Environ. Sci. Technol. 2004, 38, 2873-2878
Toxicity Increases in Ice Containing Monochlorophenols upon Photolysis: Environmental Consequences L U D Eˇ K B L AÄ H A , * , † J A N A K L AÄ N O V AÄ , † P E T R K L AÄ N , ‡ J A R O S L A V J A N O Sˇ E K , † M I C H A L Sˇ K A R E K , † A N D RADOVAN RU ° Zˇ I C ˇ KA‡ RECETOXsResearch Center for Environmental Chemistry and Ecotoxicology, Masaryk University, Kamenice 3, CZ-625 00 Brno, Czech Republic, and Department of Organic Chemistry, Faculty of Science, Masaryk University, Kotla´rˇska´ 2, CZ-611 37 Brno, Czech Republic
The toxic effects of photoproducts formed upon the photolysis of 2- and 4-chlorophenol (CP) frozen solutions in polycrystalline ice phase were determined with a bacterial luminescence test (Vibrio fisheri), and in vitro biomarker assay for dioxin-like effects (inductions of AhR-dependent luciferase in H4IIE-luc cells) and compared to the toxic effects of products of the same photoreaction in aquatic phase. Coupling photoproducts formed in ice samples (3′chlorobiphenyl-2,4′-diol and 3-chlorobiphenyl-2,2′-diol from 2-CP photolysis and 5-chlorobiphenyl-2,4′-diol from 4-CP photolysis) were found to be more toxic to V. fisheri than parent CPs and elicited significant inductions of dioxinlike effects (the effective concentrations EC50 ∼ 3 × 10-5 mol L-1 corresponded to known weaker ligands of AhR, such as nonplanar polychlorinated biphenyls or polycyclic aromatic hydrocarbons). To complete the picture, a photoproduct formed from 4-CP (5-chlorobiphenyl-2,4′-diol) was synthesized, and a detailed toxicity assessment with purified compound confirmed the results obtained with irradiated samples. Our findings support a recently proposed model according to which solar radiation can trigger the formation of new types of organic pollutants in polar ice or tropospheric ice cloud particles, presenting possibly greater risk to the environment than the parent compounds.
Introduction In the past decade, the laboratory research results have provided evidence that many organic compounds can undergo light-induced chemical transformations in the ice matrix (1-6). This may have large consequences for the environment since many secondary photoprocesses may occur in natural ice or snow (6-14) with photoproducts completely different from those obtained in liquid solutions or gas phase. 2-Chlorophenol (2-CP) and 4-chlorophenol (4-CP) are chloroorganic compounds widespread in the ecosystem because they are commonly used in paper, herbicide, and pesticide industries. They are routinely monitored in aquatic * Corresponding author phone: +420-549 493 194; fax: +420 549 492 840; e-mail:
[email protected]. † RECETOX. ‡ Department of Organic Chemistry. 10.1021/es035076k CCC: $27.50 Published on Web 04/15/2004
2004 American Chemical Society
environments as well as in soil environments (15-17) and can be transported to the polar regions via global atmospheric transport. Several research groups were focused on their photochemical behavior and on the development of a variety of destruction techniques (18, 19). We have recently reported that major phototransformations of 2- and 4-CP in an ice matrix are based on coupling reactions within the polycrystalline ice cavities in the concentration range of 1 × 10-210-7 mol L-1 (5). No photosolvolysis (i.e., an intermolecular reaction between an organic and water molecules) was observed at temperatures below -10 °C. In this work, we intend to prove our original assumption that photoproducts of chlorophenol irradiation in ice may be more toxic and that they may present a greater risk to the environment than the parent compounds. We compared the toxicity of chlorophenol solutions irradiated in liquid water and solid ice phases in this paper. We studied inhibition of natural luminescence emitted by the marine bacteria Vibrio fisheri, which is a validated ecotoxicological test for assessment of pure chemicals, their mixtures, and environmental samples (20). Beside the bacterial assay, we studied the potencies of irradiated samples to activate AhR, an important toxicity mechanism of numerous organic pollutants, such as chlorinated dibenzo-p-dioxins or dibenzofurans (PCDDs/Fs), chlorinated biphenyls (PCBs), or polycyclic aromatic hydrocarbons (PAHs) (21, 22). Endocrine disruptions, reproductive disorders, developmental toxicity, tumor promotion, and immunosuppresive effects were attributed to AhR activation (22, 23). We used an in vitro reporter luciferase assay with H4IIE-luc cells to study those effects. The assay was successfully employed in numerous environmental studies, and it is validated as a biomarker of the dioxin-like toxicity of chemicals and complex environmental samples (24-27). To prove that the major photoproduct is responsible for the toxicity increase of irradiated solutions, 5-chlorobiphenyl-2,4′-diol was synthesized and purified; its toxic effects were evaluated and compared to those of irradiated solutions. Environmental consequences are discussed in detail.
