Assessment of the genotoxic impact of pesticides on farming communities in the countryside of Santa Catarina State, Brazil

The aim of this study was to assess the use of pesticides on farms located in the Lambedor River watershed in Guatambu, State of Santa Catarina, as well as to determine, by micronucleus testing, the risk of genotoxic impact. Samples from locally collected Cyprinus carpio, Hypostomus punctatus, Rhamdia quelen and Oreochromis niloticus gave evidence of a mean increase in micronuclei frequency from 6.21 to 13.78 in 1,000 erythrocytes, a clear indication of the genotoxic potenciality of pesticide residues in regional dams, and their significant contribution to local environmental contamination.


Introduction
Agrochemicals are widely used for decades, in an attempt to protect crops against insect pests. Nevertheless, in the light of the increasing resistance, every year a vast array of new compounds is introduced into the market, with consequential negative side effects and increased costs in food production. More specifically, pesticides are poisons intentionally dispersed in the environment to control pests, and which subsequently persist in the soil, water and food, with toxicity related outcomes to both humans and animals (Schulz, 2004;Carvalho, 2006;Moraes et al., 2009). Notwithstanding, the use of agrochemicals remains a common practice, especially in tropical regions (Carvalho, 2006).
Brazil is the the third main consumer of pesticides worldwide (Agriculture and Agri-Food, Canada, 2005). Numbers from the Brazilian pesticide industry show that pesticide sales in Brazil have risen from US$ 0.98 billion in 1992 to US$ 4.5 billion in 2004, this representing an increase of 360% over the period (Brazil, 2008). Furthermore, discrepancies between Brazilian regulation of the pesticide market, and those enforced in developed countries, have paved the way to the importation of vast amounts of pesticides that are highly toxic, severely restricted, or even banned in other nations (Carvalho, 2006;Smith, 2001).
According to Peres and Moreira (2007), the widespread and growing use of pesticides for raising crops and cattle in Brazil has given rise to a number of environmental changes and problems, both by the contamination of human communities, as part of the environment, and by accumulation in biotic and abiotic segments of ecosystems. With year-round harvests, the low level of mechanization in the various phases of production, and the consequential exposure to multiple environmental contaminants (pesticides), the vulnerability of Brazilian workers and the environment itself to these genotoxic agents becomes evident.
Environmental quality control requires the monitoring of various indicators, including the assessment of pesticide residues. Research on bioindicators is instrumental in detecting the toxic effects caused by these. Processes in the bioconcentration and transformation of pesticides have been studied mainly in fishes. This is due to these animals living in direct contact with aquatic sediments adjacent to areas where pesticides are commonly used. In these water bodies, many poorly water-soluble compounds eventually settle, with the consequential increase in the degree of local contamination, when compared to the water column as a whole (Grisolia, 2005;Umbuziero et al., 2006).
In Brazil, reports on pesticide toxicity in fish are still rare. Nevertheless, in recent studies, potentially genotoxic substances have been recognized and quantified, when using fish as experimental organisms (Andrade et al., 2004). Nowadays, several tests have been well developed and standardized for assessing the genotoxic profile of a wide spectrum of substances. In comparison to other methods, the advantage of the simplicity of the micronucleus test facilitates counting the micronuclei in erythrocytes. Therefore, the test has been widely used to evaluate the mutagenic potential of new drugs and chemicals, being also indicated in routine screening and environmental monitoring (Al-Sabti et al., 1994;Bücker et al., 2006).
Micronuclei are formed during the telophase of mitosis or meiosis, when the nuclear envelope is reconstituted around the chromosomes of daughter cells (Udroiu, 2006). Micronuclei are the result of chromosome acentric fragments (clastogenic effect) or whole chromosomes that, through incomplete migration, have been excluded from the main core (aneugenic effect). Thus, micronuclei represent a loss in chromatin as a result of damage to either chromosome structure (fragmentation) or the mitotic apparatus. There may also be the formation of bilobed nuclei, thereby indicating an early change in cellular metabolism (Fenech, 2000;Grisolia and Cordeiro, 2000;Bombail et al., 2001;Grisolia and Starling, 2001).
Within this context, the aim was to survey the use of pesticides in Guatambu, Santa Catarina State, Brazil, and, through micronucleus testing, determine the risk and toxicological impact of pesticides contaminating regional dams.

Sampling
The study was undertaken in a farming community which included members of the Lambedor River Watershed Association of Guatambu, in the west Santa Catarina State, southern Brazil, during April, 2009. Fish were collected in all the dams existing in agricultural rural properties (10 sampling areas), as shown in Figure 1.
Three fishes of similar size and weight (between 700 g and 900 g) were collected with cast nets in each dam. Blood samples of approximately 40 mL were taken from each by cardiac puncturing with sterile heparinized syringes and needles. Samples were transferred to labeled Eppendorf tubes containing EDTA, and then taken to the Toxicology Laboratory of the Community University of the Region of Chapecó for micronucleus testing.

