Internat. Rev. Hydrobiol.
85
2000
5–6
663–672
JANINA BARšIENE˙ 1 and DALIA BARšYTE˙ LOVEJOY2 1
Institute of Ecology, Akademijos 2, 2600 Vilnius, Lithuania, E-mail
[email protected] 2 School of Biological Sciences, Manchester University, 3.614 Stopford Bldg., Manchester M13 9PT, UK
Environmental Genotoxicity in Klaipe˙ da Port Area key words: genotoxicity, freshwater molluscs, cytogenetic damage, aneugenic effects
Abstract Genotoxic effects were evaluated in the somatic and gonadal cells of five bivalve and gastropod mollusc species inhabiting various sites of Klaipe˙da port area, differing by ecotoxic impacts. Aneuploidy, polyploidy of cells, meiotic injures, centromere dissociation and fragmented polyploid nuclei were assessed as cytogenetic disturbances. The highest level of environmental genotoxicity was observed in the cells of Lymnaea ovata snails inhabiting Malku˛ Bay in 1995 and 1996. Dredging and removal of contamined sediments from Malku˛ Bay resulted in marked decrease of cytogenetic damage in molluscs, which were studied in 1997 and 1998. In 1997 the aneugenic effects were more frequent in the tissues of Bithynia tentaculata from Smelte˙ River than those collected from Malku˛ Bay and Klaipe˙ da passage. The level of cytogenetic damage in molluscs from Vilhelmo Channel was increasing from 1995 to 1998.
1. Introduction Urban sewage, specific industrial and agricultural effluents as well as naturally produced substances can affect aquatic organisms by genotoxic and cancerogenic compounds. Agents that cause genetic effects may be presented at very low, sublethal concentrations. However, sometimes even high concentrations of pollutants may be tolerated by organisms due to mechanisms of elimination or detoxification of harmful compounds. In this case the correlation between concentrations of pollutants in water, sediments, organisms and the biological effects might be absent. Thus, methods which are capable of indicating cumulative responses to environmental toxicants are needed. Assessment of environmental genotoxicity is an exceptional useful tool for the evaluation of long-term pollutant exposure and for identification of early biological injures (PEAKALL, 1992; ANDERSON et al., 1994; SHUGART, 1996). Klaipe˙ da port area is a very specific part of the water system “Curonian lagoon-Baltic Sea” in ecotoxicological respect. Eco-genotoxic risk can arise not only due to effluents from rivers, but also due to municipal discharge and sea-port pollution. The highest level of pollution is observed in the Malku˛ Bay, where is the most intensive port activity. The aim of the present study was to assess the cytogenetic damage in freshwater molluscs, inhabiting various sites in Klaipe˙ da port area differing by ecotoxic impacts. The feasibility of the study is based on the vicinity of Klaipe˙ da water works, municipal discharge and inflow of genotoxic compounds from tributaries.
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2. Material and Methods Cytogenetic techniques used in this study proved to be a good approach in the evaluation of peculiarities of genotoxic damage induced by heavy metals, PCBs, radionuclides and other dangerous environmental agents. As biomarkers of environmental influence the cytogenetic disturbances aneuploidy, polyploidy, meiotic injures, centromere dissociation and fragmented polyploid nuclei were evaluated in somatic and gonadal cells of molluscs. Wide distribution, abundance, relative sensitivity to environmental contamination, sedentary, filter or detritus feeding style, and different life cycles were the main criteria for selection of indicator species. Four sites were chosen for the genotoxicity assessment in Klaipe˙ da Port area. One of the site was Malku˛ Bay, which is occupied by the port dock area and urban sewage is discharged directly into this bay. The presence of high pollution loads, salt water inflow into this bay and permanent water mixing make the ecological situation here very complicated. Two rivers flow directly into Malku˛ Bay – Smelte˙ and Vilhelmo Channel (Fig. 1). In summer of 1995 intensive dredging of sediments in the bay was started. Dredging could lead to a greater possibility of salt water and chemicals intrusion from this bay into Vilhelmo Channel, which serves as the biggest drinking water source for Klaipe˙ da. This was the reason for closing the channel from the bay in June 1995. The data collected in May 1995, before the closure, and the present data on environmental genotoxicity allow a comparison and elucidation of the effects of the closure and dredging. Molluscs were collected by the special drag-net, hands, or smaller ones were screened through special device. In total 394 molluscs were collected. It was tried to collect the same species from all loca-
Figure 1.
