Toxicity of the neonicotinoid insecticides thiamethoxam and imidacloprid on tadpoles of four species of South American amphibians and effects of thiamethoxam on the metamorphosis of Rhinella arenarum

doi:10.21203/rs.3.rs-1980944/v1

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Toxicity of the neonicotinoid insecticides thiamethoxam and imidacloprid on tadpoles of four species of South American amphibians and effects of thiamethoxam on the metamorphosis of Rhinella arenarum

https://doi.org/10.21203/rs.3.rs-1980944/v1

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The present study examined the acute and chronic toxicity of the neonicotinoid insecticides imidacloprid and thiamethoxam in neotropical amphibian species from the Pampa region of Argentina. The median lethal concentration after 96h of exposure (96h-LC50) ranged between 11.28 and > 71.2 mg/L amongst all species and development stages tested; indicating that these pesticides are unlikely to cause acute toxicity in the wild. The subchronic toxicity was also low, with 21d-LC50 values ranging between 27.15 and > 71.2 mg/L. However, exposure of developing tadpoles of Rhinella arenarum to thiamethoxam from stage 27 until the completion of metamorphosis showed that treated animals present a significant reduction in metamorphic success together with a smaller size at metamorphosis and an asymmetry in the length of the left and right front arms. These effects, which are observed starting from the lowest concentration tested (9 mg/L), suggest an effect of thiamethoxam on the hypothalamic-pituitary-thyroid (HPT) axis. Given that the observed effects can clearly impair amphibian performance and survival in the wild, further studies employing the same experimental approach but looking at lower environmentally-relevant concentrations are needed.

Modern agricultural systems are highly dependent on pesticides and a leading cause of water pollution (Sanchez-Bayo, 2011; Mateo-Sagasta et al., 2018). An estimated 3 to 4 million tons of pesticides are applied annually to agricultural lands at the global level (Pimentel, 2009; Pretty and Bahrucha, 2015). A single family of insecticides, the neonicotinoids, account for more than 25% of the global insecticide market, and represent the most widely used insecticide class worldwide (Sparks, 2013; Bass et al., 2015). Imidacloprid (IMI), the first commercially available neonicotinoid, has been in use since the 1990s (Jeschke et al., 2011). Since then, additional active ingredients have also been developed. These are classified into three groups: N-nitroguanidines (IMI, thiamethoxam (TIA), clothianidin, dinotefuran), nitromethylenes (nitenpyram), and N-cyanoamidines (acetamiprid and thiacloprid) (Jeschke et al., 2011; Simon-Delso et al. 2015).

Neonicotinoid insecticides are broad-spectrum, systemic compounds that exhibit activity against sucking insects and several species of flies and moths (Kundoo et al., 2018). They are neurotoxic agents that act in the pest insect by binding to the postsynaptic nicotinic receptors causing neuronal hyper-excitation, paralysis and death (Yamamoto and Casida, 1999). Neonicotinoids are used in a wide variety of crops including soybean, wheat, rice, corn, sorghum, and cotton, as well as vegetables, fruit orchards, and in forestry (Hadlik et al., 2018a). They are employed as foliar sprays, soil applications, as well as seed treatment (Jeschke et al., 2011; Wood and Goulson, 2017; Simon-Delso et al., 2015). Seed treatment uses are possible due to the high solubility of neonicotinoids. As the seed germinates, the insecticide is incorporated into the plant and distributed systemically during growth (Miles et al., 2017).

However, the same water solubility characteristics that make neonicotinoids useful as systemic insecticides, also make them susceptible to leaching and runoff into ground and surface waters (Morissey et al., 2015; Anderson et al., 2015). The potential for water contamination is furthermore enhanced by the fact that neonicotinoids are relatively persistent and have a low binding affinity in soils (Morissey et al., 2015; Miles et al., 2017). As a consequence, a number of studies have now reported the presence of neonicotinoid insecticides natural waters worldwide (Main et al., 2014; Morrissey et al. 2015; Miles et al., 2017; Perez et al., 2017; Hadlik et al., 2018; Yi et al., 2019, Montiel-Leon et al., 2019; Milleman et al., 2020; Sandstrom et al., 2022; Silvanima et al., 2022). The European Union banned most outdoor uses of neonicotinoids in 2018 based on the widespread presence in water and the dangers posed to bees (Regulation 2018/783-785). Nevertheless, neonicotinoid insecticides remain widely used in many parts of the world, including the United States, Canada, and many South American countries.

A number of studies have examined the toxicity of neonicotinoid insecticides to non-target organisms. Risk to insects and aquatic invertebrates is of special concern, as reviewed by van der Sluijs et al. (2015), Wood and Goulson (2017) and Hladik et al. (2018a). Vertebrates are consistently less sensitive than insects because of structural differences at the level of their nicotinic receptors and their location within the cell (Tomizawa and Casida, 2005). Among vertebrates, birds feeding on seeds treated with neonicotinoids are especially at risk and may experience lethality or sublethal effects by ingesting only a few seeds (van der Sluijs et al., 2015; Poliserpi et al., 2021a). Fish, which are commonly used as surrogates for amphibians in ecological risk assessment (Ortiz-Santaliestra et al., 2018; Glaberman et al., 2019), are generally considered at low risk of direct mortality, as they present toxicity values in the range of the parts per million (Tisler et al., 2009; Gradila, 2013; Quadir et al., 2016; Wang et al., 2016; Xia et al 2016; Finnegan et al., 2017; Rosado-Berrios, 2018; Kocamaz and Oruc, 2018; Wu et al., 2018; Chang et al., 2020; Yang et al., 2021), much above the concentrations normally reported in the environment (Morrissey et al., 2014). Acute toxicity values previously reported in amphibians for neonicotinoids insecticides are given in Table 1. Most available values are for IMI and clothianidin, whereas very few studies have examined the lethality of other compounds.. Overall, based on the few data available, toxicity to amphibians appears to be similar to that observed in fish, median lethal concentration values (LC50) being in the range of the parts per million, although further data on a wider range of amphibian species and neonicotinoids are needed before a conclusion may be reached.

