Functional responses of tadpoles exposed to different concentrations of glyphosate

doi:10.21203/rs.3.rs-5017955/v1

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Functional responses of tadpoles exposed to different concentrations of glyphosate

https://doi.org/10.21203/rs.3.rs-5017955/v1

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The main causes of biodiversity decline are associated with habitat loss and degradation. This process is mainly related to the expansion of agricultural frontiers (habitat loss) combined with the intense use of pesticides (habitat degradation). Even aquatic animals are affected by agriculture due to the run-off of pesticides from plantations to water bodies. In this study, we evaluated the effects of the pesticide glyphosate (commercial name Roundup) on the morphological development of tadpoles of Boana faber (Blacksmith Treefrog) and their functional traits. We analyzed 51 tadpoles in a laboratory experiment composed of four treatments with different concentrations of glyphosate. We measured 16 morphological characters of each tadpole, which were used to determine four functional traits: feeding, locomotion, gas exchange, and sensory perception. Our results indicate that glyphosate exposure directly affects the morphology and functional traits, with potential consequences on tadpole ecology. Morphological characteristics related to locomotion (caudal fin height and dorsal fin height) and sensory perception (internal distance between the eyes) were the attributes that presented greater variation after exposure. Greater exposure to glyphosate leads to smaller functional space occupied by locomotory attributes, which could indicate a reduction in morphological plasticity and changes in the ability to explore the habitat. At the same time, functional attributes related to space use and diet occupied the functional space in a manner that allowed greater differentiation between the treatments. As we detected huge changes in tadpole functional traits after a few days of exposure (seven days) and based on the current knowledge that many Brazilian ecosystems are vulnerable to runoff water from agricultural areas, we suggest that the ecological performance of free-ranging tadpoles has been suffering continuous effects of agricultural pollutants.

Agricultural activities

Boana faber

functional attributes

malformations

pesticides.

Habitat loss and degradation associated with human interference are the main causes of biodiversity decline (Tylianakis et al. 2005; Becker et al. 2007; Hayes et al. 2010; Potts et al. 2010). Much of this loss is related to habitat changes, such as vegetation suppression and pesticide pollution (Albuquerque et al. 2016; Zabel et al. 2019; Suárez et al. 2021). This process is even more intense in countries like Brazil, whose farmlands occupy huge extensions of land subjected to intensive pesticide use (Devine and Furlong 2007; Suárez et al. 2016).

The impacts of pesticides on biodiversity have been widely studied in different types of ecosystems and involving different taxa (Agostini et al. 2013, 2020; Bridi et al. 2017; Sanchez-Domene et al. 2018; Borges et al. 2019). For example, in ecosystems associated with water, such as wetlands, the dispersion of these products tends to be facilitated by water, impacting these ecosystems even more (Benachour and Seralini 2009; Roy et al. 2016). Thus, species from aquatic or floodable systems tend to be good models for comparative studies (Stuart et al. 2008; Bridi et al. 2017). Contact with this water can affect species far from the original site of pesticide application. The effects of pesticides on fauna can be investigated under different approaches since they can generate behavioral and physiological changes and malformations and affect the survival and reproduction of species (Bridges 1999; Stuart et al. 2008; Bridi et al. 2017).

Glyphosate is the most used pesticide in agriculture worldwide, a non-selective herbicide that acts against weeds (Benbrook 2018; Marques et al. 2021). In Brazil, glyphosate is available under the commercial name Roundup, which is a combination of the acid equivalent of N-(phosphonomethyl) glycine (Glyphosate), isopropylamine salt and n-(phosphonomethyl) glycine and inert ingredients (Hentges et al. in prep.). Roundup is used in most agricultural systems in Brazil, from small/familiar crops to large soybean, corn, and wheat plantations (Amarante Jr. et al. 2002; Marques et al. 2021). As agricultural land has gotten larger in recent decades, the negative effects of pesticides on wildlife and soil and water contamination have been growing faster (Tarazona et al. 2017; Tarone 2018). The Roundup formula is toxic and synergistic with glyphosate in animals (Mikó and Hettyey 2023). Several authors have reported that the toxic effects of glyphosate on amphibians include alterations at different stages of embryonic development and in organs such as the liver, epidermis, or brain (Lanctot et al. 2014; Rissoli et al. 2016; Bach et al. 2018). As most amphibian species spend their initial life on water, they are susceptible to exposure to the contaminants that flow from plantations in water bodies. Based on laboratory experiments, tadpoles exposed to glyphosate develop embryonic and physiologic disorders such as hypertrophy, epithelial hyperplasia, and chromatid rupture in their epidermis (Rissoli et al., 2016; Riaño et al., 2020). Some experiments detected alterations in tadpoles under glyphosate concentrations lower than the minimal limit according to the Brazilian legislation regarding human drinking (maximum value of 65µg/L; Resolution of the National Council for the Environment no. 357/2005).

Although the harmful effect of pesticides is widely known, many vertebrate groups, such as amphibians (Cunha et al. 2021), birds (Gloria and Tozetti 2021), and fish (Kumari 2020) can maintain relatively abundant and apparently stable populations in agricultural systems. However, the presence of animals in areas subjected to intense pesticide application does not guarantee these populations' resilience in the long term (Sánchez-Bayo et al. 2011). At the same time, exposure to chemical compounds, especially during early life stages, could generate several changes in organisms, including malformations and other phenotypic variations. One of the ways to evaluate the relationship between phenotype and pesticides is through morphological evaluation since morphological variation in organisms may indicate the effects of habitat changes (Bach et al. 2016; Herek et al. 2020).

Many studies have related environmental characteristics to individual morphology, but most were based on natural habitat variation (Whitman and Agrawal 2009, Schuck et al. 2021). In this approach, the effects of pesticide exposure could be evaluated by the presence of variation in morphology. This variation is also called phenotypic plasticity, which means the ability of an individual to express different features under different environmental conditions (Fordyce 2006; De Avila et al. 2019; Schuck et al. 2021; Travis 2023). Herein, we propose a new approach where pesticides are an additional environmental factor affecting morphological traits. This point of view is interesting because it allows understanding that the environment and its properties are associated with phenotypic variations between individuals, taxonomic groups, or populations. When the different phenotypes are evaluated within a perspective of “functional traits” or when their variation is measured at the level of “functional-trait space”, new analytical opportunities arise. In this case, we can detect not only the differences in the traits of the treatments but also create hypotheses based on how these groups of phenotypes are distributed along the functional space (e.g., the treatments are equally diverse but occupy different niches). The evaluation of traits allows broad approaches and even evidence of ecological interactions between groups (Jones et al. 2015). However, there is a lack of data evaluating the effect of anthropogenic actions on phenotypic plasticity, including functional trait space (Mammola et al. 2021).

