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Norman, T. S.; Harns, L. L.; Loayenga, R. W. J.-Am. Water Works Assoc. 1980, 72, 176-180. Mitcham, R. P.; Shelley, M. W.; Wheadon, C. M. J.-Am. Water Works Assoc. 1983, 75, 196-199. Brodtman, N. V., Jr.; Kroffskey, W. E.; DeMarco, J. In “Water Chlorination: Environmental Impact and Health Effect”; ed. Jolley, R. L.; Brungs, W. A.; Cumming, R. B., Eds.; Ann Arbor Science Publishers, Inc.: Ann Arbor, MI, 1980; Vol. 111, pp 777-788. Hubbs, S. A,; Amundsen, D.; Olthius, P. J.-Am. Water Works Assoc. 1981, 73, 97-101. “Standard Methods for the Examination of Water and Wastewater”, 15th ed.; American Public Health Association: Washington, DC, 1980; pp 277-301. Guter, K. J.; Cooper, W. J.; Sorber, C. A. J.-Am. Water Works Assoc. 1974, 66, 38-43. Cooper, W. J.; Sorber, C. A.; Meier, E. P. J.-Am. Water Works Assoc. 1975, 67, 34-39. Cooper, W. J.; Roscher, N. M.; Slifker, R. A. J.-Am. Water Works Assoc. 1982, 74, 362-368. Johnson, J. D. In “Water Chlorination-Environmental Impact and Health Effects”; Jolley, R. L., Ed.; Ann Arbor Science Publishers, Inc.: Ann Arbor, MI, 1978; Vol. I, pp 37-63. Strupler, N. Proc. A W W A Water Qual. Technol. Conf. 1978, p2A-6. Michaelis, L. Chem. Rev. 1935,16, 243-286. Michaelis, L. Ann. N.Y. Acad. Sci. 1940, 40, 39-71. Michaelis, L. J . Am. Chem. SOC. 1931,53, 2953-2961. Michaelis, L. J . Biol. Chem. 1932, 96, 703.
Elama, B. J . Biol. Chem. 1939, 100, 149. Michaelis, L.; Schubert, M. P.; Granick, S. J . Am. Chem. SOC.1939, 61, 1981-1992. Weitz, E.; Fischer, K. Ber. Dtsch. Chem. Ges. 1926,59,432. Miller, F.; Wilkins, R. G. J. Am. Chem. SOC.1970,92,2981. Brown, H. C.; McDaniel, D. N. J . Am. Chem. SOC.1955, 77, 3752. Rehm, C.; Bodin, J. I.; Connors, K. A.; Miguahi, T. Anal. Chem. 1959, 77,483. Brode, W. R. J . Am. Chem. SOC.1924,46, 581. Wilkins, R. G. “The Study of Kinetics and Mechanisms of Reactions of Transition Metal Complexes”; Allyn and Bacon: Boston, MA, 1974; p 46. Michaelis, L.; Fetcher, E. S., Jr. J . Am. Chem. SOC.1937, 59, 1246. Michaelis, L. J . Biol. Chem. 1938, 123, 527. Jaffari, G. A.; Nunn, A. J. J. Chem. SOC.C 1971,93,823-826. Palin, A. T. J.-Am. Water Works Assoc. 1957,49,873-880. Snead, M. C.; Olivieri, V. P.; Dennis, W. H. In “Chemistry in Water Reuse”; Cooper, W. J., Ed.; Ann Arbor Science Publisher, Inc.; Ann Arbor, MI, 1981; Vol. 1, 401-427. Fiquet, J. M. Tech. Sci. Munic. 1982, 77, 243-249. Nicolson, N. . Analyst (London) 1965, 90, 187-198. Johnson, J. D.; Overby, R. Anal. Chem. 1969,41,1744-1747.
Received for review June 6,1983. Accepted November 4,1983. This work was partially supported by the Physical Sciences Department and the Drinking Water Research Center, Florida International University.
