Environ. Sci. Technol. 1999, 33, 406-409
Biomonitoring of PAH Pollution in High-Altitude Mountain Lakes through the Analysis of Fish Bile E S T E F A N I A E S C A R T IÄ N A N D CINTA PORTE* Environmental Chemistry Department, CID-CSIC, Jordi Girona 18, 08034 Barcelona, Spain
Polycyclic aromatic hydrocarbon (PAH) exposure of fish in high-altitude mountain lakes was assessed by measuring bile PAH metabolites. Trout were caught in several regions in Europe, and hydrolyzed bile samples were analyzed by (a) HPLC fluorescence at the excitation/ emission wavelength pairs of naphthol (290/335 nm) and pyrenol (345/395 nm) and (b) gas chromatography-mass spectrometry (GC-MS) for the determination of individual PAHs. The obtained results showed a good correlation between both detection techniques and showed the usefulness of the first one as a screening method. Quantitative differences among lakes were recorded; biliary levels of hydroxylated PAHs ranged from 69 ng/mL bile in trouts from Redo´ Lake (Spanish Pyrenees) to 990 ng/mL bile in those sampled in Bedøichov Lake (Czech Jizera Mountains). Qualitative differences were also evident, e.g., 1-pyrenol represented 76% of PAH metabolites detected in trouts from Gossenko¨ llesee Lake (Austrian Alps) whereas it was undetected in fish from Redo´ Lake. The obtained results confirm the long-range transport of PAHs to mountain lakes and subsequent exposure of organisms inhabiting those lakes.
Introduction Historically, the atmosphere has been a source of anthropogenic compounds to surface waters with a net flux from the atmosphere to large lakes and oceans (1). Hydrophobic organic chemicals, such as polycyclic aromatic hydrocarbons (PAHs) or polychlorobiphenyls (PCBs), are transported long distances in the atmosphere, and they enter surface waters via wet and dry deposition. These atmospheric fluxes often dominate pollutant inputs to remote lakes (2). Remotely situated mountain lakes are therefore excellent indicators of air pollution and its effects because they are not influenced by other forms of disturbance (e.g., land-use or wastewater pollution). Moreover, due to climatic and geographical factors, high mountain lakes may be more vulnerable to any input than lakes in lowland areas. Similarly, fish inhabiting those lakes will be more vulnerable to pollutants because low temperatures cause low growth rates, which may result in a higher concentration of contaminants (3). Although a certain amount of research has been carried out in some European mountain areas concerning the analysis of organic pollutants in different abiotic compartments (4-7), limited or no information is available concerning bioaccumulation of pollutants by organisms inhabiting those lakes (8). * Corresponding author phone: 34 93 4006175; fax: 34 93 2045904; e-mail:
[email protected]. 406
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 33, NO. 3, 1999
PAHs exposure in aquatic organisms is often assessed by measuring the concentration of PAHs in their tissues. However, fish caught at highly polluted sites often showed only trace levels in the tissue, due to its ability of metabolizing PAHs (9). Thus, alternative techniques have been developed in order to assess PAHs exposure in fish, viz., the determination of PAHs excreted through the bile as conjugated metabolites. Laboratory studies have demonstrated that the presence of PAH metabolites in bile is well correlated with levels of exposure (10-14), and this trend has been corroborated in a number of field studies (15-17). This paper will select trout from five high-altitude mountain lakes located all over Europe to better understand the degree of exposure to PAHs of fish inhabiting those lakes. To this end, two different methodologies have been applied and compared in order to assess levels of PAH metabolites in hydrolyzed fish bile.
