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Genotoxicity of municipal landfill leachate on root tips of Vicia faba
Mutation Research 560 (2004) 159–165
Genotoxicity of municipal landfill leachate on root tips of Vicia faba Nan Sang∗ , Guangke Li College of Environment and Resource, Shanxi University, Taiyuan, Shanxi 030006, PR China Received 26 November 2003; received in revised form 17 February 2004; accepted 26 February 2004
Abstract The genotoxicity of municipal landfill leachate was studied using the Vicia faba root-tip cytogenetic bioassay. Results show that landfill leachates collected in different seasons decreased the mitotic index (MI) and caused significant increases of micronucleus (MN) frequencies and anaphase aberration (AA) frequencies in a concentration-dependent manner (concentration expressed as ‘chemical oxygen demand’ measured by the method of potassium dichromate oxidation (CODCr )). In addition, a seasonal difference in genotoxicity induced by leachate was observed. The results confirm that leachate is a genotoxic agent in plant cells and imply that exposure to leachate in the aquatic environment may pose a potential genotoxic risk to organisms. The results also show that the V. faba cytogenetic bioassay is efficient, simple and reproducible in genotoxicity studies of leachate, and that there is a correlation between the genotoxicity and the chemical measurement (CODCr ) of leachate. © 2004 Elsevier B.V. All rights reserved. Keywords: Genotoxicity; Municipal landfill leachate; Vicia faba; Mitotic index (MI); Micronucleus (MN); Anaphase aberration (AA)
1. Introduction The use of municipal solid waste (MSW) landfills is the most widely utilized method of solid waste disposal around the world. With increasing use and public awareness of this method of disposal, there is much concern with respect to the pollution potential of the leachate from the landfill [1,2]. Landfill leachate, characterized by its high concentrations of organic matter, is mainly generated due to the penetration of precipitation through the waste mass and due to biodegradation of the waste. As leachate migrates away from a landfill, it may cause serious pollution to the groundwater aquifer as well as to adjacent surface waters. ∗ Corresponding author. Tel.: +86-351-7018696; fax: +86-351-7018699. E-mail address: sangnan [email protected] (N. Sang).
A review of previous literature on the occurrence and concentration of organic chemicals in MSW landfill leachates concluded that toxic and carcinogenic chemicals were present in the leachates of all MSW landfills studied [3]. In addition, the compounds contained in the leachates can be assimilated by any surviving aquatic species and passed through the food chain and bio-accumulate by long-term exposure. However, the exposure route and toxicity of leachates has not been well studied. The genetic toxicity of leachate has been shown in microbial bioassays, including the Salmonella/microsome mutagenicity bioassay (Ames assay), the Bacillus subtilis DNA repair bioassay, and the diploid Aspergillus nidulans chromosome damage bioassay [3]. Consequently, it has become important to study the genotoxic effects of landfill leachate in the environment to humans.
1383-5718/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2004.02.015
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N. Sang, G. Li / Mutation Research 560 (2004) 159–165
Higher plants provide a useful genetic system for screening and monitoring environmental pollutants. Mutagenic activity of chemicals has been analyzed with different plant systems such as Allium cepa, Vicia faba, Arabidopsis thaliana and Hordeum vulgare. With these plant systems, chromosomal aberration assays, mutation assays and cytogenetic tests have been performed [4–11]. Plant bioassays, which are sensitive and simple in comparison with animal bioassays, have been validated in international collaborative studies under the United Nations Environment Program (UNEP), World Health Organization (WHO) and US Environmental Protection Agency (US EPA), and proven to be efficient tests for genotoxic monitoring of environmental pollutants [12–15]. Previous studies have indicated that landfill leachate irrigation could affect the growth of plants and induce physiological changes [16,17]. To date, however, information on the genotoxic effects of landfill leachate in plants is rather scarce. The chemical oxygen demand (COD) is defined as the amount of oxidant consumed when samples were treated by the oxidant. It has been proposed as a general index of the level of organic pollution [18]. Landfill leachate is characterized by its high concentrations of organic matter. For this reason, in the present study the genotoxicity of landfill leachate was investigated with the V. faba cytogenetic bioassay, using COD as a measure of leachate concentration. This work was undertaken to probe landfill leachate with respect to its genetic damage on plants and possible mechanisms, to clarify the relevance and relationship of this measurement with genotoxicity, and eventually to offer further bio-experimental gist for discharge guidelines and environmental quality standards of leachate. 2. Materials and methods 2.1. Landfill leachate sample collection Located in the east of Taiyuan, the Xingou municipal solid waste landfill was opened in 1987 with the maximal capacity of 3.5 × 106 m3 . The waste stream entering the landfill was primarily residential refuse with very little industrial waste. According to the multi-spot collection principle [18], samples were collected at different depths and