Map of sites assessed: 1 – Klaipe˙ da passage, 2 – Vilhelmo Channel near the waterworks, 3 – Malku˛ Bay, 4 – Smelte˙ River estuary. The scale is 1 : 19,000.
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Table 1. Material for the assessment of cytogenetic damage in the cells of molluscs Mollusc species
Locality, year
Anodonta cygnea
Vilhelmo Channel near waterworks
1995 1996 1997 1998
16 20 8 10
Dreissena polymorpha
Vilhelmo Channel near waterworks
1994 1995 1997 1998
17 14 16 6
Lymnaea ovata
Malku˛ Bay
1995 1996 1997 1998 1995 1996 1997 1995 1996 1997 1998
49 14 15 20 6 30 25 9 12 16 14
Vilhelmo Channel near waterworks Estuary of Smelte˙ River Klaipe˙ da passage
No of specimens studied
Bithynia tentaculata
Malku˛ Bay Klaipe˙ da passage Estuary of Smelte˙ River
1997 1997 1997
15 15 20
Sphaerium nitidum
Estuary of Smelte˙ River Klaipe˙ da passage
1997 1997
15 12
tions. However, the sites were so different ecologically and in respect to pollution that not in all cases this attempt was successful. As one of the objectives of this study was to investigate the most suitable bioindicators for detecting freshwater pollution by genotoxic agents, the main representative species from sites assessed were collected and investigated (Table 1). Pieces of somatic and gonadal tissues were dissected from molluscs and prepared according to modified methods (BARšIENE˙ et al., 1996b). The blocking of cell divisions at metaphase was obtained by injection of a 0.1–0.2% aqueous solution of colchicine into large molluscs A. cygnea, D. polymorpha and L. ovata using ca. 1 ml per 100 g of mollusc weight 4–6 h before dissection. Small molluscs from the genera Bithynia and Sphaerium were placed directly in 0.01–0.02% solution of colchicine. Hypotonization of mollusc tissues was performed in distilled water at room temperature for 40–90 min. The material was fixed with 3 : 1 ethanol acetic acid solution, which was changed three times: after 30 min, after 1 h and after 24 h. Tissues were dissociated in 45% acetic acid, and cells were smeared on slides, slightly heated up to human body temperature on a flame. The slides were stained with 4% Giemsa for 30 – 50 minutes, using phosphate buffer solution, pH = 6.8. The mitotic metaphase and meiotic stages were examined with a Jena Med cytology microscope. Chromosome number variability p was counted as a percentage according to the formula: p = (∑ a/∑x) 100% a – abnormal/normal cell number; x – all examined cells. For the evaluation of standard deviation SD of data the following formula was used (ROKICKIJ, 1974): SD = (p(100 – p)/∑x)1/2
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For statistical analysis the differences of the percentages were tested by chi-square test employing PRISM statistical package.
