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Assessment of the genotoxicity of mine-dump material using the Tradescantia-stamen hair (Trad-SHM) and the Tradescantia-micronucleus (Trad-MCN) bioassays
Mutation Research 426 Ž1999. 173–181 www.elsevier.comrlocatermolmut Community address: www.elsevier.comrlocatermutres
Assessment of the genotoxicity of mine-dump material using the Tradescantia-stamen hair žTrad-SHM / and the Tradescantia-micronucleus žTrad-MCN / bioassays Anette Fomin a
a,)
, Albrecht Paschke b, Uwe Arndt
a
Institute of Landscape and Plant Ecology, Department of Plant Ecology and Ecotoxicology, UniÕersity of Hohenheim, Germany b Department of Chemical Ecotoxicology, UFZ-Centre for EnÕironmental Research, Leipzig-Halle, Germany Received 10 March 1998; accepted 11 August 1998
Abstract The Tradescantia-stamen hair ŽTrad-SHM. and -micronucleus ŽTrad-MCN. bioassays were used to determine the genotoxicity of two eluates derived from mine tailings. The goal was to test the suitability of the Tradescantia bioassays as screening tools for this kind of waste material. Leachates obtained using the current standard German leaching test methods ŽS4 eluate. as well as leachates obtained using a new eluation method ŽpH stat4. were tested and compared. Concentration of heavy metals in the pH stat4 eluate were much higher than in the S4 eluate. The chemical analysis corresponded well with the results of the bioassays. Exposure to solutions containing more than 1% pH stat4 eluate caused a significantly higher number of micronuclei. The Trad-SHM bioassay also showed an increased pink mutation rate when plants were exposed to 8 or 16% eluate solutions. In contrast, the S4 eluate only caused increased mutation rates when solutions containing more than 32% eluate were used. The low pH of the pH stat4 eluate was not responsible for the genotoxicity observed using both bioassays, as indicated by the lack of significant mutation rates in the nitric acid controls. This demonstrates that the Tradescantia bioassays can be used as tools to assess the genotoxic potential of environmental samples with a wide range of pH values, without the need for sample modification. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Bioassay; Leachate; Spoil heap material; Tradescantia
1. Introduction The assessment of toxic potential of individual chemicals and complex mixtures with different bioassays is undertaken by many national and international environmental regulatory agencies w1–3x. These bioassays are also being used increasingly to ) Corresponding author. Tel.: q49-711-459-2189; Fax: q49711-459-3044; E-mail: [email protected]
estimate hazard potential of industrial waste material and waste disposal sites w4–6x. Bioassays give information on the effects of complex mixtures of chemicals on organisms, which in general cannot be obtained from analytical results. Nevertheless, the analytical data are necessary for determining causality and developing conservation or remediation strategies. Thus, a combined investigation is required. A great number of bioassays encompassing a range of different biological systems and physio-
0027-5107r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 Ž 9 9 . 0 0 0 6 4 - 0
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logical and genetic endpoints currently exist. For testing mutagenic effects of chemical compounds and mixtures, bacterial tests, such as the Ames-test w7x or the umu-test w8,9x are frequently used. However, some plants are very sensitive to genotoxic compounds and are increasingly being used for monitoring of environmental pollutants w10,11x. In particular, a number of review articles on the use of Tradescantia plants for the detection of genotoxic substances in various media, with differing viewpoints, have been published w12–16x. Tradescantia can indicate mutagenic effects through the induction of micronuclei in meiotic or mitotic cells and pink mutations in stamen hairs. In many cases, such as for putative environmental pollutants, it is suitable to use these plant systems as a screening system for detecting the presence of genotoxic agents in the test medium. Vast quantities of untreated spoil heap material and mine tailings dumped over a period of several decades have created an urgent environmental problem in the former East Germany. These spoil heaps and mine tailings cause serious environmental pollution problems, especially because of acid rain deposition and subsequent drainage of leachate into the surrounding environment w17x. Heavy metals and toxic organic compounds derived from the waste material are often released into the environment in high concentrations. Recycling and dumping of industrial waste material Žslags, ashes, metal sludges, etc.. can also cause such problems. The only standardized German leaching test is that according to DIN 38414-S4 w18x specifications. It was developed to determine the leachability of heavy metals from sludges and sediments by water but is also widely used for measuring the mobilization of inorganic and organic compounds from solid waste w19,20x. This test simulates the initial contact of waste material with water, i.e., only the short-term release of harmful chemical substances. Increased environmental relevance of the leaching test was achieved with the development of the pH stat method w20,21x. In order to simulate the worst-case of acidic deposition an agitated suspension of test material and water is kept at a constant pH s 4 by an automated titration with nitric acid for a 24-h period. With such a procedure it is possible to determine the acid neutralization capacity of the material and the influ-
Fig. 1. Location of the University of Hohenheim in relation to other major cities.
ence of pH-lowering on the mobilization of harmful chemical compounds. Such concentrated leachates represent an operationally-defined worst case scenario. It is instructive to test such eluates with bioassays after considerable dilution because leachate from such heaps and other waste disposal sites will also be diluted under natural conditions by groundwater streams, etc. In this study ŽFig. 1., we present a combination of analytical and bioassay-based investigations of two leachates derived from spoil-heap material from copper-slate mining in the Mansfeld region east of the Harz Mountains. The Trad-SHM w22,23x and the Trad-MCN w24,25x bioassays were used to test the genotoxic potential of the two leachates.
