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Chromosomal aberrations in a fish, Channa punctata after in vivo exposure to three heavy metals
Mutation Research 678 (2009) 7–12
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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Chromosomal aberrations in a fish, Channa punctata after in vivo exposure to three heavy metals Kamlesh K. Yadav, Sunil P. Trivedi ∗ Environmental Toxicology Laboratory, Department of Zoology, University of Lucknow, Lucknow 226007, India
a r t i c l e
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Article history: Received 21 June 2008 Received in revised form 29 April 2009 Accepted 29 May 2009 Available online 21 June 2009 Keywords: Chromosomal aberrations Mercuric chloride Arsenic trioxide Copper sulphate Fish Channa punctata
a b s t r a c t The studies were designed to assess the extent of chromosomal aberrations (CA) under the exposure of three common heavy metalic compounds, viz. mercuric chloride, arsenic trioxide and copper sulphate pentahydrate, in vivo using fish, Channa punctata (2n = 32), as a test model. Prior acclimatized fishes were divided into five groups. Group I and II served as negative and positive control, respectively. An intramuscular injection of Mitomycin-C (@ 1 mg/kg body wt.) was administered to group II only. Fishes of groups III, IV and V were subjected to sublethal concentrations (10% of 96 h LC50 ), of HgCl2 (0.081 mg/L), As2 O3 (6.936 mg/L) and CuSO4 ·5H2 O (0.407 mg/L). Fishes of all the groups were exposed uninterrupted for 24, 48, 72, 96 and 168 h. Observations of kidney cells of exposed fishes revealed chromatid and chromosome breaks, chromatid and chromosome gaps along with ring and di-centric chromosomes. A significant increase over negative control in the frequency of chromosomal aberrations (CA) was observed in fish exposed to Mitomycin-C, Hg(II), As(III) and Cu(II). As the average ± SE total number of CA, average number of CA per metaphase and %incidence of aberrant cells in Hg(II) was 104.40 ± 8.189, 0.347 ± 0.027 and 10.220 ± 0.842, respectively; in As(III) 109.20 ± 8.309, 0.363 ± 0.027 and 10.820 ± 2.347, respectively and in Cu(II) 89.00 ± 19.066, 0.297 ± 0.028 and 8.900 ± 0.853, respectively. Hence, it reveals that the order of induction of frequency of CA was Cu < Hg < As. The findings depict genotoxic potential of these metals even in sublethal concentrations. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Metals and their compounds are natural constituents of different ecosystems [1]. They are ubiquitous contaminants in aquatic ecosystems [2–5]. Their distribution in the environment is governed by natural as well as anthropogenic activities [6]. Heavy metals received considerable attention due to their toxicity and potential to bioaccumulate in aquatic biota [7–9]. Hg(II), As(III) and Cu(II) are present in industrial and municipal waste water and in mine tailings. They also cause mutagenic and carcinogenic actions in living beings. Among heavy metals, mercuric chloride is a poisonous white crystalline compound, used since ages as a reliable ingredient in antiseptics, disinfectants and preservatives, insecticides, batteries and in metallurgical and photographic operations [10,11]. In India, about 200 ton mercury and its compounds are introduced into the environment annually as effluents from industries [12]. Exposure to high concentrations of mercury causes damage to nervous system, immune system, kidney and liver in human beings [13–15].
∗ Corresponding author. Tel.: +91 0522 2740040. E-mail addresses: kamlesh [email protected] (K.K. Yadav), [email protected] (S.P. Trivedi). 1383-5718/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2009.05.021
Exposure to mercuric chloride in human parotid salivary glands and lymphocytes has also been reported by Schmid et al. [16]. Occupational exposure of mercury vapour results in greater frequency of chromosomal aberrations [17]. Weis and Weis [18] reported teratogenic effect of inorganic mercury in killfish, Fundulus heteroclitus. Methyl mercury is known to induce chromosomal aberrations and micronuclei in the same fish [19]. Arsenic is introduced into water via weathering of rocks, minerals and ores, industrial effluents including mining wastes and atmospheric deposition [20]. Human exposure takes place via contaminated water and soil as well as from food rich in arsenic, viz., garlic, marine food, etc. and occupational activities [21,22]. Wider applications of arsenic compounds, particularly arsenic trioxide, as the starting substance for the manufacture of arsenic-based pesticides, arsenic-based pharamaceuticals (Neosalvarsan), veterinary products, decolorizing agents for glasses and enamels and wood preservatives make human beings more prone to its exposure. In spite of its high toxicity arsenic is a common contaminant in pharmaceuticals [23]. It is also used for therapeutic purposes for the treatment of chronic myelogenous leukaemia, leishmaniosis, trypanosomiasis [24] and cancer by inducing G(2)/M arrest [25]. Arsenic has long been regarded as a potential carcinogen [26–28], genotoxic both in vivo conditions [29–31] and in vitro [21,28,32,33], besides causing chromosomal abnormalities [34].
