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Microbial ecotoxicity and mutagenicity of 1-hydroxypyrene and its photoproducts
Chemosphere 45 (2001) 445±451
www.elsevier.com/locate/chemosphere
Microbial ecotoxicity and mutagenicity of 1-hydroxypyrene and its photoproducts Huey-Min Hwang
a,*
, Xiaochun Shi a, Isi Ero a, Amalee Jayasinghe a, Shiming Dong b, Hongtao Yu b
a b
Department of Biology, Jackson State University, Jackson, MS 39217, USA Department of Chemistry, Jackson State University, Jackson, MS 39217, USA Received 31 October 2000; accepted 8 January 2001
Abstract 1-Hydroxypyrene (1-HP) is a carcinogenic and slightly water-soluble polycyclic aromatic hydrocarbon. Ecotoxicity and mutagenicity of 1-HP and its photoproducts, and the eect of Mn2 and Cu2 on their mutagenicity were measured with microbial assay in this study. The assay includes spread plate counting, direct counting, microbial mineralization of 14 C-UL-D -glucose and Mutatoxâ Test. At the concentration examined (0.8 lM), the photoproducts (after 1.5 h solar irradiation) of 1-HP inhibited microbial glucose mineralization activity (by 64%) after microbial assemblages of a local reservoir site were exposed for 1 day. However, heterotrophic bacteria were able to utilize 1-HP photoproducts as the growth substrates and increase viability counts by up to 4.75-folds. 1-HP exhibited positive response to Mutatox Test in both direct medium and S-9 medium, with the lowest observable eective concentration of 0.625 lM in the test with direct medium. After photolysis, 1-HP decreased its mutagenicity. Mn2 (312.5 lM±5 mM) and Cu2 (6.25±100 lM) themselves are not mutagenic. However, addition of the metal ions before or after photolysis modi®es the light readings of 1-HP during the test. Therefore, presence of metal ions could aect the genotoxicity of 1-HP in aquatic environments, depending on timing of the addition. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: 1-Hydroxypyrene; Ecotoxicity; Mutagenicity; Photoproducts
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) enter natural environments from a variety of sources, such as forest ®res, volcanic eruption, incomplete combustion of fuel and other materials, industrial euents and seepage or spillage of petrochemical products (Hites et al., 1980; Grimmer and Pott, 1983; Johnson et al., 1985; Manahan, 2000). PAHs are toxic and some of them are known carcinogens and/or mutagens (IARC, 1983; UDHHS, 1995). They are also listed as US Environmental Protection Agency priority pollutants (Keith and Telliard, *
Corresponding author. Fax: +1-601-979-2778. E-mail address: [email protected] (H.-M. Hwang).
1979). Photolysis transforms some organic pollutants to less toxic or harmless products. However, in the presence of natural or simulated sunlight, some PAHs become more toxic to aquatic organisms at concentrations that are less than their aqueous solubility (Landrum et al., 1987; McConkey et al., 1997; Hatch and Burton, 1998). This can occur via photosensitization reactions (e.g., production of singlet oxygen) (Ankley et al., 1994) and/or photooxidation of the compounds to more toxic products such as diols and quinones (David and Boule, 1993; Huang et al., 1993). Since the water solubility of PAHs usually increases after photolysis, aquatic organisms may be exposed to higher concentrations of the photoproducts than the parent PAHs. Therefore, the photo-induced toxicity can present a greater risk to aquatic organisms and ultimately humans, because of
0045-6535/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 1 ) 0 0 0 4 6 - 7
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direct exposure and/or bioaccumulation through food chains. Biological monitoring of exposure to PAHs has been mostly performed by measuring metabolites of PAHs in urine. 1-Hydroxypyrene (1-HP), a major metabolite of pyrene, has been proposed and used as a biological marker for monitoring occupational and environmental exposure to PAHs (Jongeneelen et al., 1986; Jongeneelen, 1994; Angerer et al., 1997; Merlo et al., 1998). Recently, 1-HP was reported to be acutely toxic and genotoxic (Hauser et al., 1997) and appeared to be more toxic to the test organisms than its parent compound ± pyrene (Lambert et al., 1995). Under UVA irradiation, 1-HP has been shown to cause light-induced DNA single strand cleavage and form covalent adducts with the supercoiled plasmid phage DNA (Dong et al., 2000a,b). Toxic metals are generated from both natural and anthropogenic sources and become common contaminants in natural waters (Moore and Ramamoorthy, 1984). Their presence may signi®cantly aect potentially important microbial transformation of organic chemicals in natural environments (Said and Lewis, 1991; Kuo and Genthner, 1996). Complexation of metal ions by organic ligands is a subject of great environmental relevance, because the availability and toxicity of both metals and organic chemicals depend on their speciation (Sunda and Huntsman, 1995; Xue and Sigg, 1999). Manganese is a major constituent of sediments and copper is an essential trace element to biota. They are common component in discharges from mining activities, smelting and plating operations, and other industrial processes. More than 51.4% and 74.4% of the natural waters in the US contained elevated level of manganese and copper, respectively (Manahan, 2000). Mn and Cu have been reported to exist in mine drainages and associated receiving sediment±pore waters at concentrations up to 75 mg/l (Brumbaugh et al., 1994; Lasier et al., 2000; Markwiese and Colberg, 2000). Therefore, these two metal ions were added during the Mutatoxâ Test to study the eect of their co-occurrence on 1-HP mutagenicity. Bacterial in vitro assays have emerged as important ecotoxicological screening tools to monitor the hazards of chemical contaminants in natural environment. In this study, the possible photo-induced genotoxicity and ecotoxicity of 1-HP were measured with a variety of bacterial bioassays, including viable bacteria counting, bacterial direct counting, heterotrophic mineralization of glucose and the Mutatoxâ Test. The Mutatoxâ Test determines the genotoxicity of chemical contaminants in liquid phase from environmental samples by measuring the changes of light emitted by a dark mutant strain of bioluminescent bacteria Vibrio ®scheri strain M169. The test has been used as a rapid screening tool for detecting the mutagenicity of DNA-damaging substances (Sun and Stahr, 1993; Johnson, 1998). Glucose mineralization
re¯ects bacterial heterotrophic activities, while spread plate counting and direct counting measure viability of heterotrophic bacterial populations and total number of bacterial populations, respectively (Hwang et al., 1998). The objectives of this study are: (1) To compare the contribution of photolysis and microbial degradation to 1-HP transformation in short term incubation; (2) To determine the ecotoxicity and mutagenicity of 1-HP and its photoproducts with microbial bioassay; and (3) To assess the eect of Mn2 and Cu2 on the mutagenicity of 1-HP and its photoproducts.