According to van der Sluijs et al. (2015), short-term toxicity is not a relevant predictor of neonicotinoid-related long term mortality in most vertebrate taxa, as sublethal effects dominate. Sublethal exposures of frogs and fish to TIA and IMI have been shown to affect red and white blood cell counts (Gavel et al., 2019; 2021; Paunescu et al., 2022), endocrine systems (Lifei et al., 2019; Gavel et al. 2019; 2021; Crayton et al., 2020), glycogenesis and blood glucose levels (Stoyanova et al., 2015; Vieira et al., 2018; Paunescu et al., 2022), oxidative stress and antioxidant response (Yan et al., 2015; Vieira et al., 2018), the escape response to predators (Lee-Jenkins y Robinson, 2018; Sweeney et al., 2020), as well as DNA damage and genotoxicity (Feng et al., 2004; Perez-Iglesias et al., 2014, Arcaute et al., 2014; Yan et al., 2015; Iturburu et al., 2017; Moe, 2017; Vieira et al., 2018). For its part, clothianidin was found to induce oxidative stress and alter the leukocyte profile in tadpoles of the frog Rana pipiens (Gavel et al., 2019;2021; Robinson et al. 2021); although it did not affect the susceptibility of larval leopard frogs to infection by trematode parasites (Robinson et al., 2019).

With respect to anuran metamorphosis, delays in completion have been reported for Xenopus laevis exposed to 20 and 100 mg/L of TIA and clothianidin (Jenkins et al., 2021), Lithobates pipiens tadpoles exposed to 8.5 mg/L and 250 ng/L of IMI (Moe et al., 2007), and Lithobates sylvaticus tadpoles exposed to 1 µg/L, 10 µg/L and 100 µg/L of IMI, although, in this case, the same concentrations of TIA had no effect on development time (Robinson et al., 2017). A lack of effect on development time was also reported for tadpoles of Acris blanchardi, Lithobates clamitans and Hyla chrysosceli exposed to 1 mg/L of IMI (Boone, 2018), Rana pipiens exposed to 2.5 and 250 ug/L of clothianidin and TIA (Robinson et al., 2019), and to 0,23, 1, 10 and 100 µg/L of clothianidin (Robinson et al., 2021). Finally, larvae of the Northwestern salamander (Ambystoma gracile) presented, in contrast, accelerated development, when exposed to 100 μg/L of IMI (Danis and Marlatt, 2021).

Considering that amphibians are the most threatened and rapidly declining vertebrate group (Stuart et al., 2004; Roelants et al., 2007), and that pesticides and other contaminants are recognized drivers of the problem (Bruhl et al., 2013; Campbell Grant et al., 2016; Brodeur and Vera Candioti, 2017), the goal of the present study was to obtain further information regarding the acute and chronic toxicity of IMI and TIA in neotropical amphibian species from the Pampa region of Argentina. Neonicotinoid insecticides are widely employed in this region where extensive row crop agriculture is dominant and diffuse contamination by pesticides, including neonicotinoids, has been repeatedly reported (Brodeur et al., 2017; Castro Berman et al., 2018; Alonso et al., 2018; Andrade et al., 2021; Perez et al., 2021). A number of physiological and ecological alterations have furthermore been reported in pampean amphibian species living in close association with crop fields (Babini et al., 2016; 2018; Bionda et al., 2011; 2013; 2018; Agostini et al., 2013; 2020; 2021; Suarez et al., 2016; 2020; Brodeur et al. 2011; 2012; 2022).

2.1. Studied species

Three anuran species were studied: the toads Rhinella arenarum, and Rhinella fernandezae and the frog Scinax granulatus. All three species exhibit stable populations, which are categorized as of “Least Concern” according to the UICN (2022). Rhinella arenarum is widely distributed in Argentina, and is also present in the south of Brazil, Uruguay and Bolivia (Cei 1980). It is a large terrestrial toad species, whose snout-vent length (SVL) ranges between 8 and 11 cm. For its part, Rhinella fernandezae is a smaller toad (SVL between 5 and 8 cm) with terrestrial fossorial habits, which is present in the north-west of Argentina and Uruguay, and in the south of Paraguay and Brazil (IUCN 2022). Finally, Scinax granulatus, is a small (SVL: 3 to 4 cm) tree frog that inhabits open swampy areas with forests or scattered trees, and which is distributed in eastern South America, from the south of Brazil until Buenos Aires Province in Argentina, including Uruguay (Agostini and Roesler, 2011).

2.2. Test organisms

Amphibian eggs were collected in the spring, when breeding occurred in permanent and temporary water bodies scattered within the 650 hectares of lands (3436024S, 5839056W) pertaining to the National Center for Agricultural Research (CNIA). Pesticide applications are not normally realized within the CNIA, which is located in a residential area in the suburbs of Buenos Aires. The eggs were collected within 24 h of being laid and immediately transferred to the laboratory where they were kept in a climate-controlled room at 20 ± 2 C and 16:8 h light:dark photoperiod. In the case of R. arenarum, some of the eggs were used to carry out acute bioassays with embryos. All the other eggs were kept until they hatched and the larvae reached stage 25 (S25) and 27 (S27) (Gosner 1960). Tadpoles were fed boiled chard ad libitum upon reaching S25 (Gosner 1960), which is when they begin to feed.