at different points at the same depth from a tank, the terminal part of the collection system that drained the landfill, in February and August 2003. For each time, collected samples from different points were mixed and sealed in a clean plastic barrel with a lid and ring clamp, and promptly transported to the laboratory for analysis. Mixed samples collected in the two different seasons were defined as sample 1 (collected in February 2003) and sample 2 (collected in August 2003), respectively. The municipal landfill leachate has been shown to contain significant quantities of hazardous chemicals including chlorinated and non-chlorinated hydrocarbons, solvents and organic waste products from automobiles, such as carbon tetrachloride, chlorobenzene, ethylbenzene, toluene, chloromethane, chloroethane, chloroethylene, xylene, phenols, phthalate, camphor, decanoic acid, nonanoic acid, cresol, etc. [19]. Basic chemical properties of samples 1 and 2 were measured, with the following results. For sample 1 (pH 8.45): Pb, undetected; Cd, 0.026 mg/l; Hg, 0.065 mg/l; As, 0.773 mg/l; Cr, 0.006 mg/l; NH3 -N, 1079.52 mg/l; NO3 -N, 31.463 mg/l; NO2 -N, 0.181 mg/l; phenols, 1.893 mg/l. For sample 2 (pH 8.01): Pb, undetected; Cd, 0.012 mg/l; Hg, 0.013 mg/l; As, 0.186 mg/l; Cr, 0.002 mg/l; NH3 -N, 295.89 mg/l; NO3 -N, 22.424 mg/l; NO2 -N, 0.003 mg/l; phenols, 0.682 mg/l. With potassium dichromate as the oxidant, the resulting COD was expressed as CODCr [18]. The original CODCr values of samples 1 and 2 were 3452.55 and 1980.34 mg/l, respectively, measured by the method described by Xi et al. [18]. 2.2. Root tip preparations V. faba “qingpidou” (broad bean) was selected as the test plant. Roots of the species were germinated at 25 ± 1 ◦ C and the test was carried out according to Kanaya et al. [20]. Dry broad bean seeds were soaked for 24 h in distilled water and allowed to germinate between two layers of moist cotton. When the newly emerged roots were 1.00–2.00 cm in length, they were used in the test. 2.3. Treatments of landfill leachate For each sample, there were six groups, each consisting of at least 20 root tips. The negative control
N. Sang, G. Li / Mutation Research 560 (2004) 159–165
was exposed to distilled water. Five treatment groups were exposed to landfill leachates of different concentration, by diluting the crude leachate with distilled water so that the final CODCr was 80, 160, 320, 640 and 800 mg/l, respectively. All the experimental groups were kept in an incubator at 25 ± 1 ◦ C.
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control and treated groups was determined using the Student’s t-test. 3. Results 3.1. Mitotic index and cell cycle
2.4. Micronucleus (MN) assay The roots were treated with leachate for 24 h, and then kept in distilled water for 24 h for recovery. After that, the roots were fixed with acetic acid–ethanol (1:3, v/v) solutions freshly prepared before use. Negative control samples were fixed at the same time. After 24 h, the fixative was replaced with 70% alcohol for storage. For slide preparation and microscopic examination, the root tips were rinsed in distilled water and hydrolyzed in 1 M HC1 at 60 ◦ C for 8–12 min. After staining with Schiff’s reagent, 1 mm of the mitotic zone from well-stained root tips were immersed in a drop of distilled water on a clean slide and squashed under a cover glass. The cells with MN were evaluated under 800× magnification with a light microscope (Olympus, Japan). The MN frequency was expressed in terms of the number of cells with MN per 1000 cells scored, resulting from about 6000 examined cells in 10 separate seedlings for each group. 2.5. Cell kinetics and anaphase aberration (AA) assays The procedure used for these assays were essentially the same as for the root MN assay except that the treated roots were fixed immediately after treatment, without a recovery period. The mitotic index (MI) was determined by counting the number of mitotic cells among the total amount of scored cells per seedling. The results were expressed per 100 cells scored. The frequencies of AA were expressed per 1000 cells scored. Approximately 6000 cells were scored from 10 separate seedlings for each treatment and control group. Each experiment was run with three replications.
Table 1 shows that both samples induced mitotic delay and decreased the mitotic index in V. faba root tips. The MI decreased with increasing treatment concentrations of leachate and showed a statistically significant difference between the negative control and leachate exposure groups, after exposure to sample 1 at 160, 320, 640 and 800 mg/l, and after 320, 640 and 800 mg/l of sample 2. Compared with the negative control, reductions of the MI of 82 and 61% were detected for the highest leachate concentrations in Vicia root tips treated with samples 1 and 2, respectively. 3.2. Micronucleus formation Both samples of leachate were tested for potential clastogenicity in V. faba using the MN assay. The results on leachate-induced MN are summarized in Table 2. The control group showed a background level of 3.81 ± 2.15‰ MN. Among the exposure groups, 80 mg/l leachate induced MN formation after 24 h exposure, and the frequencies of MN significantly increased with increasing concentrations of sample 1 leachate from CODCr 80 to 320 mg/l, and from CODCr 80 to 640 mg/l after exposure to sample 2. The results indicate that leachates from both samples induced the formation of MN in a concentration (CODCr )-dependent manner. The concentration– response curves could be fitted well with the equations: y = 0.0267x + 10.21 (γ = 0.95) and y = 0.0094x + 9.3648 (γ = 0.96). However, the MN frequency decreased at CODCr 640 and 800 mg/l for sample 1 treatment, and at the highest concentration (CODCr 800 mg/l) for sample 2 treatment, because of mitotic delay and acute cell toxicity. 3.3. Anaphase aberrations
2.6. Statistical analysis of data Results are presented as mean ± S.D., and the statistical significance of the differences between the
Results of genotoxicity tests with the V. faba AA test are given in Tables 3 and 4. There were marked increases of fragments, gaps, laggards, bridges and
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Table 1 Effects on the mitotic index in Vicia root tips exposed to the leachate for 24 h Test substance
Mitotic index (%, ±S.D.)