2. Experimental 2.1. Sampling and chemical analysis Samples of surface freshwaters were collected from Ross Barnett Reservoir, Ridgeland, MS. The pH of the water samples ranged from 7.0 to 7.5, and the temperature ranged from 15°C to 28°C. All chemicals used were of analytical or HPLC grade. 1-HP (Aldrich Chemical, Milwaukee, WI) was dissolved in dimethyl sulfoxide (®nal DMSO concentration 4%; Fisher Scienti®c) with sodium phosphate buer (1 mM, pH 7.0), added to 50 ml of water sample in 150 ml quartz ¯asks (Quartz Scienti®c, Fairport Harbor, OH) and incubated in triplicate. Degradation of 1-HP was measured by quantifying disappearance of the parent compound (transformation rate). The water samples containing 1HP were exposed to irradiation from a UVA lamp (Dong et al., 2000a) indoors or solar source outdoors. At the termination of incubation, aliquots of water samples were ®ltered (0.45 lm; Schleicher and Schuell, Keene, NH) and the quanti®cation was performed by using a Waters 996 HPLC system. The system is equipped with a photodiode array detector and a reverse-phase Supelcosil LC-8 column (Supelco, Bellefonte, PA), under isocratic conditions with acetonitrile± water (50:50, v/v) at a ¯ow rate of 1 ml/min. Detection wavelength was set at 254 nm. The ®nal concentration of 1-HP ranged from 0.8±10 lM. The quartz ¯asks allowed 85% and 100% transmission of light at wavelength of 285 and P300 nm, respectively. For outdoor incubation, the ¯asks were suspended in an outdoor tub which contained continuous running water for maintaining the water temperature at 27 3°C. The water level in the ¯ask was about 3 cm below the surface of the cooling water. A research radiometer (model IL 1700, International Light, Newburyport, MA) was placed beside the tub to measure UV irradiance. The dark exposure group consisted of ¯asks being wrapped with aluminum foil. Killed control was accomplished by autoclaving at 121.1°C at 15 psi for 20 min. All bottles were capped with silicon stoppers.
H.-M. Hwang et al. / Chemosphere 45 (2001) 445±451
Dierence in experimental data between dierent treatment groups was determined with student-t-test (P 6 0:05). 2.2. Toxicity measurement In a separate experiment, the ecotoxic eects of 1-HP and its photoproducts on bacterial assemblages in reservoir water was measured with spread plate counting (nutrient agar) and heterotrophic mineralization of 14 CUL±D -glucose (S.A.: 265 mCi/mmol; 99% purity; Moravek Biochemicals, Brea, CA). 1-HP was added to 25 ml of autoclaved water in quartz ¯asks to make the ®nal concentration 1.6 lM. For outdoor experiments, all irradiated but one water samples containing 1-HP were exposed to midday solar irradiation for 1.5 h. The group of 1-HP photoproducts was constructed after 3 days of irradiation. The presence of the parent compound and/ or the photoproducts were con®rmed by use of HPLC. An equal amount of unamended freshwater was then added to the photo-exposed water to make the ®nal 1HP starting concentration 0.8 lM. Water samples were incubated in darkness for 1 day at 25°C in the laboratory. Treatments included: darkness and light controls (no 1-HP), darkness and light exposures (exposure to 1HP in darkness or light). Radiolabeled glucose (®nal concentration 1 lg/l) dissolved in ethanol was added to the water samples after the exposure and incubated at 25°C in darkness for 1 h. At the termination of the incubation, 0.5 ml of 2N H2 SO4 was added to the sample, and the 14 CO2 produced was trapped with 2-phenylethylamine-soaked ®lter papers (Hwang and Maloney, 1996). The radioactivity of the ®lter paper was measured with liquid scintillation spectrometry (Packard Instrument; model TR 1600). Total number and viability of heterotrophic bacterial assemblages were measured with acridine orange direct counting and spread plate counting, respectively (Hwang et al., 1998). Genotoxic eects of 1-HP (10 lM) were measured by use of the Mutatoxâ Test according to the Mutatoxâ genotoxicity test manual (Microbics Mutatox Manual, Microbics Corporation, 1995). Cu2 (100 lM) and Mn2 (5 mM) were added in the form of cupric chloride and manganese sulfate to assess their eect on 1-HP mutagenicity. The mutagenic response of the reconstituted bacteria with serial 2-fold dilutions was determined after 16, 20 and 24 h of incubation at 27°C. Activation with S-9 enzymes received 1 h incubation at 35°C. Light emitted from the media control, solvent (4% DMSO) control, positive controls (phenol for direct acting and bezo[a]pyrene for S-9 acting) and samples receiving dierent treatments were measured. Positive mutagenic agents are de®ned as those samples which induce, increase light levels to at least two times the average control light reading in at least two consecutive sample dilute (concentration) cuvettes from two test
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results (Microbics Mutatox Manual, Microbics Corporation, 1995). Mutatoxâ Test was conducted twice for the same treatment structure and the reading results are reported (Table 2). Because the enzymatic kinetics vary in dierent assays, comparison of the light reading numbers cannot be made across dierent assays. 