The assessment of functional diversity is an innovative approach since functional attributes can group morphological, physiological, and behavioral elements that ultimately relate to how organisms interact with the environment (Dalmolin et al. 2019, 2020, 2022). Functional diversity, which can be studied through the selection of functional attributes, is considered one of the facets of diversity, which produces more refined results regarding the structural patterns of the different levels of ecological organization besides allowing the elucidation of the associated ecological processes that maintain biodiversity (Pillar et al. 2013; Gerhold et al. 2015).

Amphibians are good models to assess the effect of pesticides on biodiversity since most species have an aquatic life stage (Sansiñena et al. 2018; Borges et al. 2019), making them susceptible to pesticide exposure (Van Meter et al. 2015). This exposure, especially during metamorphosis, may favor the occurrence of morphological abnormalities linked to tadpole development (Lajmanovich et al. 2003; Brunelli et al. 2009; Jayawardena et al. 2010; Bach et al. 2016). Although a more common approach in terms of seeking relationships between the presence of pesticides and species diversity exists (Cunha et al. 2021), there is a huge field to be explored regarding the effect of pesticides on the growth, development, and individual survival rate (Boone and Bridges 2003).

The Blacksmith Treefrog Boana faber (Wied-Neuwied 1821) is a hylid whose characteristics make it a good model for studies on the relationship between pesticide exposure and changes in functional diversity. This species inhabits forested areas close to streams and wetlands and is also found in open and agricultural areas during the reproductive period, which ends up putting it in contact with chemical agents (Moutinho et al. 2020). Females deposit eggs in the water layer in nests built by males and, after the eggs hatch, the tadpoles disperse and finish their development in the water (Rossa-Feres et al. 2004). This means that, from the embryonic stage, these animals are exposed to water and potentially to the pesticides it may contain.

We hypothesize that the pesticide glyphosate influences the variation in the occupation of functional space by tadpoles of B. faber. We expect the effects caused by exposure to the pesticide to increase the phenotypic plasticity (occupation of functional spaces) of the affected tadpoles.

Obtaining the animals

The tadpoles were obtained from spawning collected at the Pró-Mata Center for Research and Conservation of Nature (-29.48592 S, -50.21919 W), in the municipality of São Francisco de Paula in southern Brazil. The local climate is classified as superhumid to humid, with a total annual rainfall of around 2,252 mm (Backes 1999; Maluf 2000). Average monthly temperatures remain below 15°C for seven months (April to October) (Maluf 2000). The collections were carried out under the SISBIO collection license nº 78625-1 and were approved by the Ethics Committee of the University of Vale do Rio dos Sinos (UNISINOS) (PPECEUA02.2021).

The collections were made in December 2021 with a plastic collection bag dipped into the lake to obtain the eggs together with the local water. After collection, the eggs were kept in plastic bags with local water and aeration and transported to the laboratory to be submitted to the experiment.

Glyphosate treatments

All experimental procedures were carried out in the Laboratory of Conservation Physiology of the Pontifical Catholic University of Rio Grande do Sul (PUCRS). Immediately after hatching, tadpoles were transferred to experimental aquariums with different concentrations of glyphosate. We used the commercial version of glyphosate, registered as Roundup. We will only refer to “glyphosate” to facilitate the description. Aquariums had a capacity of five liters, received constant aeration, and were maintained under a room temperature of 20 ± 1 ºC, under a controlled photoperiod of 12 hours with and 12 hours without light. We controlled water parameters (pH, ammonia, O₂, temperature, H₂O and Sodium Dodecyl Sulfate - SDS) and cleaning and maintenance procedures daily. We set four treatment groups (aquariums) in which glyphosate concentrations were: low (65 µg/L), medium (260 µg/L), high (520 µg/L) and control (no glyphosate present). The glyphosate concentration in each treatment was based on the federal Brazilian norms for water quality (see Brasil, 2005). For our low-concentration treatment, we used the standard level of glyphosate allowed in drinking water; for the medium-concentration treatment, we adopted the standard level of glyphosate in water for recreation activities (see Brasil 2005). For the high-concentration treatment, we used the average glyphosate concentrations found in rivers and agricultural runoff waters at the vicinities of the study site (Lima et al. 2023). The concentrations in the aquariums were nominal and obtained from a stock solution, quantified at the Laboratory of Environmental Analytical Chemistry of PUCRS using the methodology proposed by Marques et al. (2009), resulting in 1253 mg/L with no significant decay for seven days.

Tadpoles' exposure to glyphosate

Each treatment (control − 0 µg/L, low concentration − 65 µg/L, medium concentration − 260 µg/L, and high concentration − 520 µg/L) was formed by a set of five aquariums (replicates) containing four tadpoles (pseudoreplicates) each one. A total of 80 tadpoles were fed daily with fish food (Alcon Basic). Tadpoles were kept in experimental aquariums. This time length was defined based on the short half-life of glyphosate, which could vary from a few days to 91 days (Berman et al. 2018; Lutri et al. 2020). Based on this, we adopted the strategy to perform the experiment in a short time to avoid variances in glyphosate concentration within treatments. At the end of the experiment, all tadpoles were euthanized using liquid lidocaine (2%), according to the rules of the Animal Experimentation Control Council (CONCEA 2018) and stored in 70% ethanol for further processing. It is important to highlight that, while measuring euthanized tadpoles, we damage the body parts of some individuals (e.g., during handling with tweezers). These individuals were discarded, and we considered the following number of tadpoles: N = 14 (control), N = 9 (low concentration), N = 13 (medium concentration), and N = 15 (high concentration), totaling 51 tadpoles. Despite this variation, the average body mass of these animals was 0.102 ± 0.006 mg, representing a small difference between replicas.

Morphological analysis

We measured 11 morphological characters in each tadpole: body height (BH), body length (BL), body width (BW), caudal musculature width (CMW), dorsal fin height (DFH), ventral fin height (VFH), inner eye distance (IED), eye size (ES), mouth size (MS), spiracle length (SL) and spiracle width (SW) (adapted from Dalmolin et al. 2022). Besides the measurements, we also recorded the stage of development according to Gosner (1960). The measured functional characteristics and morphological characters are related to the main aspects of resource acquisition, resource use, and life habits (Table 1). We performed the measurements using a digital caliper (Stainless Hardened), with 0.01 mm accuracy, and a stereoscope (Zeiss, 10x zoom) for better visualization of the morphological characters.