Materials and Methods Sample Collection and Preparation. Brown trout (Salmo trutta), brook trout (Salvelinus fontinalis), and arctic char (Salvelinus alpinus) were sampled during the summer period (1997) from five mountain lakes located all over Europe, namely, Øvre Neadalsvatn (Norwegian Mountains), Gossenko¨llesee (Austrian Alps), Bedøichov (Czech Jizera Mountains, Black Triangle), Aube´ (French Pyrenees), and Redo´ (Spanish Pyrenees) (Figure 1). Fish were killed by severing the spinal cord, and the gall bladder was dissected and stored in dark glass vials at -20 °C. The main characteristics of the lakes and fish samples analyzed are given in Table 1. Hydrolysis of Bile and Extraction of Metabolites. Bile samples were analyzed individually, and conjugated PAHs metabolites were hydrolyzed by a modification of the method of Krahn et al. (18). Briefly, 100 µL of bile was treated with 1 mL of 0.4 M acetic acid/sodium acetate buffer, at pH 5.0, containing 2000 units of β-glucuronidase and 50 units of sulfatase and incubated for 2 h at 40 °C. Hydrolyzed metabolites were extracted with 1 mL of ethyl acetate (×3); the extracts were recombined and reduced to 100 µL under nitrogen. An aliquot of this extract (10-20 µL) was analyzed by HPLC, and the rest was analyzed by gas chromatographymass spectrometry-electron impact mode (GC-MS-EI). Glutathione conjugates remaining in the water phase were recovered by acidic hydrolysis (14); the aqueous phase was treated with 0.1 N HCl until pH 1.0 and was extracted with ethyl acetate (×3). The combined extracts were concentrated under nitrogen to 100 µL and injected onto the HPLC system. Recovery of the extraction procedure was higher than 90% for all the compounds examined (1-naphthol, 2-phenylphenol, 9-fluorenol, and 9-phenanthrol) except for 1-pyrenol, which was 85%. Fluorescent Aromatic Compounds (FACs) in Bile. Hydrolyzed bile samples were analyzed by HPLC with fluorescence detection according to Krahn et al. (19). The analytical column was a 15 × 0.46 cm HCODS, C18, 5 µM (Perkin-Elmer), fitted with 10 × 4 mm guard cartridges of Hypersil PAH (Shandon HPLC). The column was coupled with a Kontron Instruments SFM 25 fluorescence detector. The linear gradient used was 100% water/acetic acid (5 µL/ L) to 100% methanol in 15 min at flow rate of 1 mL/min. Hydrolyzed bile samples (10 µL) were injected directly into the liquid chromatographic system, and the chromatograms were recorded at the excitation/emission wavelength pairs of 1-naphthol (290/335 nm) and 1-pyrenol (345/395 nm). Integrated peak areas eluting after 7.5 min (after peak tail of tryptophane) in the HPLC chromatograms were summed 10.1021/es980798a CCC: $18.00
1999 American Chemical Society Published on Web 12/30/1998
FIGURE 1. Location of the studied lakes. and quantified as naphthol and pyrenol equivalents, respectively. Detection limitsscalculated as signal-to-noise ratio 3:1swere 0.7 ng for 1-naphthol and 0.3 pg for 1-pyrenol. Analysis by GC-MS-EI. Individual quantification of PAHs metabolites was achieved by GC-MS-EI, using a Fisons GC 8000 series chromatograph interfaced to a Fisons MD800 mass spectrometer. The column, a 30 m × 0.25 mm i.d. HP5MS cross-linked 5% PH ME siloxane (Hewlett-Packard), was programmed from 80 to 120 °C at 15 °C/min and from 120 °C to 300 °C at 6 °C/min, holding the final temperature for 5 min. The carrier gas was helium at 80 kPa. The injector temperature was 250 °C, and the ion source and the analyzer were maintained at 200 and 250 °C, respectively. The mass spectra were obtained at 70 eV by selected ion register (SIR) mode. Metabolites were identified and quantified by comparison of retention times and spectra of reference compounds. Ions used for monitoring were as follows: m/z 144,115 for 1-naphthol; m/z 170,141 for 2-phenylphenol; m/z 182,152 for 9-fluorenol; m/z 194,165 for 9-phenanthrol, and m/z 218,189 for 1-pyrenol. 2,6-Dibromophenol (m/z 252,250) and hexamethylbenzene (m/z 162,147) were used as a surrogate standard and a GC internal standard, respectively, and their recoveries were higher than 95%. Detection limits of the GC-MS-EI techniquescalculated as signal-tonoise ratio 3:1swere at the low picogram level (4-9 pg), except for 1-naphthol (95 pg) and 1-pyrenol (68 pg). Measurement of Bile Proteins. Total biliary proteins were measured by the method of Lowry et al. (20), using bovine serum albumin as a standard.