3. Results The diploid chromosome set of Lymnaea ovata consists of 34 chromosomes. In 1996 L. ovata from Malku˛ Bay had only 53.8 % of diploid cells, which is a significantly lower quantity (p < 0.001) than in those individuals from Vilhelmo Channel (70.6 %) and those (54.8 %) collected in 1995 (p < 0.001). Molluscs, collected from Malku˛ Bay in 1997 and 1998, had significantly higher amounts of cells with normal diploid chromosome number (in 1997 – 68.0%, in 1998 – 70.2 %) than those sampled in 1996 (p < 0.01). It should be stressed, that L. ovata inhabiting Malku˛ Bay in 1996 possessed a very high amount of hypodiploid cells – up to 46.2% of studied cells. Amounts of hypodiploid and polyploid cells in the tissues of these molluscs inhabiting Malku˛ Bay significantly decreased after dredging of polluted sediments (Fig. 2). The chromosome number variation in this species was studied in Vilhelmo Channel in 1995, 1996 and 1997. In 1995 the chromosome numbers were much more stable than in 1996 and 1997 (Fig.2). Remarkable and extensive chromosome set irregularities were observed in tissues of snails L. ovata collected from Smelte˙ River estuaries near the motorboat port. Those molluscs possessed a high amount of cells with hypodiploid chromosome number. 20 % of cells with 2n = 32 in molluscs sampled in 1996 and 14.5 % of cells with 2n = 31 in L. ovata in 1995 from Smelte˙ were observed. Also, comparatively high amounts of cells, which possessed tetraploid or hexaploid chromosome sets were noticed. A high level of polyploid fragmented nuclei (25–30 %) in L. ovata from Smelte˙ was detected in 1996. L. ovata sampled in 1995 and 1996 had statistically the same number of cells, possessing hypodiploid and polyploid chromosome complements. In the tissues of L. ovata, collected from Klaipe˙ da passage near Kiaule˙ s Nugara Isles, was a comparatively low level of cytogenetic damage. Changes of chromosome numbers occurred in 23 – 24 % of studied cells (Fig. 2). Anodonta cygnea, sampled in the channel near the waterworks in 1996 had 46.7 % of cells with diploid chromosome number which is significantly less than in 1995 (77.1 %) and in 1997 (67.5%) (p < 0.0001). Also a high amount of cells with 2n = 36 (16 % in samples of
Figure 2.
Amount of hypodiploid cells in the tissues of Lymnaea ovata.
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Figure 3. Chromosome numbers in Anodonta cygnea from Vilhelmo Channel.
1996 and 8.2% in samples of 1995) and 2n = 34 (12.5 % in 1997) were noticed. In 1998 in A. cygnea only 29.9 % normal cells were found which is significantly lower than cell numbers in the same species collected from this location in any other year (p < 0.0001). Most cells in A. cygnea in 1998 were polyploid (Fig. 3). In 1994 Dreissena polymorpha from Vilhelmo Channel near waterworks had 70.1 % of diploid chromosome sets compared with statistically the same amount in 1995 (59.6 %; p < 0.05), and significantly less in 1997 (51.5 %; p = 0.0024) and in 1998 (53.5 %; p = 0.0137). The number of diploid cells in molluscs, sampled in 1997, was significantly smaller (p < 0.001) than in those collected in 1995. Molluscs sampled in 1997 had the highest amount of polyploid cells in their tissues (37.8 %) if compared with 1995 and 1994 observations (23.3% and 13 % respectively). Octoploid cells were abundant in D. polymor-
Figure 4.
Chromosome numbers in Dreissena polymorpha from Vilhelmo Channel.
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Figure 5.
Amount of aneuploid and hypodiploid cells in the tissues of Bithynia tentaculata.
pha collected in 1997. This species in 1998 had 53.5 % of normal cells in their tissues. The amount of normal diploid chromosome sets in molluscs, collected in 1998 is significantly different from that found in 1997. In 1998 most of genotoxic damage was expressed as elimination of 2 chromosomes and occurrence of hypodiploid cells, possessing 30 chromosomes (Fig. 4). The diploid chromosome set of B. tentaculata consists of 34 chromosomes. There were only 58.1% of such cells in those molluscs from Smelte˙ in 1997. Significantly higher amounts (78.9%) of diploid cells were observed in B. tentaculata inhabiting Klaipe˙ da passage near the Kiaule˙ s Nugara Isles (p = 0.0031) but statistically the same amount was found in molluscs from Malku˛ Bay in 1997 (67.0 %). The highest level of polyploid cells was found in Malku˛ Bay. The most frequent hypodiploid cells occurred in the tissues of B. tentaculata inhabiting the estuary of Smelte˙ River (Fig. 5). The normal diploid chromosome set of Sphaerium nitidum consisted of 30 chromosomes. In 1997 only 57 % of such cells were detected in the tissues of this species from Smelte˙ River. The most frequent other group were hyperdiploid cells. It is noteworthy to stress, that the hyperdiploid chromosome complements rarely occurred in the cells of molluscs. In karyotypes of S. nitidum there were additional chromosomes, and increasing hyperdiploid cells was determined. A significant lower level of cytogenetic damage was observed in the
Figure 6.