2. Materials and methods 2.1. InÕestigated material A representative sample of material from a slagheap near Hettstedt, in the Mansfeld region east of the Harz Mountains, Germany, was provided by the regional environmental protection agency ŽLandesamt fur ¨ Umweltschutz Sachsen-Anhalt, Haller Saale, Germany.. The material was ground to a final particle size of - 10 mm. The material was further
A. Fomin et al.r Mutation Research 426 (1999) 173–181
separated gravimetrically into particle fractions above and below 2.5 mm. 2.2. Leaching tests The standard leaching test according to DIN 38414-S4 w18x specifications was carried out by mixing 100 g of sample material and 1000 g distilled water in square flasks ŽPE. on a rotating shaker GFL 3040 ŽGesellschaft fur ¨ Labortechnik, Burgwedel, Germany. at 3 rpm for 24 h. The eluate will be referred to henceforth as S4-eluate. The pH stat4-experiment was carried out using TITRO-8 instruments manufactured by Wittenfeld and Cornelius, Bochum ŽGermany.. Ninety grams of sample material and 900 ml of distilled water were placed in an Erlenmeyer flask Žequipped with a pH electrode and a burette tip for a computer-controlled titration. on a horizontal shaker. The suspension was shaken vigorously for 24 h at 175 rpm. The required pH value of 4 was attained within 30 min by adding 2.5 M HNO 3 . This pH value was held constant by gradual titration during the experiment. The eluate obtained was called pH stat4 eluate. Both leaching tests were repeated at least twice, and were conducted at room temperature. The suspended solid matter was separated from the liquid phase by centrifugation and, in the case of the pH stat4 eluates, by the additional step of pressure filtration with 0.80 mm cellulose acetate membranes. Leachate samples to be used for heavymetal analysis were stabilized by adding concentrated HNO 3 to reach a pH value of less than 2 and stored at 48C until analysis. For the determination of organic compounds it was necessary to extract them from the aqueous eluate with toluene Žliquid extraction of 750 ml eluate with 2 = 35 ml toluene followed by volume reduction of the toluene phase down to 10 ml in a rotary evaporator at 458C and 50 Torr.. The leachate aliquots used for the bioassays were not stabilized, but kept refrigerated in dark glass bottles and tested within 6 weeks. 2.3. Analytical determinations The heavy-metal concentrations in the eluates, with the exception of Hg, were determined with an ICP-AES system ŽSpectra flame PrM, Spectro A.I.. following DIN 38406-E22 w26x specifications. Ar-
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senic and mercury concentrations were determined using an Atomic Absorption Spectrophotometry Žwith a PE-3100 and FIMS system, Perkin-Elmer. according to DIN 38405-D18 w27x and DIN 38405-E12 w28x specifications, respectively. Chloride and sulphate concentrations in the eluates were determined by ion chromatography according to DIN 38405-D19 w29x specifications. The quantification of dissolved organic matter ŽDOC. was achieved by oxidation with sodium peroxodisulphate according to DIN 38409-H3 w30x specifications Žwith Liqui-TOC, Heraeus. and adsorbable organic halogens ŽAOX. were determined according to DIN 38409-H14 w31x specifications Žwith ECS 2000, Euroglas.. The total amount of hydrocarbons in the eluate samples was measured with a FTIR-spectrometer ŽSystem 2000, Perkin-Elmer. according to DIN 38409-H18 w32x specifications. For the determination of polycyclic aromatic hydrocarbons ŽPAH. a HPLC system ŽGold, Beckmann. with fluorescence detector ŽRF-551, Shimadzu. was used. The detection of some other priority xenobiotics— hexachlorobenzene ŽHCB., hexachlorocyclohexaneisomers ŽHCH. and seven of the polychlorinated biphenyls ŽPCB. —was carried out according to DIN 38407-F2 w33x specifications via gas chromatography ŽHP 5890 II and MSD, Hewlett-Packard.. 2.4. Tradescantia bioassays Tradescantia clone 4430 was used exclusively in this study. The stamen-hair-mutation-bioassay ŽTradSHM. was carried out according to the specifications developed for the International Program on Chemical Safety w23x. For each eluate sample, 20–25 cuttings with inflorescence were placed in glass beakers containing various dilutions of the eluates. Dilutions of the eluate samples were prepared with distilled water. The exposure time was 24 h with a 14-h photoperiod and 25r188C dayrnight temperatures. For each sample, 10–15 flowers were evaluated daily, starting on the 6th day after treatment. The negative control for the Tradescantia cuttings was exposed to pure distilled water. Positive controls consisted of a single exposure to 1 mM maleic hydrazide as reported by Gichner et al. w34x. The frequency of pink mutation rate is the indicator of mutagenicity. The micronucleus-bioassay ŽTrad-MCN. were conducted according to the specifications developed
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for the International Program on Chemical Safety w25x. Briefly, fresh plant cuttings ŽTradescantia clone 4430. were exposed to the eluate samples for 24 h in 400 ml glass beakers, and then in Hoagland solution for 6 h. Dilutions of the eluate samples were prepared with distilled water. Subsequently, the inflorescence were fixed in ethanol–acetic acid Ž3:1. and transferred to 70% ethanol 24 h later. Early tetrad stages of the meiotic pollen mother cells were scored for MCNs under 400 = magnification. In general, five to seven slides were prepared per eluate and dilution, and 300 tetrads per slide were scored. The increase in frequency of MCN above the control value was the indicator of clastogenicity. The negative control for the Tradescantia cuttings was distilled water. The positive control consisted of a single exposure to 2 mM maleic hydrazide w35x. Since the pH stat4 eluate was eluted with nitric acid, a nitric acid control, consisting of the same amount of nitric acid as in the eluate plus distilled water at pH s 4.2 was also tested to reveal the possible adverse effect of the acid. 2.5. Statistical analysis of data All experiments with the eluates were repeated twice. Statistical analyses of Trad-MCN and TradSHM scores were performed using Statgraphics version 5.0 software. Differences between controls and treated groups were determined using the t-test at the 0.05 significance level. When the sample means of the negative control were significantly lower than that of treated groups, a positive response sign Žq. was given to the treated group.