3. Results and discussion 3.1. Degradation of 1-HP and microbial ecotoxicity test Rates of photochemical and microbial degradation of 1-HP (0.8 lM) were monitored by use of HPLC. Photolysis half-life was computed to compare the relative contribution to degradation by photolysis and microbial activity. It was also used to determine the irradiation periods required for conducting ecotoxicity and mutagenicity measurements. After 90 min 1-HP was transformed by 0±3%, 80% and 84±100% in darkness (i.e., microbial degradation only), solar photolysis only, and combined photolysis and microbial degradation, respectively. Therefore, 1-HP was resistant to microbial degradation and synergistic interaction between photolysis and microbial degradation for 1-HP was observed. After 1 day of exposure to the 90 min photoproducts of 1-HP (i.e., photoproducts generated after 90 min of photolysis under outdoor irradiation), viability counts of heterotrophic bacterial assemblages were signi®cantly enhanced by comparison to the light control group (Fig. 1). The enhancement was greatest (4.75-folds) with exposure to the 1-HP outdoor photoproducts. However, no eect of exposure to 1-HP was observed. Therefore, the net response of the bacterial assemblages is utilizing the mixture of the photoproducts as substrates. A similar case was also reported for photolysis and bacterial transformation of other organic contaminants (Hwang et al., 1998, 2000). Concurrent measurement of the total bacterial numbers indicated the similar trend in response (results not shown). The eect of 1-HP and its photoproducts on bacterial heterotrophic activity of glucose mineralization is shown (Fig. 2). With comparison to the control groups, microbial mineralization activity of D -glucose was inhibited by up to 64% and 75% after exposing to 1-HP photoproducts and 1-HP, respectively. Based on the results (Figs. 1 and 2), we speculate that the inhibition on heterotrophic activity by the photoproducts was due to substrate competition between glucose and the mixtures of 1-HP photoproducts (Hwang et al., 1998, 2000), while 1-HP inhibited bacterial metabolic activity by virtually toxicity. 3.2. Microbial mutagenicity test The eect of 1-HP was determined by use of the Mutatoxâ Test for both direct-acting (in direct growth
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Fig. 1. The eect of 1-HP and its photoproducts on bacterial viability (1 day exposure, November 1999). (I) irradiated by an indoor UVA lamp for 30 min; (O) irradiated by midday sunlight for 90 min. ( ) signi®cantly dierent from the corresponding control group (P 6 0:05; t-test). 1± Time zero; 2 ± dark control; 3 ± 1-HP (I); 4 ± 1-HP (O); 5 ± 1-HP (dark); 6 ± killed control; 7 ± light control.
Fig. 2. The eect of 1-HP and its photoproducts on bacterial mineralization of D -glucose (1 day exposure, November 1999). (I) irradiated by an indoor UVA lamp for 30 min; (O) irradiated by midday sunlight for 90 min. ( ) signi®cantly dierent from the corresponding control group (P 6 0:05; t-test). 1 ± Dark control; 2 ± 1-HP (I); 3 ± 1-HP (O); 4 ± 1-HP (dark); 5 ± killed control; 6 ± light control.
medium) and pro-mutagens (growth medium containing S-9 enzymes). 1-HP exhibited positive responses in both direct medium and S-9 medium. However, after pho-
tolysis 1-HP only scored positive responses to the test in direct medium (Table 1). Therefore, other Mutatoxâ Tests for various treatment structures were conducted with direct medium for comparison. 1-HP was also reported by Hauser et al. (1997) to respond positively in both direct medium and S-9 medium, although there is a dierence in the substrate and solvent concentrations between the two studies; i.e., 1-HP: 1.4±11.5 lM in their system vs 0.0625±0.8 lM in our system; DMSO: 2% in their system vs 4% in our system. Results of the Mutatoxâ Test on 1-HP (10 lM), photoproducts of 1-HP, mixtures of Cu2 , Mn2 and 1-HP or its photoproducts are shown in Table 2. If the test sample concentration is cytotoxic, the luminescent bacteria cannot express any light emission and a zero value of light reading is reported in this study (Microbics Mutatox Manual, Microbics Corporation, 1995). Results indicate that 1HP is mutagenic, with positive responses occurring at a wide concentration range including the lowest concentration examined (i.e., 0.625 lM) (Table 2). Phototransformation by solar irradiation for 1.5 h (1-HP irrad.) or 3 days (1-HP PP) changed the light reading intensity of 1-HP signi®cantly. According to the result of the ®rst experiment (Table 2), the lowest observable effective concentration (LOEC) of 1-HP increased from 0.625 to 1.25 lM after solar irradiation (i.e., 1-HP irrad.). At the same time, the light reading intensity at lesser concentration ranges (i.e., 0.625±2.5 lM) decreased. Therefore, mutagenicity of 1-HP was decreased after solar photo-transformation. The change in mutagenicity could be due to the decrease in 1-HP concentration or the accumulation of 1-HP photoproducts. The environmental importance of complexation derives from the fact that the complexed chemical species behave dierently from the uncomplexed forms, as well as dierently from other complexes. Chemical properties, such as solubility, attenuation behavior on soils, bioconcentration factors, and toxicity are modi®ed through complexation (Lyman, 1995). Some metal ions were reported to cause oxidative DNA damage. For example, copper can induce single-base substitutions. Cu (II) binds preferentially to guanine residues and induces in combination with H2 O2 oxidative damage at sites of two or more adjacent guanine residues (Sagripanti and Kraemer, 1989). In our study, Mn2 (312.5 lM±5 mM) and Cu2 (6.25±100 lM) were not mutagenic (Table 2). However, additions of Mn2 (5 mM) increased the cytotoxicity of 1-HP, 1-HP irradiated sample (i.e., 1-HP irrad.) and 1-HP photoproduct sample (1-HP PP) synergistically at higher concentration range (10±2.5 lM) and changed their light reading at lower concentration range. Addition of Cu2 (100 lM) produced the similar cytotoxic eect. However, no predictable trend was observed in its eect on the light reading of 1-HP, 1-HP irrad. and 1-HP PP samples. Enhancement of metal toxicity by UV-A irradiation was
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Table 1 Mutatoxâ Test of 1-HP and its photoproducts In direct medium In S-9 medium a b
Parent compound
Parent compound and photoproductsa
Photoproductsb
Positive Positive
Positive Negative
Positive Negative
1-HP was irradiated outdoors for 90 min. Presence of the parent compound and photoproducts was veri®ed with HPLC. 1-HP was irradiated outdoors for 3 days. Presence of the photoproducts was veri®ed with HPLC.