Cytogenetic damage in the cells of Sphaerium nitidum in 1997.
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tissues of S. nitidum inhabiting Klaipe˙ da passage near the Kiaule˙ s Nugara Isles (p = 0.0004). On the other hand, a comparative high frequency of hypodiploid cells was marked in snails from this part of Klaipe˙ da port area (Fig. 6).
4. Discussion Aquatic organisms may accumulate a variety of pollutants, including those with mutagenic and cancerogenic modes of action. There is a strong correlation found between chromosomal damage and the presence of petrochemical wastes (MCBEE et al., 1987), aromatic hydrocarbons, heavy metals (DIXON, 1982; BARšIENE˙, 1994) in the environment, and between neoplastic processes and pollution with dioxin which comes from some pesticide production processes (GARDNER et al., 1991; VAN BENEDEN et al., 1993). Molluscs by their distribution, filtration activity, size, sedentary style of life and other biological peculiarities are very suitable for the studies of aquatic genotoxicity. It was noticed, that cytogenetical changes may reflect a rapid response of organisms to environmental toxicant exposure and can provide early warning signs of adverse long-term effects in the populations of molluscs (DIXON, 1982; BRUNETTI et al., 1988; BARšIENE˙, 1994; BARšIENE˙ et al., 1994, 1996a; BURGEOT et al., 1996; BOLOGNESI et al., 1996; DOPP et al., 1996; BARšYTE˙, 1997). L. ovata collected from Malku˛ Bay in 1995 had a significantly lower level of cells with the diploid chromosome number than L. ovata inhabiting Vilhelmo Channel in the same year. These differences might be addressed to the presence of genotoxic pollutants in Malku˛ Bay. This was confirmed by studies of heavy metal concentrations in water, sediments and mollusc tissues from this locality. It is found that Malku˛ Bay is much more polluted with heavy metals, especially with Ni, Mn, Pb, Cu than Vilhelmo Channel (BARšYTE˙ LOVEJOY, in press). This pollution was evidently the reason for genotoxic damage occurring in the tissues of the bottom aquatic organisms. There are some studies on the mechanisms of mutagenesis and carcinogenesis of heavy metals. For instance insoluble nickel compounds are taken into cells by phagocytosis and cause neoplastic foci formation (MIURA et al., 1989). Inside the cell insoluble Ni compounds undergo dissolution and produce highly selective damage to heterochromatin. Ni ions have a much higher affinity for proteins and amino acids than for DNA, there it cross-links certain amino acids and proteins to DNA acting catalytically (COSTA et al., 1994). Ni compounds are carcinogenic and also cause chromosomal aberrations (HARTWIG, 1995). Mn acts as an enhancer mediating mispairing of nucleotides (BHANOT and SOLOMON, 1994). Mn (II) also enhances genotoxic action of UV light (HARTWIG, 1995). LANDOLPH (1994) proposed that As, Ni, Cr compounds, and oxygen radicals generated by them cause small deletions or mutations in the regulatory regions of the c-myc and other protooncogenes resulting in the activation of them and tumor promotion. Also such factors as intensive eutrophication and the lack of oxygen in Malku˛ Bay can play a role in genotoxic damage. It is known that anoxia has caused an increase in the frequency of micronuclei in molluscs (BRUNETTI et al., 1992). In L. ovata from Malku˛ Bay sampled in 1995 the same level of normal mitotic and meiotic cells was found as collected in 1996. However, like in the case of L. ovata from Vilhelmo Channel collected in 1995 and 1996 where they had the same level of numerical chromosomal aberrations but the percentage of polyploid cells was considerably higher in 1995. The mechanism of damage is expressed mostly not as the change of cells having normal chromosome number, but as the change of polyploid nuclei percentage. Therefore, in L. ovata collected in 1995 from Malku˛ Bay 13 individuals from 49 studied had about 90 % of polyploid nuclei while in the same species sampled in 1996 only 6 % of polyploid nuclei were observed. Also, in the tissues of L. ovata, sampled in 1995 a higher percentage of polyploid nuclei with irregular, fragmented shape was found.
