Introduction

Currently, the main method to control diseases, insects, and weeds that infest crops is the use of pesticides (Jardim and Caldas 2012). These products can be hazardous to the ecosystem and health, and persist in the environment or even bioaccumulate (EPA 2017). According to Carneiro et al. 2015, Brazil has become one of the largest consumers of agrochemicals in the world, a fact that has been related to the contamination of water (Albuquerque et al. 2016), soil and food. The contamination of the environment by pesticide derivatives is related, mainly, to mistaken or excessive application, allowing these chemicals to reach surface water or ground water (Hayes et al. 2003).

The pesticides used on crops for the control of diseases, weeds, and insects present active and inert ingredients in their formulations. In many cases, the inserts present in a formulation may be even more harmful than the active ingredients. However, both can cause damage to non-target organisms continuously exposed to the product (Candioti et al. 2010; Vieira et al. 2014). In this sense, several studies have been carried out using commercial formulations of agrochemicals to test their effects on wild animals, such as amphibians (e.g., Silva et al. 2013; Svartz and Pérez-Coll 2013), since producers apply commercial formulations and not the isolated active or inert components.

In Brazil, agrochemicals containing the active ingredients atrazine, cypermethrin and tebuconazole in their formulation are widely used for the control of crop pests (e.g. Pignati et al. 2014). According IBAMA (2014), atrazine is the third most commercialized herbicide in Brazil (the first is glyphosate). Cypermethrin is the most commonly used insecticide and tebuconazole is a commercially available fungicide (IBAMA 2010, 2014). Atrazine is classified as Class III—moderately toxic, cypermethrin as Class II—highly toxic and Tebuconazole as Class IV—slightly toxic. However, regarding environmental classifications, atrazine, cypermethrin, and tebuconazole are classified as Class II, i.e., they are very dangerous to the environment (IBAMA 2010).

The herbicide atrazine is a photosystem II inhibitor and belongs to the group of triazines (Silva et al. 2007). It has been banned in the European Union since 2004 because it is considered toxic (Sass and Colangelo 2006). In Brazil, it is used for the control of weeds of the main crops of agronomic interest (Silva et al. 2007). Herbicides of the triazine group have a high environmental contamination potential due to their high leaching and runoff capacity, prolonged soil persistence, slow hydrolysis, low-water solubility and moderate organic matter absorption (Grizolia 2005). The literature estimates the half-life of atrazine at between 16 and 100 days, with an average of 60 days. In rivers, the half-life is 1.5–13.3 days (Chung and Gu 2003) and in lakes the half-life can be between 124 and 365 days (Schottler and Eisenreich 1997).

Cypermethrin is a synthetic pyrethroid insecticide with a wide spectrum delivered via foliar application (Andrei 2017). It is highly toxic to aquatic animals, as it causes the disruption of endocrine system in fish (Singh and Singh 2008). Cypermethrin is categorized as restricted use by the United States Environmental Protection Agency (USEPA) due to its high toxicity to fish (Saha and Kaviraj 2009).

The fungicide tebuconazole belongs to the chemical group of triazoles and is used in agriculture to control fungi (Andrei 2017). Tebuconazole formulated fungicides are persistent in the environment (Herrero-Hernández et al. 2011), and may cause adverse effects on aquatic organisms (Sancho et al. 2016). Tebuconazole’s adsorption coefficient is 2500 mL/g, the half-life is 403 days in the soil, and it has the potential to be transported in water associated with sediment because of its high affinity with organic matter. Thus, this fungicide presents high mobility in the environment (Ferracini et al. 2001).

The presence of agrochemicals in the ecosystem has been related to the decline of amphibian populations (Bishop et al. 1999; Allran and Karasov 2000; Boone et al. 2001; Sparling and Fellers 2009; Sparling et al. 2010). Amphibians are highly sensitive to different chemicals found in the aquatic ecosystem (Silvano and Segalla 2005). There have been several studies on atrazine (e.g., Ezemonye and Tongo 2009; Hayes et al. 2002; Brodeur et al. 2009; Svartz et al. 2012) and cypermethrin (e.g., Izaguirre et al. 2000; 2006a, b; Svartz and Pérez-Coll 2013; Svartz et al. 2016; Macagnan et al. 2017), but few on tebuconazole (e.g., Bernabò et al. 2016) and their effects on amphibians.