3. Results 3.1. Chemical analysis The material under investigation was a marly, anthraconite copper slate with a high content of quartz and calcite. The results of a X-ray fluorescence analysis of the ground material are summarized in Table 1. In addition to the inorganic components the material contains considerable amounts of fixed organic carbon such as bitumen w36x. Therefore, it is possible that, in addition to the heavy
Table 1 Results of a X-ray fluorescence analysis of the spoil-heap material Ž% wwrwx or ppm. under investigation Compounds
w%x
SiO 2 Al 2 O 3 CaO MgO Na 2 O K 2O Fe 2 O 3 TiO 2 P2 O5
38.18 13.16 8.42 1.88 0.504 3.527 3.39 0.653 0.162
Elements
wppmx
S Cl Cd Cu Ni Cr Pb Zn Hg As V Mn Co Ba Rb Sr Zr U Mo Ag Sn
3402 - 20 60 7380 160 114 8200 15 933 3 268 718 1672 156 848 153 195 188 20 205 61 94
metals, harmful organic compounds such as PAH could also be mobilized in contact with water streams. Results of the chemical analysis of leachates S4 and pH stat4 are summarized in Table 2. The original pH value of the S4 leachate was approximately 8.5 due to the high content of alkaline components. This is also demonstrated by the relatively high acid neutralization capacity ŽANC. of 799 " 23 mmol wHqx rkg determined in the pH stat4 test after 24 h. This value is comparable to that of a refuse-incineration ash w21x. For a correct interpretation of the results of bioassay testing it is necessary to take into account the amount of hydrogen protons in the pH stat4 eluate, which were 10y4 molrl wHqx under the present test conditions. As expected, the heavy metal
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Table 2 The concentrations Žin mgrl( ppm. of analytes in the bioassaytested eluates Žaveraged values from two independent leaching experiments.; for analytical details see text
tion, the mobilization was greater under pH stat4 conditions.
Analyte
S4 eluate
pH stat4 eluate
3.2. Bioassays
Cd Cu Ni Pb Cr Zn Hg As Cl SO42y AOX HCB DOC Ý Hydrocarbons Ý PAH ŽEPA 610. w44x
- 0.01 0.028 0.010 0.060 0.003 0.100 - 0.0001 0.003 16.60 60.25 0.245 0.011 2.16 0.20 0.019
1.415 90.181 1.146 40.025 0.004 473.25 - 0.0001 0.004 72.45 108.10 0.032 0.004 4.54 0.44 0.07
concentrations in the pH stat4 eluates were much higher than in S4 eluates due to the mobilizing effect of the nitric acid. Of the chlorinated hydrocarbons measured Žsee Table 2., only HCB concentrations slightly exceeded the limit of detection. The total PAH’s Že.g., benzoŽ k .fluoranthene, benzoŽ a.pyrene. exceeded the limit of detection considerably. In addi-
3.2.1. Trad-SHM bioassay Results of the Trad-SHM bioassays demonstrated that solutions containing 8% and 16% pH stat4 eluate produced significantly higher mutation rates than did the controls ŽTable 3.. The plants stopped flowering altogether when exposed to 32% eluate solutions, suggesting the onset of cytotoxic reactions. Mutation rates of the nitric acid controls were no higher than in the negative controls, confirming that mutagenic effects were due to compounds in the eluate, and not due to the nitric acid. The S4 eluate was less toxic, and only induced significant mutation rates in plants exposed to solutions containing 64% eluate ŽTable 4.. Even at such high percentages of the eluate, no reduction in flowering rate, or other negative physiological effects were observed. 3.2.2. Trad-MCN bioassay The Trad-MCN bioassay proved to be a more sensitive test of genotoxicity in the leachate than the Trad-SHM bioassay. Solutions containing pH stat4
Table 3 Results of pH stat4 eluate testing with the Trad-SHM assay pH stat4 eluate Ž%.
Experiment 1
0 ŽNCU . 1 2 4 8 16 32 PCUU HNO 3-solutionUUU
3.49 " 0.52 3.30 " 2.39 4.53 " 2.36 4.30 " 2.36 6.04 " 2.00 5.82 " 1.79 overdose 9.08 " 3.44
Mr1000 hairs " SE
U
Experiment 2 Significance
Mr1000 hairs " SE
Significance
y y y q q
1.15 " 0.73 1.03 " 0.58 1.47 " 0.92 2.10 " 1.03 2.35 " 0.89 3.72 " 1.79
y y y q q
1.58 " 0.94
y
q
NC, negative control. PC, positive control; 6-h exposure with 1 mM maleic hydrazide w34x. UUU Under the present test conditions the determined acid neutralization value ŽANC. corresponds to 10y4 molrl wHqx . The nitric acid solution with pH value of 4.2 was produced in the same way as the eluate testing. Plus signs indicate a significant difference Ž a s 0.05. between control and treatment. UU
A. Fomin et al.r Mutation Research 426 (1999) 173–181
178
Table 4 Results of S4 eluate testing with the Trad-SHM assay pH stat4 eluate Ž%. 0 ŽNCU . 4 8 16 32 64 PCUU
Experiment 1
Experiment 2
Mr1000 hairs " SE
Significance
1.17 " 0.91 1.92 " 1.17 1.58 " 1.05 1.22 " 0.58 1.69 " 1.02 4.07 " 0.58 8.25 " 3.20
y y y y q q
Mr1000 hairs " SE
Significance
1.31 " 1.23 1.49 " 1.07 1.21 " 0.78 1.83 " 1.34 5.44 " 2.84 4.08 " 1.05
y y y y q
U
NC, negative control. PC, positive control; 6-h exposure with 1 mM maleic hydrazide w34x. Plus signs indicate significant difference Ž a s 0.05. between control and treatment.
UU
Table 5 Results of pH stat4 eluate testing with the Trad-MCN assay pH stat4 eluate Ž%. U
0 ŽNC . 1 2 4 8 16 PCU HNO 3-solutionUU
Experiment 1
Experiment 2
MCNr100 tetrads" SE
Significance
1.32 " 0.57 2.03 " 0.52 3.18 " 1.00 4.11 " 1.78 6.05 " 2.86 6.42 " 3.59 4.80 " 0.56
y q q q q q
MCNr100 tetrads" SE
Significance
1.19 " 1.03 1.89 " 0.61 4.00 " 0.73 4.71 " 1.55 4.38 " 1.37 6.08 " 4.50
y q q q q
1.67 " 1.00
y
U
NC, negative control. PC, positive control; 6-h exposure with 2 mM maleic hydrazide w35x. UUU Under the present test conditions the determined acid neutralization value ŽANC. corresponds to 10y4 molrl wHqx . The nitric acid solution with pH value of 4.2 was produced on the same way as the eluate testing. Plus signs indicate significant difference Ž a s 0.05. between control and treatment. UU
Table 6 Results of S4 eluate testing with the Trad-MCN assay pH stat4 eluate Ž%. 0 ŽNCU . 8 16 32 64 PCUU U
Experiment 1
Experiment 2
MCNr100 tetrads" SE
Significance
1.91 " 0.77 2.26 " 1.24 1.83 " 0.69 4.31 " 1.99 4.93 " 1.78 5.23 " 0.72
y y q q q
MCNr100 tetrads" SE
Significance
1.79 " 0.93 1.62 " 0.41 2.31 " 1.25 3.73 " 1.94 6.88 " 3.21
y y q q
NC, negative control. PC, positive control; 6-h exposure with 2 mM maleic hydrazide w35x. Plus signs indicate significant difference Ž a s 0.05. between control and treatment.
