Effects on Reproduction, Genotoxicity and DNA Methylation Pattern after Chronic Exposure of the Freshwater Snail Physaacuta (Gastropoda, Pulmonata) to Vinclozolin

Research Article

Austin J Environ Toxicol. 2016; 2(1): 1008.

Effects on Reproduction, Genotoxicity and DNA Methylation Pattern after Chronic Exposure of the Freshwater Snail Physaacuta (Gastropoda, Pulmonata) to Vinclozolin

Sanchez-Arguello P¹*, Aparicio N¹, Guevara MA², Díaz L², Cervera MT² and Fernandez C¹

¹Department of the Environment, Laboratory for Ecotoxicology, INIA, Spain

²Department of Forest Ecology and Genetics, INIACIFOR, Spain

*Corresponding author: Paloma Sanchez-Arguello, Department of the Environment, Laboratory for Ecotoxicology, INIA, Madrid, Spain

Received: January 20, 2016; Accepted: February 12, 2016; Published: February 12, 2016;


The Fungicide Vinclozolin (VZ) is an endocrine disruptor with anti-androgenic activity whose potential for disrupting epigenetic mechanisms in mammals has also been shown. In this regard, this study combined reproductive endpoints and effect assessments at the sub-individual level associated with Genotoxicity and altered DNA methylation patterns. Physaacuta was exposed to VZ at nominal concentrations from 0.0005 to 5mg/L. Fecundity (production of F1 eggs) over 45 days and fertility (viability of F1 eggs) after further 21-dayembryonic development under exposure and non-exposure conditions were monitored. The Genotoxicity of VZ was evaluated by micronuclei scoring. Potential epigenetic alterations were measured by scoring DNA methylation patterns. Although acute exposure (96h prescreening test) at 5mg/L did not caused mortality, long-term exposure (45-day reproduction test) affected the survival of snails. Reduced effects on reproduction, micronuclei induction and DNA demethylation events were observed at 5mg/L. At the other VZ concentrations no effects on these endpoints were found. Thus reproduction endpoints were as sensitive as the effects at the sub individual level. This reproduction test, with additional assessments of the hatching success of the F1 recovered eggs, assessed reliable reproduction success in freshwater snails. The use of exposed and nonexposed F1 embryos allowed us to observe if damage due to parental exposure could be maintained or recovered when suspending treatment during embryonic development. Finally, the combination of long-term ecotoxicological effects and genotoxic/epigenetic biomarkers can broaden our understanding of pollutant impacts.

Keywords: Molluscs; Reproduction test; Micronucleus induction; DNA methylation


Continuously increasing chemical pollution has led to a complex environmental scenario in which chronic tests on a wide range of species and specific endpoints are crucial tools for studying chemical effects. Chronic testing can provide information on processes such as growth, development and reproduction, which are relevant at the population level. Aquatic invertebrates are target organisms for different OECD chronic toxicity tests [1-4] including one lifecycle test [5]. Although no mollusk-based toxicity tests have been internationally standardized, an OECD project is developing a partial life-cycle test with gastropods [6,7]. The OECD/EDTA (Endocrine Disrupters Testing and Assessment) Conceptual Framework has indicated the need for mollusc-based tests, and the phylum offers species of ecological and economic relevance that are known to be uniquely sensitive to a number of Endocrine Disrupter Compounds (EDCs) [8,9] Some assays performed with freshwater gastropods have addressed the effects of xenobiotics their reproductive capacities (fecundity and fertility) after long-term exposure. Oehlmann et al. [10] reported malformations of the female genital system, and massive stimulation of oocyte and spawning mass production in Marisa cornuarietis induced by Bisphenol A and Octyl phenol with a complete life-cycle. These endpoints were also assessed on Lymnaeaacuminata after exposure to pyrethroid pesticides [11] Czech et al. [12] exposed adults of Lymnaeastagnalis to Tributyltin, β-sitosterol and 4-nonylphenol with a view to looking at the reproductive and histopathological effects on the F0 and F1 generations. Leung et al. [13,14] also exposed Lymnaeastagnalis and Physafontinalis from embryos to sexual maturity to Tributyltin. Recently, different approaches have been developed to perform embryo tests with freshwater snails to form part of mollusc lifecycle tests [8, 15-18]. All these studies have explored the life-stage specific effects of molluscs to xenobiotics, while other studies have investigated their genotoxic responses [19].

Presence of genotoxic compounds in the environment is a matter of concern given the transfer of effects across generations. The detection of chemical Genotoxicity has focused mainly on a direct change at the DNA level, such as point mutations. Cytogenetic alterations are widely accepted for detecting potential carcinogens.

The cytogenetic assay of Micronucleus (MN) induction has been applied successfully in ecotoxicological studies [20,21]. Exposure to chemicals can result in genetic alterations but many pollutants do not have the capacity to induce direct DNA damage. Non-genotoxic carcinogens are often assumed to possess an epigenetic mode of action which can induce heritable changes that cannot be explained by changes in DNA sequence [22,23]. Therefore, additional molecular mechanisms need to be considered, such as epigenetics, which has recently become a very promising target in molecular biology. DNA methylation has been the most extensively studied epigenetic mechanism. The evaluation of the global methylation status and the assessment of methylation in GC-rich regions of the genome have been proposed for both initial toxicity assessments of a compound’s toxicity potential and an earlier indication of its possible mechanism of action [24]. Genetic and epigenetic mechanisms are crucial for genome stability [25] and, regardless of the chemical-induced changes in DNA or in gene expression; both can have knock-on effects at higher biological organization levels. Therefore studying genotoxic/ epigenetics in Ecotoxicology can help clarify how these mechanisms can link to responses of ecological relevance.

