Mutation Research,
245 (1990) 231-237
231
Elsevier MUTLET 0422
The micronucleus assay in Anodonta cygnea for the detection of drinking water mutagenicity R. Scarpato, L. Migliore and R. Barale I Dipartimento di Scienze dell'Ambiente e del Territorio , Universit~ di Pisa, Pisa and l lstituto di Zoologia, Universit~ di Ferrara, Ferrara (Italy)
(Received30 April 1990) (Accepted4 July 1990)
Keywords:
Mutagenicityof drinking water; Micronucleustest; Anodonta cygnea
Summary A micronucleus test in gill cells o f the freshwater mussel A n o d o n t a c y g n e a has been proposed for the detection o f drinking water genotoxicity. Animals were exposed for 28 days to a drinking water sample and collected every week. Highly significant increases in spontaneous MN frequency were observed at each sampling, especially after 13 days of exposure. As positive control 2 doses of mytomicin C (MMC) were used (10- 8 and 10- 7 M). A second experiment was performed at a municipal waterworks in order to assess the role o f water treatment processes in the production of mutagenic compounds. The most prevalent genotoxic effects were detected after chlorination (R: 10.47%0_+ 3.05, p < 0.001).
Several classes of organic chemicals are usually present in drinking waters. Contamination of source water by industrial, agricultural and domestic effluents, water treatment processes or components of the distribution systems can be involved in the production of pollutants. Toxicological investigations have recognized many of these compounds as toxic a n d / o r potentially carcinogenic substances (for a review see Bull, 1985). In relation to this problem, the choice of shortterm tests able to detect the presence o f Correspondence: Dr. R. Scarpato, Dipartimento di Scienze dell'Ambiente e del Territorio, Universit/t di Pisa, Via S. Giuseppe 22, Pisa (Italy)
mutagenically active compounds is important. Bacterial assays, in particular the Ames test, have been frequently used (Nestmann et al., 1979; Monarca et al., 1985; Vartiainen and Liimatainen, 1986; Galassi et al., 1989), but they only reveal point mutations. On the other hand, genetic endpoints such as sister-chromatid exchanges (SCE), chromosome aberrations (CA) or micronuclei (MN) can be detected in some species of aquatic organisms exposed to reference mutagens or polluted waters (Alink et al., 1980; Siboulet et al., 1984; A1-Sabti, 1985; Brunetti et al., 1986; Das and Nanda, 1986). In consideration of its ability to detect chromosome mutations due to both clastogenic effects and
0165-7992/90/$ 03.50 © 1990Elsevier SciencePublishers B.V. (BiomedicalDivision)
232
Fig. 1. Micronucleated gill cell of A. cygnea.
alterations of the mitotic apparatus (Migliore et al., 1987), an in vivo micronucleus test seems particularly indicated for investigations of this type (Krauter et al., 1986; Jaylet et al., 1987; Fernandez et al., 1989). Micronuclei are small intracytoplasmic chromatine masses close to the main nucleus and can be easily observed in interphasic cells which have completed at least one replication cycle (Fig. 1). Among aquatic organisms, sea-water mussels are used to study biological effects caused by accumulation of exogenous substances, and therefore they are intensively employed in environmental monitoring. For this purposse a micronucleus test in gill tissue of the marine mussel Mytilus galloprovincialis has been recently carried out in our laboratory (Scarpato et al., 1990). On the basis of this experience, the same methodology was applied to the freshwater clam Anodonta cygnea (Unionidae). The ability of this family of bivalves to bioaccumulate heavy metals,
organochlorine pesticides or other xenobiotics is widely documented (Graney et al., 1983; Hemelraad et al., 1986a; Cassini et al., 1986; Holwerda and Herwig, 1986; Holwerda et al., 1989). The aim of the present study was to assess the suitability of this system for evaluating the mutagenic activity of drinking water samples and for determining the contribution of water treatments to genotoxicity.
Materials and methods
Animals Individuals of Anodonta cygnea (shell length 8-12 cm) provided by Aquarium (Pisa, Italy) were acclimatized for 2 weeks in well oxygenated aquaria containing 20 1 of synthetic fresh water at a temperature of 15°C and an initially sterile sand sediment; mussels were regularly fed with Liquifry No. 1. The same rearing conditions were maintained throughout the experiments.