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Therefore, unlike in Vilhelmo Channel the assessment of genotoxicity in vivo shows the situation in Malku˛ Bay to be better in 1997 and in 1998 than in 1996 and 1995. The main reason for the improvement was dredging of highly polluted sediments. It is noteworthy to stress, that in 1995 and 1996 molluscs from Malku˛ Bay were collected during the dredging of contaminated sediments. Significant increasing cancer frequencies in fish, which occur at elevated levels of exposure to genotoxic compounds during the dredging of contaminated sediments has been recently described by BAUMANN (1998). In 1995 and 1996 L. ovata from Smelte˙ River had more karyologically normal cells than the same species from Malku˛ Bay. However, in 1997 B. tentaculata from Smelte˙ had greater genotoxic damage than the same species from Malku˛ Bay. L. ovata from Smelte˙ possessed 25–30% of cells of an irregular shape and fragmented polyploid nuclei and 42.3 % of cells in B. tentaculata from Smelte˙ had polyploid chromosome sets in 1996 while only 6% polyploid nuclei was noted in Malku˛ Bay molluscs. The increase in a nuclear size and irregular or fragmented shapes of nuclei were noted in the cases of human colon cancer (ZUSMAN, 1995). There are a lot of studies on the relationships of tumor and chromosomal damage. Human leukaemia tend to have chromosome alterations which are aneuploid, while solid tumors chromosome numbers are often greater than tetraploid (RENO et al., 1994). Numeric and structural chromosome aberrations were described in human breast carcinomas (THOMPSON et al., 1993) and in the primary gastric cancer (ALLIENDE and ARANDA, 1994). In softshell clams, Mya arenaria with disseminated neoplasia most of cells showed the chromosome number which was polyploid. In normal clams gill cells had the chromosome number of 34. Variability of the number was from 26 to 39. Clams affected by disseminated neoplasia had chromosome numbers ranging from 44 to 80. Polyploid cells from affected clams were significantly larger in size (RENO et al., 1994). ELSTON and colaborators (1990) found polyploid populations of circulating cells in Mytilus with hemic neoplasia. Polyploid cell populations consisted of cells with 5.1 n and 10.1 n. Those populations were autonomously replicating polyploid hemocytes. Polyploid cells showed enlarged nuclei, which were irregular-shaped and fragmented. In some papers polyploid fragmented nuclei are defined as apoptotic in other they are attributed to cancer. Cancer represents uncontrollable cell proliferation while apoptosis is an attempt to prevent cancer. Therefore, it is difficult to describe nuclei found in molluscs from polluted locations to either of them. Additional assays are needed for this. However, in any case polyploid interphase nuclei, polyploid chromosome sets, irregular and fragmented nuclear shape in L. ovata and B. tentaculata from Smelte˙ and some of L. ovata from Malku˛ Bay show the presence of environmental chemicals with potential of inducing tumors. The incidence of polyploid, fragmented nuclei in Lymnaea as well as Anodonta was reported before (BARšIENE˙, 1994). The worse cytogenetic situation in Smelte˙ estuaries and therefore high pollution can mean an input of chemicals into Malku˛ Bay and as a consequence the situation worsening in it. However, L. ovata from Smelte˙ in 1996 had higher amounts of diploid