Physalaemus cuvieri is a very common species found throughout Brazil, and widely distributed in South America (Frost 2017). It is a nocturnal species of amphibian, and occurs in many habitats, including open grassland, flooded savannahs, and pastureland (Abraham et al. 2010). It is locally threatened in Argentina by the destruction of habitat for agriculture and wood extraction, land, and water pollution caused by agrochemical runoff (Abraham et al. 2010).

The objective of this study was to evaluate the acute and chronic toxicity of the pesticides atrazine, cypermethrin, and tebuconazole in P.cuvieri, a non-target organism to pesticides, which breeds in agricultural ecosystems in South America. Because, it is widely distributed and can be found in lands being used for agriculture, P. cuvieri is a good choice for evaluating the effects these pesticides have on non-target organisms—particularly amphibians.

Materials and methods

Pesticides

The pesticides studied (Table 1) were atrazine (commercial herbicide product Atrazine 500 SC), cypermethrin (commercial insecticide product Cypermethrin 250 EC) and tebuconazole (commercial fungicidal product 200 EC). All of these agrochemicals are registered with the Ministry of Agriculture and Livestock (Brasil 2017) to be used in the management of weeds, insects, and diseases, respectively.

Table 1 Pesticides used in the study
Full size table

To generate pesticide solutions, we dissolved commercial grade solution of pesticides in distilled water to create a stock solution of 500 mg/L as needed. The stock solution was added with a micropipette at the beginning of the experiment to each of the glass containers to obtain the desired concentrations for each pesticide. The pesticides were added in begin the test.

For acute exposure, concentrations of 0, 3, 12, 24, 35, 45, and 55 mg/L of atrazine, 0, 0.14, 0.4, 1.5, and 6 mg/L of cypermethrin, and 0, 0.4, 0.7, 1, 2, 3, 4, and 8 mg/L of tebuconazole were used in the tests. These concentrations were based on bibliographical references, taking into account the recommendations of the package insert for field applications. For cypermethrin, concentrations were used close to the LC50 recorded for P. biligonigerus (Izaguirre et al. 2006a). For tebuconazole, no studies were found in the literature with species similar to P. cuvieri. In this way, several concentrations were tested to find the LC50 based on the work of Toni et al. 2011. For atrazine, the reference of Moutinho (2013) was used for P. cuvieri.

For chronic exposure, the tested concentrations of atrazine were 0, 2.4, 0.48, and 0.24 mg/L, for cypermethrin 0, 0.14, 0.028, and 0.014 mg/L, and for tebuconazole 0, 0.11, 0.02, and 0.01 mg/L.

As indicated in the leaflet for each pesticide, the tested formulations were (a) atrazine 3–8 L/ha as the commercial product Atrazina 500 SC, according to the soil characteristics (for example, if the soil is sandy, the dose is lower; if it is clayey and rich in organic matter, the dose is higher) or depending on the crop to be applied and the weeds present in the crop; (b) cypermethrin, as the commercial product Cypermethrin 250 EC is indicated to be used from 40 to 600 μL/ha, depending on the crop and insect to be monitored, and (c) tebuconazole 200 EC, is applied from 300 mL/ha to 1 L/ha, depending on the disease and the crop.

Test species

The selected species was P. cuvieri, a species of frog in the Leptodactylidae family, popularly known as the dog frog, with a wide geographical distribution in Brazil, Argentina and Paraguay (Frost 2017).

Collection and maintenance of eggs and larvae

Eggs of P.cuvieri, with a maximum of 24 h of oviposition (until stage 14, according to Gosner’s classification (1960)) were collected in a pond in the municipality of Erechim/RS (27º30′50″S; 52º16′21″ W), from November 2014 to February 2015.

The eggs were transported to the Ecology and Conservation Laboratory of the Federal University of Fronteira Sul–Erechim Campus, and kept separately in aquariums of ~50 L with artesian well water and acclimatized at 25 °C until the hatching.