233
Experimental procedures Experiment 1. Animals were divided into 2 groups: one was exposed in two aquaria to drinking tap water from our laboratory (S. Piero a Grado, Pisa) and the other was kept in synthetic fresh water as a control. Tap water was slowly supplied at constant flow to maintain a water level of 20 I. Eight mussels from the exposed group (4 animals for each aquarium) were sampled every week for a total treatment of 28 days (17/11/1987-15/12/ 1987). Control experiments were performed as follows: (a) the spontaneous MN frequency of untreated mussels was assessed at the start and the end of exposure (day 0 and day 28); (b) as a positive control, 2 days before the last sampling (days 26) two samples of 6 clams each were exposed for 48 h to the reference mutagen mytomicin C (MMC) at doses of 10-8 and 10-7 M. Experiment 2. Four groups of 7 animals were transferred to the municipal waterworks of Marina di Pisa and directly exposed for 13 days (3/2/ 1988-16/2/1988) to the following types of water which represent the 4 steps of water treatment: (1) ground water; (2) chlorinated water; (3) filtered water; (4) drinking water. The raw water of this waterworks derives from an artesian well and is very rich in inorganic compounds, especially iron salts. Water disinfection is performed by adding 1.94 ml/s of sodium hypochlorite to groundwater (flow: 40 l/s). Filtered water, obtained by sand filtration, is again chlorinated before the distribution in the water mains (0.3 ppm of residual chlorine). Water addition to aquaria was as described above. A control sample of 6 mussels was harvested only at the end of the exposure.
was fixed in methanol-acetic acid (3:1) for 40 min, dropped onto clean glass slides and then stained in 3% Giemsa/distilled water. Between the 2 cell populations recovered by our technique, we performed cytogenetic analysis only in the cell type, present in the majority, that showed typical eosinophilia. 1500 cells per animal with intact cytoplasm were scored under an oil-immersion objective (× 1000 final magnification) and the MN frequency is expressed as the number of micronucleated cells per 1000 cells scored.
Statistical analysis Data were statistically processed by Student's ttest for unpaired data to analyze the effects of treatments on spontaneous MN level, and by regression analysis to determine the dose-effect relationship. Since the results of the second experiment seemed to indicate the presence of toxicity phenomena (no significant correlation was found between MN frequencies and exposure time by linear regression), we applied the method of Margolin et al. (1981). This method is based on a simple equation which allows the simultaneous estimation of mutagenic and toxicity effects in the following model: Y = (a + bX)exp(-rX) where Y is the frequency of the observed effect, X is the time of exposure, a and b are the linear regression coefficients, and r is the toxicity parameter. This equation can fit most types of biological responses, and well represents the behavior of those experimental points which deviate from linearity.
Slide preparation
Results
A small piece of gdl was excised from each animal and washed in Hanks' Balanced Salt Solution (HBSS). Tissue disaggregation was carried out by gentle rubbing of the gill on an appropriate small-meshed net (4~ 80 ~m); about 5 ml of HBSS were added to obtain a cellular suspension. After centrifugation at 1000 rpm for 10 min, the pellet
The results of MMC treatment are reported in Table 1. Both MMC concentrations significantly (p
245 (1990) 231-237
231
Elsevier MUTLET 0422
The micronucleus assay in Anodonta cygnea for the detection of drinking water mutagenicity R. Scarpato, L. Migliore and R. Barale I Dipartimento di Scienze dell'Ambiente e del Territorio , Universit~ di Pisa, Pisa and l lstituto di Zoologia, Universit~ di Ferrara, Ferrara (Italy)
(Received30 April 1990) (Accepted4 July 1990)
Keywords:
Mutagenicityof drinking water; Micronucleustest; Anodonta cygnea
Summary A micronucleus test in gill cells o f the freshwater mussel A n o d o n t a c y g n e a has been proposed for the detection o f drinking water genotoxicity. Animals were exposed for 28 days to a drinking water sample and collected every week. Highly significant increases in spontaneous MN frequency were observed at each sampling, especially after 13 days of exposure. As positive control 2 doses of mytomicin C (MMC) were used (10- 8 and 10- 7 M). A second experiment was performed at a municipal waterworks in order to assess the role o f water treatment processes in the production of mutagenic compounds. The most prevalent genotoxic effects were detected after chlorination (R: 10.47%0_+ 3.05, p < 0.001).
Several classes of organic chemicals are usually present in drinking waters. Contamination of source water by industrial, agricultural and domestic effluents, water treatment processes or components of the distribution systems can be involved in the production of pollutants. Toxicological investigations have recognized many of these compounds as toxic a n d / o r potentially carcinogenic substances (for a review see Bull, 1985). In relation to this problem, the choice of shortterm tests able to detect the presence o f Correspondence: Dr. R. Scarpato, Dipartimento di Scienze dell'Ambiente e del Territorio, Universit/t di Pisa, Via S. Giuseppe 22, Pisa (Italy)
mutagenically active compounds is important. Bacterial assays, in particular the Ames test, have been frequently used (Nestmann et al., 1979; Monarca et al., 1985; Vartiainen and Liimatainen, 1986; Galassi et al., 1989), but they only reveal point mutations. On the other hand, genetic endpoints such as sister-chromatid exchanges (SCE), chromosome aberrations (CA) or micronuclei (MN) can be detected in some species of aquatic organisms exposed to reference mutagens or polluted waters (Alink et al., 1980; Siboulet et al., 1984; A1-Sabti, 1985; Brunetti et al., 1986; Das and Nanda, 1986). In consideration of its ability to detect chromosome mutations due to both clastogenic effects and
0165-7992/90/$ 03.50 © 1990Elsevier SciencePublishers B.V. (BiomedicalDivision)
232
Fig. 1. Micronucleated gill cell of A. cygnea.
