Genotoxic risk of herbicides to Anguilla anguilla L.

Herbicides are considered among the most hazardous contaminants of water bodies, since they easily reach these ecosystems through aerial spray drift, artificial drainage systems and surface or sub-surface runoff. The occurrence of these agrochemicals in the aquatic environment has been associated to deleterious effects in non-target organisms, namely fish. However, a considerable gap is evident regarding the scientific information on their genotoxic impact. Therefore, the present thesis was designed with the intention to evaluate the genotoxic risk to fish of the herbicide formulations Roundup® (glyphosate-based) and Garlon® (triclopyr-based), representing broadly used products worldwide to manage unwanted vegetation in agriculture and forestry. It was also planned to develop of a biologically base knowledge on DNA damage mechanisms. As ultimate goal, it was intended to contribute to mitigate the effects of agrochemicals in aquatic biota, namely fish, providing scientific data able to improve forestry and agriculture managing practices. The study was carried out adopting the European eel (Anguilla anguilla L.) as test organism and performing short-term exposures (1 and 3 days) to environmentally realistic concentrations of the mentioned commercial products. Two different genotoxic endpoints were adopted: comet and erythrocytic nuclear abnormalities (ENA) assays. The comet assay measures DNA stand breaks, a repairable type of damage, whereas the ENA assay identifies chromosomal lesions, signalizing a type of damage hardly repairable. The comet assay was also upgraded with an extra-step involving incubation with repair enzymes (FPG and EndoIII), in order to detect oxidative DNA damage. In what concerns to Roundup®, the involvement of the antioxidant system as indication of a pro-oxidant status was also assessed. Once the aforementioned formulations are presented as mixtures of chemicals, the genotoxic potential of their active ingredients individually was also assessed. In the case of Roundup®, the evaluation of the risk associated to the surfactant (polyethoxylated amine; POEA) and to the major environmental breakdown product of the active principle (aminomethylphosphonic acid; AMPA) was carried out as well. The results obtained showed the Roundup® ability to induce both DNA (in blood, gills and liver cells) and chromosomal damage (in blood cells). The investigation on the causative involvement of oxidative stress demonstrated that the type of DNA damage varies with tested concentrations and exposure duration. Thus, ROS-dependent processes gained preponderance as a mechanism of DNA damage with the increase of exposure length, which was corroborated by the antioxidant activation observed in gills as well as the net FPG-sensitive sites elevation detected in liver. Glyphosate and the surfactant POEA were also found to be genotoxic. Moreover, POEA induced the highest extent of DNA damage, when compared to glyphosate and the commercial mixture. Though both components showed to contribute to the overall genotoxicity of the herbicide formulation, the sum of their individual effects was never observed, pointing out an antagonistic interaction between them, indicating that POEA does not increase the risk associated to the active ingredient. These findings also emphasized the need to define regulatory thresholds for all the formulation components, recommending, in particular, the revision of the hazard classification of POEA (classified as “inert” until date). Since the ability of the main environmental metabolite of glyphosate AMPA in exert DNA and chromosomal damage was also confirmed, it was pointed out the silent problem that the products of environmental degradation of the active ingredients can constitute. In addition, the importance to include AMPA in risk assessment studies concerning the glyphosate-based herbicides was highlighted. The formulation Garlon® and its active ingredient triclopyr also showed a clear genotoxic potential. In addition, it was demonstrated the higher genotoxicity of the formulation, in comparison to the active ingredient. However, their ability in exert oxidative DNA damage could not be demonstrated. In what concerns to the evolution of the damage progression after removal of the contamination source, neither fish exposed to Roundup® nor Garlon® achieved a complete restoration of DNA integrity in 14 days. In relation to Roundup®, the use of the DNA lesion-specific repair enzymes allowed understanding the occurrence of a late oxidative DNA damage, indicating a progressive decay of cell antioxidant protection as well as the incapacity to repair this particular type of damage. The Garlon® post-exposure period revealed a tendency to decrease damage levels, although not enough to be regarded as an effective recovery. However, an efficient intervention of DNA repair enzymes specifically directed to oxidized purines became evident. Evaluating the performance of the adopted genotoxic endpoints, it was evident that the standard comet procedure, detecting only non-specific DNA damage, displayed some limitations when compared to the methodology that includes the incubation with the repair enzymes, since the latter reduced the possibility of false negative results. The two adopted endpoints (comet and ENA assays) demonstrated complementary aptitudes, being recommended their jointly application since it allows a more effective genotoxic risk assessment. Overall, the results obtained provided useful recommendations for policymaking, contributing to (re)formulate regulatory procedures for protecting the health of aquatic environment. In this direction, the data gathered in this work point to the importance of performing a genotoxic evaluation in order to actually determine the hazard posed by herbicides and their by-products. The magnitude of risk detected for both formulations strongly advise the adoption of restrictive measures in relation to their application in the proximity of watercourses. As mitigation measures, the development of formulations incorporating adjuvants selected on the basis of their lower toxicity emerged as a recommended path.