cells than the same species in 1995. L. ovata collected in Smelte˙ in 1995 had only 2 % of polyploid nuclei which was less than in L. ovata sampled in 1996. It is confirmed by data on B. tentaculata, which had significantly more polyploid cells in 1996 than in 1995. This shows an increase in extent of cytogenetic damage in 1996. In 1997 collected B. tentaculata the amount of diploid cells was greater than 1996 molluscs but lower than in 1995 molluscs. This mollusc species lives in sediments, therefore, high degree of genotoxic damage in it implies on dangerous levels of pollution accumulation in sediments. The increase in pollution coming from Smelte˙ into Malku˛ Bay can cause genotoxicity level in Malku˛ Bay to be the same as before dredging. The cytogenetic analysis of molluscs taken from Smelte˙ before Klaipe˙ da revealed that their levels of cytogenetic damage approximately are in the range of damage in the same species from clean water bodies (BARšIENE˙, 1994). It shows that Smelte˙ is polluted in Klaipe˙ da industrial area and perhaps especially in the motorboat area. Smelte˙ was the most polluted place from all assessed in 1996 as well as 1997. Even Malku˛ Bay, which is one of the most
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polluted sites in Curonian Lagoon is less polluted than Smelte˙ River. Especially high concentrations of Mn, Ni, Pb, Zn, and Cu were found in Smelte˙ molluscs, sediments, and water (BARšYTE˙ LOVEJOY, in press). The high occurrence of polyploid fragmented nuclei in B. tentaculata and the great amount of polyploid chromosome sets in S. nitidum sampled from Smelte˙ River implies on the existence of pollution in the environment of those molluscs and biological damage due to it to this ecosystem. Also occurrence of polyploid species as P. inflatum shows this habitat to be stressful because polyploid species can more easily survive under severe conditions (BARšIENE˙ et al., 1996a). A. cygnea molluscs from Vilhelmo Channel near Klaipe˙ da water works had more diploid cells in 1995 than in 1997. This shows that ecological conditions in this water body was worse in 1997 compared with 1995, but better than 1996. Data on Anodonta also suggest that the worst conditions were present in 1998 in comparison to 1995, 1996, 1997. Higher genotoxic damage also could be the result of agrochemical and hydrocarbon pollution from Minija River which has a large agricultural drainage area and/or the leakage from the old military area near Vilhelmo Channel. It is known that chromosomal damage occurs in molluscs inhabiting springs polluted by agrochemicals from rice and orange fields (BARšIENE˙ et al., 1998). The high amount of Zn found in D. polymorpha and sediments, elevated levels of Mn, Ni in water in 1996 in comparison with 1995 may be components of pollution causing cytogenetic damage (BARšYTE˙ LOVEJOY, in press). Therefore the genotoxicity level in D. polymorpha was going up from 1994 to 1998. The process of building the dam might have had an effect on molluscs as it created conditions with less oxygen and more suspended solids. Also pollution from waterworks themselves might play a role in this. It is known that frequency of micronuclei increase after water chlorination (SCARPATO et al., 1990). However, the general trend in genotoxicity in Vilhelmo Channel near waterworks is increasing and the extent of hazard in Vilhelmo Channel to wildlife exists further.