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Copper, an essential trace metal for living organisms, is present in natural waters and sediments [35] and virtually in all media, i.e. air, water and soil [36]. Copper sulphate is the best known and most widely used among all copper salts. Today, there are more than 100 manufacturers of salt and its world consumption is around 200,000 tons per annum, of which it is estimated that approximately three-quarters are used in agriculture, principally as a fungicide to control bacterial and fungal diseases of fruit, vegetable, nut and field crops. Copper in surface water is a wellknown environmental hazard, associated with toxicity to a variety of aquatic organisms [37]. High copper levels lead to an increase in the rate of free radical formation [38,39], teratogenicity [40] and chromosomal aberrations [41,42]. Studies on aquatic organisms exposed to pollutant waters or sediments containing heavy metals have implicated DNA strand breakage [43–48] and fishes are employed as sensitive indicators for their genotoxic and mutagenic effects [49–51]. Investigations of toxic effects of metal pollutants at cellular level demonstrated cytogenetic aberrations for environmental monitoring and risk assessment. The contamination of aquatic ecosystems by heavy metals has gained increasing attention in recent decades. Chronic exposure and accumulation of these chemicals by aquatic biota can result in tissue burdens that produce adverse effects not only in the exposed organisms, but also in organisms including human beings [52,53]. In view of the above, the present study was carried out to evaluate the genotoxic potential of HgCl2 , As2 O3 and CuSO4 ·5H2 O under static renewal system by estimating the frequency and type of chromosomal aberrations in the kidney cells of Channa punctata (Bloch) after in vivo exposure to sublethal concentrations. 2. Materials and methods In the present investigations a common pond murrel, C. punctata was selected as a test animal because it is commonly found in abundance in fresh water bodies, its sensitivity and suitable karyotype consisting of small number (2n = 32) of large sized chromosomes. Live specimens of C. punctata taken from local lentic habitat were acclimatized to laboratory conditions (hardness 74.6 as CaCO3 mg/L, alkalinity 76.35 as CaCO3 mg/L, DO 6.91 mg/L, COD 62.5 mg/L, chloride 146.4 mg/L, TDS 228.33 mg/L, pH 7.02 and temperature 28 ◦ C) in 100 L well aerated glass aquaria (100 cm × 40 cm × 40 cm) for 15 days. LC50 of test chemicals against fish (size and weight ranges 13–15 cm; 28–32 g) were estimated by “Trimmed Spearman-Karber method” [54]. Apparently, 50 healthy fishes were divided into five groups having equal number of individuals. Group I representing negative control comprises of normal, untreated fishes, group II or positive control comprises of fish having a single dose, by intramuscular injection, of Mitomycin-C (Cadila Pharmaceutical Pvt. Ltd., Oncocare Division, Ahmedabad, India), @ 1 mg/kg body weight. Experimental groups III, IV and V were exposed to sublethal concentrations (0.081, 6.936 and 0.407 mg/L) of HgCl2 (S.D. Fine-chem Ltd., Mumbai, India), As2 O3 (S.D. Fine-chem Ltd., Mumbai, India) and CuSO4 ·5H2 O (S.D. Fine-chem Ltd., Mumbai, India), respectively, dissolved in two weeks aged tap water. Fishes of all groups were maintained on normal diet composed of minced goat liver and artificial fish feed – ‘Tokyo’ and exposed uninterrupted for 24, 48, 72, 96, and 168 h under static renewal system. Test medium in all sets was analyzed at onset and after termination of each experiment following standard methods [55]. Fishes were sacrificed by dipping them in 50 mg/L solution of benzocaine [56]. Before the killing of specimens, an intramuscular injection of 0.05% colchicine (Sigma Chemical Co. St. Louis, USA), @ 1 ml/100 g of body wt. was administered to fishes to arrest cell growth at metaphase stage. After 1 h the kidneys were dissected, homogenized in hypotonic solution (0.56% KCl) to prepare cell suspension and incubated for about 25–35 min at room temperature for swelling of the cells. The hypotonic action was stopped by adding 1 ml of freshly prepared, chilled Carnoy’s fixative (methanol:acetic acid in 3:1 ratio) and the cell suspension was centrifuged at 1200–1500 rpm for 10 min. The cell pellet was resuspended in 7–8 ml of chilled fixative and again centrifuged. The process of washing of the cell pellet with fixative was repeated thrice to get clear whitish pellet. The slides were prepared by the flame drying technique and stained with 4–5% Giemsa in phosphate buffer (pH 6.8). Scoring was kept limited to only those metaphase plates that contained a complete set of chromosomes (2n = 32). For the estimation of CA frequency six specimens from each group were sacrificed and 200 cells were selected out of these 50 well spread metaphases were scored per specimen amounting to total 300 metaphases/group. The common chromosomal aberrations scored were chromatid and chromosome breaks, chromatid and chromosome gaps along with ring and di-centric chromosomes. Mitotic index was estimated by counting the number of metaphases in
Fig. 1. Normal metaphase spread of C. punctata.
2000 cells/specimen or 12,000 cells per group at 40× magnification under Olympus KH microscope. The data were subjected to a non parametric test, Kruskal Wallis test i.e. H-test [57] before employing the Student’s t-test for determining the level of significance between treated and control groups (Fig. 1).
3. Results The changes in water characteristics were observed before and after dissolving Hg(II), As(II) and Cu(II). Hardness, alkalinity, COD and TDS were recorded increased in all the groups. DO was found to decrease throughout the experiment in different groups except group IV. In group I and II, chloride content was recorded constant while it was increased in group III and V, and registered a decline trend in group IV. pH was also found constant in group I and II, while it was decreased in group III and V, but increased in group IV. Temperature almost remained unchanged throughout (Table 1). Mitotic index was evaluated as a percentage of dividing cells and values are given as mean ± SE. It was recorded 4.14 ± 0.010 in group I after 24 h of exposure period and it remained almost constant and did not differ statistically for the subsequent exposure periods and on this basis data from 24 h are used to represent the whole of control values. The cytotoxicity of groups II–V was evident as the mitotic index was found to be significantly (p < 0.05) less in comparison to group I (Table 2). Significantly high (p < 0.05, p < 0.01) frequencies of chromosomal aberrations were recorded in group II (Fig. 2; Table 2). Significant (p < 0.05, p < 0.01, p < 0.001) induction of chromosomal aberrations were also observed in group III–V
Fig. 2. Metaphase spread showing chromatid gap (G ) and chromosomal break (B ) induced by Mitomycin-C (72 h exposure).
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Table 1 Physico-chemical profile of the test medium. DOb
CODb
Chlorideb
TDS
pH
Aquaria temperature (◦ C)
79.1 77.4 76.5 75.7 75.1 74.3
6.98 6.97 6.92 6.91 6.90 6.82
50.2 54.1 60.0 65.3 69.9 75.5
146.40 146.40 146.40 146.40 146.40 146.40
200 200 210 230 250 280
7.2 7.2 7.2 7.2 7.2 7.2
27.0 27.5 27.0 28.0 27.8 27.8
75.9 75.8 75.6 75.3 74.0 73.6
80.1 79.5 78.2 76.3 75.4 74.9
6.98 6.97 6.92 6.91 6.90 6.82
50.5 55.2 62.0 68.2 70.3 76.8
143.36 143.36 143.36 143.36 143.36 143.36
200 200 200 200 250 300
7.2 7.2 7.2 7.2 7.2 7.2
27.0 27.5 27.0 28.0 27.8 27.8
00 24 48 72 96 168
90.0 89.5 87.1 84.3 82.2 78.4
82.0 81.4 80.0 79.5 79.1 77.0
6.80 6.50 6.32 6.10 6.00 5.18
55.4 60.0 68.2 79.0 87.2 96.0
151.20 151.70 152.00 152.80 153.30 155.20
250 250 275 280 310 350
6.8 6.8 6.7 6.7 6.7 6.6
27.0 27.5 27.0 28.0 27.8 27.8
As2 O3 treated (group IV)
00 24 48 72 96 168
110.20 110.00 108.30 107.00 106.10 104.30
115.10 114.00 112.80 112.10 110.20 109.00
7.70 7.65 7.25 7.00 6.90 6.12
58.20 63.40 70.38 80.80 89.20 98.10
130.60 133.10 135.30 136.80 138.50 141.90
280 290 295 300 320 340
7.6 7.6 7.6 7.5 7.5 7.4
27.0 27.5 27.0 28.0 27.8 27.8
CuSO4 treated (group V)
00 24 48 72 96 168
105.10 102.00 100.00 99.20 98.00 96.50
109.30 108.10 106.10 103.50 102.10 100.60
6.80 6.65 6.26 5.90 5.75 5.56
58.10 61.30 66.80 71.10 85.90 97.20
140.62 142.30 145.10 145.50 147.10 150.00
260 290 290 300 310 325
6.5 6.5 6.3 6.2 6.0 5.9
27.0 27.5 27.0 28.0 27.8 27.8
Hardnessa
Toxicants
Exposure periods (h)
Control (group I)
00 24 48 72 96 168
75.7 75.4 74.9 74.5 74.1 73.1
Positive control (group II)
00 24 48 72 96 168
HgCl2 treated (group III)
a b
Alkalinitya
Hardness and Alkalinity as CaCo3 mg/L. DO, COD chloride and TDS in mg/L.