Concentration (mg/l)
Negative control
0
Leachate
80 160 320 640 800
Sample 1
Sample 2
24.15 ± 4.16
24.15 ± 4.16
23.03 16.63 11.75 8.18 4.30
± ± ± ± ±
2.16 4.09∗ 3.28∗∗ 2.10∗∗ 1.41∗∗
25.28 19.70 16.08 12.98 9.38
± ± ± ± ±
3.61 2.82 4.80∗ 3.54∗∗,a 3.66∗∗,b
Significant difference from sample 1 in same concentration of leachate exposure group: P < 0.05. Significant difference from sample 1 in same concentration of leachate exposure group: P < 0.01. ∗ Significant difference from negative control: P < 0.05. ∗∗ Significant difference from negative control: P < 0.01. a
b
Table 2 Frequencies of micronuclei induced by leachate in Vicia root tips after a 24 h exposure Test substance
MCN frequencies (‰, ±S.D.)
Concentration (mg/l)
Sample 1 Negative control
3.81 ± 2.15
0
Leachate
Sample 2
80 160 320 640 800
11.50 15.75 18.33 16.25 15.00
± ± ± ± ±
3.81 ± 2.15
3.51∗∗ 2.00∗∗ 3.16∗∗ 1.71∗∗ 1.63∗∗
9.50 11.00 13.25 14.98 12.05
± ± ± ± ±
3.00∗∗ 1.41∗∗,b 3.77∗∗,a 4.99∗∗ 3.56∗∗
Significant difference from sample 1 in same concentration of leachate exposure group: P < 0.05. Significant difference from sample 1 in same concentration of leachate exposure group: P < 0.01. ∗∗ Significant difference from negative control: P < 0.01. a
b
stickiness frequencies in both exposure groups, and the frequencies of AA increased significantly with increasing concentrations of sample 1 from CODCr 80 to 320 mg/l, and from CODCr 80 to 640 mg/l for sample 2, indicating that leachates induced the formation
of AA in a concentration (CODCr )-dependent manner. Among these aberrations, stickiness, fragments and gaps were prominent. Similar to the MN frequency induced by high leachate concentrations, the AA frequency also
Table 3 Anaphase aberrations (AA) induced by sample 1 in Vicia root tips after a 24 h exposure Test substance
Concentration (mg/l)
Negative
0
Leachate
80 160 320 640 800
∗
Fragments (‰, S.D.)
Gaps (‰, S.D.)
Laggards (‰, S.D.)
Bridges (‰, S.D.)
Stickiness (‰, S.D.)
1.00 ± 0.89
1.17 ± 0.75
0.67 ± 0.52
0
1.33 ± 0.82
0 1.33 1.50 1.00 0.83
3.17 4.67 5.67 5.33 4.83
4.17 5.50 6.33 6.00 5.17
± ± ± ± ±
1.17∗∗ 2.07∗∗ 2.07∗∗ 1.41∗∗ 0.98∗∗
3.00 3.50 4.83 4.17 4.50
± ± ± ± ±
1.10∗∗ 1.64∗ 0.75∗∗ 1.17∗∗ 0.84∗∗
Significant difference from negative control: P < 0.05. Significant difference from negative control: P < 0.01.
∗∗
1.50 2.67 3.83 3.50 3.33
± ± ± ± ±
0.84 1.37∗∗ 1.17∗∗ 1.05∗∗ 1.63∗∗
± ± ± ±
1.03∗ 0.55∗∗ 0.89∗ 0.75∗
± ± ± ± ±
0.98 1.03∗∗ 0.82∗∗ 1.51∗∗ 1.17∗∗
AA (‰, S.D.) 4.17 ± 0.98 11.83 17.67 22.17 20.00 18.67
± ± ± ± ±
3.06∗∗ 1.86∗∗ 3.92∗∗ 2.97∗∗ 1.97∗∗
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Table 4 Anaphase aberrations (AA) induced by sample 2 in Vicia root tips after a 24 h exposure Test substance Negative Leachate
Concentration (mg/l) 0 80 160 320 640 800
Fragments (‰, S.D.)
Gaps (‰, S.D.)
1.00 ± 0.89
1.17 ± 0.75
2.17 2.50 3.83 4.67 3.83
± ± ± ± ±
0.75∗
0.55∗∗ 1.33∗∗ 1.37∗∗ 1.60∗∗
2.83 3.83 4.00 4.33 4.17
± ± ± ± ±
∗∗
0.61 0.75 1.67∗∗ 1.21∗∗ 0.98∗∗
Laggards (‰, S.D.)