Polycyclic Aromatic Hydrocarbons in the Clam Tridacna maxima from the Great Barrier Reef, Australia J. David Smith,?John Bagg,**$and Brian M. Bycroft’ Marine Chemistry Laboratory, School of Chemistry, University of Melbourne, Parkville, Victoria 3052, Australia, and Department of Industrial Science, University of Melbourne, Parkville, Victoria 3052, Australia
The concentrations of eight polycyclic aromatic hydrocarbons (PAH), anthracene, pyrene, chrysene, benzo[klfluoranthene, benzo[a]pyrene, benzo[ghi]perylene, fluoranthene, and perylene, were measured in clams, Tridacna maxima, collected from sites on the Great Barrier Reef ranging in latitude from 14’31’ S to 23’33’ S. At most locations the concentrations of PAH were not significantly above the limit of detection, e.g., pyrene C 0.07 pg/kg wet weight, benzo[a]pryene C 0.01 pg/kg, and chrysene < 0.07 pg/kg. These levels of PAH appear to be the lowest reported for clams anywhere in the world, indicating the pristine nature of the Great Barrier Reef a t the present time. Concentrations significantly above detection levels were found at only two sites, Lizard Island First Beach (anthracene, 3.2 pg/kg; pyrene, 1.4 hg/kg) and Heron Island Harbour (pyrene, 1.2 pg/kg; benzo[a]pyrene, 0.02 pg/kg). Both sites are frequently visited by power boats which are the most likely source of hydrocarbon contamination. These low levels of contamination would not have been demonstrated by the measurement of only the most commonly studied PAH, benzo[a]pyrene. Simultaneous determination of several PAH was necessary to show clearly that some localized pollution had occurred. Introduction The Great Barrier Reef is a chain of individual reefs stretching 2000 km approximately north-south from laMarine Chemistry Laboratory, School of Chemistry. of Industrial Science.
1Department
0013-936X/84/09 18-0353$01.50/0
titude 8 O S to 2 4 O S. It lies on the eastern continental shelf of Australia facing the Coral Sea. With the exception of some small resorts, and the research stations on Lizard and Heron Islands, the reef has no permanent habitation. The coastal region adjacent to the Great Barrier Reef has a population of only 300000 and is not highly industrialized. Consequently, inputs of polycyclic aromatic hydrocarbons (PAH), generated by combustion of fuels, into the atmosphere or into rivers with waste discharges are small and would have to be transported for distances greater than 20 km in order to reach the reef. This paper describes an investigation that shows the Great Barrier Reef to be almost unaffected by coastal discharges with the most likely sources of contamination arising from human activity on the reef itself. The Great Barrier Reef is being studied with increasing intensity because of its importance as the largest coral reef system in the world and the possibility that oil reserves under the reef may be exploited. Petroleum exploration permits issued by the Queensland State Government cover more than 60% of the Great Barrier Reef region. These permits have been suspended following an inquiry by State and Federal Royal Commissions (19741, but the possibility of future exploration still exists, and such activity could lead to some contamination of the reef. The determination of background hydrocarbon levels was regarded as essential and urgent. Petroleum is a complex and variable mixture containing up to 50% aromatic hydrocarbons. Aromatic hydrocarbons have more undesirable effects on biota than do the ali-