Table 2 Mutatoxâ Test reading resultsa Medium control 11(2) 10(3) 16(3) 16(3) 15(4)
Positive control 1215(3485) 11938(6573) 381(11035) 100(2047) 28(999)
1-HP 0(0) 62(2) 12310(44) 719(5694) 171(13112)
Mn2 11(2) 8(0) 6(2) 7(2) 9(2)
1-HP + Mn2 0(0) 0(0) 0(0) 736(329) 233(20000)
(1-HP + Mn2 ) irrad. 0(2) 0(31) 0(49) 88(342) 39(13033)
Cu2 10(0) 8(2) 7(2) 8(2) 10(2)
(1-HP + Cu2 )d (0) (2) (3) (3586) (342)
(1-HP + Cu2 ) irrad. 0(2) 0(3) 631(24) 189(154) 17(89)
d
d
1-HP irrad.b 1483(2) 1652(0) 376(2) 58(882) 8(12293)
1-HP PPc 15(2) 166(2) 249(2) 102(599) 22(883)
(1-HP irrad. + Mn2 )e 0(0) 0(0) 653(12) 333(210) 14(12432)
(1-HP PP + Mn2 )c 0(2) 0(2) 0(2) 5(2) 50(2)
(1-HP irrad. + Cu2 )e 0(2) 0(0) 46(2) 11(2) 5(2)
(1-HP PP + Cu2 )c (2) (0) (2) (756) (3574)
a
The concentrations of the chemicals added (decreasing with 2-fold dilutions from the top to the bottom of each column): 1-HP: 10 lM; Mn2 : 5 mM; Cu2 : 100 lM. All irradiated samples but the group of 1-HP photoproducts were exposed to midday sunlight for 1.5 h before conducting the Mutatoxâ Test in direct medium. Results of the second experiment are given in parenthesis. b 1-HP was irradiated for 1.5 h under midday sunlight. c 1-HP was irradiated for 3 days to generate the mixture of photoproducts (PP). d The mixture of 1-HP and metal ion was irradiated for 1.5 h under midday sunlight. e Metal ion was added after 1-HP was irradiated for 1.5 h under midday sunlight.
reported to mediate through the formation of hydroxyl radicals within the cell or the complexation between DNA and metal ion radicals (Afrsten et al., 1994). However, this theory does not apply to our case because of the short longevity of the radical species in natural water (i.e., ls range; Stumm and Morgan, 1981) and the time lapse between irradiation treatment and execution of the Mutatoxâ Test. (i.e., min). Instead, we speculate that the changes in 1-HP and photoproducts mutagenicity by metal addition are due to the interactions (e.g., complexation) between the metal ions and the organic species of 1-HP and/or its photoproducts.
4. Conclusions Photo-transformation changes the ecotoxicity and mutagenicity of 1-HP to aquatic bacterial assembalges
and V. ®scheri M169 strain used in the Mutatoxâ Test. Our ®nding of bacterial capability of utilizing the test PAHs photoproducts con®rms the potential of using photolysis and biodegradation for remediating PAHs contamination. The modi®ed toxicity of the test PAHs by photolysis and metal ions indicates that the disappearance of PAHs in the environment does not always render them less ecologically hazardous. Close monitoring on the toxic or mutagenic properties of the complexed compounds and intermediates should be a necessary task for health regulatory professions.
Acknowledgements This research was supported by: (1) NIH-RCMI G12RR13459-03 and NIH MBRS S06GM08047 (to JSU); (2) Department of Energy DE-FG02-97ER62451
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to UGA with subcontract #RR100-239/4891914 to JSU; and (3) The Army HPC Research Center under the auspices of the Department of the Army, Army Research Laboratory. The content does not necessarily re¯ect the position or policy of the US government, and no ocial endorsement should be inferred. References Angerer, J., Mannschreck, C., G undel, J., 1997. Occupational exposure to polycylic aromatic hydrocarbons in a graphiteelectrode producing plant: biological monitoring of 1hydroxypyrene and monohydroxylated metabolities of phenanthrene. Int. Arch. Occup. Environ. Health 69, 323±331. Ankley, G.T., Collyard, S.A., Monson, P.D., Kosian, P.A., 1994. In¯uence of ultraviolet light on the toxicity of sediment contaminated with polycyclic aromatic hydrocarbons. Environ. Toxicol. Chem. 13, 1791±1796. Arfsten, D.P., Davenport, R., Shaeer, D.J., 1994. UV-A coexposure enhances the toxity of aromatic hydrocarbons, nutritions, and metals to Photobacterium phosphoreum. Biomed. Environ. Sci. 7, 101±108. Brumbaugh, W.J., Ingersoll, C.G., Kemble, N.E., May, T.W., Zajicek, J.L., 1994. Chemical characterization of sediments and pore water from the upper Clark Fork River and Milltown Reservoir, Montana. Environ. Toxicol. Chem. 13, 1971±1983. David, B., Boule, P., 1993. Phototransformation of hydrophobic pollutants in aqueous media I-PAHs adsorbed on silica. Chemosphere 26, 1617±1630. Dong, S., Hwang, H.-M., Harrison, C., Holloway, L., Shi, X., Yu, H., 2000a. UVA light-induced DNA cleavage by selected polycyclic aromatic hydrocarbons. Bull. Environ. Contam. Toxicol. 64, 467±474. Dong, S., Hwang, H.-M., Shi, X., Holloway, L., Yu, H., 2000b. UVA-induced DNA single-strand cleavage by 1-hydroxypyrene and formation of covalent adducts between DNA and 1-hydroxypyrene. Chem. Res. Toxicol. 13, 585±593. Grimmer, G., Pott, F., 1983. Occurrence of PAHs. In: Grimmer, G. (Ed.), Environmental Carcinogens: Polycyclic Aromatic Hydrocarbons. CRC Press, Boca Raton, FL, pp. 62±128. Hatch, A.C., Burton Jr., G.A., 1998. Eects of photoinduced toxicity of ¯uoranthene on amphibian embryos and larvae. Environ. Toxicol. Chem. 17, 1777±1785. Hauser, B., Schrader, B.H., Bahadir, M., 1997. Comparison of acute toxicity and genotoxicity concentrations of single compounds and waste elutriate using the Microtox/Mutatox Test system. Ecotoxicol. Environ. Safety 38, 227±231. Hites, R.A., La¯amme, R.E., Windsor Jr., J.G., 1980. Polycyclic aromatic hydrocarbons in marine/aquatic sediments: their ubiquity. In: Petrakis, L.F., Weiss, T. (Eds.), Petroleum in The Marine Environment. American Chemical Society, Washington, DC, pp. 289±311. Huang, X.-D., Dixon, D.G., Greenberg, B.M., 1993. Impacts of ultraviolet radiation and photomodi®cation on the toxicity of polycyclic aromatic hydrocarbons to the higher plant Lemna gibba G-3 (Duckweed). Environ. Toxicol. Chem. 12, 1067±1077.