Bridges (‰, S.D.)
Stickiness (‰, S.D.)
0.67 ± 0.52
0
1.33 ± 0.82
1.17 2.17 2.67 3.17 3.00
± ± ± ± ±
0.98 1.03∗∗ 1.21∗∗ 1.17∗∗ 1.26∗∗
1.00 1.50 1.67 2.50 2.17
± ± ± ± ±
0.63∗∗ 0.84∗∗ 1.03∗∗ 1.38∗∗ 1.47∗∗
1.83 3.17 4.50 5.17 4.33
± ± ± ± ±
0.73 0.41∗∗ 1.76∗∗ 1.17∗∗ 1.75∗∗
AA (‰, S.D.) 4.17 ± 0.98 9.00 13.17 16.67 19.83 17.50
± ± ± ± ±
1.55∗∗ 1.72∗∗,b 4.59∗∗,a 3.13∗∗ 4.76∗∗
Significant difference from sample 1 in same concentration of leachate exposure group: P < 0.05. Significant difference from sample 1 in same concentration of leachate exposure group: P < 0.01. ∗ Significant difference from negative control: P < 0.05. ∗∗ Significant difference from negative control: P < 0.01. a
b
decreased at CODCr 640 and 800 mg/l for sample 1, and at the highest concentration (CODCr 800 mg/l) for sample 2. 3.4. The seasonal difference in genotoxicity induced by leachate With increasing concentrations of leachate, significant differences of MI, MN frequency and AA frequency were observed after exposure to similar concentrations of the two samples. The reduction of MI induced by sample 1 was stronger than that induced by sample 2, and the increases in MN and AA frequencies induced by sample 1 were higher than those caused by sample 2 (Tables 1–4). The results indicate that the genetic damage in V. faba induced by sample 1 was more severe than after exposure to sample 2. Because the two samples were collected in different seasons, the results imply a seasonal difference in genotoxicity induced by the leachate.
4. Discussion With increasing use of municipal solid waste landfills as the most widely utilized method of solid waste disposal, contamination from municipal landfill leachate has become a serious problem for the aquatic environment, either due to faulty design and/or construction of the landfill. Therefore, it is important to study its toxic effects on plants, mammals and humans. The present study indicates that municipal
landfill leachates collected in different seasons (samples 1 and 2) decreased the mitotic index in V. faba, and induced significant increases in the levels of MN and AA in V. faba root tips in a concentration (CODCr )-dependent manner. The frequencies of MN and AA decreased after exposure to unusually high concentrations, due to physiological toxicity. Analysis of MN and AA in V. faba root tips is a most useful method to assess genetic effects of chemicals. However, a study on MN formation and AA induction by municipal landfill leachate, and an analysis of their concentration (CODCr )–response relationships has not previously been carried out. Although our data are still preliminary, this is the first experimental evidence that landfill leachate induces MN and AA in V. faba root-tip cells in a concentration (CODCr )-dependent manner, and that there is a correlation between this chemical measurement (CODCr ) of the leachate and its genotoxicity in a plant system. A possible mechanism for leachate-induced MN and AA may involve formation of free radicals, either via auto-oxidation or by enzyme-catalyzed oxidation of organic compounds in the leachate, such as chlorinated and non-chlorinated hydrocarbons including carbon tetrachloride, chloromethane, chloroethane, chloroethylene, decanoic acid, nonanoic acid, etc. These free radicals could attack nucleic acids, result in base substitution and breakage of DNA, and eventually induce mutation [21–25]. Further work on the formation of free radicals by the components in the leachate, and on the effects of the radicals on various kinds of biological molecules is required
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to understand the mechanisms of leachate-induced genetic toxicity in plant cells. Variations in leachate quality have been reported [19]. In our studies, a seasonal difference of genotoxicity induced by leachates was observed. Sample 1 represented the sample collected in the cold and dry season, and sample 2 was collected in the hot and rainy season. The results show that genetic damages in V. faba were more severe when the sample had been collected in the cold and dry season than in the hot and rainy season. This confirmed the variations in the presence and amount of different contaminants in the leachates over time, which might be dependent on air temperature and precipitation. Higher temperatures would tend to increase internal landfill temperatures, accelerate the microbiological decomposition processes, and result in the predominant low molecular weight nature of the organic materials. Organic contaminants in the leachate would be diluted by precipitation. The result suggests that different discharge guidelines and environmental quality standards should be established for leachates discharged from landfills in different seasons. The genetic effects of leachates have been previously examined in bacterial cells [3]. Our results confirm that leachate may act as a genotoxic agent in plant cells, which implies that landfill leachate could result in groundwater contamination even at dilute concentrations either due to faulty design and/or construction of the landfill. Exposure to leachate pollution in groundwater may thus pose a potential risk for induction of cytogenetic damage in organisms. The results also suggest that the V. faba MN and AA assays can be used as efficient genotoxicity tests of landfill leachate, and as monitors of its pollution of the aquatic environment. However, the present work is the first report, to our knowledge, to show direct evidence of genetic damage induced in plants by leachate, and more studies on the genotoxic effects of municipal landfill leachate on other plants, mammals and humans are needed.