UU
A. Fomin et al.r Mutation Research 426 (1999) 173–181
eluate caused significant increases in micronuclei in all except 1% eluate solutions ŽTable 5.. The results were the same in both experiments. As with the Trad-SHM bioassays, nitric acid controls did not have significantly higher mutation rates than negative controls. Exposure to solutions containing 32 and 64% S4 eluate lead to a significantly greater number of micronuclei in both experiments ŽTable 6.. The results of both bioassays demonstrated the greater genotoxicity of the pH stat4 eluate compared to the S4 eluate, thereby supporting the findings of the chemical analyses, which reported higher concentrations of most compounds in the pH stat4 eluate.
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Tradescantia w35x. Thus, the toxicity of single compounds to Tradescantia does not always correctly predict the genotoxicity of complex mixtures such as leachate, in which additive effects occur. The results of the study further confirm the importance of a combined analytical and toxicological approach to estimate the genotoxic potential of mining waste material in order to develop effective remediation strategies. Both the Trad-SHM and the Trad-MCN bioassays are effective tools for the assessment of genotoxicity of leachate obtained from spoil heap material.
Acknowledgements 4. Discussion The results of this study demonstrated the suitability of both the stamen-hair ŽTrad-SHM. and the micronuclei ŽTrad-MCN. bioassays to assess the genotoxic potential of leachate obtained from contaminated soils. Although these tests are not new, and have been widely used to screen for genotoxicity in various media w37–40x, they never been used to test leachate obtained from mining waste material. In addressing the question of acid samples, the Tradescantia bioassays have an important advantage over traditional mutagenic tests using bacteria. Most of the bacterial mutagenic tests require circumneutral pH conditions in the test media w41,42x, that frequently means altering the original sample. Neutralizing acidic samples can change the solubility of key compounds, thereby changing the overall toxicity of the sample. This disadvantage is absent in the Tradescantia bioassay, which can be performed at a much wider range of pH values. This was demonstrated in this study, in that the pH of the two eluates differed greatly Ž4.0 for pH stat4 eluate, 8.5 for S4 eluate.. The chemical analysis of the two leachates suggested possible sources of genotoxicity in the samples, however no definite conclusions could be drawn w43x. Although metals such as arsenic, lead and cadmium have been shown to be toxic to Tradescantia w35,44x, the observed concentrations of cadmium, copper, arsenic, zinc and lead in both eluates were well below those found to be genotoxic to
We thank Dr. Gerald Schumann and Jorg ¨ Winkler ŽLandesamt fur ¨ Umweltschutz Sachsen-Anhalt. for supplying the sample material and determining AOX, ŽUFZ. is thanked for carrespectively. Elke Buttner ¨ rying out the leaching experiments. For the analytical determinations in the Department of Analytical Chemistry of UFZ we are grateful to Dr. Rainer Wennrich, Dr. Johannes Flachowsky and Dr. Peter Popp. The assistance of Simone Ressler and Frank Bohnsack ŽHohenheim. in conducting the SHM bioassays is also gratefully acknowledged.
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desamt fur ¨ Wasser und Abfall Nordrhein-Westfalen, Dusseldorf, Bd. 6, 1992. ¨ A.G. Underbrink, L.A. Schairer, A.H. Sparrow, Tradescantia stamen hairs: a radiological test system applicable to chemical mutagenesis, in: A. Hollaender ŽEd.., Chemical Mutagens, Principle and Methods for their Detection, Vol. 3, Plenum, New York, 1973, pp. 171–207. T.H. Ma, G.L. Cabrera, A. Cebulska-Wasilewska, R. Chen, F. Loarca, A.L. Vanderberg, M.F. Salomone, Tradescantia stamen hair bioassay, Mutat. Res. 310 Ž1994. 211–220. T.H. Ma, Tradescantia cytogenetic tests Žroot-tip mitosis, pollen mitosis, pollen mother cell meiosis., Mutat. Res. 99 Ž1982. 293–302. T.H. Ma, G.L. Cabrera, R. Chen, B.S. Gill, S.S. Sandhu, A.L. Vanderberg, M.F. Salomone, Tradescantia micronucleus bioassay, Mutat. Res. 310 Ž1994. 221–230. DIN 38406-E22: German standard methods for the estimation of water, waste water and sludges; cations Žgroup E.; Determination of 33 elements by inductively coupled plasma emission spectrometry ŽICP-OES., March 1988. DIN 38405-D18: German standard methods for the estimation of water, waste water and sludges; anions Žgroup D.; Determination of arsenic by atomic absorption spectrometry, September 1985. DIN 38405-E12: German standard methods for the estimation of water, waste water and sludges; cations Žgroup E.; Determination of mercury, July 1980. DIN 38405-D19: German standard methods for the estimation of water, waste water and sludges; anions Žgroup D.; Determination of fluoride, chloride, nitrite, orthophosphate, bromide, nitrate and sulphate by ion chromatography, February 1988. DIN 38409-H3: German standard methods for the estimation of water, waste water and sludges; general measures of effects and substances Žgroup H.; Determination of total organic carbon ŽTOC., June 1983. DIN 38408-H14: German standard methods for the estimation of water, waste water and sludges; general measures of effects and substances Žgroup H.; Determination of adsorbable organic halogens ŽAOX., March 1985. DIN 38409-H18: German standard methods for the estimation of water, waste water and sludges; general measures of effects and substances Žgroup H.; Determination of hydrocarbons, February 1981. DIN 38407-F2: German standard methods for the estimation of water, waste water and sludges; jointly determinable substances Žgroup F.; Determination of low volatile halogenated hydrocarbons by gas chromatography, February 1993. T. Gichner, C. Langebartels, H. Sandermann, Ozone is not mutagenic in the Tradescantia and tobacco mutagenicity assays, Mutat. Res. 281 Ž1992. 203–206. H. Steinkellner, Vergleichende Untersuchungen zur Gentoxizitat ¨ von Schwermetallen und schwermetallbelasteten Boden ¨ mit Kleinkerntests bei Pflanzen, Unveroffentlichte Diplomar¨ beit der Universitat ¨ Wien, 1996, 60 S. R. Jubelt, P. Schreiter, Gesteinsbestimmungsbuch, Dt. Verlag fur ¨ Grundstoffindustrie, Leipzig, Germany, 8. Aufl., 1982.