Table 2 Frequencies of chromosomal aberrations induced by Mitomycin-C (MC), mercuric chloride (HgCl2 ), arsenic trioxide (As2 O3 ) and copper sulphate (CuSO4 ·5H2 O) in kidney cells of Channa punctata (n = 6) (cells examined for mitotic index – 2000 cells/fish, for CA frequency 50 metaphases/fish). Toxicants
Exposure periods (h)
Mitotic index ± SE
Relative mitotic index (RMI)
Absolute frequencies of CA G
4.08 ± 0.010
Control
G
B
B
Ring
Average number of aberrations per metaphase DIC
%Incidence of aberrant cells
Total number of aberrations
2
1
7
3
0
0
13
0.043
1.30
MC
24 48 72 96 168
3.60 2.91 2.26 2.27 2.90
± ± ± ± ±
0.006* 0.034* 0.006* 0.096* 0.007*
0.882 0.713 0.553 0.556 0.710
15 23 33 31 27
14 15 18 17 15
48 45 62 73 70
13 25 25 16 15
3 2 7 2 5
0 7 4 2 1
93∗∗ 117* 149* 141* 133*
0.310 0.390 0.496 0.470 0.443
9.30 11.70 14.90 14.10 13.30
Hg(II)
24 48 72 96 168
2.10 1.99 1.43 1.60 1.80
± ± ± ± ±
0.026* 0.014* 0.017* 0.019* 0.031*
0.514 0.487 0.350 0.392 0.441
15 19 15 20 20
12 19 19 15 14
27 43 52 52 49
17 20 33 26 16
2 2 1 2 5
1 1 1 1 3
74∗∗∗ 104* 121* 116* 107*
0.246 0.346 0.403 0.386 0.356
7.40 9.40 12.20 11.40 10.70
As(III)
24 48 72 96 168
2.43 2.18 1.75 2.10 2.28
± ± ± ± ±
0.020* 0.006* 0.050* 0.015* 0.021*
0.594 0.534 0.428 0.514 0.558
12 20 22 20 19
11 15 16 14 14
39 48 58 56 55
16 21 28 26 22
0 1 2 2 2
1 1 2 1 1
79** 107* 128∗∗∗ 119∗∗ 113∗∗
0.263 0.356 0.426 0.396 0.376
7.90 10.00 13.00 11.90 11.30
Cu(II)
24 48 72 96 168
2.21 2.01 1.58 1.76 1.95
± ± ± ± ±
0.004* 0.010* 0.030* 0.010* 0.018*
0.541 0.492 0.387 0.431 0.477
10 14 17 15 15
9 10 12 8 7
24 37 53 49 45
15 21 26 25 21
1 1 1 1 1
1 1 2 1 1
60∗∗ 84∗∗ 111∗∗∗ 99∗∗∗ 91∗∗∗
0.200 0.280 0.370 0.330 0.303
6.00 8.40 11.10 9.90 9.10
G : chromatid gaps, G : chromosome gaps: B : chromatid breaks, B : chromosome breaks, ring and DIC: Di-centric chromosomes. The data were subjected to a non parametric test, Kruskal Wallis test i.e. H-test before employing the Student’s t-test for determining the level of significance between treated and control groups. * p < 0.05. ** p < 0.01. *** p < 0.001.
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4. Discussion
Fig. 3. Metaphase spread showing chromatid breaks (B ), chromatid gap (G ) and chromosome breaks (B ) induced by mercury (72 h exposure).
Fig. 4. Metaphase spread showing chromatid breaks (B ) and chromosome breaks (B ) induced by arsenic (72 h exposure).
in comparison to group I. The frequency of chromosomal aberrations increased up to 72 h of exposure and decreased thereafter (Figs. 3–5; Table 2). In all groups, frequency of chromosomal aberrations was found to decrease after 96 and 168 h. Similar trend in %incidence of aberrant cells was also recorded.
Fig. 5. Metaphase spread showing chromatid break (B ) and chromosomal breaks (B ) induced by copper (72 h exposure).
