Experiments in L. latinasus adults simulating real-life exposure scenarios demonstrate the potential adverse effect on the physiology and individual parameters of Neotropical anurans. Particularly, as mentioned in the introduction in other groups of organisms, few laboratory studies have evaluated adverse biological effects by IMZT and less in realistic exposure routes in juvenile and adult anurans, some examples are Rana esculenta (Quaranta et al., 2009), Sclerophrys regularis (Ezemonye and Tongo, 2010), Rana temporaria (Leiva-Presa and Munro Jenssen, 2006; Brühl et al., 2013), Rana pipiens (Selcer and Verbanic, 2014; Abercrombie et al., 2020), Acris crepitans, Gastrophryne carolinensis and Hyla versicolor (Van Meter et al., 2014), Hyla gratiosa and Hyla cinerea (Van Meter et al., 2014; Van Meter et al., 2015), Lithobates sphenocephala and Anaxyrus fowleri (Van Meter et al., 2014; Glinski et al., 2018), Lithobates clamitans (Edge et al., 2011; Van Meter et al., 2018), Anaxyrus americanus (Van Meter et al., 2015; Abercrombie et al., 2020). However, studies in Neotropical anurans affected by environmental stressors using various biomarkers at different organization levels are scarce and using other species such as Boana pulchella (Brodeur et al., 2011; Brodeur et al., 2012) and Rhinella arenarum (Castillo et al., 1991; Castillo et al., 2005; Lajmanovich et al., 2018). At this point, this is the first laboratory work evaluating and integrating the effects of pesticidas at multiple biological scales in adults bioindicator species from agricultural environments and to be applied as early detection tools.
Evaluating the individual parameters, a decrease in L. latinasus HSI was observed due to exposure to IMZT in both scenarios. These results reinforce the idea of using these end points as biomarkers at the individual level in adult anurans that are in stressful situations in the environments they inhabit (Brodeur et al., 2012). In particular, these authors report that adults of B. pulchella that inhabit agroecosystems present a reduction in HSI and explain that this particular response is consistent with stress situations such as droughts, poor nutrition or even exposure to different pesticides. The results presented in this study provide new evidence on L. latinasus as a model species to evaluate this type of biomarker not only in disturbed environments but also in laboratory bioassays.
The histological effects of this study demonstrate for the first time that IMZT produces liver lesions such as HV and AS and affects hepatic MMC in L. latinasus. Specifically, IMZT induced alterations in the area occupied by the pigments melanin and hemosiderin in the adult liver of L. latinasus, corroborating the usefulness of using these pigments in MMC as histological biomarkers against the action of pesticides as previously reported for this species (Pérez-Iglesias et al., 2016). It is important to mention that other authors have highlighted the importance of determine the effects of environmental stressors in detoxification organs due to maintain the homeostasis in aquatic vertebrates (McDiarmid and Altig, 1999; Păunescu et al., 2010; Çakici, 2015). In this study, we observed an increase in the frequency of liver alterations (HV and ES) for both scenarios. According to Gürkan and Hayretdağ (2012), this situation may be an indication of irreversible damage that consequently ends in hepatocellular degeneration. Several studies carried out in anurans support this idea since they have shown that high concentrations of pesticides cause damage to liver structures that lead to an increase in the rate of hepatic cell apoptosis and it has even been pointed out that principles of carcinogenesis may be associated with processes such as hypervascularization (Păunescu et al., 2010; Zaya et al., 2011; Çakici, 2015; Pérez-Iglesias et al., 2016). In addition, the analysis of the histological biomarkers in adults showed alterations in the pigments of the hepatic MMC by exposure to IMZT in S1 and S2 after 96 h exposure. The MMC are widely used to describe effects of environmental stressors (De Oliveira et al., 2017), and particularly in Leptodactylidae adults species (Pérez-Iglesias et al., 2016; Fanali et al., 2018). These results are consistent with previous studies carried out in anurans that demonstrate the alteration produced in the hepatic pigmentary system (MMC) by environmental stressors (Păunescu et al., 2010; Zaya et al., 2011; Gürkan and