Acknowledgements This study was supported by the National Natural Science Foundation of China (No. 20177014).
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Genotoxicity of municipal landfill leachate on root tips of Vicia faba Nan Sang∗ , Guangke Li College of Environment and Resource, Shanxi University, Taiyuan, Shanxi 030006, PR China Received 26 November 2003; received in revised form 17 February 2004; accepted 26 February 2004
Abstract The genotoxicity of municipal landfill leachate was studied using the Vicia faba root-tip cytogenetic bioassay. Results show that landfill leachates collected in different seasons decreased the mitotic index (MI) and caused significant increases of micronucleus (MN) frequencies and anaphase aberration (AA) frequencies in a concentration-dependent manner (concentration expressed as ‘chemical oxygen demand’ measured by the method of potassium dichromate oxidation (CODCr )). In addition, a seasonal difference in genotoxicity induced by leachate was observed. The results confirm that leachate is a genotoxic agent in plant cells and imply that exposure to leachate in the aquatic environment may pose a potential genotoxic risk to organisms. The results also show that the V. faba cytogenetic bioassay is efficient, simple and reproducible in genotoxicity studies of leachate, and that there is a correlation between the genotoxicity and the chemical measurement (CODCr ) of leachate. © 2004 Elsevier B.V. All rights reserved. Keywords: Genotoxicity; Municipal landfill leachate; Vicia faba; Mitotic index (MI); Micronucleus (MN); Anaphase aberration (AA)
1. Introduction The use of municipal solid waste (MSW) landfills is the most widely utilized method of solid waste disposal around the world. With increasing use and public awareness of this method of disposal, there is much concern with respect to the pollution potential of the leachate from the landfill [1,2]. Landfill leachate, characterized by its high concentrations of organic matter, is mainly generated due to the penetration of precipitation through the waste mass and due to biodegradation of the waste. As leachate migrates away from a landfill, it may cause serious pollution to the groundwater aquifer as well as to adjacent surface waters. ∗ Corresponding author. Tel.: +86-351-7018696; fax: +86-351-7018699. E-mail address: sangnan [email protected] (N. Sang).
A review of previous literature on the occurrence and concentration of organic chemicals in MSW landfill leachates concluded that toxic and carcinogenic chemicals were present in the leachates of all MSW landfills studied [3]. In addition, the compounds contained in the leachates can be assimilated by any surviving aquatic species and passed through the food chain and bio-accumulate by long-term exposure. However, the exposure route and toxicity of leachates has not been well studied. The genetic toxicity of leachate has been shown in microbial bioassays, including the Salmonella/microsome mutagenicity bioassay (Ames assay), the Bacillus subtilis DNA repair bioassay, and the diploid Aspergillus nidulans chromosome damage bioassay [3]. Consequently, it has become important to study the genotoxic effects of landfill leachate in the environment to humans.
1383-5718/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2004.02.015
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Higher plants provide a useful genetic system for screening and monitoring environmental pollutants. Mutagenic activity of chemicals has been analyzed with different plant systems such as Allium cepa, Vicia faba, Arabidopsis thaliana and Hordeum vulgare. With these plant systems, chromosomal aberration assays, mutation assays and cytogenetic tests have been performed [4–11]. Plant bioassays, which are sensitive and simple in comparison with animal bioassays, have been validated in international collaborative studies under the United Nations Environment Program (UNEP), World Health Organization (WHO) and US Environmental Protection Agency (US EPA), and proven to be efficient tests for genotoxic monitoring of environmental pollutants [12–15]. Previous studies have indicated that landfill leachate irrigation could affect the growth of plants and induce physiological changes [16,17]. To date, however, information on the genotoxic effects of landfill leachate in plants is rather scarce. The chemical oxygen demand (COD) is defined as the amount of oxidant consumed when samples were treated by the oxidant. It has been proposed as a general index of the level of organic pollution [18]. Landfill leachate is characterized by its high concentrations of organic matter. For this reason, in the present study the genotoxicity of landfill leachate was investigated with the V. faba cytogenetic bioassay, using COD as a measure of leachate concentration. This work was undertaken to probe landfill leachate with respect to its genetic damage on plants and possible mechanisms, to clarify the relevance and relationship of this measurement with genotoxicity, and eventually to offer further bio-experimental gist for discharge guidelines and environmental quality standards of leachate. 2. Materials and methods 2.1. Landfill leachate sample collection Located in the east of Taiyuan, the Xingou municipal solid waste landfill was opened in 1987 with the maximal capacity of 3.5 × 106 m3 . The waste stream entering the landfill was primarily residential refuse with very little industrial waste. According to the multi-spot collection principle [18], samples were collected at different depths and