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Stumm, W., Morgan, J.J., 1981. Aquatic Chemistry: An Introduction Emphasizing Chemical Equilibria in Natural Waters, second ed. Wiley, New York. Sun, T.S.C., Stahr, H.M., 1993. Evaluation and application of a bioluminescent bacterial genotoxicity test. J. AOAC Int. 76, 893±898. Sunda, W.G., Huntsman, S.A., 1995. Regulation of copper concentration in the oceanic nutricline by phytoplankton uptake and regeneration cycles. Limnol. Oceanogr. 40, 132± 137. US Department of Health and Human Services, P.H.S., ATSDR 1995. Toxicological Pro®le for Polycyclic Aromatic Hydrocarbons (PAHs), Atlanta, GA. Xue, H., Sigg, L., 1999. Comparison of the complexation of Cu and Cd by humic or fulvic acids and by ligands observed in lake waters. Aqua. Geochem. 5, 313±335. Huey-Min Hwang is a Professor of biology and a microbial ecologist at the Jackson State University. He has been extensively involved in a variety of research projects in the areas of environmental toxicology, aquatic photochemistry and bioremediation. Dr. Hwang has published 20 refereed publications on the fates and eects of various contaminants including polycyclic aromatic hydrocarbons.
www.elsevier.com/locate/chemosphere
Microbial ecotoxicity and mutagenicity of 1-hydroxypyrene and its photoproducts Huey-Min Hwang
a,*
, Xiaochun Shi a, Isi Ero a, Amalee Jayasinghe a, Shiming Dong b, Hongtao Yu b
a b
Department of Biology, Jackson State University, Jackson, MS 39217, USA Department of Chemistry, Jackson State University, Jackson, MS 39217, USA Received 31 October 2000; accepted 8 January 2001
Abstract 1-Hydroxypyrene (1-HP) is a carcinogenic and slightly water-soluble polycyclic aromatic hydrocarbon. Ecotoxicity and mutagenicity of 1-HP and its photoproducts, and the eect of Mn2 and Cu2 on their mutagenicity were measured with microbial assay in this study. The assay includes spread plate counting, direct counting, microbial mineralization of 14 C-UL-D -glucose and Mutatoxâ Test. At the concentration examined (0.8 lM), the photoproducts (after 1.5 h solar irradiation) of 1-HP inhibited microbial glucose mineralization activity (by 64%) after microbial assemblages of a local reservoir site were exposed for 1 day. However, heterotrophic bacteria were able to utilize 1-HP photoproducts as the growth substrates and increase viability counts by up to 4.75-folds. 1-HP exhibited positive response to Mutatox Test in both direct medium and S-9 medium, with the lowest observable eective concentration of 0.625 lM in the test with direct medium. After photolysis, 1-HP decreased its mutagenicity. Mn2 (312.5 lM±5 mM) and Cu2 (6.25±100 lM) themselves are not mutagenic. However, addition of the metal ions before or after photolysis modi®es the light readings of 1-HP during the test. Therefore, presence of metal ions could aect the genotoxicity of 1-HP in aquatic environments, depending on timing of the addition. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: 1-Hydroxypyrene; Ecotoxicity; Mutagenicity; Photoproducts
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) enter natural environments from a variety of sources, such as forest ®res, volcanic eruption, incomplete combustion of fuel and other materials, industrial euents and seepage or spillage of petrochemical products (Hites et al., 1980; Grimmer and Pott, 1983; Johnson et al., 1985; Manahan, 2000). PAHs are toxic and some of them are known carcinogens and/or mutagens (IARC, 1983; UDHHS, 1995). They are also listed as US Environmental Protection Agency priority pollutants (Keith and Telliard, *
Corresponding author. Fax: +1-601-979-2778. E-mail address: [email protected] (H.-M. Hwang).
1979). Photolysis transforms some organic pollutants to less toxic or harmless products. However, in the presence of natural or simulated sunlight, some PAHs become more toxic to aquatic organisms at concentrations that are less than their aqueous solubility (Landrum et al., 1987; McConkey et al., 1997; Hatch and Burton, 1998). This can occur via photosensitization reactions (e.g., production of singlet oxygen) (Ankley et al., 1994) and/or photooxidation of the compounds to more toxic products such as diols and quinones (David and Boule, 1993; Huang et al., 1993). Since the water solubility of PAHs usually increases after photolysis, aquatic organisms may be exposed to higher concentrations of the photoproducts than the parent PAHs. Therefore, the photo-induced toxicity can present a greater risk to aquatic organisms and ultimately humans, because of
0045-6535/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 1 ) 0 0 0 4 6 - 7
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direct exposure and/or bioaccumulation through food chains. Biological monitoring of exposure to PAHs has been mostly performed by measuring metabolites of PAHs in urine. 1-Hydroxypyrene (1-HP), a major metabolite of pyrene, has been proposed and used as a biological marker for monitoring occupational and environmental exposure to PAHs (Jongeneelen et al., 1986; Jongeneelen, 1994; Angerer et al., 1997; Merlo et al., 1998). Recently, 1-HP was reported to be acutely toxic and genotoxic (Hauser et al., 1997) and appeared to be more toxic to the test organisms than its parent compound ± pyrene (Lambert et al., 1995). Under UVA irradiation, 1-HP has been shown to cause light-induced DNA single strand cleavage and form covalent adducts with the supercoiled plasmid phage DNA (Dong et al., 2000a,b). Toxic metals are generated from both natural and anthropogenic sources and become common contaminants in natural waters (Moore and Ramamoorthy, 1984). Their presence may signi®cantly aect potentially important microbial transformation of organic chemicals in natural environments (Said and Lewis, 1991; Kuo and Genthner, 1996). Complexation of metal ions by organic ligands is a subject of great environmental relevance, because the availability and toxicity of both metals and organic chemicals depend on their speciation (Sunda and Huntsman, 1995; Xue and Sigg, 1999). Manganese is a major constituent of sediments and copper is an essential trace element to biota. They are common component in discharges from mining activities, smelting and plating operations, and other industrial processes. More than 51.4% and 74.4% of the natural waters in the US contained elevated level of manganese and copper, respectively (Manahan, 2000). Mn and Cu have been reported to exist in mine drainages and associated receiving sediment±pore waters at concentrations up to 75 mg/l (Brumbaugh et al., 1994; Lasier et al., 2000; Markwiese and Colberg, 2000). Therefore, these two metal ions were added during the Mutatoxâ Test to study the eect of their co-occurrence on 1-HP mutagenicity. Bacterial in vitro assays have emerged as important ecotoxicological screening tools to monitor the hazards of chemical contaminants in natural environment. In this study, the possible photo-induced genotoxicity and ecotoxicity of 1-HP were measured with a variety of bacterial bioassays, including viable bacteria counting, bacterial direct counting, heterotrophic mineralization of glucose and the Mutatoxâ Test. The Mutatoxâ Test determines the genotoxicity of chemical contaminants in liquid phase from environmental samples by measuring the changes of light emitted by a dark mutant strain of bioluminescent bacteria Vibrio ®scheri strain M169. The test has been used as a rapid screening tool for detecting the mutagenicity of DNA-damaging substances (Sun and Stahr, 1993; Johnson, 1998). Glucose mineralization