Table 1

Functional characteristics and morphological characters measured in *B. faber* tadpoles. Legend: BH = body height; BL = body length; BW = body width; CMW = caudal musculature width; DFH = dorsal fin height; VFH = ventral fin height; IED = inner eye distance; ES = eye size; MS = mouth size; ND = nostril diameter; SL = spiracle length; SW = spiracle width. References: (1) Altig and Johnston 1989; (2) Altig and McDiarmid 1999; (3) Buskirk 2009; (4) Jordani et al., 2019; (5) Johnson et al., 2015; (6) Queiroz et al., 2015; (7) Gollman and Gollman 1999; (8) Altig 2006.
Functional traits	Variation	Ecological relevance	Ecological association of the attribute
BH	Continuous	Associated with tadpole biomass; determines the position of the species in the water column (1, 2, 3, 4).	Habitat use (e.g., foraging, shelter, use of the water column).
BL	Continuous	Associated with tadpole biomass; determines the position of the species in the water column (1, 2, 3, 4).	Habitat use (e.g., foraging, shelter, use of the water column).
BW	Continuous	Associated with tadpole biomass; determines the position of the species in the water column (1, 2, 3, 4).	Habitat use (e.g., foraging, shelter, use of the water column).
CMW	Continuous	Related to the species’ swimming capacity, microhabitat use, and, in the case of nektonic tadpoles, the maintenance of balance when at rest (1, 2, 4, 5, 6).	Locomotion.
DFH	Continuous	Related to the species’ swimming capacity, microhabitat use, and, in the case of nektonic tadpoles, the maintenance of balance when at rest (1, 2, 4, 5, 6).	Locomotion.
VFH	Continuous	Related to the species’ swimming capacity, microhabitat use, and, in the case of nektonic tadpoles, the maintenance of balance when at rest (1, 2, 4, 5, 6).	Locomotion.
IED	Continuous	Related to habitat use and the species’ ability to detect predators (1, 2, 4).	Sensory.
ES	Continuous	Related to habitat use and the species’ ability to detect predators (1, 2, 4).	Sensory.
MS	Continuous	Directly related to the ability to exploit food resources (1, 2).	Diet.
SL	Continuous	The spiracle, together with the operculum, is associated with the regulation and control of respiratory currents and feeding (7, 8).	Gas exchange.
SW	Continuous	The spiracle, together with the operculum, is associated with the regulation and control of respiratory currents and feeding (7, 8).	Gas exchange .

Table 2

Mean values (in mm) of the measured morphological characters per treatment of *Boana faber*. Values in parentheses represent the standard deviation.
Functional traits	G1 (N = 14)	G2 (N = 9)	G3 (N = 13)	G4 (N = 15)
Body height	2.59 (0.40)	2.70 (0.98)	2.51 (0.34)	2.56 (0.31)
Body length	6.05 (0.62)	6.33 (0.72)	6.22 (0.55)	6.25 (0.50)
Body width	3.59 (0.36)	3.85 (0.37)	3.67 (0.43)	3.81 (0.25)
Caudal musculature width	1.31 (0.29)	1.49 (0.18)	1.26 (0.23)	1.26 (0.13)
Dorsal fin height	1.11 (0.24)	1.14 (0.23)	1.08 (0.17)	1.16 (0.18)
Ventral fin height	0.76 (0.09)	0.79 (0.24)	0.81 (0.14)	0.92 (0.16)
Inner eye distance	1.63 (0.36)	1.81 (0.33)	1.87 (0.20)	1.95 (0.22)
Eye size	0.79 (0.11)	0.68 (0.05)	0.63 (0.06)	0.74 (0.08)
Mouth size	1.58 (0.21)	1.73 (0.16)	1.63 (0.23)	1.74 (0.19)
Spiracle length	1.33 (0.26)	1.58 (0.34)	1.34 (0.19)	1.34 (0.20)
Spiracle width	0.59 (0.13)	0.62 (0.13)	0.66 (0.08)	0.61 (0.09)

Statistical analyses

To assess changes in functional space occupied by tadpoles along glyphosate exposure gradients, we used the method proposed by Blonder et al. (2014), which consists of measuring the hypervolumetric niche based on multi-attribute approaches. We calculated the maximum estimate of the functional hypervolume occupied by the control group and each treatment group exposed to glyphosate using the “expectation_maximal” function of the hypervolume package (Blonder et al. 2014). This function generates an expectation of the hypervolume corresponding to a polygon that minimally involves the observed data (points). Furthermore, the sampling algorithm generates random points within a hyperbox that encloses the points and then sequentially tests whether each is inside or outside the convex polygon based on a dot product test.

Before measuring the maximum expectation of the functional spaces, we performed an analysis to assess the contribution of each functional attribute to the hypervolume using the “Hypervolume variable importance” function of the hypervolume package (Blonder et al. 2014) using R version 3.2.2 (R Core Team 2023). To quantify the amplitude and the overlapping of the occupation of each group of tadpoles in the functional space, we used the “hypervolume_overlap_statistics” function, which is calculated between pairs of hypervolumes. This function uses the Sørensen index to quantify the similarity in the functional space occupied by two hypervolumes (therefore, the overlapping), as well as the unique fraction of each functional space (i.e., the portions in which there were no overlaps). For this, the unique fraction of a hypervolume consists of dividing the unique fraction of the unique volume by the total hypervolume occupied by the species (Blonder et al. 2014).