2.3. Culture water

Well water used in holding tanks, controls and treatments exhibited the following characteristics: pH 8,15; total dissolved solids 424 mg/L; hardness 170 mg CaCO3/L; chlorine < 0.1 mg/L; fluor 1.14 mg/L; vanadium < 0.05 mg/L; total iron < 0.01 mg/L; arsenic 0.016 mg/L; lead < 0.01 mg/L; calcium 18.0 mg/L; sulfates < 200 mg/L; nitrates 50 mg/L; nitrite < 0.02 mg/L; ammonium < 0.02 mg/L. All values fall within accepted guidelines for tadpole testing (ASTM, 1998). Water was kept in drums in the test room for 12 hours before being used, so that it was stable at the experimental temperature.

2.4. Test substances

Two commercial formulations of neonicotinoid insecticides were used: Actara® y Confidor®. Actara is a water dispersible granule containing 25% of TIA 3-(2-cloro-tiazol-5-ilmetil)-5-metil-[1,3,5] oxadiazinan-4-ilideno-N-nitroamina; CAS No. 153719-23-4. Confidor is a concentrated suspension containing 20% of IMI 1-(6-Chloro-3-pyridinyl-methyl)-N-nitro-2-imidazolidinimine (C.A.); CAS No. 138261-41-3. Both formulations contain proprietary surfactants, additives or emulsifiers of undisclosed molecular nature.

2.5. Stock solutions

In the case of TIA, a stock solution of 750 mg/L and pH 7 was prepared once a week and stored at 4 ^oC in darkness. Every stock solution served for two renewals of the test solutions. For IMI, a 200 mg/L stock solution was prepared before each renewal of the test solutions. The diluent used for these stock solutions was the culture water described above.

The concentration of TIA and IMI present in the stock solution was verified by ultraperformance liquid chromatography and mass spectrometry (UPLC-MS/MS). In the case of TIA, determinations were performed using a BEH C18 column (2.5 µm 2.1 x 150 mm). The mobile phases consisted of water (phase A) and methanol (phase B), both containing ammonium acetate at a concentration of 0.1 mM and 0.01% formic acid. The following gradient was employed: (90:10)-(90:10) 0-0.5 min, (90:10)-(50:50) 0.5-2 min, (50:50)-(0:100) 2–6 min, (0:100)-(0:100) 6–7 min, (0:100)-(90:10) 7–8 min, (90:10)-( 90:10) 8–10 min. The column was maintained at 45°C and the flow was set at 0.25 ml/min. For MS/MS analysis, the ion 211 m/z was used for quantification, whereas the ion 292 m/z was used for confirmación. Retention time was 4.2 min.

For IMI, determinations were performed using a BEH C18 column (1.7 µm, 100 x 2.1 mm). Flow was set at 0.2 mL/min. The mobile phases consisted of water (phase A) and methanol (phase B), both containing ammonium acetate at a concentration of 0.01% formic acid. The mobile phases consisted of water (phase A) and methanol (phase B), both containing ammonium acetate at a concentration of 0.1 mM and 0.01% formic acid. A gradient in which phase B varied in the following manner was employed: 0 min, 0%; 0.5 min, 0%; 3.5 min, 100%; 4.5 min, 100%; 5.0 min, 0%; and 6 min, 0%. The column was maintained at 35°C. For MS/MS analysis, the positive ionization mode was performed using a capillary voltage of 3.5 kV. The collision gas was Argon 99.995%, with a pressure of 4.04×10^− 3 mbar in the T-Wave cell. Conic gas and desolvation were optimized at 60 L/h and 600 L/h, respectively. The temperature of gas desolvation was 400°C and the temperature of the source was 120°C.

2.6. Experimental Protocols

2.6.1. Acute toxicity tests

For the toads R. arenarum and R. fernandezae, the experimental units consisted of two superposed 10 cm-diameter acid-washed glass petri dishes containing 70 mL of test solution. Four replicates, each with ten tadpoles, were performed for each treatment, including the control group. In the case of Scinax granulatus, because these tadpoles prefer to remain in a vertical position, fixed to the glass through their mouthpiece, the experimental chamber consisted of glass test tubes (1.7 cm of diameter and 12 cm of length) containing 15 mL of test solution. In all cases, test animals were maintained at a temperature of 20 ± 2 ^oC and a photoperiod of 16:8 h light:darkness throughout the exposure, which lasted 96 h. Test solutions were entirely replaced every 24 h, with the exception of the test using R. arenarum in S27, which was renewed every 48 h. Dead tadpoles were removed and survival was evaluated when renewing the solutions.

The following acute toxicity tests were performed:

- Toxicity of thiamethoxam and imidacloprid formulations in embryos of R. arenarum: For both formulations, the nominal concentrations tested corresponded to 20, 40, 60, 80, 100, 120, 140 and 160 mg/L. The eggs were exposed in the presence of their gelatinous protection, as would occur in nature. Embryos were in stage 1 according to Gosner (1960) when exposure began. In the case of TIA, exposures were conducted on two different opportunities: at 20 ^oC and 25 ^oC.
- Toxicity of thiamethoxam and imidacloprid formulations in tadpoles of R. arenarum in S25 and S27. The nominal concentrations tested corresponded to 20, 40, 60, 80, 100, 120, 140 and 160 mg/L for TIA and 0.1, 0.5, 1, 5, 10, 20, 40, 60, 80, 100, 120, 140 and160 mg/L for IMI. In the case of TIA, exposures of S25 R. arenarum tadpoles were conducted on two different opportunities: at 20 ^oC and 25 ^oC.
- Toxicity of a formulation of thiamethoxam in tadpoles of R. fernandezae tadpoles in S25. Nominal concentrations of TIA tested were 80, 100, 120, 140 y 160 mg/L.
- Toxicity of a formulation of imidacloprid in tadpoles of S. granulatus tadpoles in S25. Nominal concentrations of IMI tested were 1, 5, 10, 20 y 40 mg/L.