The larvae were fed every 24 h with complete feed in flakes for fish (Alcon Basic) ad libitum every other day. The acute test was performed with the animals fasted and not fed during the test. In the chronic test, feeding was maintained daily after the start of the experiment, with three flakes of balanced fish food per glass container.

The tadpoles that were not used in the experiments were released at the same site where eggs were collected.

Experimental design

Individuals from six breedings of P. cuvieri were used. Individuals were selected with a body mass between 0.0803 and 0.1186 g, and between stages 24 and 27 of development, according to Gosner’s classification (1960) in order to reduce the variation of the measured parameters. Only organisms considered healthy, with normal swimming movements, and with the ability to move through the aquarium without any deformity or apparent injury were used in the experiments.

Artesian well water was used for the tests and for the development of larvae. Water quality parameters were recorded for all aquaria and before the test solutions were applied. The well water used in the aquaria, controls and treatments had the following characteristics: 23 °C (±1 °C), pH 7.0 (±0.5), dissolved oxygen 5.0 (±1.0 mg/L), turbidity <5, conductivity 160 (±10) μS/cm, alkalinity 9.74 mg CaCO3/L, Ca 6.76 mg/L, Na 44.1 mg/L, Mg 1.35 mg/L, Fe 0.08 mg/L, Ni <0.001 mg/L.

Acute exposure

The laboratory ecotoxicological tests of acute exposure lasted for 96 h. All tests were performed in triplicate, including the control test. The temperature and oxygen of the water were evaluated every 24 h. Dead individuals were counted and withdrawn every 12 h until the end of the tests.

First, the tadpoles were arranged randomly in glass containers, in groups of 10, to be assigned later in the tests. For the classification of compounds according to the acute toxicity (LC50) for aquatic organisms, the present work used the classification of the Globally Harmonized System of Classification and Labeling of Chemicals (GHS Criteria, GHS 2011). Therefore, the compounds can be classified into three categories after exposure for 96 h: high toxicity (LC50 < 1 mg/L), moderate toxicity (LC50 between 1 and 10 mg/L) and low toxicity (LC50 > 10 mg/L).

The containers used in the tests had a capacity of 10 L, containing one tadpole per liter of water. The size of the vessel simulated a water pool ~10 cm deep, similar to the environment where the eggs were collected.

Chronic exposure

The static test, with daily exposure, was carried out for 7 days using larvae raised in the same manner as in the previous experiment. The laboratory conditions and the water evaluations were the same as described for the acute test. Every 24 h, the mortality of the individuals was assessed, as well as the effect of the chronic concentration on the swimming activity of surviving tadpoles.

To test the swimming activity of the tadpoles during the chronic test, every 24 h, movements were made in the water of the aquarium with the aid of a glass rod, three times in a circle. This movement stimulated the movement of tadpoles in the aquarium without causing stress. As a way of comparison, the standard of swimming activity established was: (3) swimming movements equal to control, that is, without changes in tadpole swimming, when compared to the control group, (2) swimming movements different from control, i.e., in circles or half the speed of the control group, (1) swimming movements only after a touch stimulus to some part of the body and (0) static, but alive. In the latter case, the tadpoles did not present any movement with the stimulus or touch of the rod. However, when they were removed from the aquarium with a sieve, if they moved they were then returned to the aquarium and classified as zero (0).

Data analysis

Median lethal concentration (LC50) values and their respective 95% confidence intervals were statistically estimated by Trimmed Spearman-Karber method. We used a one-way analysis of variance (ANOVA) to evaluated acute and chronic effects, and Tukey o Dunnet post hoc test when p < 0.05. For chronic test, we calculated the no observable-effect concentration (NOEC) and the lowest observable-effect concentration (LOEC) by analysis of variance with mean comparison made by Dunnett’s test, when p < 0.05. The maximum acceptable toxicant concentration (MATC) was calculated from NOEC and LOEC and expressed mathematically as the geometric mean of the NOEC and LOEC. This analysis was performed with the use of the Statistica software 8.0.