Our results revealed that glyphosate exposure leads to morphological variations in tadpoles of Boana faber. A set of measured attributes contributed to morphological variation between treatments, indicating that glyphosate acts over a set of morphological characteristics, affecting the shape of tadpoles. Our results indicate that the morphological characteristics dorsal and ventral fin height, which are related to locomotion, had the greatest importance for the formation of functional hypervolume in both the control group and the treatments (Control = 30.1% and 17.3%, respectively; Low concentration = 20.5% and 26.3%; Medium concentration = 22.3% and 16.8%; High concentration = 20.5% and 23.3%) (Fig. 1). Furthermore, the caudal musculature width, also related to locomotion, was important for the formation of functional hypervolume in the control group (10.4%) and in the group exposed to medium concentration of the pesticide (10.2%). The sensorial attribute that most contributed to the formation of hypervolume was the inner eye distance (Control = 10.1%; Low concentration = 11%; Medium concentration = 10.6; High concentration = 8.5%) (Fig. 1). Meanwhile, the attributes associated with habitat use (i.e., body height, length, and width) were more important for the formation of hypervolume in the high (23.5%) and medium (17.2%) concentration groups than in the control (12.4%) and low concentration groups (12.6%) (Fig. 1). Among the two attributes related to gas exchange, spiracle length had a greater contribution to the hypervolume of all four groups compared to spiracle width (Control = 7.6% and 1.6%, respectively; Low concentration = 10.2% and 1.4%; Medium concentration = 8.1% and 1.2%; High concentration = 6.9% and 1.3%) (Fig. 1).

Overlap and single fraction analyzes revealed differences in the manner that tadpoles from each treatment occupied the functional space. The Sørensen index indicated that the control and low concentration groups had the lowest overlap in the functional space related to locomotion attributes (37%), while the greatest overlap was between the medium and high concentration groups (70%) (Fig. 2.1a). Considering the functional space, the control group showed the greatest amplitude of exclusive space (around 70% compared to the three treatments) (Fig. 2.1b), while tadpoles exposed to medium (10%) and high (19%) concentrations occupied lower exclusive portions of the whole hypervolume (Fig. 2.1b and 3), which reinforces the direct relationship between exposure to the pesticide and changes in the functional space occupied by individuals. Regarding the sensory attributes, niche overlap was lower among the control group and medium (37%) and high (46%) concentration groups. At the same time, the greatest overlap was between the medium and low (72%) as well between control and low concentration groups (65%) (Fig. 2.2a). The control group occupied the most distinct position among treatments in the functional space associated with sensory attributes (Fig. 4B and 2.2b), with high percentages exclusive to hypervolume compared to most other groups. The amplitude in the occupation of functional spaces determined by attributes linked to gas exchange showed that tadpoles with medium and high glyphosate exposure had a very similar position (Fig. 4A), where they had the greatest niche overlap in the space formed by gas exchange attributes (75%) (Fig. 2.3a). The greatest overlap occurred between the medium and high concentration groups (75%) and the smallest between the low and medium concentration groups (57%) and between the control and high concentration groups (58%) (Fig. 2.3a). Attributes related to space use and diet occupied the functional space in a manner that allowed greater differentiation between treatments, where the high concentration group presented the greatest amplitude of occupation of this functional space, while the other groups occupied more similar portions of the space (Fig. 5). The high concentration group occupied the smallest exclusive spaces compared to all other groups, as well as the medium concentration group compared to the control group (Fig. 2.3b). The greatest overlap in the functional space associated with the feeding attributes was between the control and low concentration groups (60%), while the low and high concentration groups had the smallest hypervolume overlap (33%) (Fig. 2.4a). Furthermore, the medium (71%) and high concentration (71%) groups occupied a much larger exclusive portion of the functional space than the control group (25% and 24%, respectively) (Fig. 2.4b)

Our results demonstrate that glyphosate exposure influences the development of Boana faber tadpoles. Considering that even a short exposure time (seven days) was enough to affect tadpole morphology, we suggest that wild populations have been suffering dramatic effects of pesticides, especially because many Brazilian ecosystems are vulnerable to runoff water from agricultural areas (Lima et al. 2023). It is important to highlight that our results were not limited to morphometric comparisons between treatments. Here, we offered a new approach where the spatial range of functional traits was used as a proxy for the glyphosate effects on the ecological performance of tadpoles. Apparently, higher levels of glyphosate can promote a wide variation in tadpole body shape. This hypothesis is based on the fact that the tadpoles exposed to the highest concentration of glyphosate (520 µg/L) presented the greatest amplitude of functional space for attributes of space use (i.e., body height, body length and body width) and feeding (i.e., mouth size). Furthermore, the group exposed to high concentration of glyphosate occupied larger exclusive spaces in the functional space. This suggests that the exposition on higher concentration of glyphosate favored the development of unique morphological characteristics. These results may suggest that at a concentration of 520 µg/L of glyphosate (similar to that found in rivers and agricultural runoff waters at the vicinities of the study site; Lima et al. 2023), tadpoles present a wide variation in body shape and mouth size, which can affect their ability of foraging and space exploration. At the same time, under the highest glyphosate concentration, tadpoles tend to occupy smaller ranges of the functional space related to locomotion attributes (i.e., width of the caudal muscles, height of the dorsal fin, height of the ventral fin). The smaller exclusive spaces of higher concentration group, compared to the control group, indicating that high concentrations of glyphosate lead to a reduction in the functional diversity of locomotion attributes in tadpoles. Furthermore, the high overlap between the medium and high concentration groups related to locomotion attributes compared to the control and low concentration groups may indicate that increasing glyphosate concentration leads to morphological homogenization in locomotion attributes. Thus, we can argue that glyphosate tends to limit the phenotypic variations on locomotory structures.

Our results are important since they point out to the effects of the pesticide on some morphological attributes and establish a link to the ecological functions of traits. Furthermore, we have seen that the effects of glyphosate vary according to their concentration in water. In our experiment, the maximum concentration of glyphosate used (520 µg/L) was already found in water bodies in Brazil (Lima et al. 2023). However, this value may be higher in environments close to or connected with plantations. As a consequence, some populations could be exposed to even higher concentrations of glyphosate (Boone and Semlitsch 2002; Mineau and Whiteside 2013). It is important to emphasize that other factors can also interfere with the effects caused by this pesticide, such as the pH of the local water, which can increase the toxicity and half-life of glyphosate, in addition to mixing with other pesticides (Edge et al. 2014).