2.6.2. Subchronic toxicity tests

This test constitutes the prolongation for 21 d of the acute test with tadpoles of R. arenarum in S27. The test included four replicates of ten tadpoles in each treatment, including the controls. The experimental units consisted of two superposed 10 cm-diameter acid-washed glass petri dishes containing 70 mL of test solution. Test solutions were completely renewed every 48h and experiments were conducted at a temperature of 20 ± 2 ^oC and a photoperiod of 16:8 h light:darkness. A piece of boiled swiss chard of approximately 2 cm² was added to the test vessels after changing the solutions so as to provide tadpoles with food ad libitum. Nominal concentrations tested were 20, 40, 60, 80, 100, 120, 140 y 160 mg/L in the case of TIA and 0.5, 1, 5, 10, 20, 40 y 60 mg/L for IMI. R. arenarum tadpoles were in S27 according to Gosner (1960) when exposures began. Every 48 h, before the renewal of the test solutions, dead tadpoles were removed and survival was registered.

On the day of the experiment, before exposition began, the total body weight of all ten tadpoles belonging to each replicate was measured after removing the extra water by quickly posing the small red containing the group of tadpoles on a paper towel. Body weight was again measured in a similar manner at the end of the experiment. The variation in body weight was expressed in the form of a percentage of the initial body weight according to the following equation:

$$\frac{(Final mass of the group of tadpoles - Initial mass of the group tadpoles / n}{Initial mass of the group of tadpoles / n} x 100$$

where n represents the number of individual tadpoles present in the replicate when the measure was taken.

2.6.3 Effect of a formulation with thiamethoxam on the metamorphosis of Rhinella arenarum tadpoles

This test constitutes the prolongation until completion of metamorphosis of the 4 d and 21 d tests with tadpoles of R. arenarum in S27. After completing the first 21 d of exposure, and thereby obtaining subchronic toxicity values, the ambient temperature was gradually raised (1^oC per day) from 20 ^oC until 25 ^oC, so as to simulate a springtime increase in temperature and reach conditions more similar those present in nature when metamorphosis occurs. Exposure to TIA was continued as described above: four replicates per treatment in superposed 10 cm diameter petri dishes with ten individuals each one in 70 mL of test solutions. Test solutions were completely renewed every 48h and experiments were conducted at a temperature of 20 ± 2 ^oC and a photoperiod of 16:8 h light:darkness. A piece of boiled swiss chard of approximately 2 cm² was added to the test vessels after changing the solutions so as to provide tadpoles with food ad libitum. Nominal concentrations tested were 20, 40, 60, 80, 100, 120, 140 and 160 mg/L. Dead individuals were removed every 48h before renewing test solutions. Survival and development stages according to Gosner (1960) were also registered at this time.

Each new metamorph was anesthetized individually in culture water containing 100 mg/L of tricaine methanesulfonate, closely examined for malformations, weighed with a precision of 1 mg, and photos of the dorsal and ventral views were taken. Animals were sacrificed by cutting the neural cord behind the brain when fully anesthetized. Snout-vent length and the length of left and right arms and forearms were measured using the photos previously mentioned using the software ImageJ (National Institutes of Health).

2.7. Statistical Analysis

Pesticide concentrations inducing mortality in 50% of the individuals (LC50) were calculated by fitting a four parameters logistic regression equation to the survival data using the GraphPad Prism software version 5.03. In the subchronic tests, average body weight at 21 d and average gain in body mass were compared amongst treatments by using a one-way analysis of variance (ANOVA) followed by a Holm-Sidak test for multiple comparisons, after verifying that the data presented normality and equal variance.

The effect of TIA on metamorphosis was analyzed as previously described by Brodeur et al. (2009; 2013). A four-parameter logistic regression equation was fitted for every treatment to the cumulative numbers of animals reaching stage 39 (S39), stage 42 (S42) or completing metamorphosis (MET) in function of exposure duration using GraphPad Prism software version 3.02. With each curve fitted, the software calculates the time for 50 percent of the individuals to reach the stage being examined S39 (T50-S39), S42 (T50-S42) or completion of metamorphosis (T50-MET). After corroborating the normality and equal variance of the data, the values of T50-S39, T50-S42 and T50-MET, the snout-vent length (SVL) and body weight of the metamorphs, as well as, the length of their arms were compared amongst treatments using a one-way ANOVA followed by a Holm–Sidak test for multiple comparisons, All ANOVAs and multiple comparison tests were conducted using SigmaStat 3.11 statistical software (SPSS, Chicago, IL, USA). The criterion for significance was set at po0.05 in all cases

3.1. Pesticide concentrations in stock solutions

The analytic concentration of TIA determined in stock solutions was 416 ± 66 mg/L (N = 5, mean ± standard deviation (S.D.)), which corresponds to 55.5 % of the nominal value. Because this number is lower than 80 % of the nominal value, TIA concentrations corresponding to 55.5 % of nominal concentrations were used in data analysis and LC50 calculations as generally recommended in standardized test protocols (OECD, 1992; USEPA, 2012). Actual concentrations of 9, 18, 26.7, 35.6, 44.5, 53.4, 62.3 and 71.2 mg/L of TIA were therefore considered to have been employed instead of nominal concentrations of 20, 40, 60, 80, 100, 120, 140 y 160 mg/L. For their part, the analytic concentration of IMI determined in stock solutions was 206 ± 45 mg/L (N = 3 mean ± S.D.), which represents 103 % of the nominal value. Previously stated nominal concentrations of IMI were used for calculations and analysis, considering the high level of correspondence between nominal and analytic concentrations found in stock solutions.

