Evidence of mutagenic and lethal effects of herbicides on Amazonian frogs

Amphibians are excellent bioindicators because they are sensitive to chemical pollution and can indicate ecosystem changes due to the presence of or exposure to chemical compounds. Here we report evidence of the impact of herbicides, including glyphosate, on amphibians in a locality in the central Brazilian Amazon and compare it with data from other biomes in Brazil. We observed malformations in three species of Leptodactylus and local extinctions of Scinax ruber and Rhinella marina from reproductive sites close to an area where herbicides had been applied. The observations in the Amazon are similar to reports from Brazil’s Atlantic forest on morphological anomalies and mortality in amphibians exposed to herbicides. We warn of the threat of expanding crops for the production of biofuels in the Amazon due to their cultivation being associated with agrochemicals, including glyphosate, posing a threat to the biodiversity of the Amazon biome.

PALAVRAS-CHAVE: agrotóxicos, anuros, bioindicadores, ecotóxicologia, floresta amazônica, impacto ambiental Ecotoxicological research on the harmful effects of agricultural chemicals on fauna is necessary given the expansion of crops associated with the use of these products (Schiesari and Grillitsch 2011). Agricultural landscapes can harbor many amphibian species (e.g., Ferrante et al. 2017), which tend to respond negatively to agricultural chemicals at different life stages, such as tadpoles (Orton and Routledge 2011) or post-metamorphics (Brühl et al. 2013). Their sensitivity to pesticides makes amphibians good bioindicator organisms (Gonçalves et al. 2017). The influence of agriculture on these animals is so strong that entire anuran assemblages within forest fragments are influenced by the type of agricultural crops around the fragments (Ferrante et al. 2017;). Here we report evidence of the effect of herbicides, including glyphosate, on anurans on a farm in the central Brazilian Amazon.
All observations were made at the experimental farm of Universidade Federal do Amazonas − UFAM (2°39'15.98"S, 60°3'16.09"W), in the municipality of Manaus, Amazonas state. The observations are part of a study on amphibians conducted in four deforested areas with a predominance of grasses. The areas were close to artificial ponds that had been installed for pisciculture ( Figure 1). The study consisted of eight three-night visual-encounter surveys (Crump and  According to the technicians responsible for the farm, six months prior to the observations, a 3.6-ha area of pasture close to our sampling sites and a nearby sheep corral were sprayed with 2 L of glyphosate (Roundup®) at a dosage of 200 mL per 20 L of water. The pasture was also sprayed with 120 mL of Disparo®, a herbicide containing 2,4-D and Picloram, at a dosage of 60 mL per 20 L of water. A 0.5-ha area of the pasture that was cultivated to produce silage was sprayed with 1 L of Select 240 EC®, a herbicide containing Cletodim and Alquilbenzene, at a dosage of 35 mL per 20 L of water. According to the instructions for the three products, Roundup® is "moderately toxic" and Select 240 EC® and Disparo® are "extremely toxic." All have classifications ranging from "potentially hazardous" to "Class III," that is, products that are dangerous to the environment.
On 1 March 2018, during the first survey after the herbicide application, we observed the absence of two anuran species and found individuals with malformations in areas where amphibians had been continuously monitored since July 2016. Five individuals with malformations were found in temporary puddles and a pond, respectively 2 and 250 m from the pasture area where herbicides had been applied (Figure 1, areas 1 and 3). The individuals with malformations belonged to three Leptodactylidae species: Leptodactylus fuscus (Figure 2), L. macrosternum and L. podicipinus (Figure 3). Three individuals of L. fuscus and one of L. macrosternum had brachydactyly (digit shortened) (Figure 2). One individual of L. podicipinus had microphtalmia (one small eye) (Figure 3). In one other area further away from the areas where herbicides were applied we found the same species, but without any anomalies (Figure 1, area 4).