Regarding diet/feeding attributes (i.e., body height, body length, body width and mouth size), individuals exposed to the highest concentration of pesticide occupied most of the functional space, reinforcing the potential of glyphosate in affecting anuran development, morphology and, consequently, the functional traits. Previous studies reported malformations in the oral region in tadpoles promoted by glyphosate exposure (Bach et al. 2016; Herek et al. 2020). In fact, mouth structures of Boana faber tadpoles, such as denticulate and lip papillae, show high sensitivity to glyphosate (Pavan et al. 2021). As a consequence, it is plausible to suppose that morphological abnormalities prompted by pesticides may start chain effects related to food acquisition and, at the same time, promote an expansion of the functional space occupied by individuals since they show a wide variation of morphological measurements. For example, the abnormalities reported by Bach et al. (2016) and Herek et al. (2020) may adversely affect food consumption since morphological changes in the oral apparatus can limit the type and amount of food consumed, causing deleterious consequences for the growth and development of some of these individuals (Mathew et al. 2010; Venesky et al. 2010). Glyphosate promoted a reduction of occupied functional space of some attributes related to locomotion (i.e., caudal muscle width, dorsal fin height, ventral fin height) suggesting a flatting process on functional space occupation. From an ecological perspective, this may indicate that the greater the exposure to the pesticide, the lower the phenotypic plasticity related to locomotory structures and, probably, the lower the locomotion capacity. This result is worrying in terms of survival since locomotion is fundamental in the process of habitat exploration, dispersal, and escape from predators (Van Buskirk and McCollum 2000). Studies have investigated that alterations in attributes related to tadpole locomotion reduce locomotion speed and distance covered, besides promoting events of spasmodic contractions, tremors, or seizures (Azevedo-Ramos et al. 1992; Bach et al. 2016; Herek et al. 2020; Pavan et al. 2021). For example, although we did not assess locomotion ability, Bridi et al. (2017) showed that exposure to glyphosate leads to a reduction in locomotor capacity (covered distance and speed) of zebrafish (Danio rerio).

Functional attributes related to sensorial skills (i.e., internal distance between the eyes) in individuals exposed to the pesticide occupied a greater functional space than in the control group. Although somewhat speculative, this result may indicate that tadpoles exposed to glyphosate have greater phenotypic plasticity regarding this attribute than tadpoles not exposed to the pesticide. Taking this into consideration, exposure to the pesticide may lead to alterations in sensory attributes, making some individuals less able to detect predators (Rohr and Madison 2003; Moore et al. 2015; Mikó et al. 2017), which may interfere with population dynamics in more contaminated areas (Jefferson et al. 2013).

The results found in this study indicate that the exposure of tadpoles to glyphosate affects the studied functional attributes differently, with consequences for basic aspects of their survival, such as foraging, locomotion and ability to recognize predators. Thus, it is important to emphasize that even at sub-lethal concentrations glyphosate can negatively affect the maintenance of amphibian populations in habitats influenced by agriculture.

I, Gabriela Taiza de Souza, as well as the other authors, declare that we have no conflicts of interest concerning this article.

Acknowledgments: We thank the Research Support Foundation of the State of RS (FAPERGS) and the National Council for Scientific and Technological Development (CNPq) for financial support.

Author contributions: All authors contributed to the study conception and design. Material preparation and data collection were performed by Gabriela Taiza de Souza, Carolina de Abreu Caberlon and Guendalina Turcato de Oliveira. Data analysis was performed by Diego Anderson Dalmolin. The first draft of the manuscript was written by Gabriela Taiza de Souza and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding Declaration: We have no funds to declare.