In the present studies, all the three metallic compounds viz. Hg(II), As(III) and Cu(II) registered a significant decrease in MI vis-a-vis negative control (group I) even against sublethal exposures for shortest exposure period of 24 h. Moreover, even lesser RMI than positive control (group II) coupled with elevated frequencies of CA for above said compounds form a valid ground for potentially cytotoxic nature of these metallic compounds. Further, a reduction of RMI values is indicative of delay of interphasic stage of cell cycle on account of activation of DNA damage and repair processes. This is evident by increasing frequency of chromosomal damage within the subsequent time interval. These genotoxic anomalies increased gradually up to 72 h, after which they registered a declining trend. Similar observations were documented by Yadav and Trivedi [51] in C. punctata following exposer to sublethal concentration of chromium (VI) up to 168 h. In another study, De Lemos et al. [58] accounted significant induction of micronucleus in fish erythrocytes exposed to chromium (VI) for 21 days but with a decrease thereafter. Brunetti et al. [59] reported that the higher concentration of toxicant might inhibit normal cell division, damage chromosome and interdict DNA duplication, thus cytotoxic damage more or less declined. Exposure to high concentrations of mercury is known to cause damage to the nervous system, kidney and liver in human beings [13–15]. It has been reported that mercury interferes with numerous cellular activities, such as cellular repair enzymes to enhance genotoxicity [60] and significantly decreased DNA repair efficiency with the duration of exposure [17]. Mercury compounds are known to be potent chemical agents that cause DNA damage in cells. Strand breaks in DNA, unlike the breaks caused by X-rays, cannot be easily repaired [61]. In the present study, it was found that fish exposed even to sublethal concentration of Hg(II) resulted in a significant (p < 0.05, p < 0.001) increase in the frequency of chromosomal aberrations after all the exposure periods compared with the group I, but lower than the group II. As the exposure time increased, the percentage of aberrations per metaphase and percentage incidence of aberrant cells was also increased upto 72 h and then decreased subsequently (Table 2). Single-strand breaks may arise directly from the binding of mercury with DNA. Inorganic mercury was found to be teratogenic [18] and mutagenic [19] in the killfish, Fundulus heteroclitus. In another study, mercury was found to induce significant cytogenetic damage in terms of micronuclei in hepatocytes of salmonid rainbow trout, Oncorhynchus mykiss [62] and, in renal erythrocytes of European minnow Phoxinus phoxinus and mollie Poecilia latipinna [63]. All mercury compounds interfere with thiol metabolism, causing inhibition or inactivation of proteins containing thiol ligands and ultimately leading to mitotic disturbances [64–66]. Exposing C. punctata to sublethal concentration of arsenic trioxide also caused significant induction of chromosomal aberrations (Table 2). Arsenic has long been regarded as a potential carcinogen [26–28]. Its genotoxicity has been analyzed extensively both in vivo [29–31] and in vitro [21,28,32,33]. Chou et al. [34] also documented that arsenic exposure causes chromosomal abnormalities, with a preponderance of end-to-end fusions. These chromosomal end fusions suggested that telomerase activity may be inhibited by arsenic. Sodium arsenite has been demonstrated to induce chromosome aberrations in different cell types. The aberrations found in many studies were chromatid gaps, fragmentations, endoreduplications and chromosomal breaks [67]. Huang et al. [68] found that arsenic significantly delays mitotic division, inhibits assembly of the mitotic spindle and induction of chromosome endoreduplication. The genotoxicity of arsenic occurs due to generation of reactive oxygen species and the inhibition of DNA repair [69].
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In the present study, copper sulphate also induced a significant frequency of chromosomal aberrations in the kidney cells of C. punctata (Table 2). Similar observations were also reported in Swiss albino mice after oral, subcutaneous and intraperitoneal exposures of copper sulphate [41,42]. A single intraperitoneal injection of copper (II) sulphate pentahydrate in mice induced a dose-related increase in the incidence of chromatid-type chromosome aberrations in the bone marrow after 6 h [70]. Bhunya and Pati [41] reported a significant dose-related increase in the incidence of micronuclei in the bone marrow of mice after two injections at doses between 1.3 and 5 mg Cu/kg body weight. Stouthart et al. [40] reported teratogenic effects of copper in carp at much lower copper concentrations (0.051 mg/L at pH 7.6 and 0.019 mg/L at pH 6.3). They reported deformed head, spinal column, upper jaw and a small or absent swim bladder. Copper toxicity is believed to be due to nonspecific binding of the reactive metal cation (Cu++ ) to biologically important molecules. In addition, high copper levels lead to an increase in the rate of free radical formation [38,39]. It may, thus, be concluded that mercury, arsenic and copper are highly deleterious environmental pollutants. Their disposal into aqueous ecosystems leads to their accumulation in sediments, benthic and pelagic food chains. Human beings are thus exposed throughout their lifetime to their low levels through potable water and aquatic food. Therefore, the present study highlights the genotoxic hazards of these metals to fish by inducing chromosomal aberrations even in sublethal exposures. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgments One of us, K.K. Yadav is thankful to CSIR, New Delhi for the award of SRF and financial support. We are grateful to Prof. N. Agrawal, Head, Department of Zoology, University of Lucknow, Lucknow for providing laboratory facilities and her keen interest in present research work and Dr. Omkar, Executive Editor, Journal of Applied Bioscience, for critically reviewing the manuscript and useful suggestions. References [1] R. Bargagli, Trace metals in Antarctica related to climate change and increasing human impact, Rev. Environ. Contam. Toxicol. 166 (2000) 129–173. [2] E. Tarifeno-Silva, L. Kawasaki, D.P. Yn, M.S. Gordon, D.J. Chapman, Aquacultural approaches to recycling dissolved nutrients in secondarily treated domestic wastewaters: uptake of dissolved heavy metals by artificial food chains, Water Res. 16 (1982) 59–65. [3] L. Marcano, O. Nusetti, J. Rodriguez-Grau, J. Vilas, Uptake and depuration of copper and zinc in relation to metal-binding protein in the Polychaete Eurythoe complanata, Comp. Biochem. Physiol. 114 (1996) 179–184. [4] H. Roche, G. Boge, Fish blood parameters as a potential tool for identification of stress caused by environmental factors and chemical intoxication, Mar. Environ. Res. 41 (1996) 27–43. [5] P.V. Winger, P.J. Lasier, D.H. White, J.T. Seginak, Effect of contaminants in dredge material from the lower Savannah river, Arch. Environ. Contam. Toxicol. 28 (2000) 128–136. [6] A.M. Florea, E. Dopp, G. Obe, A.W. Rettenmeier, Genotoxicity of organometallic species, in: A.V. Hirner, H. Emons (Eds.), Organic Metal and Metalloid Species in the Environment: Analysis, Distribution, Processes and Toxicological Evaluation, Springer-Verlag, Heidelberg, 2004, pp. 205–219. [7] B.N. Gupta, A.K. Mathur, Toxicity of heavy metals, Indian J. Med. Sci. 37 (1983) 236–240. [8] R.D. Blevins, Metal concentrations in muscle of fish from aquatic systems in East Tennessee, USA, Water Air Soil Pollut. 29 (1985) 361–371. [9] P. Szefer, K. Szefer, B. Skwarzec, Distribution of trace metals in some representative fauna of the Southern Baltic, Mar. Pollut. Bull. 21 (1990) 60–62. [10] L. Goldberg, A history of pest control measures in the anthropology collections, national museum of natural history. Smithsonian institution, JAIC 35 (1996) 23–43. [11] M.H. Freeman, T.F. Shupe, R.P. Vlosky, H.M. Barnes, Past present and future of the wood preservation industry, Forest Products J. 53 (2003) 8–15.