Materials and Methods Sample Irradiation, Extraction, and Analysis. The compound studied, 2-CP or 4-CP (99.9%, Sigma), was dissolved in water of the Milli-Q quality (degassing the solutions by sonication) with the final concentration of 2.5 × 10-4 mol L-1. The samples in Pyrex (transparent over 280 nm) or quartz (>250 nm) tubes were UV-irradiated either as aqueous solutions at 20 °C (up to 120 min) or solid matrix at -10 °C (up to 15 h). Methods of irradiation as well as identification and quantification of the photoproducts were described thoroughly in a previous work (5). 5-Chlorobiphenyl-2,4′-diol Synthesis, Purification, and NMR Characterization. The major photoproduct of 4-CP irradiation in ice (5-chlorobiphenyl-2,4′-diol) was synthesized by UV photolysis (a 400-W medium-pressure Hg lamp equipped with a quartz filter enabling multiwavelength irradiation at >250 nm) of a frozen aqueous 4-CP solution (c ) 2 × 10-3 mol L-1) in a cryostat box at -20 °C for 30 h (5). When the conversion reached 25% (GC Shimadzu GC17A apparatus), the only photoproduct was isolated by a preparative HPLC (a petroleum ether/ethyl acetate mixture was used as a mobile phase). 1H and 13C NMR spectra of the isolated product were obtained for solutions in CDCl3 on a Bruker 300 MHz spectrometer: 1H NMR (300 MHz, CDCl3) δ (ppm): 6.90 (d, 1Harom, J ) 8.3 Hz), 6.96 (d, 2Harom, J ) 8.6 Hz), 7.19-7.16 (m, 2Harom), 7.32 (d, 2Harom, J ) 8.3 Hz); 13C VOL. 38, NO. 10, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Photochemistry of 2-chlorophenol and 4-chlorophenol in liquid water (A) and solid ice (B) samples according to ref 5. NMR (75.5 MHz, CDCl3) δ (ppm): 116.5, 117.3, 125.6, 128.2 (broad), 128.6, 130.0, 130.6, 151.6, 156.3. The mass spectrum of 5-chlorobiphenyl-2,4′-diol matched that of the compound was prepared according to the literature (28). Assessment of Acute Toxicity. The toxicity of irradiated samples was assessed with marine bacterium V. fischeri NRRL B-11177 (toxicity testing of irradiated ice samples was performed immediately after melting of the solid media). Salinity (2% w/v) and pH (6-8) of the samples were adjusted according to standard conditions (29). Inhibition of a natural bacterial luminescence after a 30-min exposure (cuvette luminometer Lumino M90a) was used as an end point, and the results were expressed as a percentage (%) of the negative control. Median inhibition concentrations (IC50) were calculated with a simple log-linear regression model. Determination of AhR-Mediated Toxicity. Prior to in vitro dioxin-like toxicity assessment, the organic compounds present in irradiated water or ice (chlorophenols and their photoproducts) were extracted to dichloromethane, DCM (15 mL of water samples were extracted with a liquid-liquid extraction to 2 mL of DCM), concentrated under a stream of nitrogen and diluted with 50 µL of dimethyl sulfoxide (DMSO). Rat hepatoma H4IIE-luc cells stably transfected with a luciferase reporter gene under the control of dioxinresponsive enhancers (a kind gift of prof. J. Giesy, Michigan State University, East Lansing, MI) were used for assessment of AhR-mediated effects. The assay was performed as described previously (27). The concentration-induction curves obtained after 6- and 24-h exposure periods were extrapolated using log-linear regression, and the EC50 values were estimated and compared with the reference toxicant 2,3,7,8-tetrachloro-p-dibenzodioxin (TCDD).