at different points at the same depth from a tank, the terminal part of the collection system that drained the landfill, in February and August 2003. For each time, collected samples from different points were mixed and sealed in a clean plastic barrel with a lid and ring clamp, and promptly transported to the laboratory for analysis. Mixed samples collected in the two different seasons were defined as sample 1 (collected in February 2003) and sample 2 (collected in August 2003), respectively. The municipal landfill leachate has been shown to contain significant quantities of hazardous chemicals including chlorinated and non-chlorinated hydrocarbons, solvents and organic waste products from automobiles, such as carbon tetrachloride, chlorobenzene, ethylbenzene, toluene, chloromethane, chloroethane, chloroethylene, xylene, phenols, phthalate, camphor, decanoic acid, nonanoic acid, cresol, etc. [19]. Basic chemical properties of samples 1 and 2 were measured, with the following results. For sample 1 (pH 8.45): Pb, undetected; Cd, 0.026 mg/l; Hg, 0.065 mg/l; As, 0.773 mg/l; Cr, 0.006 mg/l; NH3 -N, 1079.52 mg/l; NO3 -N, 31.463 mg/l; NO2 -N, 0.181 mg/l; phenols, 1.893 mg/l. For sample 2 (pH 8.01): Pb, undetected; Cd, 0.012 mg/l; Hg, 0.013 mg/l; As, 0.186 mg/l; Cr, 0.002 mg/l; NH3 -N, 295.89 mg/l; NO3 -N, 22.424 mg/l; NO2 -N, 0.003 mg/l; phenols, 0.682 mg/l. With potassium dichromate as the oxidant, the resulting COD was expressed as CODCr [18]. The original CODCr values of samples 1 and 2 were 3452.55 and 1980.34 mg/l, respectively, measured by the method described by Xi et al. [18]. 2.2. Root tip preparations V. faba “qingpidou” (broad bean) was selected as the test plant. Roots of the species were germinated at 25 ± 1 ◦ C and the test was carried out according to Kanaya et al. [20]. Dry broad bean seeds were soaked for 24 h in distilled water and allowed to germinate between two layers of moist cotton. When the newly emerged roots were 1.00–2.00 cm in length, they were used in the test. 2.3. Treatments of landfill leachate For each sample, there were six groups, each consisting of at least 20 root tips. The negative control
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was exposed to distilled water. Five treatment groups were exposed to landfill leachates of different concentration, by diluting the crude leachate with distilled water so that the final CODCr was 80, 160, 320, 640 and 800 mg/l, respectively. All the experimental groups were kept in an incubator at 25 ± 1 ◦ C.
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control and treated groups was determined using the Student’s t-test. 3. Results 3.1. Mitotic index and cell cycle
2.4. Micronucleus (MN) assay The roots were treated with leachate for 24 h, and then kept in distilled water for 24 h for recovery. After that, the roots were fixed with acetic acid–ethanol (1:3, v/v) solutions freshly prepared before use. Negative control samples were fixed at the same time. After 24 h, the fixative was replaced with 70% alcohol for storage. For slide preparation and microscopic examination, the root tips were rinsed in distilled water and hydrolyzed in 1 M HC1 at 60 ◦ C for 8–12 min. After staining with Schiff’s reagent, 1 mm of the mitotic zone from well-stained root tips were immersed in a drop of distilled water on a clean slide and squashed under a cover glass. The cells with MN were evaluated under 800× magnification with a light microscope (Olympus, Japan). The MN frequency was expressed in terms of the number of cells with MN per 1000 cells scored, resulting from about 6000 examined cells in 10 separate seedlings for each group. 2.5. Cell kinetics and anaphase aberration (AA) assays The procedure used for these assays were essentially the same as for the root MN assay except that the treated roots were fixed immediately after treatment, without a recovery period. The mitotic index (MI) was determined by counting the number of mitotic cells among the total amount of scored cells per seedling. The results were expressed per 100 cells scored. The frequencies of AA were expressed per 1000 cells scored. Approximately 6000 cells were scored from 10 separate seedlings for each treatment and control group. Each experiment was run with three replications.
Table 1 shows that both samples induced mitotic delay and decreased the mitotic index in V. faba root tips. The MI decreased with increasing treatment concentrations of leachate and showed a statistically significant difference between the negative control and leachate exposure groups, after exposure to sample 1 at 160, 320, 640 and 800 mg/l, and after 320, 640 and 800 mg/l of sample 2. Compared with the negative control, reductions of the MI of 82 and 61% were detected for the highest leachate concentrations in Vicia root tips treated with samples 1 and 2, respectively. 3.2. Micronucleus formation Both samples of leachate were tested for potential clastogenicity in V. faba using the MN assay. The results on leachate-induced MN are summarized in Table 2. The control group showed a background level of 3.81 ± 2.15‰ MN. Among the exposure groups, 80 mg/l leachate induced MN formation after 24 h exposure, and the frequencies of MN significantly increased with increasing concentrations of sample 1 leachate from CODCr 80 to 320 mg/l, and from CODCr 80 to 640 mg/l after exposure to sample 2. The results indicate that leachates from both samples induced the formation of MN in a concentration (CODCr )-dependent manner. The concentration– response curves could be fitted well with the equations: y = 0.0267x + 10.21 (γ = 0.95) and y = 0.0094x + 9.3648 (γ = 0.96). However, the MN frequency decreased at CODCr 640 and 800 mg/l for sample 1 treatment, and at the highest concentration (CODCr 800 mg/l) for sample 2 treatment, because of mitotic delay and acute cell toxicity. 3.3. Anaphase aberrations
2.6. Statistical analysis of data Results are presented as mean ± S.D., and the statistical significance of the differences between the
Results of genotoxicity tests with the V. faba AA test are given in Tables 3 and 4. There were marked increases of fragments, gaps, laggards, bridges and
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Table 1 Effects on the mitotic index in Vicia root tips exposed to the leachate for 24 h Test substance
Mitotic index (%, ±S.D.)