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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Chromosomal aberrations in a fish, Channa punctata after in vivo exposure to three heavy metals Kamlesh K. Yadav, Sunil P. Trivedi ∗ Environmental Toxicology Laboratory, Department of Zoology, University of Lucknow, Lucknow 226007, India
a r t i c l e
i n f o
Article history: Received 21 June 2008 Received in revised form 29 April 2009 Accepted 29 May 2009 Available online 21 June 2009 Keywords: Chromosomal aberrations Mercuric chloride Arsenic trioxide Copper sulphate Fish Channa punctata
a b s t r a c t The studies were designed to assess the extent of chromosomal aberrations (CA) under the exposure of three common heavy metalic compounds, viz. mercuric chloride, arsenic trioxide and copper sulphate pentahydrate, in vivo using fish, Channa punctata (2n = 32), as a test model. Prior acclimatized fishes were divided into five groups. Group I and II served as negative and positive control, respectively. An intramuscular injection of Mitomycin-C (@ 1 mg/kg body wt.) was administered to group II only. Fishes of groups III, IV and V were subjected to sublethal concentrations (10% of 96 h LC50 ), of HgCl2 (0.081 mg/L), As2 O3 (6.936 mg/L) and CuSO4 ·5H2 O (0.407 mg/L). Fishes of all the groups were exposed uninterrupted for 24, 48, 72, 96 and 168 h. Observations of kidney cells of exposed fishes revealed chromatid and chromosome breaks, chromatid and chromosome gaps along with ring and di-centric chromosomes. A significant increase over negative control in the frequency of chromosomal aberrations (CA) was observed in fish exposed to Mitomycin-C, Hg(II), As(III) and Cu(II). As the average ± SE total number of CA, average number of CA per metaphase and %incidence of aberrant cells in Hg(II) was 104.40 ± 8.189, 0.347 ± 0.027 and 10.220 ± 0.842, respectively; in As(III) 109.20 ± 8.309, 0.363 ± 0.027 and 10.820 ± 2.347, respectively and in Cu(II) 89.00 ± 19.066, 0.297 ± 0.028 and 8.900 ± 0.853, respectively. Hence, it reveals that the order of induction of frequency of CA was Cu < Hg < As. The findings depict genotoxic potential of these metals even in sublethal concentrations. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Metals and their compounds are natural constituents of different ecosystems [1]. They are ubiquitous contaminants in aquatic ecosystems [2–5]. Their distribution in the environment is governed by natural as well as anthropogenic activities [6]. Heavy metals received considerable attention due to their toxicity and potential to bioaccumulate in aquatic biota [7–9]. Hg(II), As(III) and Cu(II) are present in industrial and municipal waste water and in mine tailings. They also cause mutagenic and carcinogenic actions in living beings. Among heavy metals, mercuric chloride is a poisonous white crystalline compound, used since ages as a reliable ingredient in antiseptics, disinfectants and preservatives, insecticides, batteries and in metallurgical and photographic operations [10,11]. In India, about 200 ton mercury and its compounds are introduced into the environment annually as effluents from industries [12]. Exposure to high concentrations of mercury causes damage to nervous system, immune system, kidney and liver in human beings [13–15].
∗ Corresponding author. Tel.: +91 0522 2740040. E-mail addresses: kamlesh [email protected] (K.K. Yadav), [email protected] (S.P. Trivedi). 1383-5718/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2009.05.021
Exposure to mercuric chloride in human parotid salivary glands and lymphocytes has also been reported by Schmid et al. [16]. Occupational exposure of mercury vapour results in greater frequency of chromosomal aberrations [17]. Weis and Weis [18] reported teratogenic effect of inorganic mercury in killfish, Fundulus heteroclitus. Methyl mercury is known to induce chromosomal aberrations and micronuclei in the same fish [19]. Arsenic is introduced into water via weathering of rocks, minerals and ores, industrial effluents including mining wastes and atmospheric deposition [20]. Human exposure takes place via contaminated water and soil as well as from food rich in arsenic, viz., garlic, marine food, etc. and occupational activities [21,22]. Wider applications of arsenic compounds, particularly arsenic trioxide, as the starting substance for the manufacture of arsenic-based pesticides, arsenic-based pharamaceuticals (Neosalvarsan), veterinary products, decolorizing agents for glasses and enamels and wood preservatives make human beings more prone to its exposure. In spite of its high toxicity arsenic is a common contaminant in pharmaceuticals [23]. It is also used for therapeutic purposes for the treatment of chronic myelogenous leukaemia, leishmaniosis, trypanosomiasis [24] and cancer by inducing G(2)/M arrest [25]. Arsenic has long been regarded as a potential carcinogen [26–28], genotoxic both in vivo conditions [29–31] and in vitro [21,28,32,33], besides causing chromosomal abnormalities [34].