0 1984 American Chemical Society
Environ. Sci. Technol., Vol. 18, No. 5, 1984 353
Table I. Location of Sample Sites and Size of Clams Taken dimensions of clams sample no. site weight, g length x breadth, cm Heron Island (23"27' S 151"55' E), Dec 1980 1 1 760 16.5 X 9.0
740 16.0 x 8.5 740 16.0 X 8.5 1660 18.4 x 10.0 970 16.0 X 11.0 970 18.0 X 8.6 I 1030 18.0 X 9.0 Polmaise Reef (23"33' S 157"41' E), Dec 1980 8 4 2310 20X 14 Masthead Island (23'32' S 157'44' E), Dec 1980 9 5 2250 21X 15 Wistari Reef (23"27' S 157"51' E), Dec 1980 10 6 2980 24X 15 One Tree Island (23"29' S 152"Ol' E), Dec 1980 11 7 2370 19X 13 Wreck Island (23"20' S 157"57' E), Dec 1980 12 8 2580 20X 14 Lizard Island (14"40' S 145"24' E), May 1 9 8 1 13 9 1100 1 9 x 11 14 l o b 1700 2 1 X 1 2 15 10 1380 17.5 X 11.5 16 10 1160 1 8 X 11 Carter Reef (14"31' S 145'34' E), May 1 9 8 1 17 11 2970 20X 13 Flinders Reef (17'55' S 148"30' E), Aug 1 9 8 1 18 12 540 soft tissue sample; 2 3 4 5 6
Flgure 1. Great Barrier Reef showing sampling regions. (A) Heron, Masthead, One Tree, and Wreck Islands and Polmaise and Wistari Reefs, (B) Flinders Reef, (C) Orpheus Island, and (D) Lizard Island, Carter Reef.
phatic hydrocarbons. Compared to the aliphatics, most aromatics degrade more slowly, persist in tissues for longer, and are usually more toxic. One group of aromatics, the PAH, includes some compounds that are toxic or carcinogenic. One compound,benzo[a]pyrene(B[a]P), is highly carcinogenic, is widely distributed in the environment, and is one of the US.EPA Priority Pollutants. PAH can act by interfering with metabolic processes and tend to be concentrated and retained by tissues more effectively than other hydrocarbons (1). Low concentrations of PAH are present in crude and refined oil, and greater amounts are produced during the high-temperature combustion of fuels. Natural bush fires are a potential source of PAH, and in Queensland sugar cane crops are burned before harvesting each year. Some PAH can apparently be formed by the transformation of biogenic precursors during relatively short times. Of the PAH determined in this work, perylene is the only one for which there is substantial evidence of biogenic production in sediments (2). On the Great Barrier Reef actively growing organisms occur mainly in shallow water, between the low-water mark and about 5-m depth. This location makes them especially susceptible to oil pollution which commonly occurs at the air/water interface through spillage, through discharges from boats, or from deposited aerosols. The earliest indications of contamination might be expected to be found in species that live in this vulnerable zone. The clam, Tridacna maxima, chosen as an indicator species, is found in adequate numbers along the entire length of the reef and yields enough tissue to permit the detection of PAH at very low concentrations. During collection, the shells close so that the likelihood of significant contamination occurring in subsequent transporation is very small. Tridacna maxima is readily identified, being visibily different from the other four species of Tridacna, and 354
Environ. Sci. Technol., Vol. 18, No. 5, 1984
1 1 1 2a 2 3
dimensions not known Orpheus Island (18'33' S 146'30' E), Aug 1982 19 13 290 soft tissue sample; 20 13 450 dimensions not known 21
13
430
a Site 2 is Heron Island Harbor. Island First Beach.
Site 10 is Lizard
confusion could only occur between T. maxima and juvenile specimens of the giant clam T. gigas ( 3 ) .