A. Fomin et al.r Mutation Research 426 (1999) 173–181 w37x T.H. Ma, S.S. Sandhu, Y. Peng, T.D. Chen, T. Kim, Synergistic and antagonistic effects on genotoxicity of chemicals commonly found in hazardous waste sites, Mutat. Res. 270 Ž1992. 71–77. w38x E.F. Ruiz, V.M.E. Rabago, S.U. Lecona, A.B. Perez, T.H. Ma, Tradescantia-micronucleus ŽTrad-MCN. bioassay on clastogenicity of wastewater and in situ monitoring, Mutat. Res. 270 Ž1992. 45–51. w39x S. Knasmuller, T.W. Kim, T.H. Ma, Synergistic effects ¨ between tannic acid and X-rays detected by the Tradescantia-micronucleus assay, Mutat. Res. 270 Ž1992. 31–37. w40x B.N. Ames, J. McCann, E. Yamasaki, Methods for detecting carcinogens and mutagens with Salmonella mammalian microsome mutagenicity test, Mutat. Res. 31 Ž1975. 347–364. w41x Y. Oda, S.I. Nakamura, I. Oki, T. Kato, H. Shinagawa,
w42x
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Evaluation of the new system Ž umu-test. for the detection of environmental mutagens and carcinogens, Mutat. Res. 147 Ž1985. 219–229. U. Arndt, W. Nobel, B. Schweizer, BioindikatorenMoglichkeiten, Grenzen und neue Erkenntnisse, Ulmer Ver¨ lag, Stuttgart, 1987, 388 S. H. Steinkellner, M.S. Kong, C. Helma, T.H. Ma, S. Knasmuller, Vergleichende Untersuchungen zur Kleinkernin¨ duktion in Pflanzen ŽTradescantia paludosa Klon 4430, Allium cepa und Vicia faba. durch Schwermetalle. Abstract 15. Jahrestagung der Gesellschaft fur ¨ Umweltmutagene ŽGUM.: Tierversuche in der Mutations- und Krebsforschung: Notwendigkeit und in vitro Alternativen. Gmunden am ¨ Traunsee, Osterreich 1995, p. 84. EPA, Polynuclear aromatic hydrocarbons—methods 610, Fat. Reg. 44 Ž1979. 69514.
Assessment of the genotoxicity of mine-dump material using the Tradescantia-stamen hair žTrad-SHM / and the Tradescantia-micronucleus žTrad-MCN / bioassays Anette Fomin a
a,)
, Albrecht Paschke b, Uwe Arndt
a
Institute of Landscape and Plant Ecology, Department of Plant Ecology and Ecotoxicology, UniÕersity of Hohenheim, Germany b Department of Chemical Ecotoxicology, UFZ-Centre for EnÕironmental Research, Leipzig-Halle, Germany Received 10 March 1998; accepted 11 August 1998
Abstract The Tradescantia-stamen hair ŽTrad-SHM. and -micronucleus ŽTrad-MCN. bioassays were used to determine the genotoxicity of two eluates derived from mine tailings. The goal was to test the suitability of the Tradescantia bioassays as screening tools for this kind of waste material. Leachates obtained using the current standard German leaching test methods ŽS4 eluate. as well as leachates obtained using a new eluation method ŽpH stat4. were tested and compared. Concentration of heavy metals in the pH stat4 eluate were much higher than in the S4 eluate. The chemical analysis corresponded well with the results of the bioassays. Exposure to solutions containing more than 1% pH stat4 eluate caused a significantly higher number of micronuclei. The Trad-SHM bioassay also showed an increased pink mutation rate when plants were exposed to 8 or 16% eluate solutions. In contrast, the S4 eluate only caused increased mutation rates when solutions containing more than 32% eluate were used. The low pH of the pH stat4 eluate was not responsible for the genotoxicity observed using both bioassays, as indicated by the lack of significant mutation rates in the nitric acid controls. This demonstrates that the Tradescantia bioassays can be used as tools to assess the genotoxic potential of environmental samples with a wide range of pH values, without the need for sample modification. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Bioassay; Leachate; Spoil heap material; Tradescantia
1. Introduction The assessment of toxic potential of individual chemicals and complex mixtures with different bioassays is undertaken by many national and international environmental regulatory agencies w1–3x. These bioassays are also being used increasingly to ) Corresponding author. Tel.: q49-711-459-2189; Fax: q49711-459-3044; E-mail: [email protected]
estimate hazard potential of industrial waste material and waste disposal sites w4–6x. Bioassays give information on the effects of complex mixtures of chemicals on organisms, which in general cannot be obtained from analytical results. Nevertheless, the analytical data are necessary for determining causality and developing conservation or remediation strategies. Thus, a combined investigation is required. A great number of bioassays encompassing a range of different biological systems and physio-
0027-5107r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 Ž 9 9 . 0 0 0 6 4 - 0
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logical and genetic endpoints currently exist. For testing mutagenic effects of chemical compounds and mixtures, bacterial tests, such as the Ames-test w7x or the umu-test w8,9x are frequently used. However, some plants are very sensitive to genotoxic compounds and are increasingly being used for monitoring of environmental pollutants w10,11x. In particular, a number of review articles on the use of Tradescantia plants for the detection of genotoxic substances in various media, with differing viewpoints, have been published w12–16x. Tradescantia can indicate mutagenic effects through the induction of micronuclei in meiotic or mitotic cells and pink mutations in stamen hairs. In many cases, such as for putative environmental pollutants, it is suitable to use these plant systems as a screening system for detecting the presence of genotoxic agents in the test medium. Vast quantities of untreated spoil heap material and mine tailings dumped over a period of several decades have created an urgent environmental problem in the former East Germany. These spoil heaps and mine tailings cause serious environmental pollution problems, especially because of acid rain deposition and subsequent drainage of leachate into the surrounding environment w17x. Heavy metals and toxic organic compounds derived from the waste material are often released into the environment in high concentrations. Recycling and dumping of industrial waste material Žslags, ashes, metal sludges, etc.. can also cause such problems. The only standardized German leaching test is that according to DIN 38414-S4 w18x specifications. It was developed to determine the leachability of heavy metals from sludges and sediments by water but is also widely used for measuring the mobilization of inorganic and organic compounds from solid waste w19,20x. This test simulates the initial contact of waste material with water, i.e., only the short-term release of harmful chemical substances. Increased environmental relevance of the leaching test was achieved with the development of the pH stat method w20,21x. In order to simulate the worst-case of acidic deposition an agitated suspension of test material and water is kept at a constant pH s 4 by an automated titration with nitric acid for a 24-h period. With such a procedure it is possible to determine the acid neutralization capacity of the material and the influ-
Fig. 1. Location of the University of Hohenheim in relation to other major cities.