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Copper, an essential trace metal for living organisms, is present in natural waters and sediments [35] and virtually in all media, i.e. air, water and soil [36]. Copper sulphate is the best known and most widely used among all copper salts. Today, there are more than 100 manufacturers of salt and its world consumption is around 200,000 tons per annum, of which it is estimated that approximately three-quarters are used in agriculture, principally as a fungicide to control bacterial and fungal diseases of fruit, vegetable, nut and field crops. Copper in surface water is a wellknown environmental hazard, associated with toxicity to a variety of aquatic organisms [37]. High copper levels lead to an increase in the rate of free radical formation [38,39], teratogenicity [40] and chromosomal aberrations [41,42]. Studies on aquatic organisms exposed to pollutant waters or sediments containing heavy metals have implicated DNA strand breakage [43–48] and fishes are employed as sensitive indicators for their genotoxic and mutagenic effects [49–51]. Investigations of toxic effects of metal pollutants at cellular level demonstrated cytogenetic aberrations for environmental monitoring and risk assessment. The contamination of aquatic ecosystems by heavy metals has gained increasing attention in recent decades. Chronic exposure and accumulation of these chemicals by aquatic biota can result in tissue burdens that produce adverse effects not only in the exposed organisms, but also in organisms including human beings [52,53]. In view of the above, the present study was carried out to evaluate the genotoxic potential of HgCl2 , As2 O3 and CuSO4 ·5H2 O under static renewal system by estimating the frequency and type of chromosomal aberrations in the kidney cells of Channa punctata (Bloch) after in vivo exposure to sublethal concentrations. 2. Materials and methods In the present investigations a common pond murrel, C. punctata was selected as a test animal because it is commonly found in abundance in fresh water bodies, its sensitivity and suitable karyotype consisting of small number (2n = 32) of large sized chromosomes. Live specimens of C. punctata taken from local lentic habitat were acclimatized to laboratory conditions (hardness 74.6 as CaCO3 mg/L, alkalinity 76.35 as CaCO3 mg/L, DO 6.91 mg/L, COD 62.5 mg/L, chloride 146.4 mg/L, TDS 228.33 mg/L, pH 7.02 and temperature 28 ◦ C) in 100 L well aerated glass aquaria (100 cm × 40 cm × 40 cm) for 15 days. LC50 of test chemicals against fish (size and weight ranges 13–15 cm; 28–32 g) were estimated by “Trimmed Spearman-Karber method” [54]. Apparently, 50 healthy fishes were divided into five groups having equal number of individuals. Group I representing negative control comprises of normal, untreated fishes, group II or positive control comprises of fish having a single dose, by intramuscular injection, of Mitomycin-C (Cadila Pharmaceutical Pvt. Ltd., Oncocare Division, Ahmedabad, India), @ 1 mg/kg body weight. Experimental groups III, IV and V were exposed to sublethal concentrations (0.081, 6.936 and 0.407 mg/L) of HgCl2 (S.D. Fine-chem Ltd., Mumbai, India), As2 O3 (S.D. Fine-chem Ltd., Mumbai, India) and CuSO4 ·5H2 O (S.D. Fine-chem Ltd., Mumbai, India), respectively, dissolved in two weeks aged tap water. Fishes of all groups were maintained on normal diet composed of minced goat liver and artificial fish feed – ‘Tokyo’ and exposed uninterrupted for 24, 48, 72, 96, and 168 h under static renewal system. Test medium in all sets was analyzed at onset and after termination of each experiment following standard methods [55]. Fishes were sacrificed by dipping them in 50 mg/L solution of benzocaine [56]. Before the killing of specimens, an intramuscular injection of 0.05% colchicine (Sigma Chemical Co. St. Louis, USA), @ 1 ml/100 g of body wt. was administered to fishes to arrest cell growth at metaphase stage. After 1 h the kidneys were dissected, homogenized in hypotonic solution (0.56% KCl) to prepare cell suspension and incubated for about 25–35 min at room temperature for swelling of the cells. The hypotonic action was stopped by adding 1 ml of freshly prepared, chilled Carnoy’s fixative (methanol:acetic acid in 3:1 ratio) and the cell suspension was centrifuged at 1200–1500 rpm for 10 min. The cell pellet was resuspended in 7–8 ml of chilled fixative and again centrifuged. The process of washing of the cell pellet with fixative was repeated thrice to get clear whitish pellet. The slides were prepared by the flame drying technique and stained with 4–5% Giemsa in phosphate buffer (pH 6.8). Scoring was kept limited to only those metaphase plates that contained a complete set of chromosomes (2n = 32). For the estimation of CA frequency six specimens from each group were sacrificed and 200 cells were selected out of these 50 well spread metaphases were scored per specimen amounting to total 300 metaphases/group. The common chromosomal aberrations scored were chromatid and chromosome breaks, chromatid and chromosome gaps along with ring and di-centric chromosomes. Mitotic index was estimated by counting the number of metaphases in
Fig. 1. Normal metaphase spread of C. punctata.
2000 cells/specimen or 12,000 cells per group at 40× magnification under Olympus KH microscope. The data were subjected to a non parametric test, Kruskal Wallis test i.e. H-test [57] before employing the Student’s t-test for determining the level of significance between treated and control groups (Fig. 1).
3. Results The changes in water characteristics were observed before and after dissolving Hg(II), As(II) and Cu(II). Hardness, alkalinity, COD and TDS were recorded increased in all the groups. DO was found to decrease throughout the experiment in different groups except group IV. In group I and II, chloride content was recorded constant while it was increased in group III and V, and registered a decline trend in group IV. pH was also found constant in group I and II, while it was decreased in group III and V, but increased in group IV. Temperature almost remained unchanged throughout (Table 1). Mitotic index was evaluated as a percentage of dividing cells and values are given as mean ± SE. It was recorded 4.14 ± 0.010 in group I after 24 h of exposure period and it remained almost constant and did not differ statistically for the subsequent exposure periods and on this basis data from 24 h are used to represent the whole of control values. The cytotoxicity of groups II–V was evident as the mitotic index was found to be significantly (p < 0.05) less in comparison to group I (Table 2). Significantly high (p < 0.05, p < 0.01) frequencies of chromosomal aberrations were recorded in group II (Fig. 2; Table 2). Significant (p < 0.05, p < 0.01, p < 0.001) induction of chromosomal aberrations were also observed in group III–V
Fig. 2. Metaphase spread showing chromatid gap (G ) and chromosomal break (B ) induced by Mitomycin-C (72 h exposure).
K.K. Yadav, S.P. Trivedi / Mutation Research 678 (2009) 7–12
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Table 1 Physico-chemical profile of the test medium. DOb
CODb
Chlorideb
TDS
pH
Aquaria temperature (◦ C)
79.1 77.4 76.5 75.7 75.1 74.3
6.98 6.97 6.92 6.91 6.90 6.82
50.2 54.1 60.0 65.3 69.9 75.5
146.40 146.40 146.40 146.40 146.40 146.40
200 200 210 230 250 280
7.2 7.2 7.2 7.2 7.2 7.2
27.0 27.5 27.0 28.0 27.8 27.8
75.9 75.8 75.6 75.3 74.0 73.6
80.1 79.5 78.2 76.3 75.4 74.9
6.98 6.97 6.92 6.91 6.90 6.82
50.5 55.2 62.0 68.2 70.3 76.8
143.36 143.36 143.36 143.36 143.36 143.36
200 200 200 200 250 300
7.2 7.2 7.2 7.2 7.2 7.2
27.0 27.5 27.0 28.0 27.8 27.8
00 24 48 72 96 168
90.0 89.5 87.1 84.3 82.2 78.4
82.0 81.4 80.0 79.5 79.1 77.0
6.80 6.50 6.32 6.10 6.00 5.18
55.4 60.0 68.2 79.0 87.2 96.0
151.20 151.70 152.00 152.80 153.30 155.20
250 250 275 280 310 350
6.8 6.8 6.7 6.7 6.7 6.6
27.0 27.5 27.0 28.0 27.8 27.8
As2 O3 treated (group IV)
00 24 48 72 96 168
110.20 110.00 108.30 107.00 106.10 104.30
115.10 114.00 112.80 112.10 110.20 109.00
7.70 7.65 7.25 7.00 6.90 6.12
58.20 63.40 70.38 80.80 89.20 98.10
130.60 133.10 135.30 136.80 138.50 141.90
280 290 295 300 320 340
7.6 7.6 7.6 7.5 7.5 7.4
27.0 27.5 27.0 28.0 27.8 27.8
CuSO4 treated (group V)
00 24 48 72 96 168
105.10 102.00 100.00 99.20 98.00 96.50
109.30 108.10 106.10 103.50 102.10 100.60
6.80 6.65 6.26 5.90 5.75 5.56
58.10 61.30 66.80 71.10 85.90 97.20
140.62 142.30 145.10 145.50 147.10 150.00
260 290 290 300 310 325
6.5 6.5 6.3 6.2 6.0 5.9
27.0 27.5 27.0 28.0 27.8 27.8
Hardnessa
Toxicants
Exposure periods (h)
Control (group I)
00 24 48 72 96 168
75.7 75.4 74.9 74.5 74.1 73.1
Positive control (group II)
00 24 48 72 96 168
HgCl2 treated (group III)
a b
Alkalinitya
Hardness and Alkalinity as CaCo3 mg/L. DO, COD chloride and TDS in mg/L.