3.2. Acute toxicity

According to the Globally Harmonized System of Classification and Labelling of Chemicals (United Nations, 2019), the acute toxicity of both commercial formulations corresponds to Category #3 of lowest short-term aquatic hazard (LC50 > 10 and ≤ 100 mg/L) for all amphibian species and developmental stages tested (Table 2). In R. arenarum, embryos and S25 tadpoles presented a similar sensitivity to TIA, whereas S27 tadpoles were less sensitive than earlier stages (Table 2). The variation of testing temperatures from 20 to 25 ^oC had little influence on the toxicity results obtained in R. arenarum. IMI was found to be more toxic to S25 and S27 R. arenarum tadpoles than TIA, whereas the toxicity of both products was similar in embryos (Table 2). S25 tadpoles of Scinax granulatus presented the largest sensitivity, with an LC50 of 11.28 mg/L for IMI. R. fernandezae appeared to be less sensitive to TIA than R. arenarum based on results obtained with S25 tadpoles (Table 2).

3.3. Subchronic toxicity

3.3.1. Thiamethoxam

More than 90 % of S27 tadpoles of R. arenarum survived 21 days of exposure to concentrations of TIA up to 71.2 mg/L. The subchronic 21d-LC50 of this species is therefore greater than 71.2 mg/L (Data not shown). There was a clear tendency for the natural increase in body weight of treated tadpoles to be inferior to that of controls in exposures of 35.6 mg/L of TIA and above, the effect being statistically significant starting from 53.4 mg/L (Fig. 1).

3.3.2. Imidacloprid

All S27 tadpoles of R. arenarum died within the first 24h of exposure when exposed to 40 and 60 mg/L of IMI. Survival was, nevertheless, greater than 95 % in tadpoles exposed for 21-days to concentrations of IMI of 20 mg/L or lower (Data not shown). Values of LC50 calculated for S27 R. arenarum tadpoles after 7, 14 and 21 days of exposure to IMI were, therefore, all equal at 27,15 mg/L (Data not shown). The natural increase in body weight was found to be greater than controls in tadpoles exposed for 21 days to concentrations of IMI of 1, 10 and 20 mg/L (Fig. 2).

3.4. Effect of a formulation with thiamethoxam on the metamorphosis of Rhinella arenarum tadpoles

3.4.1. Survival

Figure 3 illustrates the survival of tadpoles exposed to a formulation of TIA until completing metamorphosis. Less than 20% of the tadpoles exposed to the highest TIA concentration (71,2 mg/L) survived, most of the mortality occurring between 25 and 50 days of exposure (Fig. 3). In all the other experimental groups, a gradual decrease in survival was observed from day 75 onward, but this decrease was equivalent between controls and treatments until day 125. Only in the last 25 days of the experiment, did a tendency for a lower survival appeared in groups treated with 26.7 mg/L of TIA and up; their survival at end of the experiment being of approximately 20-30 %, whereas the control group and the groups treated with the two lowest concentrations of TIA had a survival of 40 - 50% at this time. However, the difference was only statistically significant at 53.4 mg/L (Fig. 3) and, as will be described below, most tadpoles had already completed metamorphosis when the mortality of treated animals began to diverge from that of the controls.

3.4.2. Timings of metamorphosis

Curves describing the progression of metamorphosis until S39 (complete back legs), S42 (four complete legs) and MET (complete tail resorption) are illustrated in Fig. 6, 7 and 8, respectively. The first individuals to reach S39 did so after 20 days of exposure (counting from S27) and by day 60, all animals that were to eventually reach S39 had done so in all treatment groups (Fig. 4). The proportion of individuals reaching S39 was slightly larger than controls in most treatment groups, with the exception of animals treated with 53,4 and 71,2 mg/L of TIA, which were significantly less successful at reaching S39 (Table 3). For both of these groups, the lower success at reaching S39 is linked to a lower survival between days 25 and 50 (i.e. when progression to S39 is in progress) compared to other groups. Finally, the time needed for half of the tadpoles to reach S39 (T50-S39), was approximately 10 days longer than in controls at 18, 35.6, 53.4, 62.3 mg/L, whereas no significant differences were observed at the other concentrations of TIA (Table 3). This delay in metamorphosis to S39 was due to the fact that the transformation process began later in these treatments, since the transformation rate (slope) was the same for all groups (Table 3) .

In contrast to observations made at S39, where most of the treatments were proceeding through metamorphosis in a similar manner, some with even more success than the control group; the proportion of individuals reaching S42 was significantly lower in all groups treated with TIA compared to the controls (Fig. 5). Indeed, whereas 50% of the controls reached S42, this value varied between 15 and 38% in the exposed groups, without a concentration-response relationship being observed (Table 4). The time for the individuals to reach S42 extended over a longer period than for S39, occurring between days 30 and 100 (Fig. 5). The lower success of treated individuals in reaching S42 is not explained by the mortality rate, as survival at T50-S42 is not significantly different from controls (Table 4). The effect of TIA on the time to reach S42 (T50-S42) was not as clear as its effect on the capacity to reach S42, as it varied with treatment concentrations: equivalent to controls at 9, 18 and 62.3 mg/L, greater than controls at 26.7 mg/L, and reduced at 35.6, 44.5 y 53.4 mg/L of TIA (Table 4). This variability is probably due to the fact that a reduced number of treated individuals reached S42 over a larger period of time, so the shape of the curve is highly variable. In spite of this, the transformation rate was only significantly accelerated at the lower concentration tested (Table 4).