Concentration (mg/l)
Negative control
0
Leachate
80 160 320 640 800
Sample 1
Sample 2
24.15 ± 4.16
24.15 ± 4.16
23.03 16.63 11.75 8.18 4.30
± ± ± ± ±
2.16 4.09∗ 3.28∗∗ 2.10∗∗ 1.41∗∗
25.28 19.70 16.08 12.98 9.38
± ± ± ± ±
3.61 2.82 4.80∗ 3.54∗∗,a 3.66∗∗,b
Significant difference from sample 1 in same concentration of leachate exposure group: P < 0.05. Significant difference from sample 1 in same concentration of leachate exposure group: P < 0.01. ∗ Significant difference from negative control: P < 0.05. ∗∗ Significant difference from negative control: P < 0.01. a
b
Table 2 Frequencies of micronuclei induced by leachate in Vicia root tips after a 24 h exposure Test substance
MCN frequencies (‰, ±S.D.)
Concentration (mg/l)
Sample 1 Negative control
3.81 ± 2.15
0
Leachate
Sample 2
80 160 320 640 800
11.50 15.75 18.33 16.25 15.00
± ± ± ± ±
3.81 ± 2.15
3.51∗∗ 2.00∗∗ 3.16∗∗ 1.71∗∗ 1.63∗∗
9.50 11.00 13.25 14.98 12.05
± ± ± ± ±
3.00∗∗ 1.41∗∗,b 3.77∗∗,a 4.99∗∗ 3.56∗∗
Significant difference from sample 1 in same concentration of leachate exposure group: P < 0.05. Significant difference from sample 1 in same concentration of leachate exposure group: P < 0.01. ∗∗ Significant difference from negative control: P < 0.01. a
b
stickiness frequencies in both exposure groups, and the frequencies of AA increased significantly with increasing concentrations of sample 1 from CODCr 80 to 320 mg/l, and from CODCr 80 to 640 mg/l for sample 2, indicating that leachates induced the formation
of AA in a concentration (CODCr )-dependent manner. Among these aberrations, stickiness, fragments and gaps were prominent. Similar to the MN frequency induced by high leachate concentrations, the AA frequency also
Table 3 Anaphase aberrations (AA) induced by sample 1 in Vicia root tips after a 24 h exposure Test substance
Concentration (mg/l)
Negative
0
Leachate
80 160 320 640 800
∗
Fragments (‰, S.D.)
Gaps (‰, S.D.)
Laggards (‰, S.D.)
Bridges (‰, S.D.)
Stickiness (‰, S.D.)
1.00 ± 0.89
1.17 ± 0.75
0.67 ± 0.52
0
1.33 ± 0.82
0 1.33 1.50 1.00 0.83
3.17 4.67 5.67 5.33 4.83
4.17 5.50 6.33 6.00 5.17
± ± ± ± ±
1.17∗∗ 2.07∗∗ 2.07∗∗ 1.41∗∗ 0.98∗∗
3.00 3.50 4.83 4.17 4.50
± ± ± ± ±
1.10∗∗ 1.64∗ 0.75∗∗ 1.17∗∗ 0.84∗∗
Significant difference from negative control: P < 0.05. Significant difference from negative control: P < 0.01.
∗∗
1.50 2.67 3.83 3.50 3.33
± ± ± ± ±
0.84 1.37∗∗ 1.17∗∗ 1.05∗∗ 1.63∗∗
± ± ± ±
1.03∗ 0.55∗∗ 0.89∗ 0.75∗
± ± ± ± ±
0.98 1.03∗∗ 0.82∗∗ 1.51∗∗ 1.17∗∗
AA (‰, S.D.) 4.17 ± 0.98 11.83 17.67 22.17 20.00 18.67
± ± ± ± ±
3.06∗∗ 1.86∗∗ 3.92∗∗ 2.97∗∗ 1.97∗∗
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Table 4 Anaphase aberrations (AA) induced by sample 2 in Vicia root tips after a 24 h exposure Test substance Negative Leachate
Concentration (mg/l) 0 80 160 320 640 800
Fragments (‰, S.D.)
Gaps (‰, S.D.)
1.00 ± 0.89
1.17 ± 0.75
2.17 2.50 3.83 4.67 3.83
± ± ± ± ±
0.75∗
0.55∗∗ 1.33∗∗ 1.37∗∗ 1.60∗∗
2.83 3.83 4.00 4.33 4.17
± ± ± ± ±
∗∗
0.61 0.75 1.67∗∗ 1.21∗∗ 0.98∗∗
Laggards (‰, S.D.)
Bridges (‰, S.D.)
Stickiness (‰, S.D.)