Ecological risk evaluation

The ecological risk was assessed using the hazard quotient (HQ) approach, which was calculated as EEC/LC50 of lethality for acute hazard quotient (AHQ) and EEC/NOEC for chronic risk (CHQ). The EEC is an estimated (or maximum) environmental contaminant concentration next to geographic range of the specie. The maximum level of atrazine reported in Brazil in literature was 0.075 mg/L (Moreira et al. 2012), the maximum level cypermethrin reported in Argentina in literature was 0.194 mg/L (Marino and Ronco 2005) and maximum level of tebuconazole was 0.026 mg/L (Toni et al. 2011). After the HQ was calculated, it was compared to the USEPA (United States Environmental Protection Agency) level of concern (LOC), where the risk presumption for aquatic animals is LOC = 0.5 (acute high risk) and LOC = 1 (chronic risk). If the HQ >1, harmful effects are likely due to the contaminant in question.

For comparison, we also use toxicity exposure ratio (TER; Damalas and Eleftherohorinos 2011). TER is the inverse of HQ and that is calculated by dividing the indicated toxic dose with the predicted environmental concentration or EEC (LC50/EEC and NOEC/EEC). When TER is below 100 for acute risk, is needed a detailed higher tier risk assessment, whereas TER <10 for chronic risk, assessment is required.

Results

The commercial formulations of cypermethrin and tebuconazole were more toxic than atrazine regarding the mortality of P. cuvieri tadpoles. The LC50 of the acute test was 0.24 mg/L for cypermethrin, 0.98 mg/L for tebuconazole and 19.69 mg/L for atrazine (Table 2).

Table 2 Mortality of Physalaemus cuvieri exposed to acute concentrations of atrazine, cypermethrin, and tebuconazole
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The chronic test showed gradual mortality during 7 days of exposure to the three pesticides evaluated. At the end of the test, survival was from 63 to 100% for atrazine and tebuconazole and between 23 and 53% for cypermethrin. In the chronic test, mortality started at 0.11 mg/L of the LC50 of tebuconazole in the first 24 h. For cypermethrin and atrazine, mortality started within 48 h of testing. Cypermethrin had the highest number of dead tadpoles at the end of the test (Table 3).

Table 3 Mortality of Physalaemus cuvieri tadpoles exposed to sublethal doses of atrazine, cypermethrin, and tebuconazole at different concentrations and exposure periods up to 7 days
Full size table

The different concentrations of atrazine (F2,18 = 0.16, p = 0.84), cypermethrin (F2,18 = 0.76, p = 0.48) and tebuconazole (F2,18 = 1.90, p = 0.17) did not significant for the mortality of the individuals analyzed. In other words, mortality occur in all concentrations of the three pesticides.

The swimming activity of tadpoles was affected by chronic (sublethal) doses of atrazine (F3,80 = 4.71, p = 0.004, significant for 0.24 and 0.48 mg/L, Dunnet test, p < 0.05) and cypermethrin (F3,83 = 50.81, p = 0.00, significant for all concentrations, Dunnet test, p < 0.05), but not of tebuconazole (F3,80 = 1, p = 0,41). For atrazine, the NOEC was swimming activity was 0.24 mg/L and LOEC was 0.48 mg/L, and MATC was 0.36 mg/L. For cypermetrhrin, the NOEC was 0.014 mg/L and LOEC was 0.028 mg/L, and MATC was 0.021 mg/L.

In the presence of atrazine in the water, the tadpoles swam in pattern 3, equal to the control from the first to the fourth day. On the fifth, sixth, and seventh days of the test, swimming activity was standard 2 (different from the control) at the three concentrations of atrazine tested. With exposure to cypermethrin, tadpoles demonstrated swimming activity 2, different and smaller than the control since the first day, at the three tested concentrations. On the third day of testing, it was observed that, at the concentration 0.14 mg/L, some tadpoles were upside down before stirring the aquarium. On the fourth day, at the highest dose (0.14 mg/L), the tadpoles displayed standard 1, while at doses of 0.028 and 0.014 mg/L remained at standard 2. From the fifth to the seventh day, standard 1 was seen with 0.14 mg/L, 1 with 0.028 mg/L, and 2 at 0.014 mg/L. The tadpoles that remained alive until the end of the test responded with slow movements after a single stimulus.