Results HPLC chromatograms of enzymatically hydrolyzed bile recorded at the excitation/emission wavelength pairs of 1-naphthol (290/335 nm) showed a complex mixture of fluorescent compounds. Areas of peaks eluting after 7.5 min in the chromatograms were integrated, summed, and quantified as naphthol equivalents (Table 2). Trouts from Bedøichov Lake presented the highest levels of FACs in terms of naphthol equivalents (243 µg/mL bile), followed by those from Øvre Neadalsvatn and Redo´ Lakes (153 and 115 µg/mL bile, respectively), whereas the lowest levels were recorded
in trouts from Gossenko¨llesee and Aube´ Lakes (75 and 82 µg/mL bile, respectively). Conversely, when hydrolyzed bile samples were analyzed at the fluorescence excitation/ emission wavelengths of pyrenol (345/395 nm), 1-pyrenol was the major peak detected in the chromatogram and the only one quantified (Table 2). Trouts from Bedøichov Lake presented again the highest levels of FACs (463 ng/mL bile); intermediate levels were recorded in fish from Gossenko¨llesee, Øvre Neadalsvatn, and Aube´ Lakes (114 to 229 ng/mL, respectively); and very low levels were detected in organisms from Redo´ Lake (13 ng/mL bile). Samples from acidic hydrolysis, which corresponded to glutathione conjugates, did not give any fluorescence at the wavelength pairs tested (naphthol and pyrenol); thus, the metabolized PAHs appear to be excreted primarily via glucuronidation or sulfatation in trout. The different concentrations detected in terms of naphthol or pyrenol equivalents are indicative of the presence of different mixtures of PAH metabolites in fish bile and hence different patterns of exposure. This fact was further investigated by analyzing hydrolyzed bile samples by GC-MS-EI (Table 2). In agreement with FACs data, trout from Bedøichov Lake presented the highest levels of total identified PAH metabolites (991 ng/mL bile) followed by those from Øvre Neadalsvatn Lake (454 ng/mL bile). Intermediate levels were observed in fish from Gossenko¨llesee and Aube´ Lakes (196 and 169 ng/mL), and the lowest concentration of PAHs was detected in trouts from Redo´ Lake (69 ng/mL). Looking at the metabolite profiles, 9-phenanthrol and 1-pyrenol were the major metabolites detected in trout from Øvre Neadalsvatn Lake, which represented 35% and 45% of total PAH metabolites, respectively. A similar distribution was found in trouts from Bedøichov Lake, where 9-phenanthrol and 1-pyrenol respectively represented also 38% and 46% of the detected metabolites. Conversely, 1-pyrenol was the predominant metabolite in trouts from Gossenko¨llesee Lake; it represented 76% of the total PAH metabolites. Trout from the Pyrenees lakes (Aube´ and Redo´) were characterized by a relative enrichment in low molecular weight PAHs.
Discussion Levels of hydroxylated PAHs in bile detected by HPLC fluorescence (290/335 nm) and quantified as naphthol equivalents are in good agreement with the sum of concentrations of 1-naphthol and 2-phenylphenol determined by GC-MS-EI (r 2 ) 0.78; n ) 18) (Figure 2). 2-Phenylphenol fluoresces at the same excitation/emission wavelength as naphthol, and it is consequently quantified among the unresolved mixture of compounds detected by fluorescence. Similarly, bile residues of 1-pyrenol determined by HPLC (345/395 nm) showed a good correlation with the amount of 1-pyrenol determined by GC-MS-EI (r 2 ) 0.89; n ) 18) (Figure 2). These results together with the elevated sensitivity of the fluorescence detection highlight the usefulness of the HPLC technique as a first screening method. The GC-MS technique, although it can provide more information about which particular metabolites are present in the hydrolyzed sample, is limited by the few metabolite standards available and by the need to use derivatization techniques. Attempts were made to silanize samples and standards, but the sensitivity and accuracy of the method was too low to work with field samples with low concentrations of PAH metabolites. Considering that hydroxylated PAHs in fish bile reflect levels of exposure, the obtained quantitative results showed that Bedøichov Lake (Jizera Mountains, Black Triangle) suffers the highest PAH contamination, whereas Redo ´ Lake is among the less polluted. This is in agreement with previous data on sediments from several remote lakes in Europe that showed the highest concentration of PAHs in Central Alps and Tatra VOL. 33, NO. 3, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
407
TABLE 1. Geographical and Biological Data of Studied Lakes and Fisha altitude lake area (m) (ha)
lake
situation
Øvre Neadalsvatn Bedøichov Gossenko¨ llesee Aube´ Redo´
62°46′ N/9°0′ E 50°33′ N/13°29′ E 47°13′ N/11°5′ E 42°44′ N/1°20′ E 42°38′ N/0°46′ E
a
728 775 2413 2091 2240
50 37 8.6 24
fish species
weight (g)
length (nm)
CF (g/cm3)
bile proteins (mg/mL)
Salmo trutta Salvelinus fontinalis Salmo trutta Salvelinus alpinus Salmo trutta
318 ( 41 171 ( 39 164 ( 19 298 ( 22 222 ( 9
295 ( 15 252 ( 18 250 ( 9 298 ( 8 288 ( 9
1.24 ( 0.02 1.01 ( 0.02 1.02 ( 0.04 1.12 ( 0.03 0.85 ( 0.03
3.7 ( 0.2 6.9 ( 1.5 1.6 ( 0.1 5.0 ( 1.2 18.0 ( 1.9
CF is the condition factor calculated as [weight/(length)3] × 100; N ) 4 specimens analyzed per lake. Values are mean ( SEM.