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BHANOT, O. S. and J. J. SOLOMON, 1994: The role of mutagenic metal ions in mediating in vitro mispairing by alkylpyrimidines. – Environ. Health Perspect. 102: 81–90. BOLOGNESI, C., R. RABBONI and P. ROGGIERI, 1996: Genotoxicity biomarkers in Mytilus galloprovincialis as indicators of marine pollutants. – Comp. Biochem. 113C, No 2: 319–323. BRUNETTI, R., I. GOLA and F. MAJONE, 1988: The test of micronuclei: examples of application to marine ecology. – Mar. Ecol. Prog. Ser. 44: 65–68. BRUNETTI, R., O. FUMAGALLI, P. VALERIO and M. GABRIELE, 1992: Genotoxic effects of anoxia on Mytilus galloprovincialis. – Marine Ecol. Progress, Series 83: 71–74. BURGEOT, T., S. WOLL and F. GALGANI, 1996: Evaluation of the micronucleus test on Mytilus galloprovincialis for monitoring applications along French coasts. – Marine Poll. Bull. 32: 39–46. COSTA, M., K. SALNIKOW, S. COSENTINO, C. B. KLEIN, X. HUANG and Z. ZHUANG, 1994: Molecular mechanisms of nickel carcinogenesis. – Environ. Health Perspect. 102: 127–130. DIXON, D. R., 1982: Aneuploidy in mussel embryos (Mytilus edulis L.) originated from a polluted dock. – Marine Biol. Letters 3: 155–161. DOPP, E., C. M. BARKER, D. SCHIFFMANN and C. L. REINISCH, 1996: Detection of micronuclei in hemocytes of Mya arenaria: association with leukaemia an induction with an alkylating agent. – Mutat. Res. 245: 231–237. ELSTON, R. A., A. S. DRUM and S. K. ALLEN, 1990: Progressive development of circulating polyploid cells in Mytilus with hemic neoplasia. – Diseases of Aquatic Organisms 8: 51–59. GARDNER, G. R., P. P. YEVICH, J. HURST, P. THAYLER, S. BENYI, J. C. HARSHBARGER and R. J. PRUELL, 1991: Germinomas and teratoid siphon anomalies in softshell clams Mya arenaria, environmentally exposed to herbicides. – Environm. Health Persp. 90: 43–51. HARTWIG, A., 1995: Current aspects in metal genotoxicity. – Biometals 8: 3–11. LANDOLPH, J. R., 1994: Molecular mechnisms of transformation of C3H/10T1/2C18 mouse embryo cells and diploid human fibroblasts by carcinogenic metal compounds. – Environ. Health Perspect. 102: 119–125. MCBEE, K., J. W. BICKHAM, K. W. BROWN and K. C. DONNELY, 1987: Chromosomal aberrations in native small mammals (Peromyscus leucopus and Sigmodon hispidus) at a petrochemical waste disposal site: I Standard karyology. – Arch. Environ. Contam. Toxicol. 16: 681–688. MIURA, T., S. R. PATIERNO, T. SAKURAMOTO and J. R. LANDOLPH, 1989: Morphological and neoplastic transformation of C3H/10T1/2Cl8 mouse embryo cells by insoluble carcinogenic nickel compounds. – Environ. Molecular Mutagenesis 14: 65–78. PEAKALL, D., 1992: Animal biomarkers as pollution indicators, ed. by M. H. DEPLEDGE, B. SANDERS, Chapman and Hall, London, pp. 291. RENO, P. W., M. HOUSE and A. ILLINGWORTH, 1994: Flow cytometric and chromosome analysis of softshell clams Mya arenaria, with disseminated neoplasia. – J. Invertebrate Pathol. 64: 163–172. ROKICKIJ, N. F., 1974: The introduction into statistical genetics, Minsk, pp. 415. SCARPATO, R., L. MIGLIORE, G. ALFINITO-COGNETTI and R. BARALE, 1990: Induction of micronuclei in gill tissue of Mytilus galloprovincialis exposed to polluted marine waters. – Mar. Pollut. Bull. 21: 74–80. SHUGART, L. R., 1996: Molecular Markers to Toxic Agents. – In: Ecotoxicology. A Hierarchical Treatment, ed. by M. C. NEWMAN and C. H. JAGOE, Lewis Publishers, Boca Raton, FL. pp. 133–161. THOMPSON, F., J. EMERSON, W. DALTON, J. YANG, D. MCGEE, H. VILLAR, S. KNOX, K. MASSEY, R.WEINSTEIN, A. BHATTACHARYYA and J. TRENT, 1993: Clonal chromosome abnormalities in human breast carcinomas I. Twenty-eight cases with primary disease. – Genes, Chromosomes and Cancer 7: 185–193. VAN BENEDEN, R. J., G. R. GARDNER, N. J. BLAKE and D. G. BLAIR, 1993: Implications for the presence of transforming genes in gonadal tumors in two bivalve mollusc species. – Cancer Res. 53: 2976–2979. ZUSMAN, I., 1995: Variability of neoplastic parameters in colon tumours, and its significance in diagnostic practice. – Biol. Rev. 70: 107–160. Manuscript received January 24th, 2000; revised May 22nd, 2000; accepted June 19th, 2000