Results Photosolvolysis of 2-CP (nucleophilic aromatic substitution of chlorine atom by the OH group) to pyrocatechol is a known major degradation pathway in liquid aquatic environment (30-33). On the other hand, photolysis of 4-CP gives a mixture of products (e.g., hydroquinone, benzoquinone, 5-chlorobiphenyl-2,4′-diol), the composition of which depends on the reaction conditions (31, 32, 34-37). 2874
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In this work, aqueous as well as ice matrix solutions of CP were photolyzed to different reaction conversions: 30 min of irradiation was sufficient for a total degradation of the liquid CP solutions while ice samples at -10 °C required prolonged irradiation periods (a detectable amount of chlorophenols still remained in the ice samples after a 10-h irradiation, for example). A lower photochemical efficiency in the solid medium was caused partly by a lower polycrystalline ice transparency. The major photoproducts found in liquid solutions were dihydroxybenzenes (Figure 1A, Figure 2); dimeric compoundssmonochlorobiphenyldiols (Figure 1B)sprevailed in ice. Photolysis of 2-CP provided two major products: 3′-chlorobiphenyl-2,4′-diol (the chemical yield was approximately 55-65%) and 3-chlorobiphenyl-2,2′-diol (approximately 20-30%); irradiation of 4-CP led almost exclusively to the formation of a single compound, 5-chlorobiphenyl-2,4′-diol (the chemical yield was approximately 95%). A detailed description of these photochemical transformations was recently published (5). It was found that the photoproducts obtained in ice were more photochemically stable than the parent compounds or those produced in liquid samples. Benzenediols, the main products in water, degraded with about the same efficiency as the original chlorophenols; their concentrations reached the maximum in 5-10 min and were fully consumed after 30 min. The secondary (subsequent) photoproducts in ice were produced notably less efficiently that the primary ones: in the case of 4-CP, concentration of 5-chlorobiphenyl-2,4′-diol did not start to decrease even after 15 h of irradiation in ice. Due to very low concentrations of the secondary photoproducts and their large number, we were unable to characterize them fully. The mass spectra, nevertheless, showed that dechlorination, cyclization, and coupling reactions dominated in the subsequent photodestruction process. The major goal of this work was to provide the first experimental data on the toxicity changes of monochlorophenol solutions after irradiation in two phases, the knowledge of which may be useful for better understanding the environmental photochemical behavior of chloroorganic contaminants under various ambient temperatures.
FIGURE 2. Effects of 2-chlorophenol (2-CP, panel A) and 4-chlorophenol (4-CP, panel B) during irradiation of aqueous solutions (2.5 × 10-4 mol L-1) in the bacterial luminescence assay with Vibrio fisheri (inhibition of bacterial luminescence after a 30-min exposure); the toxicity of 10× diluted 4-CP samples is presented. The results are compared with the conversion of chlorophenols (continuous line with circles) and the formation of major photoproducts (pyrocatechol (PC) and hydroquinone (HQ); dotted line with triangles).
TABLE 1. Toxicity of 2-Chlorophenol and 4-Chlorophenol and Their Irradiation Photoproducts in Liquid Water and Solid Icea dioxin-like toxicity Vibrio fisheri IC50 EC50 in H4IIE-luc -1 [30 min, mol L ] cells [6 h, mol L-1] parent: 2-chlorophenol water: pyrocatechol ice: 3′-chlorobiphenyl-2,4′-diol 3-chlorobiphenyl-2,2′-diol parent: 4-chlorophenol water: hydroquinone ice: 5-chlorobiphenyl-2,4′-diol a
2.9 × 10-4 4.8 × 10-4 (39)
no effect no effect
∼0.7-1 × 10-5
∼2-5 × 10-5
7.8 × 10-6 4.5 × 10-7 (39) 3.5 × 10-6
no effect no effect 3.2 × 10-5
Reference numbers are in parentheses.
Toxicity of Monochlorophenol Solutions Irradiated in Liquid Phase. Figure 2 summarizes the acute toxicity of samples irradiated in liquid water as determined with the bacterial V. fisheri assay. Toxicity results (IC50/EC50 values) are summarized in Table 1. Differences in the acute toxicity of nonirradiated 2-CP and 4-CP were also examined. The highest tested concentration of 2-CP (2.5 × 10-4 mol L-1) elicited a relatively weak effect (45-50% inhibition of the control luminescence) while 4-CP was significantly more toxic with IC50 ) 7.8 × 10-6 mol L-1. Because nonirradiated 4-CP (2.5 × 10-4 mol L-1) elicited strong 95% inhibition of bacterial luminescence and a further increase in the toxicity of samples irradiated in the ice was not apparent (that is a decrease in luminiscence below 5% of the original value), the effects of 10× diluted samples are presented to describe reasonably the trends in the toxicity (Figure 2B, Figure 3B, dark bars). After a short (