Hayretdağ, 2012; Franco-Belussi et al., 2013; Çakici, 2015; Pérez-Iglesias et al., 2016; De Oliveira et al., 2017). In general, MMC and its associated pigments such as melanin and hemosiderin are important in catabolic processes as an immune response, as detoxifiers and as protectors against ROS (Fenoglio et al., 2005; Franco-Belussi et al., 2013). However, an increase in MMC may be due to increased oxidative damage that occurs in cell membranes, increased cell death processes by the accumulation of lipofuscin within MMC (Couillard and Hodson, 1996; Fenoglio et al., 2005; Franco-Belussi et al., 2013) or due to the ability of this cellular defense system to eliminate waste due to an increase in phagocytic activity (Agius and Roberts, 2003; Franco-Belussi et al., 2013). Moreover, the results obtained in this work show that at 96 h there is a significant decrease in the area of the pigments melanin and hemosiderin in the hepatic MMC. According to previous studies, the decrease in these pigments is related with a reduction in phagocytic and protective activity against oxidizing agents of MMC in the liver (Fenoglio et al., 2005; Pérez-Iglesias et al., 2016; Fanali et al., 2018; Franco-Belussi et al., 2020). Focusing on the response of the catabolic pigment hemosiderin in S1, this work again reports similar results to those previously described for us in this species after herbicide exposure (Pérez-Iglesias et al., 2016). The decrease of hemosiderin pigment, due to the herbicide exposure, may be related to more severe damages that involve alterations in the hepatic catabolic processes inhibiting the recycling of ferric compounds related to the erythrocatetic function (Agius and Roberts, 2003). This statement can be corroborated by the significant increase in the frequency of MNs in erythrocytes of circulating blood cells, which would indicate alterations in erythrocateresis making cell renewal impossible, a function that depends on hepatic MMC in anurans (Agius and Roberts, 2003; Fenoglio et al., 2005). In this sense, the physiology and well-being or fitness of anurans exposed to pesticides would be negatively affected by the inability of the liver to eliminate or detoxify xenobiotics and damaged cellular components of the body (Pérez-Iglesias et al., 2016). In this context, we consider that MMC are biomarkers of the health status of amphibians at the histological and cellular level in adults.
Analyzing the biochemical aspect, IMZT is also capable of inducing alterations in AChE activity in L. latinasus adults. In this sense, our results corroborate the same effects reported in fish by Pasha and Singh (2005), Moraes et al. (2011) and Pasha (2013), where IMZT alters the cholinergic system of local aquatic vertebrates. Oxidative stress refers to the imbalance due to excess ROS or oxidants over the cell's ability to carry out an effective antioxidant response. ROS are highly reactive species that can affect membrane lipids and nucleic acids, so regulating the delicate balance between ROS production and the activation of antioxidant cell defenses, such as CAT and GST enzymes, is essential to maintain cell viability (because they participate in the elimination of ROS) (Ferrari et al., 2008). In particular, the significant increase in CAT in adults of L. latinasus suggests that this enzyme acts as one of the main antioxidant defenses for these organisms against the action of a herbicide (Ferrari et al., 2011) with pro-oxidant characteristics such as IMZT in its Pivot® H formulation (Moraes et al., 2011). Added to this, the increase in CAT would be considered a beneficial response in a stress situation since it prevents oxidation in cells before the respiratory system takes an active role in the gas exchange of anti-oxidative defense (Ferrari et al., 2011). On the other hand, the decrease in GST activity demonstrates an enzymatic inhibition of the dominant antioxidant defense system of these organisms (Ferrari et al., 2011). The decrease in GST would lead to the deactivation of detoxifying systems that counteract the damaging effects of ROS. Specifically, GST activity is involved in phase II biotransformation of xenobiotics in conjunction with the CYP-450 dependent monooxygenase system and thus prevents oxidative damage after acute exposure (Attademo et al., 2007; Ferrari et al., 2008; Ferrari et al., 2011). In this case, the role of GST is inhibited at 48 h, which could lead to an increase in oxidative stress by IMZT exposure.