re¯ects bacterial heterotrophic activities, while spread plate counting and direct counting measure viability of heterotrophic bacterial populations and total number of bacterial populations, respectively (Hwang et al., 1998). The objectives of this study are: (1) To compare the contribution of photolysis and microbial degradation to 1-HP transformation in short term incubation; (2) To determine the ecotoxicity and mutagenicity of 1-HP and its photoproducts with microbial bioassay; and (3) To assess the eect of Mn2 and Cu2 on the mutagenicity of 1-HP and its photoproducts.
2. Experimental 2.1. Sampling and chemical analysis Samples of surface freshwaters were collected from Ross Barnett Reservoir, Ridgeland, MS. The pH of the water samples ranged from 7.0 to 7.5, and the temperature ranged from 15°C to 28°C. All chemicals used were of analytical or HPLC grade. 1-HP (Aldrich Chemical, Milwaukee, WI) was dissolved in dimethyl sulfoxide (®nal DMSO concentration 4%; Fisher Scienti®c) with sodium phosphate buer (1 mM, pH 7.0), added to 50 ml of water sample in 150 ml quartz ¯asks (Quartz Scienti®c, Fairport Harbor, OH) and incubated in triplicate. Degradation of 1-HP was measured by quantifying disappearance of the parent compound (transformation rate). The water samples containing 1HP were exposed to irradiation from a UVA lamp (Dong et al., 2000a) indoors or solar source outdoors. At the termination of incubation, aliquots of water samples were ®ltered (0.45 lm; Schleicher and Schuell, Keene, NH) and the quanti®cation was performed by using a Waters 996 HPLC system. The system is equipped with a photodiode array detector and a reverse-phase Supelcosil LC-8 column (Supelco, Bellefonte, PA), under isocratic conditions with acetonitrile± water (50:50, v/v) at a ¯ow rate of 1 ml/min. Detection wavelength was set at 254 nm. The ®nal concentration of 1-HP ranged from 0.8±10 lM. The quartz ¯asks allowed 85% and 100% transmission of light at wavelength of 285 and P300 nm, respectively. For outdoor incubation, the ¯asks were suspended in an outdoor tub which contained continuous running water for maintaining the water temperature at 27 3°C. The water level in the ¯ask was about 3 cm below the surface of the cooling water. A research radiometer (model IL 1700, International Light, Newburyport, MA) was placed beside the tub to measure UV irradiance. The dark exposure group consisted of ¯asks being wrapped with aluminum foil. Killed control was accomplished by autoclaving at 121.1°C at 15 psi for 20 min. All bottles were capped with silicon stoppers.
H.-M. Hwang et al. / Chemosphere 45 (2001) 445±451
Dierence in experimental data between dierent treatment groups was determined with student-t-test (P 6 0:05). 2.2. Toxicity measurement In a separate experiment, the ecotoxic eects of 1-HP and its photoproducts on bacterial assemblages in reservoir water was measured with spread plate counting (nutrient agar) and heterotrophic mineralization of 14 CUL±D -glucose (S.A.: 265 mCi/mmol; 99% purity; Moravek Biochemicals, Brea, CA). 1-HP was added to 25 ml of autoclaved water in quartz ¯asks to make the ®nal concentration 1.6 lM. For outdoor experiments, all irradiated but one water samples containing 1-HP were exposed to midday solar irradiation for 1.5 h. The group of 1-HP photoproducts was constructed after 3 days of irradiation. The presence of the parent compound and/ or the photoproducts were con®rmed by use of HPLC. An equal amount of unamended freshwater was then added to the photo-exposed water to make the ®nal 1HP starting concentration 0.8 lM. Water samples were incubated in darkness for 1 day at 25°C in the laboratory. Treatments included: darkness and light controls (no 1-HP), darkness and light exposures (exposure to 1HP in darkness or light). Radiolabeled glucose (®nal concentration 1 lg/l) dissolved in ethanol was added to the water samples after the exposure and incubated at 25°C in darkness for 1 h. At the termination of the incubation, 0.5 ml of 2N H2 SO4 was added to the sample, and the 14 CO2 produced was trapped with 2-phenylethylamine-soaked ®lter papers (Hwang and Maloney, 1996). The radioactivity of the ®lter paper was measured with liquid scintillation spectrometry (Packard Instrument; model TR 1600). Total number and viability of heterotrophic bacterial assemblages were measured with acridine orange direct counting and spread plate counting, respectively (Hwang et al., 1998). Genotoxic eects of 1-HP (10 lM) were measured by use of the Mutatoxâ Test according to the Mutatoxâ genotoxicity test manual (Microbics Mutatox Manual, Microbics Corporation, 1995). Cu2 (100 lM) and Mn2 (5 mM) were added in the form of cupric chloride and manganese sulfate to assess their eect on 1-HP mutagenicity. The mutagenic response of the reconstituted bacteria with serial 2-fold dilutions was determined after 16, 20 and 24 h of incubation at 27°C. Activation with S-9 enzymes received 1 h incubation at 35°C. Light emitted from the media control, solvent (4% DMSO) control, positive controls (phenol for direct acting and bezo[a]pyrene for S-9 acting) and samples receiving dierent treatments were measured. Positive mutagenic agents are de®ned as those samples which induce, increase light levels to at least two times the average control light reading in at least two consecutive sample dilute (concentration) cuvettes from two test
447