Experimental Methods Sample Collection. Specimens of T. maxima were collected from reef flats or slopes during the period Dec 1980 to Aug 1982 at 13 different sites ranging in latitude from 14'31's to 23"33'S (Figure 1 and Table I). Heron, Lizard, and Orpheus Islands each have a population of less than 30 persons for most of the year which rises to approximately 100 during the holiday season. The remaining sites are uninhabited islands or reefs. Heron Island Harbour is a channel blasted from the coral, suitable only for small vessels. Site 2 is in the boat channel and is adjacent to a small sewage outfall. The other sites on Heron Island, 1and 3, are at points 500 m from the boat channel on the reef edge. Lizard Island First Beach, site 10, is close to a small resort and is a landing point for power boats. Site 9, Lizard Island, is approximately 3 km from site 10 and is visited infrequently by boats. At sites 1, 2, 10, and 13 several clams were collected within a 1-m2area, and each clam was analyzed separately. A comparison of results, for example, samples 14-16, provides a measure of the interspecimen variability. Immediately after collection, clams were wrapped in aluminum foil (prewashed with cyclohexane),stored on ice, and transported to the laboratory within 48 h after collection. In the laboratory clams were stored at -10 "C prior to analysis. To minimize possible contamination in the laboratory, no purging or separation of the organs was
performed. Any sediment, plankton, or other material entrained in the gut was therefore included in the analysis, and reported levels of PAH are an upper estimate of the body burden. The shell dimensions and total weight of individual specimens are shown in Table I. Analytical Method. The analytical method ( 4 ) has been developed in this laboratory over several years and comprises the following steps: saponification of homogenized clam tissue in alkaline methanol; extraction of PAH into cyclohexane;preliminary separation on a Florisil column; final separation of the PAH fraction by reversephase high-performance liquid chromatography (HPLC) using fluorescence to identify individual compounds. All solvents and other chemicals were analytical or spectroscopic grade. Methanol and cyclohexane were redistilled once and water was distilled twice before use. Clams were allowed to thaw overnight and shucked. The entire soft tissue was cut into small pieces and homogenized in an Omni blender (Sorrel1Model 17106). Approximately 20 g of homogenate was refluxed in alkaline methanol (7 g of KOH, 150 mL of MeOH) for 2 h in darkness. After reflux the mixture was centrifuged and the supernatant liquid mixed with 50 mL of water in a separating funnel and then extracted with cyclohexane (3 X 50 mL). The cyclohexane was reduced to 20 mL by rotary evaporation below 60 "C and dried over sodium sulfate. The dried extract was passed through a deactivated Florisil column (2.5 X 30 cm, 100-200-mesh particle size) which had been prewashed with 50 mL of cyclohexane/dichloromethane mixture (1:l v/v). The eluate was reduced to approximately 3 mL by rotary evaporation and then to dryness under a stream of dry nitrogen below 40 "C. The residue was redissolved in 1 mL of methanol in preparation for the final separation. Aliquots of the methanol solution (10-100 pL) were injected (Rheodyne 7105 loop injector) onto a bonded octadecylsilane column (3.9 X 300 mm pBondapack CI8, Water Associates) and eluted isocratically with a mixture of water/methanol/acetonitrile (1:2:2 v/v/v, 1 mL/min flow rate) at ambient temperature. Individual PAH eluting from the column were detected by fluorescence (Hitachi Model 204 spectrofluorometer with 180-pL flow cell). Excitation (AEX) and emission (AEM) wavelengths were changed during elution to achieve optimum resolution of peaks. Wavelength settings for the first sample injection were chosen to detect anthracene (Anth), pyrene (Pyr), chrysene (Chry), benzo[k]fluoranthene (B[k]F), benzo[alpyrene (B[a]P), and benzo[ghi]perylene (B[ghi]P). For the second injection conditions were changed to detect fluoranthene (Fn) and perylene (Per). LEX,
1st injection 2nd injection
nm 262 300 288 407
nm
elution time, min
390 430 465 430
0-12.5 212.5 0-12.5 >12.5
hEM,
Recovery. A solution in methanol containing known quantities of the eight PAH was mixed with clam tissue at the beginning of sample workup. A comparison of the initial and measured concentrations gave an estimate of the recovery through the total analytical procedure. Most of the clams contained PAH a t concentrations close to the detection limits so that standard additions at similar levels would not permit the estimation of recovery values with adequate precision. Similar additions were made at higher levels, for example, 1.0-2.0 yg/kg of B[a]P compared to