In contrast to the other pesticides tested, when the sublethal doses of tebuconazole were evaluated, swimming activity remained the same as the control from the first to the seventh day. The control tadpoles did not change throughout the test period for the three pesticides.

Results for acute risk quotient (AHQ) was 0.003 for tadpoles for P. cuvieri exposed to atrazine, 0.80 for tadpoles exposed to cypermethrin and 0.02 for tadpoles exposed to tebuconazole. Only the AHQ of cypermethrin is higher than the level of concern (LOC = 0.5). For chronic risk, CHQ was 0.31 for swimming activity for P. cuvieri exposed to atrazine and 13.85 for cypermethrin.

For TER, results for acute risk was 262.5 for tadpoles exposed to atrazine, 1.23 for cypermethrin and 37.69 for tebuconazole. For swimming activity, TER was 3.2 for atrazine and 0.072 for cypermethrin.

Discussion

This study demonstrated differences in the toxicity of three pesticides evaluated in relation to acute toxicity, chronic toxicity and swimming activity in P. cuvieri. The LC50 96 h of cypermethrin and tebuconazole showed higher toxicity than atrazine, according to the GHS categories (2011). Cypermethrin and tebuconazole showed high toxicity, whereas atrazine showed low toxicity.

Atrazine is being investigated as a potential endocrine disruptor in amphibians, and there is evidence that atrazine may adversely affect the reproductive capacity of anurans (Hayes et al. 2002; Tavera-Mendoza et al. 2002a, b) and, as a consequence, potentially contribute to population decline. In recent research on atrazine, using the concentrations of 1.5, 3.0, 6.0, 12.5, 19.0, and 25.0 mg/L for Rhinella schneideri and Physalaemus nattereri at 96 h of exposure, the LC50 was 22.18 and 33.71 mg/L, respectively (Pérez Iglesias 2015). The result for atrazine tested on P. cuvieri in the present study (19.69 mg/L) is similar to the values found for other amphibians (e.g., Brodeur et al. 2009; Ezemonye and Tongo 2009).

Amphibians show differences in sensitivity to cypermethrin (Biga and Blaustein 2013), which should also occur for other pesticides. In comparison with other species, P. cuvieri is more tolerant to cypermethrin (0.24 mg/L, this study). In Physalaemus biligonigerus, the LC50 was 0.12 mg/L (Izaguirre et al. 2000) and 0.11 mg/L in Rhinela arenarum (Izaguirre et al. 2006a). During acute exposure at concentrations of 10 μg/L of cypermethrin (0.01 mg/L), Rana arvalis presented 100% mortality, again demonstrating the high sensitivity of amphibians to this insecticide (Greulich and Pflugmacher 2003).

The fungicide tebuconazole has the ability to accumulate in amphibian tissues (Smalling et al. 2015). In fish, several papers detail the lethal concentration of tebuconazole, considered the closest group for comparison. For Cyprinus carpio, the LC50 was 2.37 mg/L (Toni et al. 2011), for Rhamdia quelen it was 5.3 mg/L (Kreutz et al. 2008) and for Danio rerio it was 19.6 mg/L (Sancho et al. 2010) of tebuconazole. These results show that tebuconazole is more toxic to P. cuvieri (0.98 mg/L) than to fish species tested in other studies.

In the chronic analysis, cypermethrin can be considered to be the most toxic, as it presented the highest mortality and caused a significant change in swimming activity. It has already been shown that this insecticide can causes high-death rates (Izaguirre et al. 2006a, b) and massive apoptosis in the central nervous system of anuran larvae (Izaguirre et al. 2000, 2001, 2006b).

