toxic effect (Figure 5). Later-eluting aromatic UCM HPLC fractions 4 and 5 from Sutton Harbour mussels also produced feeding rate depressions (more than 30%). These depressions were only significant at the 90% confidence level. Again the corresponding fractions (F4, F5) from the Whitsand mussels were unweighable and nontoxic (Figures 5 and 6), containing only traces of biogenic squalene and a hexaunsaturated highly VOL. 37, NO. 21, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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In summary, we feel that the demonstrable toxicity of at least one aromatic hydrocarbon UCM (9), the high concentrations of aromatic hydrocarbon UCMs in some mussels exhibiting reduced SfG (9), the recovery in health of mussels which have depurated UCM hydrocarbons from their tissues, the toxicity of steam-distillation extracts of UCM-contaminated mussel tissues, and the toxicity of an aromatic hydrocarbon UCM fraction derived from such distillates all suggest that UCMs are an environmental burden worthy of further examination.
Acknowledgments We thank the U.K. Department for Environment Food and Rural Affairs (DEFRA) for funding this study (Contract CDEP 84/5/267) and Dr. J. Widdows and Mr. F. J. Staff of the Plymouth Marine Laboratory for helpful discussions. S.J.R. and E.L.S. dedicate this paper to the memory of Dr. Peter Donkin, a respected and principled scientist and friend, who died suddenly a few days before the submission of this manuscript.
Literature Cited FIGURE 6. GC-MS total ion current chromatograms of the normalphase HPLC fractions 1-5 of the combined steam distillates from a polluted (Sutton) and a clean (Whitsand) site. Fractions from Sutton mussels represent 8-17% by mass of the total distillate. Fractions F3-F5 were toxic to juvenile mussels (see Figure 5). The corresponding fractions from the Whitsand mussels contained unweighable amounts of material and were nontoxic. UCM ) unresolved complex mixture, S ) squalene, and H ) highly branched alkenes from diatoms (19). branched alkene, probably originating from diatoms in the mussel diet (20). HPLC fraction 1 from Sutton Harbour mussels, though more abundant (17% of the HPLC eluate compared to 13%) than toxic F3, was nontoxic (Figure 6). This is as expected since F1 and F2 comprise nonaromatic hydrocarbons which were shown previously to be nontoxic to mussels (8). F1 and F2 from Whitsand clean mussels were also nontoxic (Figure 5). F1 contained a nonaromatic UCM and a series of waxy n-alkanes (Figure 6) and F2 a mixture of biogenic di- to pentaunsaturated highly branched alkenes (Figure 6) originating from diatomaceous algae (20). These data suggest that, of those toxic components in the steam distillates that were eluted from the HPLC column, part of the UCM aromatic hydrocarbons in the polluted Sutton Harbour mussels produced the greatest relative toxic response. Of course, the response could have also been due in part to the resolved components (other than squalene) in this fraction (Figure 6), but the concentration of these was very low relative to that of the total UCM compounds. We have suggested previously that among the possible compounds in hydrocarbon UCMs are disubstituted alkyltetralins (17). Such compounds appear to have oxidation products (18, 19), aqueous solubilities, sublethal narcotic toxicities (17), and a resistance to biodegradation consistent with some aromatic hydrocarbon UCM fractions. In the context of the present findings it should also be noted that such compounds also have six double bond equivalents and elute in the HPLC retention time window of the most toxic fraction, F3.
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(1) Jernelov, A. Sci. Total Environ. 1996, 188, S37-44. (2) Sericano, J. L.; Wade T. L.; Jackson, T. J.; Brooks J. M.; Farrington J. W.; Mee L. D.; Readman, J. W.; Villeneuve, J. P.; Goldberg E. D. Mar. Pollut. Bull. 1995, 31, 214-225. (3) Goldberg, E. D.; Bowen, V. T.; Farrington, J. W.; Harvey, G. R.; Martin, J. H.; Parker, P. L.; Risebrough, R. W.; Robertson, W. Scheider, E.; Gamble, E. Environ. Conserv. 1978, 5, 101-125. (4) The International Mussel Watch; NRC/National Academy of Sciences: Washington, D.C., 1980. (5) Widdows, J.; Donkin, P. In The Mussel Mytilus: Ecology, Physiology, Genetics and Culture; Gosling, E., Ed.; Elsevier: Amsterdam, 1992; pp 383-424. (6) Murray, A. P.; Gibbs, C. F.; Kavanagh, P. E. Int. J. Environ. Anal. Chem. 1983, 16, 167-195. (7) Gough, M. A.; Rowland, S. J. Nature 1990, 344, 648-650. (8) Thomas, K. V.; Donkin, P.; Rowland, S. J. Water Res. 1995, 29, 379-382. (9) Rowland, S. J.; Smith, E. L.; Wraige, E. J.; Donkin, P. Environ. Sci. Technol. 2001, 35, 2640-2644. (10) Widdows, J.; Donkin, P.; Brinsley, M. D.; Evans, S. V.; Salkeld, P. N.; Franklin, A.; Law, R. J.; Waldock, M. J. Mar. Ecol.: Prog. Ser. 1995, 127, 131-148. (11) Widdows, J.; Donkin, P.; Staff, F. J.; Matthiessen, P.; Law, R. J.; Allen, Y. T.; Thain, J. E.; Allchin, C. R.; Jones, B. R. Mar. Environ. Res. 2002, 53, 327-356. (12) Crowe, T.; Smith, E. L.; Donkin, P.; Barnaby, D.; Rowland, S. J. Manuscript in preparation. (13) Donkin, P.; Widdows, J.; Evans, S. V.; Worrall, C. M.; Carr, M. Aquat. Toxicol. 1989, 14, 277-294. (14) Widdows, J. Mar. Biol. 1973, 20, 269-276. (15) Donkin, P.; Evans, S. V. Anal. Chim. Acta 1984, 156, 207-219. (16) Donkin, P.; Widdows, J. ; Evans, S. V.; Brinsley M. D. Sci. Total Environ. 1991, 109/110, 461-476. (17) Smith, E.; Wraige, E.; Donkin, P.; Rowland, S. Environ. Toxicol. Chem. 2001, 20, 2428-2432. (18) Thomas, K. V. Characterisation and environmental effects of unresolved complex mixtures of hydrocarbons. Ph.D. Dissertation, University of Plymouth, U.K., 1995. (19) Warton, B.; Alexander, R.; Kagi, R. I. Org. Geochem. 2000, 30, 1255-1272. (20) Belt, S. T.; Allard, W. G.; Masse´, G.; Robert, J.-M.; Rowland, S. J. Geochim. Cosmochim. Acta 2000, 64, 3839-3851.
Received for review December 4, 2002. Revised manuscript received June 5, 2003. Accepted June 25, 2003. ES021053E