TABLE 2. Biliary Levels of Hydroxylated PAHs in Trout from Five Remote Lakes in Europe Determined by (a) HPLC Fluorescence and Expressed as Naphthol (µg/mL) or Pyrenol (ng/mL) Equivalents and (b) by GC-MS-EI SIR Mode (ng/mL)a Øvre Neadalsvatn 152.7 ( 12.8 229.0 ( 60.1
Bedøichov 243.0 ( 59.4 463.5 ( 75.5
Gossenko1 llesee 75.1 ( 12.9 158.9 ( 68.2
Aube´ 82.3 ( 12.6 114.0 ( 33.9
Redo´ 114.6 ( 40.9 12.6 ( 10.4
1-naphthol 2-phenylphenol 9-fluorenol 9-phenanthrol 1-pyrenol
18.7 ( 10.5 55.7 ( 19.2 12.9 ( 5.4 148.9 ( 3.7 218.3 ( 96.1
50.0 ( 19.3 70.4 ( 45.6 42.6 ( 15.0 362.0 ( 80.7 465.8 ( 141.4
9.8 ( 3.0 25.2 ( 3.4 7.0 ( 2.1 nd 154.2 ( 45.1
32.6 ( 21.7 20.6 ( 9.2 51.0 ( 31.7 nd 64.6 ( 7.3
10.3 ( 2.0 28.6 ( 1.3 30.2 ( 10.1 nd nd
ΣPAHs (ng/mL) ΣPAHs (ng/mg protein)
454.5 ( 19.7 122.2 ( 22.2
990.8 ( 34.2 158.2 ( 24.8
196.2 ( 9.1 127.6 ( 20.5
168.8 ( 8.0 49.0 ( 23.3
69.1 ( 2.1 4.1 ( 0.6
FACs (naphthol equiv) FACs (pyrenol equiv)
a
nd, not detected. Values are means ( SEM (n ) 4).
Øvre Neadalsvatn (Norway) Lakes showed high levels of hydroxylated PAHs and similar PAH patterns, dominated by the presence of pyrenol and phenanthrol. Both lakes are located below an 800 m altitude, but whereas Øvre Neadalsvatn is a remote lake, Bedøichov receives water flows from the river EÅ erna´ Desna´, thus anthropogenic local inputs cannot be discarded. Moreover, trout from Bedøichov Lake probably reflect the influence of airborne pollution from the Black Triangle, a fairly polluted area located on the border of the Czech Republic, Poland, and Germany. Concerning the Øvre Neadalsvatn, Rose (4) studied the deposition enhancement of carbonaceous particles in its sediments, and he suggested that these particles were most probably from the United Kingdom; thus, airborne pollutants may have a similar origin.