alterations of the mitotic apparatus (Migliore et al., 1987), an in vivo micronucleus test seems particularly indicated for investigations of this type (Krauter et al., 1986; Jaylet et al., 1987; Fernandez et al., 1989). Micronuclei are small intracytoplasmic chromatine masses close to the main nucleus and can be easily observed in interphasic cells which have completed at least one replication cycle (Fig. 1). Among aquatic organisms, sea-water mussels are used to study biological effects caused by accumulation of exogenous substances, and therefore they are intensively employed in environmental monitoring. For this purposse a micronucleus test in gill tissue of the marine mussel Mytilus galloprovincialis has been recently carried out in our laboratory (Scarpato et al., 1990). On the basis of this experience, the same methodology was applied to the freshwater clam Anodonta cygnea (Unionidae). The ability of this family of bivalves to bioaccumulate heavy metals,
organochlorine pesticides or other xenobiotics is widely documented (Graney et al., 1983; Hemelraad et al., 1986a; Cassini et al., 1986; Holwerda and Herwig, 1986; Holwerda et al., 1989). The aim of the present study was to assess the suitability of this system for evaluating the mutagenic activity of drinking water samples and for determining the contribution of water treatments to genotoxicity.
Materials and methods
Animals Individuals of Anodonta cygnea (shell length 8-12 cm) provided by Aquarium (Pisa, Italy) were acclimatized for 2 weeks in well oxygenated aquaria containing 20 1 of synthetic fresh water at a temperature of 15°C and an initially sterile sand sediment; mussels were regularly fed with Liquifry No. 1. The same rearing conditions were maintained throughout the experiments.
233
Experimental procedures Experiment 1. Animals were divided into 2 groups: one was exposed in two aquaria to drinking tap water from our laboratory (S. Piero a Grado, Pisa) and the other was kept in synthetic fresh water as a control. Tap water was slowly supplied at constant flow to maintain a water level of 20 I. Eight mussels from the exposed group (4 animals for each aquarium) were sampled every week for a total treatment of 28 days (17/11/1987-15/12/ 1987). Control experiments were performed as follows: (a) the spontaneous MN frequency of untreated mussels was assessed at the start and the end of exposure (day 0 and day 28); (b) as a positive control, 2 days before the last sampling (days 26) two samples of 6 clams each were exposed for 48 h to the reference mutagen mytomicin C (MMC) at doses of 10-8 and 10-7 M. Experiment 2. Four groups of 7 animals were transferred to the municipal waterworks of Marina di Pisa and directly exposed for 13 days (3/2/ 1988-16/2/1988) to the following types of water which represent the 4 steps of water treatment: (1) ground water; (2) chlorinated water; (3) filtered water; (4) drinking water. The raw water of this waterworks derives from an artesian well and is very rich in inorganic compounds, especially iron salts. Water disinfection is performed by adding 1.94 ml/s of sodium hypochlorite to groundwater (flow: 40 l/s). Filtered water, obtained by sand filtration, is again chlorinated before the distribution in the water mains (0.3 ppm of residual chlorine). Water addition to aquaria was as described above. A control sample of 6 mussels was harvested only at the end of the exposure.
was fixed in methanol-acetic acid (3:1) for 40 min, dropped onto clean glass slides and then stained in 3% Giemsa/distilled water. Between the 2 cell populations recovered by our technique, we performed cytogenetic analysis only in the cell type, present in the majority, that showed typical eosinophilia. 1500 cells per animal with intact cytoplasm were scored under an oil-immersion objective (× 1000 final magnification) and the MN frequency is expressed as the number of micronucleated cells per 1000 cells scored.
Statistical analysis Data were statistically processed by Student's ttest for unpaired data to analyze the effects of treatments on spontaneous MN level, and by regression analysis to determine the dose-effect relationship. Since the results of the second experiment seemed to indicate the presence of toxicity phenomena (no significant correlation was found between MN frequencies and exposure time by linear regression), we applied the method of Margolin et al. (1981). This method is based on a simple equation which allows the simultaneous estimation of mutagenic and toxicity effects in the following model: Y = (a + bX)exp(-rX) where Y is the frequency of the observed effect, X is the time of exposure, a and b are the linear regression coefficients, and r is the toxicity parameter. This equation can fit most types of biological responses, and well represents the behavior of those experimental points which deviate from linearity.
Slide preparation
Results
A small piece of gdl was excised from each animal and washed in Hanks' Balanced Salt Solution (HBSS). Tissue disaggregation was carried out by gentle rubbing of the gill on an appropriate small-meshed net (4~ 80 ~m); about 5 ml of HBSS were added to obtain a cellular suspension. After centrifugation at 1000 rpm for 10 min, the pellet
The results of MMC treatment are reported in Table 1. Both MMC concentrations significantly (p