results (Microbics Mutatox Manual, Microbics Corporation, 1995). Mutatoxâ Test was conducted twice for the same treatment structure and the reading results are reported (Table 2). Because the enzymatic kinetics vary in dierent assays, comparison of the light reading numbers cannot be made across dierent assays. 3. Results and discussion 3.1. Degradation of 1-HP and microbial ecotoxicity test Rates of photochemical and microbial degradation of 1-HP (0.8 lM) were monitored by use of HPLC. Photolysis half-life was computed to compare the relative contribution to degradation by photolysis and microbial activity. It was also used to determine the irradiation periods required for conducting ecotoxicity and mutagenicity measurements. After 90 min 1-HP was transformed by 0±3%, 80% and 84±100% in darkness (i.e., microbial degradation only), solar photolysis only, and combined photolysis and microbial degradation, respectively. Therefore, 1-HP was resistant to microbial degradation and synergistic interaction between photolysis and microbial degradation for 1-HP was observed. After 1 day of exposure to the 90 min photoproducts of 1-HP (i.e., photoproducts generated after 90 min of photolysis under outdoor irradiation), viability counts of heterotrophic bacterial assemblages were signi®cantly enhanced by comparison to the light control group (Fig. 1). The enhancement was greatest (4.75-folds) with exposure to the 1-HP outdoor photoproducts. However, no eect of exposure to 1-HP was observed. Therefore, the net response of the bacterial assemblages is utilizing the mixture of the photoproducts as substrates. A similar case was also reported for photolysis and bacterial transformation of other organic contaminants (Hwang et al., 1998, 2000). Concurrent measurement of the total bacterial numbers indicated the similar trend in response (results not shown). The eect of 1-HP and its photoproducts on bacterial heterotrophic activity of glucose mineralization is shown (Fig. 2). With comparison to the control groups, microbial mineralization activity of D -glucose was inhibited by up to 64% and 75% after exposing to 1-HP photoproducts and 1-HP, respectively. Based on the results (Figs. 1 and 2), we speculate that the inhibition on heterotrophic activity by the photoproducts was due to substrate competition between glucose and the mixtures of 1-HP photoproducts (Hwang et al., 1998, 2000), while 1-HP inhibited bacterial metabolic activity by virtually toxicity. 3.2. Microbial mutagenicity test The eect of 1-HP was determined by use of the Mutatoxâ Test for both direct-acting (in direct growth
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Fig. 1. The eect of 1-HP and its photoproducts on bacterial viability (1 day exposure, November 1999). (I) irradiated by an indoor UVA lamp for 30 min; (O) irradiated by midday sunlight for 90 min. ( ) signi®cantly dierent from the corresponding control group (P 6 0:05; t-test). 1± Time zero; 2 ± dark control; 3 ± 1-HP (I); 4 ± 1-HP (O); 5 ± 1-HP (dark); 6 ± killed control; 7 ± light control.
Fig. 2. The eect of 1-HP and its photoproducts on bacterial mineralization of D -glucose (1 day exposure, November 1999). (I) irradiated by an indoor UVA lamp for 30 min; (O) irradiated by midday sunlight for 90 min. ( ) signi®cantly dierent from the corresponding control group (P 6 0:05; t-test). 1 ± Dark control; 2 ± 1-HP (I); 3 ± 1-HP (O); 4 ± 1-HP (dark); 5 ± killed control; 6 ± light control.
medium) and pro-mutagens (growth medium containing S-9 enzymes). 1-HP exhibited positive responses in both direct medium and S-9 medium. However, after pho-
tolysis 1-HP only scored positive responses to the test in direct medium (Table 1). Therefore, other Mutatoxâ Tests for various treatment structures were conducted with direct medium for comparison. 1-HP was also reported by Hauser et al. (1997) to respond positively in both direct medium and S-9 medium, although there is a dierence in the substrate and solvent concentrations between the two studies; i.e., 1-HP: 1.4±11.5 lM in their system vs 0.0625±0.8 lM in our system; DMSO: 2% in their system vs 4% in our system. Results of the Mutatoxâ Test on 1-HP (10 lM), photoproducts of 1-HP, mixtures of Cu2 , Mn2 and 1-HP or its photoproducts are shown in Table 2. If the test sample concentration is cytotoxic, the luminescent bacteria cannot express any light emission and a zero value of light reading is reported in this study (Microbics Mutatox Manual, Microbics Corporation, 1995). Results indicate that 1HP is mutagenic, with positive responses occurring at a wide concentration range including the lowest concentration examined (i.e., 0.625 lM) (Table 2). Phototransformation by solar irradiation for 1.5 h (1-HP irrad.) or 3 days (1-HP PP) changed the light reading intensity of 1-HP signi®cantly. According to the result of the ®rst experiment (Table 2), the lowest observable effective concentration (LOEC) of 1-HP increased from 0.625 to 1.25 lM after solar irradiation (i.e., 1-HP irrad.). At the same time, the light reading intensity at lesser concentration ranges (i.e., 0.625±2.5 lM) decreased. Therefore, mutagenicity of 1-HP was decreased after solar photo-transformation. The change in mutagenicity could be due to the decrease in 1-HP concentration or the accumulation of 1-HP photoproducts. The environmental importance of complexation derives from the fact that the complexed chemical species behave dierently from the uncomplexed forms, as well as dierently from other complexes. Chemical properties, such as solubility, attenuation behavior on soils, bioconcentration factors, and toxicity are modi®ed through complexation (Lyman, 1995). Some metal ions were reported to cause oxidative DNA damage. For example, copper can induce single-base substitutions. Cu (II) binds preferentially to guanine residues and induces in combination with H2 O2 oxidative damage at sites of two or more adjacent guanine residues (Sagripanti and Kraemer, 1989). In our study, Mn2 (312.5 lM±5 mM) and Cu2 (6.25±100 lM) were not mutagenic (Table 2). However, additions of Mn2 (5 mM) increased the cytotoxicity of 1-HP, 1-HP irradiated sample (i.e., 1-HP irrad.) and 1-HP photoproduct sample (1-HP PP) synergistically at higher concentration range (10±2.5 lM) and changed their light reading at lower concentration range. Addition of Cu2 (100 lM) produced the similar cytotoxic eect. However, no predictable trend was observed in its eect on the light reading of 1-HP, 1-HP irrad. and 1-HP PP samples. Enhancement of metal toxicity by UV-A irradiation was
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Table 1 Mutatoxâ Test of 1-HP and its photoproducts In direct medium In S-9 medium a b
Parent compound
Parent compound and photoproductsa
Photoproductsb
Positive Positive
Positive Negative
Positive Negative
1-HP was irradiated outdoors for 90 min. Presence of the parent compound and photoproducts was veri®ed with HPLC. 1-HP was irradiated outdoors for 3 days. Presence of the photoproducts was veri®ed with HPLC.