FIGURE 2. Correlation between (A) levels of hydroxylated PAHs in fish bile detected by HPLC fluorescence (290/335 nm) and quantified as naphthol equivalents vs the sum of 1-naphthol and 2-phenylphenol determined by GC-MS-EI and (B) levels of pyrenol detected by HPLC fluorescence (345/395 nm) vs concentration of pyrenol measured by GC-MS-EI. Mountains (Black Triangle) and a significant decrease when moving away to more peripheral areas (e.g., the Iberian Peninsula) (21). Given the fact that the studied lakes are all located far from urban areas, the hydroxylated PAHs pattern detected in fish bile may be a consequence of atmospheric transport of PAHs. Organisms from Bedøichov (Czech Republic) and 408
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 33, NO. 3, 1999
Apart from the influence of regional sources of pollution, long-range atmospheric transport and deposition of PAHs cannot be ruled out, particularly in remote lakes. Several studies have demonstrated that chemicals emitted in low latitudes may be transported to higher latitudes as part of the moving air mass, where due to cooler temperatures they condense (22, 23). The atmospheric transport will be affected by the physicochemical properties of the compounds, and any persistent chemical with a vapor pressure in the range 0.001-0.1 Pa may actually show higher concentrations in arctic ecosystems than in temperate environments (23). In the present study, the more volatile compounds naphthol and phenylphenol (vapor pressure 10.9 and 5.5 Pa at 25 °C) were detected in fish bile from all the lakes, and differences in concentration among lakes were not striking. Due to its elevated vapor pressure, these compounds can be easily transported through the atmosphere for long distances. Conversely, stronger differences among lakes were detected in terms of relative exposure to less volatile compounds (phenanthrol and pyrenol; vapor pressure 0.018 and 0.0009, respectively), with higher levels in northern lakes and very low concentration in Aube´ and Redo´ Lakes (French and Spanish Pyrenees). For comparison purposes, Salmo trutta from a fish farm located in the pre-Pyrenees area at an altitude of 700 m were analyzed. These organisms showed a concentration of hydroxylated PAHs in bile of 1422 ng/mL, 20fold higher than those from Redo´ Lake with 1-pyrenol being
the most abundant metabolite detected (76-83% of total), which indicates the existence of local sources of PAHs. Apart from atmospheric inputs, many other factors can contribute to the distribution of contaminants in remote lakes and the subsequent detection in fish bile, viz., properties of terrestrial catchments, food web characteristics, etc. Among others, the feeding status of fish could certainly affect levels of biliary metabolites (10, 24), and consequently, methods to establish this status should be developed in order to improve the accuracy of the PAHs analysis. In this study, we have calculated the condition factor (CF) of the analyzed organisms as a general measure of their nutritional status (25). Differences among lakes were observed in terms of the calculated CF (Table 1); however, no clear relationship among levels of PAHs in fish bile and CF could be established. In addition, levels of protein per milliliter of bile were determined (Table 1), as it has been observed that the amount of proteins increases markedly in nonfeeding fish (10). Organisms from Redo´ Lake showed an extremely elevated concentration of proteins in bile in comparison with fish from other lakes and showed the lowest condition factor, which suggest that they were in a bad nutritional status. When the concentration of proteins was used to normalize residues of hydroxylated PAHs, the observed concentrations did not differ strongly from data expressed per milliliter of bile (Table 2). It is possible to clearly distinguish two groups: (a) fish sampled in Øvre Neadalsvatn, Gossenko¨llesee, and Bedøichov Lakes with very similar levels of hydroxylated PAHs, ranging from 122 to 158 ng/mg protein; (b) fish sampled in Aube´ (49 ( 23 ng/mg protein) and Redo´ Lake (4.1 ( 0.6 ng/mg protein) with a significantly lower concentration of PAHs. Overall, levels of hydroxylated PAHs detected in trout from mountain lakes are in the range of those found in Mullus barbatus (90-1600 ng/mL bile), a benthic coastal fish sampled from different stations along the NW Mediterranean and directly affected by anthropogenic activities. Despite the number of studied lakes and fish being rather small, the obtained results could be understood as a first indication that fish from high-altitude mountain lakes in Europe may be exposed to similar levels of PAHs than fish from coastal areas. This fact certainly needs further research given the high susceptibility/fragility of these ecosystems (3). Moreover, future improvements in analytical instrumentation would hopefully lead to the detection of a greater number of metabolites and a better characterization of the atmospheric inputs.
Acknowledgments The authors acknowledge Dr. J. Grimalt, Dr. P. Ferna´ndez, Dr. R. Lackner, Dr. J. C. Massabuau, and Dr. E. Stuchlik as well as other partners of EC Project MOLAR (ENV4-CT950007) for kindly providing the samples and information on lakes and fish characteristics. This work was supported by
the Spanish National Plan for Research (PLANYCIT) under Project Refs. AMB96-0926 and AMB97-1800-CE.