0.67 ± 0.52
0
1.33 ± 0.82
1.17 2.17 2.67 3.17 3.00
± ± ± ± ±
0.98 1.03∗∗ 1.21∗∗ 1.17∗∗ 1.26∗∗
1.00 1.50 1.67 2.50 2.17
± ± ± ± ±
0.63∗∗ 0.84∗∗ 1.03∗∗ 1.38∗∗ 1.47∗∗
1.83 3.17 4.50 5.17 4.33
± ± ± ± ±
0.73 0.41∗∗ 1.76∗∗ 1.17∗∗ 1.75∗∗
AA (‰, S.D.) 4.17 ± 0.98 9.00 13.17 16.67 19.83 17.50
± ± ± ± ±
1.55∗∗ 1.72∗∗,b 4.59∗∗,a 3.13∗∗ 4.76∗∗
Significant difference from sample 1 in same concentration of leachate exposure group: P < 0.05. Significant difference from sample 1 in same concentration of leachate exposure group: P < 0.01. ∗ Significant difference from negative control: P < 0.05. ∗∗ Significant difference from negative control: P < 0.01. a
b
decreased at CODCr 640 and 800 mg/l for sample 1, and at the highest concentration (CODCr 800 mg/l) for sample 2. 3.4. The seasonal difference in genotoxicity induced by leachate With increasing concentrations of leachate, significant differences of MI, MN frequency and AA frequency were observed after exposure to similar concentrations of the two samples. The reduction of MI induced by sample 1 was stronger than that induced by sample 2, and the increases in MN and AA frequencies induced by sample 1 were higher than those caused by sample 2 (Tables 1–4). The results indicate that the genetic damage in V. faba induced by sample 1 was more severe than after exposure to sample 2. Because the two samples were collected in different seasons, the results imply a seasonal difference in genotoxicity induced by the leachate.
4. Discussion With increasing use of municipal solid waste landfills as the most widely utilized method of solid waste disposal, contamination from municipal landfill leachate has become a serious problem for the aquatic environment, either due to faulty design and/or construction of the landfill. Therefore, it is important to study its toxic effects on plants, mammals and humans. The present study indicates that municipal
landfill leachates collected in different seasons (samples 1 and 2) decreased the mitotic index in V. faba, and induced significant increases in the levels of MN and AA in V. faba root tips in a concentration (CODCr )-dependent manner. The frequencies of MN and AA decreased after exposure to unusually high concentrations, due to physiological toxicity. Analysis of MN and AA in V. faba root tips is a most useful method to assess genetic effects of chemicals. However, a study on MN formation and AA induction by municipal landfill leachate, and an analysis of their concentration (CODCr )–response relationships has not previously been carried out. Although our data are still preliminary, this is the first experimental evidence that landfill leachate induces MN and AA in V. faba root-tip cells in a concentration (CODCr )-dependent manner, and that there is a correlation between this chemical measurement (CODCr ) of the leachate and its genotoxicity in a plant system. A possible mechanism for leachate-induced MN and AA may involve formation of free radicals, either via auto-oxidation or by enzyme-catalyzed oxidation of organic compounds in the leachate, such as chlorinated and non-chlorinated hydrocarbons including carbon tetrachloride, chloromethane, chloroethane, chloroethylene, decanoic acid, nonanoic acid, etc. These free radicals could attack nucleic acids, result in base substitution and breakage of DNA, and eventually induce mutation [21–25]. Further work on the formation of free radicals by the components in the leachate, and on the effects of the radicals on various kinds of biological molecules is required
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to understand the mechanisms of leachate-induced genetic toxicity in plant cells. Variations in leachate quality have been reported [19]. In our studies, a seasonal difference of genotoxicity induced by leachates was observed. Sample 1 represented the sample collected in the cold and dry season, and sample 2 was collected in the hot and rainy season. The results show that genetic damages in V. faba were more severe when the sample had been collected in the cold and dry season than in the hot and rainy season. This confirmed the variations in the presence and amount of different contaminants in the leachates over time, which might be dependent on air temperature and precipitation. Higher temperatures would tend to increase internal landfill temperatures, accelerate the microbiological decomposition processes, and result in the predominant low molecular weight nature of the organic materials. Organic contaminants in the leachate would be diluted by precipitation. The result suggests that different discharge guidelines and environmental quality standards should be established for leachates discharged from landfills in different seasons. The genetic effects of leachates have been previously examined in bacterial cells [3]. Our results confirm that leachate may act as a genotoxic agent in plant cells, which implies that landfill leachate could result in groundwater contamination even at dilute concentrations either due to faulty design and/or construction of the landfill. Exposure to leachate pollution in groundwater may thus pose a potential risk for induction of cytogenetic damage in organisms. The results also suggest that the V. faba MN and AA assays can be used as efficient genotoxicity tests of landfill leachate, and as monitors of its pollution of the aquatic environment. However, the present work is the first report, to our knowledge, to show direct evidence of genetic damage induced in plants by leachate, and more studies on the genotoxic effects of municipal landfill leachate on other plants, mammals and humans are needed.
Acknowledgements This study was supported by the National Natural Science Foundation of China (No. 20177014).
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