Similar to what occurred at S42, the proportion of individuals that completed metamorphosis was significantly lower in all treatments compared to the control group (Fig. 6). Forty-four percent of the individuals in the control group completed metamorphosis, whereas this value ranged between 10 and 22% in groups exposed to the TIA (Table 5). Again, this observation is not caused by a larger rate of mortality, as survival at T50-MET is not significantly different from controls (Table 5). Passage from E42 to completion of metamorphosis took an average of 2 days (data not shown). As was observed at S42, tadpoles treated with 35.6, 44.5 and 53.4 mg/L needed less time than the control group to complete metamorphosis (Table 5). Again, the transformation rate was only significantly accelerated at the lower concentration tested (Table 5). Overall, these observations indicate that TIA causes a clear reduction of the number of individuals completing metamorphosis in R. arenarum, and that this trend is set during the transition from S39 and S42.

3.4.3. Weight and length at metamorphosis

Newly metamorphosed toads that had been exposed to thiamethoxam during their development had a significantly lower SVL and body weight at completion of metamorphosis compared to the control group (Fig. 7). Effects on body weight were observed starting from the lowest concentration of thiamethoxam tested (9 mg/L), whereas effects on SVL were seen from 18 mg/L. In both cases, the reduction in size was not concentration-dependent (Fig. 7).

3.4.4. Morphological alterations

Because of a variation in the standing position of control and treated animals (Fig. 8), the length of the arms of the newly metamorphosed toads was measured for the two lowest concentrations of thiamethoxam and expressed as a proportion of SVL length, in order to normalize for the size of the animal. Although no significant differences were found between the length of the left and right arms of the treated animals of a given group (Fig. 9), the left arm of the treated animals was lower than the left arm of the controls at both concentrations (Fig. 9). A tendency for the right arm to also be lower in treated animals vs controls was also noted, although this difference was not statistically significant (Fig. 9). The fact that the reduction in the left arm was consistently greater than the reduction in the right arm may explain the difference in standing position of the treated frogs.

To our knowledge, this is the second study to evaluate the acute toxicity of TIA to amphibian tadpoles. The previously available data was 2 mg/L and corresponded to a 48h-LC50 for the frog Lithobates sylvaticus (Pochini and Hoverman 2016). Here, 96h-LC50s ranging between 50 and 60 mg/L were obtained for both embryos and S25 tadpoles of R. arenarum; the two stages presenting a similar sensitivity to TIA. In the case of R. fernadezae, the 96h-LC50 was found to be superior, as it was greater than 71.2 mg/L (Table 2). In the subchronic exposure, S27 tadpoles of R. arenarum were found to survive up to 21 days to concentrations of up to 71.2 mg/L of TIA, although severe anorexia and reduced body weight were observed from 35.6 mg/L of TIA and above. Altogether, these results demonstrate the low acute toxicity of TIA to amphibian tadpoles; similar to the findings previously reported in fish (El Euony et al 2020, Yang et al 2021).

Regarding IMI, the present study provides the lowest values of 96h-LC50s reported to date in amphibians tadpoles, with 11.28 mg/L for S25 tadpoles of S. granulatus and 25–27 mg/L for S25 and S27 tadpoles of R. arenarum, respectively. Interestingly, as opposed to what was observed for TIA, where embryos and tadpoles presented a similar sensitivity, S25 and S27 tadpoles of R. arenarum were found to be more sensitive to IMI than embryos of the same species (Table 2). The acute IMI toxicity data reported here for amphibians ubicates their sensitivity in a range similar to that of the most sensitive fish (Desai and Parikh, 2014, Islam et al., 2019, Naiel et al., 2020).

Overall, the toxicity data obtained in this study indicates that, as previously concluded for fish, IMI and TIA do not present a risk of acute or subchronic mortality for amphibians in larval stages in nature, because the concentrations causing mortality (Tables 1 and 2) are well above the concentrations normally reported in aquatic environments. Indeed, these usually range between 0.004–9140 ng/L (mean 119.5 ng/L) for IMI and 0.002–3820 ng/L (mean 59.7 ng/L) for TIA, according to a recent meta-analysis of worldwide field data (Wang et al., 2022). Similarly, in the Pampa region, the highest concentrations of IMI measured so far were 430 and 560 ng/L (Caprile, 2019; Perez et al., 2017), while no values were found for aquatic concentrations of TIA in the Pampa.

Exposure to TIA during development significantly reduced the proportion of individuals completing metamorphosis in all treatments; although the effect was not dependent on test concentration (Fig. 6 and Table 5). In addition, exposure to TIA resulted in smaller metamorphs, lower body weights being observed at all concentrations tested, while SVL was significatively lower starting from 18 mg/L (Fig. 7). The time needed to complete metamorphosis was also significantly reduced at the largest concentrations evaluated (Table 5). By evaluating the progression through metamorphosis in terms of the succecutive progression into S39, S42 and then MET, it was possible to demonstrate that the lower metamorphic success of the treated animals is determined during the transition from S39 and S42. Indeed, up to S39, the treated group metamorphosed in a similar manner and even with a greater success than the control group (Fig. 4 and Table 5).