Atrazine affected swimming activity, but not mortality, and tebuconazole did not affect any of these parameters. Thus, with chronic exposure, tebuconazole was the least toxic. These two agrochemicals are frequently detected in water (Gerónimo et al. 2014; Albuquerque et al. 2016), but at low concentrations. For atrazine, the maximum level reported in Brazil was 0.075 mg/L (Moreira et al. 2012) and for tebuconazole this was 0.026 mg/L (e.g., Toni et al. 2011). However, these concentrations do not refer to amphibian habitats and shows the need for further studies on the sublethal effects of concentrations found at development sites.

In the natural environment, the reduced mobility of tadpoles means less escape capacity, greater susceptibility to predation and reduced survival (Azevedo-Ramos et al. 1992). In addition, the physical structure is reduced due to a lack of nutrition, which can lead to death. Thus, changes in swimming due to the sublethal effects of agrochemicals can cause interruptions in the interactions between amphibian species and in the functioning of communities, because they affect trophic chains due to the absence of predators and prey (Howe et al. 1998; Peltzer et al. 2013). Thus, atrazine and cypermethrin are potential causes of reduced swimming activity in P. cuvieri, as has already been reported for R. schneiderie, P. nattereri (Pérez Iglesias 2015) and P. gracilis (Macagnan et al. 2017).

The calculated risk evaluation analysis indicated risks for acute and chronic toxicity (HQ) for tadpoles exposed to cypermethrin, and low risk for exposed to atrazine and tebuconazole in its natural habitat. But, analyzing the toxicity exposure ratio (TER), the situation is more complicated. Cypermethrin and Tebuconazole have risks for acute toxicity, and cypermethrin and atrazine for chronic toxicity, considered swimming activity. That is, cypermethrin appeared in all risk analyzes. The sublethal concentrations of cypermethrin tested in this study (0.014–0.14 mg/L) and the maximum acceptable toxicant concentration (0.021 mg/L) are close to those found in the environment (0.194 mg/L reported in Argentina; Marino and Ronco 2005), which leads to a real concern regarding the survival of P. cuvieri populations.

The cypermethrin is the one with the lower half-life among the tested pesticides. It is not only more toxic, but also in exerts its toxicity in a shorter period after application. This insecticide has several uses in agriculture, veterinary medicine and in public health (insects control) (Montanha and Pimpão 2012), because this, is applied all year round. It has been detected in aquatic environment at concentrations sufficient to cause toxicity to anuran larvae. Atrazine and tebuconazole can be less toxic, but can exert their effects all over the year after the application.

The tested agrochemicals have possibility of coming into contact with non-target species, in this case amphibians in the natural environment. Physalaemus cuvieri is a common and widely distributed pond-breeding frog that reproduces in altered environments and agroecosystems (Frost 2017; Silva et al. 2009). Because of this, it comes into constant contact with pesticides. In Brazil, the legislated maximum permitted level of atrazine in any water is 0.002 mg/L (Brasil 2005), and 0.18 mg/L for tebuconazole in water for human consumption (Brasil 2011); there is no set limit for cypermethrin. Only in the state of Rio Grande do Sul is there a limit for cypermethrin in water for human consumption, which is 0.3 mg/L (Brasil 2014). The chronic concentrations tested in this study are close to the maximum permitted levels for tebuconazole and cypermethrin.

Conclusions

In this study, in addition to providing acute toxicity data for three pesticides, it was possible to observe chronic effects in terms of swimming activity in P. cuvieri, related to exposure to atrazine and cypermethrin. Atrazine was weakly toxic and had an effect on the swimming activity of P. cuvieri. The use of this herbicide was banned in the European Union in 2004 (Bethsass and Colangelo 2006), but it is used in Brazil and other countries. The presence of atrazine may not affect the immediate survival of tadpoles, but may decrease fitness and reduce the ability to feed and escape from predators, causing populations to decrease over time. The risk evaluation analysis indicated risks for tadpoles exposed to three tested pesticides, specially cypermethrin. Cypermethrin was the most toxic pesticide tested, with high mortality at the environmental contaminant concentrations allowed by legislation in water for human consumption and found in rivers. This means that the environmental contaminant concentration of cypermethrin can has effects in a non-target vertebrate.

Of the three pesticides studied, we call special attention to the insecticide cypermethrin and the possibility of environmental contamination causing localized declines in amphibian populations.