Table 2 Frequencies of chromosomal aberrations induced by Mitomycin-C (MC), mercuric chloride (HgCl2 ), arsenic trioxide (As2 O3 ) and copper sulphate (CuSO4 ·5H2 O) in kidney cells of Channa punctata (n = 6) (cells examined for mitotic index – 2000 cells/fish, for CA frequency 50 metaphases/fish). Toxicants
Exposure periods (h)
Mitotic index ± SE
Relative mitotic index (RMI)
Absolute frequencies of CA G
4.08 ± 0.010
Control
G
B
B
Ring
Average number of aberrations per metaphase DIC
%Incidence of aberrant cells
Total number of aberrations
2
1
7
3
0
0
13
0.043
1.30
MC
24 48 72 96 168
3.60 2.91 2.26 2.27 2.90
± ± ± ± ±
0.006* 0.034* 0.006* 0.096* 0.007*
0.882 0.713 0.553 0.556 0.710
15 23 33 31 27
14 15 18 17 15
48 45 62 73 70
13 25 25 16 15
3 2 7 2 5
0 7 4 2 1
93∗∗ 117* 149* 141* 133*
0.310 0.390 0.496 0.470 0.443
9.30 11.70 14.90 14.10 13.30
Hg(II)
24 48 72 96 168
2.10 1.99 1.43 1.60 1.80
± ± ± ± ±
0.026* 0.014* 0.017* 0.019* 0.031*
0.514 0.487 0.350 0.392 0.441
15 19 15 20 20
12 19 19 15 14
27 43 52 52 49
17 20 33 26 16
2 2 1 2 5
1 1 1 1 3
74∗∗∗ 104* 121* 116* 107*
0.246 0.346 0.403 0.386 0.356
7.40 9.40 12.20 11.40 10.70
As(III)
24 48 72 96 168
2.43 2.18 1.75 2.10 2.28
± ± ± ± ±
0.020* 0.006* 0.050* 0.015* 0.021*
0.594 0.534 0.428 0.514 0.558
12 20 22 20 19
11 15 16 14 14
39 48 58 56 55
16 21 28 26 22
0 1 2 2 2
1 1 2 1 1
79** 107* 128∗∗∗ 119∗∗ 113∗∗
0.263 0.356 0.426 0.396 0.376
7.90 10.00 13.00 11.90 11.30
Cu(II)
24 48 72 96 168
2.21 2.01 1.58 1.76 1.95
± ± ± ± ±
0.004* 0.010* 0.030* 0.010* 0.018*
0.541 0.492 0.387 0.431 0.477
10 14 17 15 15
9 10 12 8 7
24 37 53 49 45
15 21 26 25 21
1 1 1 1 1
1 1 2 1 1
60∗∗ 84∗∗ 111∗∗∗ 99∗∗∗ 91∗∗∗
0.200 0.280 0.370 0.330 0.303
6.00 8.40 11.10 9.90 9.10
G : chromatid gaps, G : chromosome gaps: B : chromatid breaks, B : chromosome breaks, ring and DIC: Di-centric chromosomes. The data were subjected to a non parametric test, Kruskal Wallis test i.e. H-test before employing the Student’s t-test for determining the level of significance between treated and control groups. * p < 0.05. ** p < 0.01. *** p < 0.001.
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4. Discussion
Fig. 3. Metaphase spread showing chromatid breaks (B ), chromatid gap (G ) and chromosome breaks (B ) induced by mercury (72 h exposure).
Fig. 4. Metaphase spread showing chromatid breaks (B ) and chromosome breaks (B ) induced by arsenic (72 h exposure).
in comparison to group I. The frequency of chromosomal aberrations increased up to 72 h of exposure and decreased thereafter (Figs. 3–5; Table 2). In all groups, frequency of chromosomal aberrations was found to decrease after 96 and 168 h. Similar trend in %incidence of aberrant cells was also recorded.
Fig. 5. Metaphase spread showing chromatid break (B ) and chromosomal breaks (B ) induced by copper (72 h exposure).