Clearly, this result suggests an effect of TIA on the hypothalamic-pituitary-thyroid (HPT) axis, given that, in anurans, metamorphic progression from S39 to S42 takes place under the strict control of the thyroid hormones (TH), thyroxine (T4) and triiodothyronine (T3), whereas evolution to S39 is independent of this hormonal system (Tata 2006; Brown and Cai, 2007; Thambirajah et al 2019). Although the present study was not designed to elucidate specific mechanisms and modes of action of TIA on the HPT, previous reports of neonicotinoids impacting the thyroid system of other vertebrates supports this view. Indeed, neonicotinoids have been shown to alter plasma levels of TSH, T4 and T3 in birds, rats and fish (Pandey and Mohanty 2015; Ibrahim et al., 2015; Mohanty et al., 2017; El Okle et al., 2016), and Wang et al. (2020) recently demonstrated that neonicotinoids cause morphological damage and an impairment of the transformation of T4 to T3 in the thyroid glands of lizards.

Although a number of studies had previously examined the impact of the neonicotinoids on amphibian tadpoles, this is the first time that the progression through the different stages is closely monitored and an impact on the thyroid system can be pinpointed. Most previous studies focused on the time to metamorphosis and a variety of results including retardement, acceleration or lack of effect have been reported (Moe et al., 2007; Robinson et al., 2017; 2019; 2021; Boone et al., 2018; Jenkins et al., 2021; Danis and Marlatt, 2021). Here, although a slight acceleration of T50-MET is observed at the largest exposure concentrations, it is shown that the main effects of TIA is a systematic decrease in the metamorphic success and smaller size at metamorphosis; two effects that can clearly impair amphibian performance and survival in the wild. Given that these effects were observed starting from the lowest concentration tested, which was 9 mg/L, further studies employing the same experimental approach but looking at lower and environmentally-relevant concentrations are needed.

The same can be said of the asymmetry found in the length of the left and right front arms of treated metamorphs. This effect, which was observed starting from the lowest concentration tested, can also impact escape performance and survival of the toads in the wild. Interestingly, this observation represents a typical example of fluctuating asymmetry (FA), which is defined as a long-term change in the body state of the organisms when extreme temperatures or contaminants cause instability during autogeny (Gondim et al., 2020). Fluctuating asymmetry is frequently reported in developing amphibians and has been proposed as a tool for assessing developmental stability of population and environmental quality of amphibian habitat (Zhelev et al., 2022).

In conclusion, the current study showed that, whereas neonicotinoids insecticides are unlikely to cause acute toxicity in neotropical amphibians in the field, further studies looking at the impacts of these pesticides on the metamorphic process should be performed at environmentally-relevant concentrations, considering that fluctuating asymmetry and reduced body size and metamorphic success were described at the lowest concentration examined.

FUNDING

This research was funded by the Instituto Nacional de Tecnologia Agropecuaria (Project number I054).

COMPETING INTEREST

The authors have no relevant financial or non-financial interests to disclose.

DATA AVAILABILITY

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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Table 1. Acute toxicity (CL50) of neonicotinoid insecticides reported for anuran species. A.I.= Active Ingredient, C.I. = Confidence Interval. G = Gosner Stage. NF = Nieuwkoop and Faber Stage

Species	Chemical Grade	Stage	Exposure duration	LC50 mg/L	Reference
Tiametoxam
Lithobates sylvaticus	Commercial Optigard Flex. 21.6 % A.I.	G28	48h	̴ 2	Pochini y Hoverman 2017

Dinotefuran

Silurana tropicalis	A.I. > 98 %	NF 49-50	96h	> 100	Saka y Tada 2020
Acetamiprid
Silurana tropicalis	A.I. > 98 %	NF 49-50	96h	> 100	Saka y Tada 2020
Imidacloprid
Silurana tropicalis	A.I. > 98 %	NF 49-50	96h	> 100	Saka y Tada 2020
Hypsiboas pulchellus	Commercial Glacoxan Imida 35 % A.I.	G 35-37	96h	52.62	Pérez-Iglesias et al 2014
Hypsiboas pulchellus	95.1 %	G 35-37	96h	84.91	Ruiz de Arcaute et al. 2014
Pelophylax nigromaculatus	A.I. > 95%	45 days-old	96h	129	Feng et al 2004
Rana limnocharis	A.I..> 95%	30 days-old	96h	82	Feng et al 2004

Clothianidin
Silurana tropicalis	A.I. > 98 %	NF 49-50	96h	> 100	Saka y Tada 2020
Hyla vesicolor	Commercial Arena 0.25% granules		48h	>327	Miles et al 2017
Lithobates clamitans	Commercial Arena 0.25% granules		48h	>327	Miles et al 2017
Lithobates pipiens	Commercial Arena 0.25% granules		48h	>327	Miles et al 2017

Table 2. Acute toxicity of thiamethoxam- and imidacloprid-based commercial formulations to developmental stages 1, 25 and 27 (according to Gosner, 1960) of Rhinella arenarum, Rhinella fernandezae and Scinax granulatus. Acute toxicity is expressed in terms of the concentrations (in mg/L) causing the death of 50 % of tested individuals after 96h of exposition (LC50 - 96h). Confidence intervals 95 % (C.I.) are indicated in parenthesis. Tests were performed at both 20 and 25 ^oC in the case of thiamethoxam in R. arenarum. Remaining tests were performed only at 20 ^oC.