The two absent species were Scinax ruber (Hylidae) and Rhinella marina (Bufonidae), both of which had been found to be abundant in the same area in all other surveys since 2016 (Figure 1). In all surveys before herbicide application, more than 150 individuals of R. marina (see  and approximately 100 individuals of S. ruber (L. Ferrante, personal observation) were recorded in areas of temporary puddles and artificial ponds (area 1,2 and 3 in Figure 1). The absence of these species in more than 250 m from the area of herbicide application (area 3 in Figure 1) is consistent with the results of a controlled study by Carvalho et al. (2019),  Although there are records in the northern hemisphere of malformations in amphibians caused by parasites or by the interaction of parasites with pesticides (Haas et al. 2017), the parasite that causes such anomalies (Ribeiroia ondatrae) is restricted to the northern hemisphere. This suggests that the malformations we observed were caused by the herbicides that were applied, as has been observed in various other studies of amphibian malformations (e.g., Ouellet et al. 1997;Lannoo 2008;Egea-Serrano et al. 2012;Wagner et al. 2013;Sparling et al. 2015;Koleska and Jablonski 2016;Mônico et al. 2019). Amphibians have high philopatry and low vagility (Wells 2007), making it possible to map the impact of herbicides on the landscape through the occurrence of individuals with malformations and the absence of species that were previously abundant. In addition, the absence of certain species even six months after herbicide application demonstrates the longterm persistence of effects on amphibian populations. Based on the shortest distance between the sprayed field and the sampled areas where no malformations or local extinctions were observed, we can conclude that the impact radius of these herbicides did not exceed 300 m.
Alternative hypotheses fail to explain the presence of individuals with morphological anomalies exclusively in proximity to the target area of the agrochemicals, while all individuals of the same species in more distant areas appeared healthy. The occurrence of malformations in three species that are abundant throughout the landscape rules out the possibility of morphological effects linked to population phenomena, as has been observed for anuran populations with genetic erosion due to inbreeding (Bessa-Silva et al. 2016).
Our observations are similar to the impacts observed in other biomes in Brazil, such as those in the Atlantic Forest discussed by . Forest fragments in the Atlantic Forest surrounded by large areas of crops such as sugarcane and coffee, with intensive use of glyphosate and other agrochemicals, harbor a low diversity of anuran generalist species that use both the agricultural area and the remaining forest (Ferrante et al. 2017). Among these species were Leptodactylus fuscus, Leptodactylus latrans and Scinax fuscovarius, which belong to the ruber group (see Ferrante et al. 2017: Table 1 and Figure 2b).
In central Amazonia vegetable producers in the lowland areas along the great rivers do not follow the recommendations provided in the inserts that are included in the packaging of agricultural chemicals, and dozens of agrochemicals are used at higher doses than suggested by their manufacturers (Schiesari et al. 2013). The areas close to rivers and streams that are preferred for vegetable production are also of vital importance to amphibian conservation, including many species that occur exclusively in these habitats and depend on the protection of the remaining natural vegetation (Moraes et al. 2016).
In 2019 alone, the Brazilian government allowed use of at least 474 new agrochemicals, many of which are banned in other countries . The doses used in Brazil are up to 5000 times greater than those recommended by European environmental agencies . The Amazon faces one of its most critical moments due to pressure from Brazilian agribusiness and its impact on the forest and biodiversity . Expansion of biofuel crops represents a new threat to the biome (Ferrante and Fearnside 2020), and these crops are already a major threat to biodiversity in biomes such as the Cerrado (central Brazilian savanna) due to use of agrochemicals (Schiezari and Grillitsch 2011;Schiezari et al. 2013). Glyphosate and other herbicides used in sugarcane and corn plantations in Brazil have been shown to be lethal to amphibians, regardless of species (Wagner et al. 2013;Daam et al. 2019). Our data suggest that the expansion of agricultural crops in the Amazon, as biofuels crops and the dependence of these crops on glyphosate and other herbicides could have devastating effects on biodiversity in the region.

ACKNOWLEDGMENTS
We thank UFAM's Experimental Farm for the logistical support for this study. LF thanks Conselho Nacional de Desenvolvimento Científico e Tecnológico − CNPq for the resources that made it possible to carry out this sampling, and Thainá Najar for help in the field. Thanks to Claudia Keller for constructive comments on this manuscript.