Agostini MG, Kacoliris F, Demetrio P, Natale GS, Bonetto C, Ronco AE (2013) Abnormalities in amphibian populations inhabiting agroecosystems in Northeastern Buenos Aires Province, Argentina. Dis Aquat Org 104:163–171. https://doi.org/10.3354/dao02592
Agostini MG, Roesler I, Bonetto C, Ronco AE, Bilenca D (2020) Pesticides in the real world: the consequences of GMO-based intensive agriculture on native amphibians. Biol Conserv 241:108355 https://doi.org/10.1016/j.biocon.2019.108355
Albuquerque AF, Ribeiro JS, Kummrow F, Nogueira AJA, Montagner CC, Umbuzeiro GA (2016) Pesticides in Brazilian freshwaters: a critical review. Environ Sci Process Impacts 18:779–787. https://doi.org/10.1039/C6EM00268D
Altig R, Johnston GF (1989) Guilds of anuran larvae: Relationships among modes, morphologies, and habitats. Herpetol Monogr 3:81–109. https://doi.org/10.2307/1466987
Altig R, McDiarmid RW (1999) Body plan: Development and Morphology. In Altig R, McDiarmid RW (ed) Tadpoles: The Biology of Anuran Larvae. University of Chicago Press, Chicago
Altig R (2006) Tadpoles evolved and frogs are the default. Herpetologica 62:1–10. https://doi.org/10.1655/05-23.1
Amarante Jr OP, Santos TCR, Brito NM, Ribeiro ML (2002) Glifosato: propriedades, usos e legislação. Química Nova 25(4):589–593. https://doi.org/10.1590/S0100-40422002000400014 Azevedo-Ramos C, Van Sluys M, Hero J, Magnusson WE (1992) Influence of tadpole movement on predation by Odonate Naiads. J Herpetol 26(3):335–338. https://doi.org/10.2307/1564891
Bach NC, Marino DJG, Natale G, Somoza GM (2018) Effects of Glyphosate and its commercial formulation, Roundup ® Ultramax, on liver histology of tadpoles of the neotropical frog, Leptodactylus latrans (amphibia: Anura). Chemosphere 202:289e297. https://doi.org/10.1016/j.chemosphere.2018.03.110
Bach NC, Natale GS, Somoza GM, Ronco AE (2016) Effect on the growth and development and induction of abnormalities by a glyphosate commercial formulation and its active ingredient during two developmental stages of the South-American Creole frog, Leptodactylus latrans. Environ Sci Pollut Res 23:23959–23971. https://doi.org/10.1007/s11356-016-7631-z
Backes A (1999) Condicionamento climático e distribuição geográfica de Araucaria angustifolia (Bertol.) Kuntze no Brasil: 2. Pesquisas, Botânica 49:31–51.
Becker CG, Fonseca CR, Haddad CFB, Batista RF, Prado PI (2007) Habitat split and the global decline of amphibians. Science 318(5857):1775–1777. https://doi.org/10.1126/science.1149374
Benbrook CM (2018) Why regulators lost track and control of pesticide risks: lessons from the case of glyphosate-based herbicides and genetically engineered-crop technology. Curr Envir Health Rpt 5:387–395. https://doi.org/10.1007/s40572-018-0207-y
Benachour N, Seralini GE (2009) Glyphosate formulations induce apoptosis and necrosis in human umbilical, embryonic, and placental cells. Chem Res Toxicol 22:97–105. http://dx.doi.org/10.1021/tx800218n
Berman MC, Marino D, Quiroga MV, Zagarese H (2018) Occurrence and levels of glyphosate and AMPA in shallow lakes from the Pampean and Patagonian regions of Argentina. Chemosphere 200:513–522. https://doi.org/10.1016/j.chemosphere.2018.02.103
Boone MD, Bridges CM (2003) Effects of pesticides on amphibian populations. In: Semlitsch RD (ed.) Amphibian Conservation. Smithsonian Institution, Washington
Boone MD, Semlitsch RD (2002) Interactions of an insecticide with competition and pond drying in amphibian communities.
Ecol Appl 12(1):307–316. https://doi.org/10.1890/1051-0761(2002)012[0307:IOAIWC]2.0.CO;2
Borges RE, Santos LRS, Assis RA, Benvindo-Souza M, Franco-Belussi L, Oliveira C (2019) Monitoring the morphological integrity of neotropical anurans. Environ Sci Pollut R 26:2623–2634. https://doi.org/10.1007/s11356-018-3779-z
Blonder B, Lamanna C, Violle C, Enquist BJ (2014) The n-dimensional hypervolume. Glob Ecol Biogeogr 23:595–609. https://doi.org/10.1111/geb.12146
Brasil (2005) Conselho Nacional do Meio Ambiente. Resolução CONAMA nº 357, de 17 de março de 2005. Dispõe sobre a classificação de águas, doces, salobras e salinas do Território Nacional e dá outras providências. Diário Oficial da União, Brasília, DF.
Brasil (2011) Portaria do Ministério da Saúde, 2914 de 12 de dezembro de 2011. http://bvsms.saude.gov.br/bvs/saudelegis/gm/ 2011/prt2914_12_12_2011.html. Accessed 12 June 2023
Bridges CM (1999) Effects of a pesticide on tadpole activity and predator avoidance behavior. J Herpetol 33:303–306. https://doi.org/10.2307/1565728
Bridi D, Altenhofen S, Gonzalez JB, Reolon GK (2017) Glyphosate and Roundup® alter morphology and behavior in zebrafish. Toxicology 392:32–39. https://doi.org/10.1016/j.tox.2017.10.007
Brunelli E, Bernabò I, Berg C, Lundstedt-Enkel K, Bonacci A, Tripepi S (2009) Environmentally relevant concentrations of endosulfan impair development, metamorphosis and behaviour in Bufo bufo tadpoles. Aquat Toxicol 91(2):135–142. https://doi.org/10.1016/j.aquatox.2008.09.006
Buskirk JV (2009) Natural variation in morphology of larval amphibians: Phenotypic plasticity in nature? Ecol Monogr 79:681–705. https://doi.org/10.1890/08-1692.1
Conselho Nacional para o Controle de Experimentação Animal (CONCEA) (2018) Normative Resolution nº 37. National Council for Animal Experimentation Euthanasia Practice Directive. Official Diary, Brasilia, Brazil.
Cunha GC, Dalzochio MS, Tozetti AM (2021) Anuran diversity in ponds associated with soybean plantations. An Acad Bras Cienc 93:e20201926. https://doi.org/10.1590/0001-3765202120201926
Dalmolin DA, Tozetti AM, Ramos Pereira MJ (2019) Taxonomic and functional anuran beta diversity of a subtropical metacommunity respond differentially to environmental and spatial predictors. PloS One 14(11):e0214902. https://doi.org/10.1371/journal.pone.0214902
Dalmolin DA, Tozetti AM, Pereira MJR (2020) Turnover or intraspecific trait variation: explaining functional variability in a neotropical anuran metacommunity. Aquat Sci 82:62. https://doi.org/10.1007/s00027-020-00736-w
Dalmolin DA, dos Santos TG, Tozetti AM, Pereira MJR (2022) Environmental descriptors and reproductive modes drive multiple facets of tadpole diversity in subtropical temporary ponds. Aquat Ecol 56:951–971. https://doi.org/10.1007/s10452-022-09977-3
De Avila F, Oliveira J, De Oliveira M, Borges-Martins M, Valiati VH, Tozetti AM (2019) Does color polymorphism affect the predation risk on Phalotris lemniscatus (Duméril, Bibron and Duméril, 1854) (Serpentes, Dipsadidae)? Acta Herpetol 14(1):57–63. https://doi.org/10.13128/Acta_Herpetol-24274