In the present studies, all the three metallic compounds viz. Hg(II), As(III) and Cu(II) registered a significant decrease in MI vis-a-vis negative control (group I) even against sublethal exposures for shortest exposure period of 24 h. Moreover, even lesser RMI than positive control (group II) coupled with elevated frequencies of CA for above said compounds form a valid ground for potentially cytotoxic nature of these metallic compounds. Further, a reduction of RMI values is indicative of delay of interphasic stage of cell cycle on account of activation of DNA damage and repair processes. This is evident by increasing frequency of chromosomal damage within the subsequent time interval. These genotoxic anomalies increased gradually up to 72 h, after which they registered a declining trend. Similar observations were documented by Yadav and Trivedi [51] in C. punctata following exposer to sublethal concentration of chromium (VI) up to 168 h. In another study, De Lemos et al. [58] accounted significant induction of micronucleus in fish erythrocytes exposed to chromium (VI) for 21 days but with a decrease thereafter. Brunetti et al. [59] reported that the higher concentration of toxicant might inhibit normal cell division, damage chromosome and interdict DNA duplication, thus cytotoxic damage more or less declined. Exposure to high concentrations of mercury is known to cause damage to the nervous system, kidney and liver in human beings [13–15]. It has been reported that mercury interferes with numerous cellular activities, such as cellular repair enzymes to enhance genotoxicity [60] and significantly decreased DNA repair efficiency with the duration of exposure [17]. Mercury compounds are known to be potent chemical agents that cause DNA damage in cells. Strand breaks in DNA, unlike the breaks caused by X-rays, cannot be easily repaired [61]. In the present study, it was found that fish exposed even to sublethal concentration of Hg(II) resulted in a significant (p < 0.05, p < 0.001) increase in the frequency of chromosomal aberrations after all the exposure periods compared with the group I, but lower than the group II. As the exposure time increased, the percentage of aberrations per metaphase and percentage incidence of aberrant cells was also increased upto 72 h and then decreased subsequently (Table 2). Single-strand breaks may arise directly from the binding of mercury with DNA. Inorganic mercury was found to be teratogenic [18] and mutagenic [19] in the killfish, Fundulus heteroclitus. In another study, mercury was found to induce significant cytogenetic damage in terms of micronuclei in hepatocytes of salmonid rainbow trout, Oncorhynchus mykiss [62] and, in renal erythrocytes of European minnow Phoxinus phoxinus and mollie Poecilia latipinna [63]. All mercury compounds interfere with thiol metabolism, causing inhibition or inactivation of proteins containing thiol ligands and ultimately leading to mitotic disturbances [64–66]. Exposing C. punctata to sublethal concentration of arsenic trioxide also caused significant induction of chromosomal aberrations (Table 2). Arsenic has long been regarded as a potential carcinogen [26–28]. Its genotoxicity has been analyzed extensively both in vivo [29–31] and in vitro [21,28,32,33]. Chou et al. [34] also documented that arsenic exposure causes chromosomal abnormalities, with a preponderance of end-to-end fusions. These chromosomal end fusions suggested that telomerase activity may be inhibited by arsenic. Sodium arsenite has been demonstrated to induce chromosome aberrations in different cell types. The aberrations found in many studies were chromatid gaps, fragmentations, endoreduplications and chromosomal breaks [67]. Huang et al. [68] found that arsenic significantly delays mitotic division, inhibits assembly of the mitotic spindle and induction of chromosome endoreduplication. The genotoxicity of arsenic occurs due to generation of reactive oxygen species and the inhibition of DNA repair [69].
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In the present study, copper sulphate also induced a significant frequency of chromosomal aberrations in the kidney cells of C. punctata (Table 2). Similar observations were also reported in Swiss albino mice after oral, subcutaneous and intraperitoneal exposures of copper sulphate [41,42]. A single intraperitoneal injection of copper (II) sulphate pentahydrate in mice induced a dose-related increase in the incidence of chromatid-type chromosome aberrations in the bone marrow after 6 h [70]. Bhunya and Pati [41] reported a significant dose-related increase in the incidence of micronuclei in the bone marrow of mice after two injections at doses between 1.3 and 5 mg Cu/kg body weight. Stouthart et al. [40] reported teratogenic effects of copper in carp at much lower copper concentrations (0.051 mg/L at pH 7.6 and 0.019 mg/L at pH 6.3). They reported deformed head, spinal column, upper jaw and a small or absent swim bladder. Copper toxicity is believed to be due to nonspecific binding of the reactive metal cation (Cu++ ) to biologically important molecules. In addition, high copper levels lead to an increase in the rate of free radical formation [38,39]. It may, thus, be concluded that mercury, arsenic and copper are highly deleterious environmental pollutants. Their disposal into aqueous ecosystems leads to their accumulation in sediments, benthic and pelagic food chains. Human beings are thus exposed throughout their lifetime to their low levels through potable water and aquatic food. Therefore, the present study highlights the genotoxic hazards of these metals to fish by inducing chromosomal aberrations even in sublethal exposures. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgments One of us, K.K. Yadav is thankful to CSIR, New Delhi for the award of SRF and financial support. We are grateful to Prof. N. Agrawal, Head, Department of Zoology, University of Lucknow, Lucknow for providing laboratory facilities and her keen interest in present research work and Dr. Omkar, Executive Editor, Journal of Applied Bioscience, for critically reviewing the manuscript and useful suggestions. References [1] R. Bargagli, Trace metals in Antarctica related to climate change and increasing human impact, Rev. Environ. Contam. Toxicol. 166 (2000) 129–173. [2] E. Tarifeno-Silva, L. Kawasaki, D.P. Yn, M.S. Gordon, D.J. Chapman, Aquacultural approaches to recycling dissolved nutrients in secondarily treated domestic wastewaters: uptake of dissolved heavy metals by artificial food chains, Water Res. 16 (1982) 59–65. [3] L. Marcano, O. Nusetti, J. Rodriguez-Grau, J. Vilas, Uptake and depuration of copper and zinc in relation to metal-binding protein in the Polychaete Eurythoe complanata, Comp. Biochem. Physiol. 114 (1996) 179–184. [4] H. Roche, G. Boge, Fish blood parameters as a potential tool for identification of stress caused by environmental factors and chemical intoxication, Mar. Environ. Res. 41 (1996) 27–43. [5] P.V. Winger, P.J. Lasier, D.H. White, J.T. Seginak, Effect of contaminants in dredge material from the lower Savannah river, Arch. Environ. Contam. Toxicol. 28 (2000) 128–136. [6] A.M. Florea, E. Dopp, G. Obe, A.W. Rettenmeier, Genotoxicity of organometallic species, in: A.V. Hirner, H. Emons (Eds.), Organic Metal and Metalloid Species in the Environment: Analysis, Distribution, Processes and Toxicological Evaluation, Springer-Verlag, Heidelberg, 2004, pp. 205–219. [7] B.N. Gupta, A.K. Mathur, Toxicity of heavy metals, Indian J. Med. Sci. 37 (1983) 236–240. [8] R.D. Blevins, Metal concentrations in muscle of fish from aquatic systems in East Tennessee, USA, Water Air Soil Pollut. 29 (1985) 361–371. [9] P. Szefer, K. Szefer, B. Skwarzec, Distribution of trace metals in some representative fauna of the Southern Baltic, Mar. Pollut. Bull. 21 (1990) 60–62. [10] L. Goldberg, A history of pest control measures in the anthropology collections, national museum of natural history. Smithsonian institution, JAIC 35 (1996) 23–43. [11] M.H. Freeman, T.F. Shupe, R.P. Vlosky, H.M. Barnes, Past present and future of the wood preservation industry, Forest Products J. 53 (2003) 8–15.
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