ence of pH-lowering on the mobilization of harmful chemical compounds. Such concentrated leachates represent an operationally-defined worst case scenario. It is instructive to test such eluates with bioassays after considerable dilution because leachate from such heaps and other waste disposal sites will also be diluted under natural conditions by groundwater streams, etc. In this study ŽFig. 1., we present a combination of analytical and bioassay-based investigations of two leachates derived from spoil-heap material from copper-slate mining in the Mansfeld region east of the Harz Mountains. The Trad-SHM w22,23x and the Trad-MCN w24,25x bioassays were used to test the genotoxic potential of the two leachates.
2. Materials and methods 2.1. InÕestigated material A representative sample of material from a slagheap near Hettstedt, in the Mansfeld region east of the Harz Mountains, Germany, was provided by the regional environmental protection agency ŽLandesamt fur ¨ Umweltschutz Sachsen-Anhalt, Haller Saale, Germany.. The material was ground to a final particle size of - 10 mm. The material was further
A. Fomin et al.r Mutation Research 426 (1999) 173–181
separated gravimetrically into particle fractions above and below 2.5 mm. 2.2. Leaching tests The standard leaching test according to DIN 38414-S4 w18x specifications was carried out by mixing 100 g of sample material and 1000 g distilled water in square flasks ŽPE. on a rotating shaker GFL 3040 ŽGesellschaft fur ¨ Labortechnik, Burgwedel, Germany. at 3 rpm for 24 h. The eluate will be referred to henceforth as S4-eluate. The pH stat4-experiment was carried out using TITRO-8 instruments manufactured by Wittenfeld and Cornelius, Bochum ŽGermany.. Ninety grams of sample material and 900 ml of distilled water were placed in an Erlenmeyer flask Žequipped with a pH electrode and a burette tip for a computer-controlled titration. on a horizontal shaker. The suspension was shaken vigorously for 24 h at 175 rpm. The required pH value of 4 was attained within 30 min by adding 2.5 M HNO 3 . This pH value was held constant by gradual titration during the experiment. The eluate obtained was called pH stat4 eluate. Both leaching tests were repeated at least twice, and were conducted at room temperature. The suspended solid matter was separated from the liquid phase by centrifugation and, in the case of the pH stat4 eluates, by the additional step of pressure filtration with 0.80 mm cellulose acetate membranes. Leachate samples to be used for heavymetal analysis were stabilized by adding concentrated HNO 3 to reach a pH value of less than 2 and stored at 48C until analysis. For the determination of organic compounds it was necessary to extract them from the aqueous eluate with toluene Žliquid extraction of 750 ml eluate with 2 = 35 ml toluene followed by volume reduction of the toluene phase down to 10 ml in a rotary evaporator at 458C and 50 Torr.. The leachate aliquots used for the bioassays were not stabilized, but kept refrigerated in dark glass bottles and tested within 6 weeks. 2.3. Analytical determinations The heavy-metal concentrations in the eluates, with the exception of Hg, were determined with an ICP-AES system ŽSpectra flame PrM, Spectro A.I.. following DIN 38406-E22 w26x specifications. Ar-
175
senic and mercury concentrations were determined using an Atomic Absorption Spectrophotometry Žwith a PE-3100 and FIMS system, Perkin-Elmer. according to DIN 38405-D18 w27x and DIN 38405-E12 w28x specifications, respectively. Chloride and sulphate concentrations in the eluates were determined by ion chromatography according to DIN 38405-D19 w29x specifications. The quantification of dissolved organic matter ŽDOC. was achieved by oxidation with sodium peroxodisulphate according to DIN 38409-H3 w30x specifications Žwith Liqui-TOC, Heraeus. and adsorbable organic halogens ŽAOX. were determined according to DIN 38409-H14 w31x specifications Žwith ECS 2000, Euroglas.. The total amount of hydrocarbons in the eluate samples was measured with a FTIR-spectrometer ŽSystem 2000, Perkin-Elmer. according to DIN 38409-H18 w32x specifications. For the determination of polycyclic aromatic hydrocarbons ŽPAH. a HPLC system ŽGold, Beckmann. with fluorescence detector ŽRF-551, Shimadzu. was used. The detection of some other priority xenobiotics— hexachlorobenzene ŽHCB., hexachlorocyclohexaneisomers ŽHCH. and seven of the polychlorinated biphenyls ŽPCB. —was carried out according to DIN 38407-F2 w33x specifications via gas chromatography ŽHP 5890 II and MSD, Hewlett-Packard.. 2.4. Tradescantia bioassays Tradescantia clone 4430 was used exclusively in this study. The stamen-hair-mutation-bioassay ŽTradSHM. was carried out according to the specifications developed for the International Program on Chemical Safety w23x. For each eluate sample, 20–25 cuttings with inflorescence were placed in glass beakers containing various dilutions of the eluates. Dilutions of the eluate samples were prepared with distilled water. The exposure time was 24 h with a 14-h photoperiod and 25r188C dayrnight temperatures. For each sample, 10–15 flowers were evaluated daily, starting on the 6th day after treatment. The negative control for the Tradescantia cuttings was exposed to pure distilled water. Positive controls consisted of a single exposure to 1 mM maleic hydrazide as reported by Gichner et al. w34x. The frequency of pink mutation rate is the indicator of mutagenicity. The micronucleus-bioassay ŽTrad-MCN. were conducted according to the specifications developed
176
A. Fomin et al.r Mutation Research 426 (1999) 173–181
for the International Program on Chemical Safety w25x. Briefly, fresh plant cuttings ŽTradescantia clone 4430. were exposed to the eluate samples for 24 h in 400 ml glass beakers, and then in Hoagland solution for 6 h. Dilutions of the eluate samples were prepared with distilled water. Subsequently, the inflorescence were fixed in ethanol–acetic acid Ž3:1. and transferred to 70% ethanol 24 h later. Early tetrad stages of the meiotic pollen mother cells were scored for MCNs under 400 = magnification. In general, five to seven slides were prepared per eluate and dilution, and 300 tetrads per slide were scored. The increase in frequency of MCN above the control value was the indicator of clastogenicity. The negative control for the Tradescantia cuttings was distilled water. The positive control consisted of a single exposure to 2 mM maleic hydrazide w35x. Since the pH stat4 eluate was eluted with nitric acid, a nitric acid control, consisting of the same amount of nitric acid as in the eluate plus distilled water at pH s 4.2 was also tested to reveal the possible adverse effect of the acid. 2.5. Statistical analysis of data All experiments with the eluates were repeated twice. Statistical analyses of Trad-MCN and TradSHM scores were performed using Statgraphics version 5.0 software. Differences between controls and treated groups were determined using the t-test at the 0.05 significance level. When the sample means of the negative control were significantly lower than that of treated groups, a positive response sign Žq. was given to the treated group.