Devine GJ, Furlong MJ (2007) Insecticide use: Contexts and ecological consequences. Agric Human Values 24:281–306. https://doi.org/10.1007/s10460-007-9067-z
Edge C, Thompson D, Hao C, Houlahan J (2014) The response of amphibian larvae to exposure to a glyphosate-based herbicide (Roundup WeatherMax) and nutrient enrichment in an ecosystem experiment. Ecotoxicol Environ Saf 109(2014):124–132. https://doi.org/10.1016/j.ecoenv.2014.07.040
Fordyce JA (2006) The evolutionary consequences of ecological interactions mediated through phenotypic plasticity. J Exp Biol 209:2377–2383. https://doi.org/10.1242/jeb.02271
Gerhold P, Cahill JF, Winter M, Bartish IV, Prinzing A (2015) Phylogenetic patterns are not proxies of community assembly mechanisms (they are far better). Funct Ecol 29:600–614. https://doi.org/10.1111/1365-2435.12425
Gloria CM, Tozetti AM (2021) Bird visits and resource use in Butia odorata (Arecaceae) palm groves in southern Brazil. Iheringia Ser Zool 111:e2021032. https://doi.org/10.1590/1678-4766e2021032
Gollmann B, Gollmann G (1999) Plasticity of operculum development in Bombina orientalis (Amphibia, Anura). Folia Zool 48:233–236
Gosner KL (1960) A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16(3):183–190.
Hayes TB, Falso P, Gallipeau S, Stice M (2010) The cause of global amphibian declines: A developmental endocrinologist’s perspective. J Exp Biol 213(6):921–933. https://doi.org/10.1242/jeb.040865
Herek JS, Vargas L, Trindade SAR, Rutkoski CF, Macagnan N, Hartmann PA, Hartmann MT (2020) Can environmental concentrations of glyphosate affect survival and cause malformation in amphibians? Effects from a glyphosate-based herbicide on Physalaemus cuvieri and P. gracilis (Anura: Leptodactylidae). Environ Sci Poll R 27:22619–22630. https://doi.org/10.1007/s11356-020-08869-z
Jayawardena UA, Rajakaruna RS, Navaratne AN, Amerasinghe PH (2010) Toxicity of agrochemicals to common hourglass tree frog (Polypedates cruciger) in acute and chronic exposure. Int J Agric Biol 12(5):641–648. https://doi.org/10–209/SAE/2010/12–5–641–648
Jefferson DM, Hobson KA, Chivers DP (2013) Understanding the information value of repeated exposure to chemical alarm cues: what can growth patterns tell us? Annal. Zool. Fenni. BioOne 50:237–246. https://doi.org/10.5735/085.050.0401
Johnson JB, Saenz D, Adams CK, Hibbitts TJ (2015) Naturally occurring variation in tadpole morphology and performance linked to predator regime. Ecol Evol 5:2991–3002. https://dx.doi.org/10.1002/ece3.1538
Jones MB, Finnan J, Hodkinson TR (2015) Morphological and physiological traits for higher biomass production in perennial rhizomatous grasses grown on marginal land. Gcb Bioenergy 7(2):375–385. https://doi.org/10.1111/gcbb.12203
Jordani MX, Mouquet N, Casatti L, Menin M, Rossa‐Feres DC, Albert CH (2019) Intraspecific and interspecific trait variability in tadpole meta-communities from the Brazilian Atlantic rainforest. Ecol Evol 9:4025–4037. https://doi.org/10.1002/ece3.5031
Josende ME, Tozetti AM, Alalan MT, Filho VM, Ximenez S, da Silva Júnior FMR, Martins SE (2015) Genotoxic evaluation in two amphibian species from Brazilian subtropical wetlands. Ecol Indic 49:83–87. https://doi.org/10.1016/j.ecolind.2014.10.007
Kumari K (2020) Pesticides toxicity in fishes: A review. J Entomol Zool Stud 8(5):1640–1642.
Lajmanovich RC, Sandoval MT, Peltzer P (2003) Induction of mortality and malformation in Scinax nasicus tadpoles exposed by glyphosate formulations. Bull Environ Contam Toxicol 70:612–618. https://doi.org/10.1007/s00128-003-0029-x
Lanctot C, Navarro-Martín L, Robertson C, Park B, Jackman P, Pauli BD, Trudeau VL (2014) Effects of glyphosate-based herbicides on survival, development, growth and sex ratios of wood frog (Lithobates sylvaticus) tadpoles. II: agriculturally relevant exposures to Roundup Weathe Max® and vision® under laboratory conditions. Aquat Toxicol 154:291e303. https://doi.org/10.1016/j.aquatox.2014.05.025
Lima IB, Boëchat IG, Fernandes MD, Monteiro JAF, Rivaroli L, Gücker B (2023) Glyphosate pollution of surface runoff, stream water, and drinking water resources in Southeast Brazil. Environ Sci Pollut Res Int 30(10):27030–27040. https://doi.org/10.1007/s11356-022-24167-2.
Lutri VF, Matteoda E, Blarasin M, Aparicio V, Giacobone D, Maldonado L, Albo JG (2020) Hydrogeological features affecting spatial distribution of glyphosate and AMPA in groundwater and surface water in an agroecosystem. Córdoba, Argentina. Sci Total Environ 711:134557. https://doi.org/10.1016/j.scitotenv.2019.134557
Maluf JT (2000) Nova classificação climática do Estado do Rio Grande do Sul. Revista Brasileira Agrometeorologia 8:141–150.
Mammola S, Carmona CP, Guillerme T, Cardoso P (2021) Concepts and applications in functional diversity. Funct Ecol 35(9):1869–1885. https://doi.org/10.1111/1365-2435.13882
Marques JGDC, Veríssimo KJDS, Fernandes BS, Ferreira SRDM, Montenegro SMGL, Motteran F (2021) Glyphosate: A review on the current environmental impacts from a Brazilian perspective. Bull Environ Contam Toxicol 107(3):385–397. https://doi.org/10.1007/s00128-021-03295-4
Marques MN, Passos EA, Silva MTS, Correia FO, Santos AMO, Gomes SS, Alves JPH (2009) Determination of Glyphosate in Water Samples by IC. J Chromatogr Sci 47:822–824. https://doi.org/10.1093/chromsci/47.9.822
Mathew DV, Wassersug RJ, Parris MJ (2010) How does a change in labial tooth row number affect feeding kinematics and foraging performance of a ranid tadpole Lithobates sphenocephalus? Biol Bull 218:160–168. https://doi.org/10.2307/25664517
Mikó Z, Hettyey A (2023) Toxicity of POEA-containing glyphosate-based herbicides to amphibians is mainly due to the surfactant, not to the active ingredient. Ecotoxicology 32(2):150–159. https://doi.org/10.1007/s10646-023-02626-x
Mikó Z, Ujszegi J, Gál Z, Hettyey A (2017) Effects of a glyphosate-based herbicide and predation threat on the behaviour of agile frog tadpoles. Ecotoxicol Environ Saf 140(2017):96–102. https://doi.org/10.1016/j.ecoenv.2017.02.032
Mineau P, Whiteside M (2013) Pesticide acute toxicity is a better correlate of US grassland bird declines than agricultural intensification. PloS one 8(2):e57457. https://doi.org/10.1371/journal.pone.0057457