Table 2 Mutatoxâ Test reading resultsa Medium control 11(2) 10(3) 16(3) 16(3) 15(4)
Positive control 1215(3485) 11938(6573) 381(11035) 100(2047) 28(999)
1-HP 0(0) 62(2) 12310(44) 719(5694) 171(13112)
Mn2 11(2) 8(0) 6(2) 7(2) 9(2)
1-HP + Mn2 0(0) 0(0) 0(0) 736(329) 233(20000)
(1-HP + Mn2 ) irrad. 0(2) 0(31) 0(49) 88(342) 39(13033)
Cu2 10(0) 8(2) 7(2) 8(2) 10(2)
(1-HP + Cu2 )d (0) (2) (3) (3586) (342)
(1-HP + Cu2 ) irrad. 0(2) 0(3) 631(24) 189(154) 17(89)
d
d
1-HP irrad.b 1483(2) 1652(0) 376(2) 58(882) 8(12293)
1-HP PPc 15(2) 166(2) 249(2) 102(599) 22(883)
(1-HP irrad. + Mn2 )e 0(0) 0(0) 653(12) 333(210) 14(12432)
(1-HP PP + Mn2 )c 0(2) 0(2) 0(2) 5(2) 50(2)
(1-HP irrad. + Cu2 )e 0(2) 0(0) 46(2) 11(2) 5(2)
(1-HP PP + Cu2 )c (2) (0) (2) (756) (3574)
a
The concentrations of the chemicals added (decreasing with 2-fold dilutions from the top to the bottom of each column): 1-HP: 10 lM; Mn2 : 5 mM; Cu2 : 100 lM. All irradiated samples but the group of 1-HP photoproducts were exposed to midday sunlight for 1.5 h before conducting the Mutatoxâ Test in direct medium. Results of the second experiment are given in parenthesis. b 1-HP was irradiated for 1.5 h under midday sunlight. c 1-HP was irradiated for 3 days to generate the mixture of photoproducts (PP). d The mixture of 1-HP and metal ion was irradiated for 1.5 h under midday sunlight. e Metal ion was added after 1-HP was irradiated for 1.5 h under midday sunlight.
reported to mediate through the formation of hydroxyl radicals within the cell or the complexation between DNA and metal ion radicals (Afrsten et al., 1994). However, this theory does not apply to our case because of the short longevity of the radical species in natural water (i.e., ls range; Stumm and Morgan, 1981) and the time lapse between irradiation treatment and execution of the Mutatoxâ Test. (i.e., min). Instead, we speculate that the changes in 1-HP and photoproducts mutagenicity by metal addition are due to the interactions (e.g., complexation) between the metal ions and the organic species of 1-HP and/or its photoproducts.
4. Conclusions Photo-transformation changes the ecotoxicity and mutagenicity of 1-HP to aquatic bacterial assembalges
and V. ®scheri M169 strain used in the Mutatoxâ Test. Our ®nding of bacterial capability of utilizing the test PAHs photoproducts con®rms the potential of using photolysis and biodegradation for remediating PAHs contamination. The modi®ed toxicity of the test PAHs by photolysis and metal ions indicates that the disappearance of PAHs in the environment does not always render them less ecologically hazardous. Close monitoring on the toxic or mutagenic properties of the complexed compounds and intermediates should be a necessary task for health regulatory professions.
Acknowledgements This research was supported by: (1) NIH-RCMI G12RR13459-03 and NIH MBRS S06GM08047 (to JSU); (2) Department of Energy DE-FG02-97ER62451
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Stumm, W., Morgan, J.J., 1981. Aquatic Chemistry: An Introduction Emphasizing Chemical Equilibria in Natural Waters, second ed. Wiley, New York. Sun, T.S.C., Stahr, H.M., 1993. Evaluation and application of a bioluminescent bacterial genotoxicity test. J. AOAC Int. 76, 893±898. Sunda, W.G., Huntsman, S.A., 1995. Regulation of copper concentration in the oceanic nutricline by phytoplankton uptake and regeneration cycles. Limnol. Oceanogr. 40, 132± 137. US Department of Health and Human Services, P.H.S., ATSDR 1995. Toxicological Pro®le for Polycyclic Aromatic Hydrocarbons (PAHs), Atlanta, GA. Xue, H., Sigg, L., 1999. Comparison of the complexation of Cu and Cd by humic or fulvic acids and by ligands observed in lake waters. Aqua. Geochem. 5, 313±335. Huey-Min Hwang is a Professor of biology and a microbial ecologist at the Jackson State University. He has been extensively involved in a variety of research projects in the areas of environmental toxicology, aquatic photochemistry and bioremediation. Dr. Hwang has published 20 refereed publications on the fates and eects of various contaminants including polycyclic aromatic hydrocarbons.