The piscine micronucleus test
Two blood smears per individual were prepared on clean microscope slides, to then be air-dried at room temperature. Subsequently, they were first fixed with absolute methanol for 10 min and then air-dried for at least 24 h. After fixation, the slides were treated with 1N HCl for 11 min in a water-bath at 60°C. After washing with distilled water, they were then stained with a Schiff solution in the dark. In sequence, the slides were removed and left to dry at room temperature, for subsequent microscopic examination.
According to criteria already described by Ayllon and Garcia-Varquez (2000), only rounded non-refractive structures that had separated from the main nucleus were taken into account for micronuclei scoring. 3000 erythrocytes from each fish were counted (1000 erythrocytes on each slide) at 1000 x magnification under an oil-immersion objective, end examined for micronucleated cells.

Results
The responses from the questionnaires showed regular use of pesticides in 100% of sampling areas. As regards equipment washing, it was evident that 70%, of the water is normally returned to the farm itself, the remainder being discarded onto the soil. The responses also revealed that farmers were uncertain as to the correct destination of empty containers.
Cyprinus carpio, Hypostomus punctatus, Rhamdia quelen and Oreochromis niloticus figured among the animals collected in this study. Micronucleate erythrocytes ( Figure 2) were found Mat different frequencies among the captured fish species, as shown in Figure 3, the highest mean values being observed in Cyprinus carpio (15.33 in Weir 4, 15.00 and 14.00 in Weir 8. Figure 4 shows the means of micronuclei per dam, being especially perceptible Salvagni et al. 123  in the larger sized sampling areas of Dams 4 (12.44) and Dam 8 (13.78). However, they were close to the overall average in Dam 5 (12.33), where the species collected were Rhamdia quelen and Oreochromis niloticus.

Discussion
The significant contribution to the environmental impact in the region, through the paramount, current use and handling of pesticides in small and middle-sized farms in the community, was amply proven. Induction of micronuclei was previously reported for fish collected in dams or rivers (Udroiu, 2006) and in situ quantification of micronuclei in piscine erythrocytes has been demonstrated to be an adequate biomarker in the evaluation of aquatic ecosystems quality (Ayllón and Garcia-Vazquez, 2000;Çavas and Ergene-Gözükara, 2003;Ergene et al., 2007).
The water used both in the preparation of the pesticide solutions and in the washing of utensils, is a relevant factor and possible source of poisoning. As reported by respondents, in the past the whole washing procedure was carried out near water sources or streams. It is known that, depending on the characteristics of the soil where disposal occurs, the contamination of both surface and ground water is a possibility, thereby constituting a contributing factor to environmental contamination (Peres and Moreira, 2007).
Of late, the effects of pesticides on aquatic organisms, especially when using non-lethal doses, have been amply demonstrated Veiga et al., 2006;Glusczak et al., 2007;Marques et al. 2007;Moraes et al., 2007;Fonseca et al., 2008;Miron et al., 2008). The processes of transport and impact on non-target organisms are governed by the rates of degradation and bioavailability of pesticides in soil or water, bioavailability itself depending on local physico-chemical and climatic conditions. Several factors influence the effects of pesticides on fishes, among others, the fish-species studied, the class, dose and concentration of the pesticide, and exposure time Crestani et al., 2007;Moraes et al., 2007;, Cattaneo et al., 2008. Lemos et al. (2005), on examining differences in response between species by in situ analysis, noted the appropriateness of the species Tilapia rendalli as a bioindicator of genotoxicity in a lake environment. According to Grisolia (2002), on exposure to different concentrations of glyphosate in the commercial formulation Roundup®, there was an increase in the frequency of micronuclei in erythrocytes.
According to Grisolia (2005) and Udroiu (2006), the prevailing, extensive farming procedures, on inducing considerable impacts on the environment, are directly related to a reduction in biodiversity. The subsequent increase in mutation rate would lead to an increase in genetic load and a reduction in adaptive potential, with the consequential elimination of susceptible genotypes.
Apparently the action of any chemical genotoxic agent may give rise to an increase in micronucleus frequency. Consequently, based on the fact that spontaneous formation of micronuclei is normally low and nearly uniform among species (Siu et al., 2004), in environmental monitoring, micronucleus assaying has emerged as a simple, inexpensive and rapid method for detecting genotoxic effects.
Spontaneous formation of micronuclei in fish is normally very rare. In our study, however, significant frequencies were observed in the captured specimens from each dam, with micronucleus testing revealing the rate of micronucleated erythrocytes to be high, with a minimum of 6.21 and a maximum of 13.78 per 1,000 erythrocytes evaluated. When compared with the results obtained by Rodriguez-Cea et al. (2003), with an average 3 micronuclei per 1000 erythrocytes examined, the above indices can be considered high. The data also further corroborated other studies in Brazil which showed a high incidence of micronuclei and nuclear abnormalities in organisms exposed to various chemicals (Matsumoto et al., 2006;Ventura et al., 2008).
In conclusion, it was proven through in vivo piscine micronucleus testing, that water from the Lambedor water-shed can be considered genotoxic, with emphasis on the degree of genotoxicity from pollution in the area. This implies the possibility of pesticide effluents discharged into 124 Pesticide genotoxic impact on fish in southern Brazil  the river constituting a disease-hazard to local populations. It is recommended that the river water be analyzed chemically, with a mind to identifying additional classes of toxicants that may also be contributing to genotoxicity in this specific water-shed.