	Thiamethoxam		Imidacloprid
Developmental Stage	*Rhinella arenarum*	*Rhinella fernandezae*	*Rhinella arenarum*	*Scinax granulatus*
Stage 1 Embryos	25°C 50,5 (C.I. 36.47 - 67.72) 20°C 62,92 (C.I. 62.03 - 63.81)		62,35 (C.I. not calculated)
Stage 25 Tadpoles	25°C 61,45 (C.I. 58.74 - 64.3) 20°C 53,8 (C.I. 52.19 - 55.44)	>71,2 mg/L	25,98 (C.I. not calculated)	11,28 (C.I. 7.67-16.60)
Stage 27 Tadpoles	> 71.2		27.15 (C.I. 9.68-44.62)

Table 3. Time required for 50% of tadpoles to reach Stage 39 (T50-S39) and survival at this time. Transformation rate and proportion of individuals reaching stage 39 (S39) in R. arenarum tadpoles exposed to different concentrations of thiamethoxam (TIA). * significantly different from controls (P < 0.05). Values in dark shaded cells are significantly greater than control. Values in light shaded cells are significantly lower than controls.

Concentration of TIA (mg/L)	T50 – S39 (days)	Survival at T50 – S39 (%)	Transformation rate (%/day)	Proportion of individuals reaching S39 (%)
Control	33.41 ± 0.75	90 ± 10	0.08 ± 0.01	80.18 ± 1.02
9	36.59 ± 0.44	90 ± 7.07	0.09 ± 0.007	*87.47 ± 0.68
18	*41.22 ± 0.45	92.5 ± 4.78	0.06 ± 0.003	*84.91 ± 0.59
26.7	34.89 ± 0.30	90 ± 7.07	0.13 ± 0.01	*88.80 ± 0.56
35.6	*42.99 ± 0.71	97.5 ± 2.5	0.04 ± 0.003	*91.82 ± 0.87
44.5l	37.87± 0.24	92.5 ± 4.78	0.08 ± 0.003	*88.93 ± 0.36
53.4	*40.38 ± 2.02	72.5 ± 17.96	0.07 ± 0.02	*59.84 ± 1.93
62.3	*43.10 ± 0.68	97.5 ± 2.5	0.07 ± 0.008	*89.71 ± 1.03
71.2	36.04 ± 3.99	*25 ± 25	0.12 ± 0.11	*24.60 ± 1.96

Table 4. Time required for 50% of tadpoles to reach Stage 39 (T50-S42) and survival at this time. Transformation rate and proportion of individuals reaching stage 42 (S42) in R. arenarum tadpoles exposed to different concentrations of thiamethoxam (TIA). * significantly different from controls (P < 0.05). Values in dark shaded cells are significantly greater than control. Values in light shaded cells are significantly lower than controls.

Concentration of TIA (mg/L)	T50 – S42 (days)	Survival at T50 – S42 (%)	Transformation rate (%/day)	Proportion of individuals reaching S42 (%)
Control	77.73 ± 2.63	80 ± 8.16	0.02 ± 0.003	50.61 ± 1.81
9	64.33 ± 0.95	77.5 ± 11.08	*0.16 ± 0.05	*24.43 ± 0.59
18	65.55 ± 3.40	87.5 ± 7.5	0.03 ± 0.006	*20.71 ± 0.91
26.7	*113.25 ± 11.2	67.5 ± 7.5	0.01 ± 0.001	*38.07 ± 5.41
35.6	*49.55 ± 4.5	97.5 ± 2.5	0.05 ± 0.02	*20.41 ± 1.29
44.5	*59.44 ± 3.03	92.5 ± 4.78	0.03 ± 0.007	*25.72 ± 1
53.4	*59.52 ± 4.9	55 ± 20.20	0.04 ± 0.02	*24.44 ± 1.73
62.3	83.33 ± 10.11	71.3 ± 5.78	0.01 ± 0.003	*14.05 ± 1.68

Table 5. Time required for 50% of tadpoles to complete metamorphosis (T50-MET) and survival at this time. Transformation rate and proportion of individuals completing metamorphosis (MET) in R. arenarum tadpoles exposed to different concentrations of thiamethoxam (TIA). * significantly different from controls (P < 0.05). Values in dark shaded cells are significantly greater than control. Values in light shaded cells are significantly lower than controls. ND = not determined

Concentration of TIA (mg/L)	T50 – MET (days)	Survival at T50 – MET (%)	Transformation rate (%/day)	Proportion of individuals reaching MET (%)
Control	76.01 ± 2.77	80 ± 8.16	0.03 ± 0.004	44.17 ± 1.71
9	66.95 ± 0.94	77.5 ± 11.08	*0.15 ± 0.04	*23.65 ± 0.56
18	70.6 ± 1.84	87.5 ± 7.5	0.04 ± 0.008	*15.68 ± 0.44
26.7	77.06 ± 5.46	85 ± 5	0.02 ± 0.005	*19.38 ± 1.37
35.6	*51.12 ± 3.98	95 ± 5	0.05 ± 0.02	*19.86 ± 1.19
44.5	*55.07 ± 3.42	92.5 ± 4.78	0.05 ± 0.01	*19.17 ± 0.96
53.4	*61.85 ± 5.82	55 ± 20.20	0.04 ± 0.02	*21.91 ± 1.79
62.3	ND	ND	0.01 ± 0.003	*14.72 ± 5.44
71.2	ND	ND	0.01 ± 0.009	*9.53 ± 4.7

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Toxicity of the neonicotinoid insecticides thiamethoxam and imidacloprid on tadpoles of four species of South American amphibians and effects of thiamethoxam on the metamorphosis of Rhinella arenarum

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Abstract

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1. Introduction

2. Materials And Methods

2.1. Studied species

2.2. Test organisms

2.3. Culture water

2.4. Test substances

2.5. Stock solutions

2.6. Experimental Protocols

2.6.1. Acute toxicity tests

2.6.2. Subchronic toxicity tests

2.6.3 Effect of a formulation with thiamethoxam on the metamorphosis of Rhinella arenarum tadpoles

2.7. Statistical Analysis

3. Results

4. Discussion

Declarations

References

Tables

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