3. Results 3.1. Chemical analysis The material under investigation was a marly, anthraconite copper slate with a high content of quartz and calcite. The results of a X-ray fluorescence analysis of the ground material are summarized in Table 1. In addition to the inorganic components the material contains considerable amounts of fixed organic carbon such as bitumen w36x. Therefore, it is possible that, in addition to the heavy
Table 1 Results of a X-ray fluorescence analysis of the spoil-heap material Ž% wwrwx or ppm. under investigation Compounds
w%x
SiO 2 Al 2 O 3 CaO MgO Na 2 O K 2O Fe 2 O 3 TiO 2 P2 O5
38.18 13.16 8.42 1.88 0.504 3.527 3.39 0.653 0.162
Elements
wppmx
S Cl Cd Cu Ni Cr Pb Zn Hg As V Mn Co Ba Rb Sr Zr U Mo Ag Sn
3402 - 20 60 7380 160 114 8200 15 933 3 268 718 1672 156 848 153 195 188 20 205 61 94
metals, harmful organic compounds such as PAH could also be mobilized in contact with water streams. Results of the chemical analysis of leachates S4 and pH stat4 are summarized in Table 2. The original pH value of the S4 leachate was approximately 8.5 due to the high content of alkaline components. This is also demonstrated by the relatively high acid neutralization capacity ŽANC. of 799 " 23 mmol wHqx rkg determined in the pH stat4 test after 24 h. This value is comparable to that of a refuse-incineration ash w21x. For a correct interpretation of the results of bioassay testing it is necessary to take into account the amount of hydrogen protons in the pH stat4 eluate, which were 10y4 molrl wHqx under the present test conditions. As expected, the heavy metal
A. Fomin et al.r Mutation Research 426 (1999) 173–181
177
Table 2 The concentrations Žin mgrl( ppm. of analytes in the bioassaytested eluates Žaveraged values from two independent leaching experiments.; for analytical details see text
tion, the mobilization was greater under pH stat4 conditions.
Analyte
S4 eluate
pH stat4 eluate
3.2. Bioassays
Cd Cu Ni Pb Cr Zn Hg As Cl SO42y AOX HCB DOC Ý Hydrocarbons Ý PAH ŽEPA 610. w44x
- 0.01 0.028 0.010 0.060 0.003 0.100 - 0.0001 0.003 16.60 60.25 0.245 0.011 2.16 0.20 0.019
1.415 90.181 1.146 40.025 0.004 473.25 - 0.0001 0.004 72.45 108.10 0.032 0.004 4.54 0.44 0.07
concentrations in the pH stat4 eluates were much higher than in S4 eluates due to the mobilizing effect of the nitric acid. Of the chlorinated hydrocarbons measured Žsee Table 2., only HCB concentrations slightly exceeded the limit of detection. The total PAH’s Že.g., benzoŽ k .fluoranthene, benzoŽ a.pyrene. exceeded the limit of detection considerably. In addi-
3.2.1. Trad-SHM bioassay Results of the Trad-SHM bioassays demonstrated that solutions containing 8% and 16% pH stat4 eluate produced significantly higher mutation rates than did the controls ŽTable 3.. The plants stopped flowering altogether when exposed to 32% eluate solutions, suggesting the onset of cytotoxic reactions. Mutation rates of the nitric acid controls were no higher than in the negative controls, confirming that mutagenic effects were due to compounds in the eluate, and not due to the nitric acid. The S4 eluate was less toxic, and only induced significant mutation rates in plants exposed to solutions containing 64% eluate ŽTable 4.. Even at such high percentages of the eluate, no reduction in flowering rate, or other negative physiological effects were observed. 3.2.2. Trad-MCN bioassay The Trad-MCN bioassay proved to be a more sensitive test of genotoxicity in the leachate than the Trad-SHM bioassay. Solutions containing pH stat4
Table 3 Results of pH stat4 eluate testing with the Trad-SHM assay pH stat4 eluate Ž%.