Finally, if we consider the response of histological biomarkers, we could think that since antioxidant enzyme systems do not respond (the case of CAT) or are inhibited (the case of GST) for that concentration and at that time, the antioxidant response would be in charge of MMC and its pigments, at least after 48 h of exposure. On the other hand, after 96 h, in S1 there is an increase in CAT that would indicate that this is the antioxidant system that prevails over the function of MMC since the concentration of the stressor is lower. However, the situation is different in S2 (higher concentration) since the antioxidant enzymes evaluated do not respond to the action of the herbicide, there is only a cytoprotective function and liver damage such as HV and AS are recorded. In summary, in high concentrations of IMZT the liver damage is severe. In this situation, the hepatic enzyme systems and MMC lose their physiological response capacities to maintain cell renewal processes and antioxidant and detoxifying functions (Brodkin et al., 2007).
It should also be note that IMZT produces cytogenetic damage in L. latinasus adults. It is important to highlight that genomic instability plays a fundamental role in the decrease of the state of health or well-being in the populations of aquatic vertebrates (Barni et al., 2007; Jha, 2008). When DNA damage occurs, either due to an increase in MNs frequencies, nuclear alterations or damage direct in the DNA, this situation can end with cell death and lead to severe pathophysiological situations that increase physiological stress (Barni et al., 2007; Çavaş and Könen, 2007; Jha, 2008). However, another scenario can occur if the damaged cells survive. In this case, damaged cells that are not repaired or that have undergone poor repair will also have immediate adverse effects on the well-being and health of these organisms, which may ultimately result in decreased survival and long-term population effects (Barni et al., 2007; Jha, 2008).
In recent years, Newman (2014) highlighted the importance of evaluating the correlation of biomarkers as a whole and not separately. This information helps to understand not only the susceptibility of organisms to environmental stressors but also their mode of action and toxicity, which can later be used as early warning signals in environments that are disturbed or contaminated by the presence of environmental stressors (Newman, 2014; Pérez-Iglesias et al., 2020). In this sense, the ecotoxicological information that evaluates the correlation of adverse effects at different levels of biological organization is scarce. In this work, the multivariate analysis using a battery of biomarkers related to the responses at different levels of biological organization allows generate a novel information not provided by the individual and separate analysis of each biomarker. Furthermore, these results show that the endpoints evaluated respond to the concept of biomarkers proposed by Walker et al. (2009) who affirms that the analyzed end points are useful biomarkers to use.
Added to this, the PCA analysis from this study clearly allowed us to observe the separation between those frogs exposed to IMZT and the control group. This situation shows that the adverse effects of IMZT induce alterations in the physiological responses evidenced when evaluating biomarkers at different levels of biological organization. The holistic approach of multivariate analysis allows the evaluation of multiple endpoints simultaneously and facilitates the general and particular understanding of the various effects of experimental exposure to an environmental stressor. This is an advantage for this type of analysis in contrast to the separate information provided by conventional analyzes. In conclusion, and in agreement with other authors (Van der Oost et al., 2003; Newman, 2014), we recommend the use of this type of approach for ecotoxicological studies since it allows us to discern the groups of anurans that were exposed to environmental stressors from those that were not exposed.
Focusing on the ecological importance of experimental design, a first acute scenario (S1) simulate surface runoff of pesticide occurs and contaminated water income to breeding sites (caves in the ground) of the frogs. It is important to note that the highest concentrations of IMZT in runoff water occurred near the site of herbicide application, when the amount of IMZT transported by runoff did not exceed 3% and storage of rainwater inside the soil (Dias Martini et al., 2013). A second scenario (S2) considered the worst-case scenario exposure where a direct application to the frog occurs to a concentration of the herbicide recommended by the manufacturer on the letterhead (e.g., the frog emerging from the cave and sprayed with the herbicide) (Van Meter et al., 2014). In these contexts, it is observed that after a realistic acute exposure of L. latinasus adults, there was a significant increase in cytogenetic damage in the proposed situation of surface runoff (S1) and the species presented more severe effects (biochemical damage, histological and individual) in the worst exposure situation considered, direct exposure or S2. We understand that the cytogenetic biomarker evaluated on this occasion (MNs) is presented as an early signal, responding in situations of low herbicide concentrations, before irreversible damage occurs at higher levels of organization. In addition to this, if the particular habit of life of this species is considered, we can understand that MNs in this case would be a valid tool to use in monitoring with L. latinasus given that a surface runoff event is the most likely exposure situation after runoff events due to heavy rains. At this point, IMZT could be a risk factor for L. latinasus populations. Finally, further research of this kind should be developed to understand the impact of pesticides on local populations of anurans.