Vinclozolin (VZ) is a nonsystemic fungicide used to control fungal pathogens. Concern about toxicity in mammals is due to its antiandrogenic activity [26,27]. VZ exerts its effects most dramatically during the development stages of animals, and ultimately result in reproductive effects [28]. VZ has been found to produce infertility of F1 males in multigenerational studies with rats [29]. Transgenerational effects in mammals through epigenetic mechanisms after VZ exposure have been observed [30]. Nevertheless, other studies have failed to find this mechanism of action [31]. The likelihood of VZ being released into surface waters, together with its known endocrine disruption effect, justifies undertaking chronic exposure studies with aquatic organisms. Martinovic et al. [32] studied the reproductive toxicity of VZ in fish and showed a concentration-dependent reduction in fecundity (production of fewer eggs) whereas hatching success of the deposited eggs (fertility) was not affected. VZ did not elicit an effect on the reproduction of the freshwater invertebrate Daphnia magna, whereas overall DNA methylation rate consistently decreased [33,34] previously observed an earlier sexual repose and morphological alterations of sex organs in the males of two species of prosobranch snails. Nevertheless, it was not possible to compare these data with fecundity data since spawning did not occur. Effects of VZ on the reproduction of the freshwater snail Lymnaeastagnalis were subsequently investigated by Ducrot et al., [35] who showed reduced in fecundity (cumulated number of eggs produced per individual) with a significant number of non-fertilized eggs as observed microscopically. Viability of eggs was not determined. Theseauthors recommended the complementary assessment of hatching success in the offspring of exposed snails (fertility) in the case of fecundity impairment when studying endocrine disruptors which may have epigenetic effects.

The present study evaluated the fecundity and fertility of the freshwater snail Physaacuta. The effects of VZ after 45-dayexposure, along with a complementary assessment of offspring embryonic development (F1 embryos) and hatching success were monitored. The F1 endpoints were evaluated for both non-exposed and exposed embryos in order to distinguish effects due to parental exposure. A combination of genetic and epigenetic endpoints to study the effects associated with mechanisms of inheritance was simultaneously assessed. The Genotoxicity of adults and F1 embryos was assessed by micronucleus test. The post-exposure profile alterations of global DNA methylation in adults was the epigenetic mechanism studied. The results were compared with our previous studies of VZ on Physaacuta embryos whose parents were not exposed [15]. Finally since long-term exposure was needed, actual VZ concentrations were measured.

Material and Methods

Acute and reproduction tests

Physaacuta individuals came from our own laboratory breeding stocks. Culture conditions are described by Sánchez-Argüello et al. [36].

In order to stablish the exposure range of the PLC test, a prescreening test with adults was performed at two concentrations (0.5 and 5mg/L) for 96h. Mortality and reproduction (only the number of egg masses) were monitored in triplicate.

For the definitive test, the adults from a single cohort cultured in our laboratory, and not previously exposed, were used for the PLC test. Groups of ten snails (size of test animals 7±2mm) were exposed to VZ in 60 ml of test medium using acetone (0.1%) as a solvent carrier at 20°C, with a photoperiod of 16h:8h light/darkness. Vessels were covered to prevent snail escaping. Maximal VZ concentration detected in surface water was 0.0005mg/L [35]. The range of exposure covered this measured environmental concentration and 1000 times higher. The VZ test treatments were: Control (reconstituted test water as used in 15); Acetone; 0.0005; 0.005; 0.05; 0.5 and 5mg/L. VZ was added by medium renewal and after spiking nominal concentrations on all the other days to maintain nominal concentrations. Test medium renewal, food supply (1.2 mg/snail of Shrimps food flakes -Sera®) and monitoring the endpoints (mortality and fecundity) were done twice-weekly over a 45-day period. Fecundity was assessed as the egg masses deposited per adult. The number of eggs inside each egg mass was also counted with a stereomicroscope (Olympus SZX12). Four replicates per treatment were used. Snails were recovered at the end of tests for the micronuclei induction study and DNA isolation and methylation analyses. Measures of chemical body burden were also taken.

F1 embryo toxicity tests

Oviposition was observed on the first day of monitoring (3 days after the test began) in all the treatments. Once a week spawned egg masses from Oviposition were sampled and washed through a stainless steel strainer tore move jelly-envelopment. This treatment allows single eggs to be obtained, which are easily observed under a stereomicroscope during the embryonic development instead of the three-dimensional arrangement of eggs in the egg mass [15]. Single eggs were used for testing embryo toxicity. F1 eggs from each treatment and control were divided into two batches: one batch was transferred to the 12-well plate with reconstituted water (nonexposed embryos), while the other batch was exposed to the same concentrations as their parents (exposed embryos). These differences in the exposure conditions for offspring help differentiate the effects due only to parental exposure from the effects observed by continuous exposure (F0-adults and F1-embryos), respectively. Embryo toxicity tests were performed in duplicate per treatment, and for both the embryos non-exposed and exposed to VZ, in accordance with the conditions described in Sánchez-Arguello et al. [15]. Briefly, a density of 30 eggs/well in 4ml/well was used. The embryo toxicity test lasted 21, for this time mal formed and dead embryos were monitored daily by stereomicroscope scoring. (Figure 1) shows differences between normal embryos, malformed embryos and dead embryos at different embryonic stages. Test medium was renewed twice weekly. Hatching success was measured until the test ended.