Experiment 1
0 ŽNCU . 1 2 4 8 16 32 PCUU HNO 3-solutionUUU
3.49 " 0.52 3.30 " 2.39 4.53 " 2.36 4.30 " 2.36 6.04 " 2.00 5.82 " 1.79 overdose 9.08 " 3.44
Mr1000 hairs " SE
U
Experiment 2 Significance
Mr1000 hairs " SE
Significance
y y y q q
1.15 " 0.73 1.03 " 0.58 1.47 " 0.92 2.10 " 1.03 2.35 " 0.89 3.72 " 1.79
y y y q q
1.58 " 0.94
y
q
NC, negative control. PC, positive control; 6-h exposure with 1 mM maleic hydrazide w34x. UUU Under the present test conditions the determined acid neutralization value ŽANC. corresponds to 10y4 molrl wHqx . The nitric acid solution with pH value of 4.2 was produced in the same way as the eluate testing. Plus signs indicate a significant difference Ž a s 0.05. between control and treatment. UU
A. Fomin et al.r Mutation Research 426 (1999) 173–181
178
Table 4 Results of S4 eluate testing with the Trad-SHM assay pH stat4 eluate Ž%. 0 ŽNCU . 4 8 16 32 64 PCUU
Experiment 1
Experiment 2
Mr1000 hairs " SE
Significance
1.17 " 0.91 1.92 " 1.17 1.58 " 1.05 1.22 " 0.58 1.69 " 1.02 4.07 " 0.58 8.25 " 3.20
y y y y q q
Mr1000 hairs " SE
Significance
1.31 " 1.23 1.49 " 1.07 1.21 " 0.78 1.83 " 1.34 5.44 " 2.84 4.08 " 1.05
y y y y q
U
NC, negative control. PC, positive control; 6-h exposure with 1 mM maleic hydrazide w34x. Plus signs indicate significant difference Ž a s 0.05. between control and treatment.
UU
Table 5 Results of pH stat4 eluate testing with the Trad-MCN assay pH stat4 eluate Ž%. U
0 ŽNC . 1 2 4 8 16 PCU HNO 3-solutionUU
Experiment 1
Experiment 2
MCNr100 tetrads" SE
Significance
1.32 " 0.57 2.03 " 0.52 3.18 " 1.00 4.11 " 1.78 6.05 " 2.86 6.42 " 3.59 4.80 " 0.56
y q q q q q
MCNr100 tetrads" SE
Significance
1.19 " 1.03 1.89 " 0.61 4.00 " 0.73 4.71 " 1.55 4.38 " 1.37 6.08 " 4.50
y q q q q
1.67 " 1.00
y
U
NC, negative control. PC, positive control; 6-h exposure with 2 mM maleic hydrazide w35x. UUU Under the present test conditions the determined acid neutralization value ŽANC. corresponds to 10y4 molrl wHqx . The nitric acid solution with pH value of 4.2 was produced on the same way as the eluate testing. Plus signs indicate significant difference Ž a s 0.05. between control and treatment. UU
Table 6 Results of S4 eluate testing with the Trad-MCN assay pH stat4 eluate Ž%. 0 ŽNCU . 8 16 32 64 PCUU U
Experiment 1
Experiment 2
MCNr100 tetrads" SE
Significance
1.91 " 0.77 2.26 " 1.24 1.83 " 0.69 4.31 " 1.99 4.93 " 1.78 5.23 " 0.72
y y q q q
MCNr100 tetrads" SE
Significance
1.79 " 0.93 1.62 " 0.41 2.31 " 1.25 3.73 " 1.94 6.88 " 3.21
y y q q
NC, negative control. PC, positive control; 6-h exposure with 2 mM maleic hydrazide w35x. Plus signs indicate significant difference Ž a s 0.05. between control and treatment.
UU
A. Fomin et al.r Mutation Research 426 (1999) 173–181
eluate caused significant increases in micronuclei in all except 1% eluate solutions ŽTable 5.. The results were the same in both experiments. As with the Trad-SHM bioassays, nitric acid controls did not have significantly higher mutation rates than negative controls. Exposure to solutions containing 32 and 64% S4 eluate lead to a significantly greater number of micronuclei in both experiments ŽTable 6.. The results of both bioassays demonstrated the greater genotoxicity of the pH stat4 eluate compared to the S4 eluate, thereby supporting the findings of the chemical analyses, which reported higher concentrations of most compounds in the pH stat4 eluate.
179
Tradescantia w35x. Thus, the toxicity of single compounds to Tradescantia does not always correctly predict the genotoxicity of complex mixtures such as leachate, in which additive effects occur. The results of the study further confirm the importance of a combined analytical and toxicological approach to estimate the genotoxic potential of mining waste material in order to develop effective remediation strategies. Both the Trad-SHM and the Trad-MCN bioassays are effective tools for the assessment of genotoxicity of leachate obtained from spoil heap material.
Acknowledgements 4. Discussion The results of this study demonstrated the suitability of both the stamen-hair ŽTrad-SHM. and the micronuclei ŽTrad-MCN. bioassays to assess the genotoxic potential of leachate obtained from contaminated soils. Although these tests are not new, and have been widely used to screen for genotoxicity in various media w37–40x, they never been used to test leachate obtained from mining waste material. In addressing the question of acid samples, the Tradescantia bioassays have an important advantage over traditional mutagenic tests using bacteria. Most of the bacterial mutagenic tests require circumneutral pH conditions in the test media w41,42x, that frequently means altering the original sample. Neutralizing acidic samples can change the solubility of key compounds, thereby changing the overall toxicity of the sample. This disadvantage is absent in the Tradescantia bioassay, which can be performed at a much wider range of pH values. This was demonstrated in this study, in that the pH of the two eluates differed greatly Ž4.0 for pH stat4 eluate, 8.5 for S4 eluate.. The chemical analysis of the two leachates suggested possible sources of genotoxicity in the samples, however no definite conclusions could be drawn w43x. Although metals such as arsenic, lead and cadmium have been shown to be toxic to Tradescantia w35,44x, the observed concentrations of cadmium, copper, arsenic, zinc and lead in both eluates were well below those found to be genotoxic to
We thank Dr. Gerald Schumann and Jorg ¨ Winkler ŽLandesamt fur ¨ Umweltschutz Sachsen-Anhalt. for supplying the sample material and determining AOX, ŽUFZ. is thanked for carrespectively. Elke Buttner ¨ rying out the leaching experiments. For the analytical determinations in the Department of Analytical Chemistry of UFZ we are grateful to Dr. Rainer Wennrich, Dr. Johannes Flachowsky and Dr. Peter Popp. The assistance of Simone Ressler and Frank Bohnsack ŽHohenheim. in conducting the SHM bioassays is also gratefully acknowledged.
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