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 . 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 ) . . 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 ; bcker 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 . 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 . 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 l 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 chapec for micronucleus testing . 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  magnification under an oil - immersion objective , end examined for micronucleated cells . 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 l 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 chapec for micronucleus testing . 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  magnification under an oil - immersion objective , end examined for micronucleated cells . 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 . the most widely used pesticides were roundup ( glyphosate ) , karate ( lambda cyhalothrin ) , herbimix ( atrasine and simazine ) and priori xtra ( azoxystrobin ) . 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 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 . 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 ( aylln and garcia - vazquez , 2000 ; avas and ergene - gzkara , 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 ( crestani et al . , 2006 ; veiga et al . , 2006 ; glusczak et al . , 2007 ; marques et al . 2007 ; 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 ( glusczak et al . , 2007 ; fonseca et al . ; 2008 , cattaneo et al . , 2008 ) . ( 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 , 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 . , 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 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 .
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 .