Literature Cited (1) Baker, J. E.; Eisenreich, S. J. Environ. Sci. Technol. 1990, 24, 342-352. (2) Swackhamer, D. L.; Hites, R. Environ. Sci. Technol. 1988, 22, 543-548. (3) Schindler, S. W.; Kidd, K. A.; Muir, D. C. G.; Lockhart, W. L. Sci. Total Environ. 1995, 160/161, 1-17. (4) Rose, N. L. Sci. Total Environ. 1995, 160/161, 487-496. (5) Ferna´ndez, P.; Vilanova, R.; Grimalt, J. O. Polycyclic Aromat. Compd. 1996, 9, 121-128. (6) Vartiainen, T.; Nannio, J.; Korhonen, M.; Kinnunen, K.; Strandman, T. Chemosphere 1997, 34, 1341-1350. (7) Vilanova, R.; Ferna´ndez, P.; Grimalt, J. O. In Sea-Air Exchange: Processes and Modelling; Pacyma, J. M., Broman, D., Lipiatou, E., Eds.; Office for Official Publications of the European Communities: Luxembourg, 1998; pp 209-215. (8) Sa´nchez, J.; Sole´, M.; Albaige´s, J. Int. J. Environ. Anal. Chem. 1993, 50, 269-284. (9) Varanasi, U.; Stein, J. E.; Nishimoto, M. In Metabolism of Polycyclic Aromatic Hydrocarbon in the Aquatic Environment; Varanasi, U., Ed.; CRC Uniscience Series; CRC Press: Boca Raton, FL, 1989; pp 93-150. (10) Collier, T. K.; Varanasi, U. Arch. Environ. Contam. Toxicol. 1991, 20, 462-473. (11) Britvic, S.; Lucic, D.; Kurelec, B. Environ. Toxicol. Chem. 1993, 12, 765-773. (12) Upshall, C.; Payne, J. F.; Hellou, J. Environ. Toxicol. Chem. 1993, 12, 2105-2112. (13) van der Oost, R.; van Schooten, F. J.; Ariese, F.; Heida, H.; Satumalay, K.; Vermeulen, N. P. E. Environ. Toxicol. Chem. 1994, 13, 859-870. (14) Yu, Y.; Wade, T. L.; Fang, J.; McDonald, S.; Brooks, J. M. Arch. Environ. Contam. Toxicol. 1995, 29, 241-246. (15) Krahn, M.; Burrows, D. G.; Ylitalo, G. M.; Brown, D. W.; Wigren, C. A.; Collier, T. K.; Chan, S. L.; Varanasi, U. Environ. Sci. Tecnol. 1992, 26, 116-126. (16) Krahn, M.; Ylitalo, G. M.; Buzitis, J.; Bolton, J. L. Mar. Pollut. Bull. 1993, 27, 285-292. (17) McDonald, S. J.; Kennicutt, M. C., II; Liu, H.; Safe, S. H. Arch. Environ. Contam. Toxicol. 1995, 29, 232-240. (18) Krahn, M.; Burrows, D. G.; MacLeod, W. D., Jr.; Malins, D. C. Arch. Environ. Contam. Toxicol. 1987, 16, 511-522. (19) Krahn, M.; Myers, M. S.; Burrows, D. G.; Malins, D. C. Xenobiotica 1984, 14, 633-646. (20) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265-275. (21) European Community’s Acidification of Mountain Lakes: Paleaeolimnology and Ecology (AL:PE) Programme, 1995. (22) Wania, F.; Mackay, D. Ambio 1993, 22, 10-18. (23) Mackay, D.; Wania, F. Sci. Total Environ. 1995, 160/161, 25-38. (24) Brumley, C. M.; Haritos, V. S.; Ahokas, J. T.; Holdway, D. A. Ecotoxicol. Environ. Saf. 1998. 39, 147-153. (25) Bagenal, T. B.; Tesch, F. W. In Age and Grow; Bagenal T. B., Ed.; Blackwell Scientific Publications: Oxford, 1978; pp 101-136.
Received for review August 6, 1998. Revised manuscript received November 9, 1998. Accepted November 9, 1998. ES980798A
VOL. 33, NO. 3, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
409