Moore H, Chivers DP, Ferrari MC (2015) Sub-lethal effects of Roundup™ on tadpole anti-predator responses. Ecotoxicol Environ Saf 111:281–285. https://doi.org/10.1016/j.ecoenv.2014.10.014
Moutinho MF, de Almeida EA, Espíndola EL, Daam MA, Schiesari L (2020) Herbicides employed in sugarcane plantations have lethal and sublethal effects to larval Boana pardalis (Amphibia, Hylidae). Ecotoxicol 29:1043–1051. https://doi.org/10.1007/s10646-020-02226-z
Pavan FA, Samojeden CG, Rutkoski CF, Folador A, Fré SP, Müller C, Hartmann PA, Hartmann MT (2021) Morphological, behavioral and genotoxic effects of glyphosate and 2,4-D mixture in tadpoles of two native species of South American amphibians. Environ Tox and Pharm 85(2021):103637. https://doi.org/10.1016/j.etap.2021.103637
Pillar VD, Blanco CC, Müller SC, Sosinski E, Joner F, Duarte LD (2013) Functional redundancy and stability in plant communities. J Veg Sci 24:963–974. https://doi.org/10.1111/jvs.12047
Potts SG, Biesmeijer JC, Kremen C, Neumann P, Schweiger O, Kunin WE (2010) Global pollinator declines: trends, impacts and drivers. Trends Ecol Evol 25(6):345–353. https://doi.org/10.1016/j.tree.2010.01.007
Queiroz CS, Silva FR, Rossa-Feres DC (2015) The relationship between pond habitat depth and functional tadpole diversity in an agricultural landscape. R Soc Open Sci 2:150165. https://doi.org/10.1098/rsos.150165
R Core Team (2023) R: A language and environment for statistical computing, version 3.2.2.
Resolution of the National Council for the Environment (2005) Resolução nº 357, de 17 de março de 2005. Diário Oficial da União, Brasília, DF.
Riaño C, Ortiz-Ruiz M, Pinto-Sánchez NR, Gómez-Ramírez E (2020) Effect of glyphosate (Roundup Active®) on liver of tadpoles of the colombian endemic frog Dendropsophus molitor (amphibia: Anura). Chemosphere 250:126287. https://doi.org/10.1016/j.chemosphere.2020.126287
Rissoli RZ, Abdalla FC, Costa MJ, Rantin FT, McKenzie DJ, Kalinin AL (2016) Effects of glyphosate and the glyphosate based herbicides Roundup Original® and Roundup Transorb® on respiratory morphophysiology of bullfrog tadpoles. Chemosphere 156:37e44. https://doi.org/10.1016/j.chemosphere.2016.04.083.
Rossa-Feres DDC, Jim J, Fonseca MG (2004) Diets of tadpoles from a temporary pond in southeastern Brazil (Amphibia, Anura). Braz J Zool 21:745–754. https://doi.org/10.1590/S0101-81752004000400003
Rohr JR, Madison DM (2003) Dryness increases predation risk in efts: support for an amphibian decline hypothesis. Oecologia 135:657–664. https://doi.org/10.1007/s00442-003-1206-7
Roy NM, Carneiro B, Ochs J (2016) Glyphosate induces neurotoxicity in zebra fish. Environ Toxicol Pharmacol 42:45–54. http://dx.doi.org/10.1016/j.etap.2016.01.003
Sánchez-Bayo F, Brink PJ, Mann RM (2011) Ecological impacts of toxic chemicals. Bentham Science Publishers.
Sanchez-Domene D, Navarro-Lozano A, Acayaba R, Picheli K, Montagner C, De EA, Cerqueira Rossa-Feres D, Rodrigues da Silva F, Alves de Almeida E (2018) Eye malformation baseline in Scinax fuscovarius larvae populations that inhabit agroecosystem ponds in southern Brazil. Amphibia-Reptilia 39:325–334. https://doi.org/10.1163/15685381-20181038
Sansiñena JA, Peluso L, Costa CS, Demetrio PM, Loughlin TMM, Marino DJG, Alcalde L, Natale GS (2018) Evaluation of the toxicity of the sediments from an agroecosystem to two native species, Hyalella curvispina (Crustacea: Amphipoda) and Boana pulchella (AMPHIBIA: ANURA), as potential environmental indicators. Ecol Indic 93:100–110. https://doi.org/10.1016/j.ecolind.2018.04.061
Schuck LK, Neely WJ, Farina RK, Oliveira JM, Tozetti AM (2021) Morphological trait variation between two populations of Cercosaura schreibersii in Southern Brazil: Insights on Habitat-Driven Adaptation. Herpetol Conserv Biol 16(2):337–344.
Stuart SN, Hoffmann M, Chanson JS, Cox NA, Berridge RJ, Ramani P, Young BE (2008) Threatened Amphibians of the World. Lynx Editions, Barcelona.
Suárez RP, Goijman AP, Cappelletti S, Solari LM, Cristos D, Rojas D, Krug P, Babbitt KJ, Gavier-Pizarro GI (2021) Combined effects of agrochemical contamination and forest loss on anuran diversity in agroecosystems of east-central Argentina. Sci Total Environ 759:143435. https://doi.org/10.1016/j.scitotenv.2020.143435
Suárez RP, Zaccagnini ME, Babbitt KJ, Calamari NC, Natale GS, Cerezo A, Codugnello N, Boca T, Damonte MJ, Vera-Candioti J, Gavier-Pizarro GI (2016). Anuran responses to spatial patterns of agricultural landscapes in Argentina. Landscape Ecol 31:2485. https://doi.org/10.1007/s10980-016-0426-2.
Tarazona JV, Court-Marques D, Tiramani M, Reich H, Pfeil R, Istace F, Crivellente F (2017) Glyphosate toxicity and carcinogenicity: a review of the scientifc basis of the European Union assessment and its differences with IARC. Arch Toxicol 91:2723e2743. https://doi.org/10.1007/s00204-017-1962-5.
Tarone RE (2018) On the International Agency for Research on Cancer classification of glyphosate as a probable human carcinogen. Eur J Canc Prev 27:82e87. https://doi.org/10.1097/CEJ.0000000000000289
Travis J (2023) Phenotypic plasticity. Oxford University Press.
Tylianakis JM, Klein AM, Tscharntke T (2005) Spatiotemporal variation in the diversity of Hymenoptera across a tropical habitat gradient. Ecology 86(12):3296–3302. https://doi.org/10.1890/05-0371
Van Buskirk J, McCollum A (2000) Influence of tail shape on tadpole swimming performance. J Exp Zool 203:2149–2158. https://doi.org/10.1242/jeb.203.14.2149
Van Meter RJ, Glinski DA, Henderson WM, Garrison AW, Cyterski M, Purucker ST (2015) Pesticide uptake across the amphibian dermis through soil and overspray exposures. Arch Environ Contam Toxicol 69:545–556. https://doi.org/10.1007/s00244-015-0183-2
Venesky MD, Wasserug RJ, Parris MJ (2010) The impact of variation in labial tooth number on the feeding kinematics of tadpoles of southern leopard frog (Lithobates sphenocephalus). Copeia 3:481–486. https://doi.org/10.1643/CG-09-093
Zabel F, Delzeit R, Schneider JM, Seppelt R, Mauser W, Václavík T (2019) Global impacts of future cropland expansion and intensification on agricultural markets and biodiversity. Nat Commun 10:28. https://doi.org/10.1038/s41467-019-10775-z
Whitman DW, Agrawal AA (2009) What is phenotypic plasticity and why is it important. Phenotypic plasticity of insects: Mechanisms and consequences. Science Publishers 1–63.

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Functional responses of tadpoles exposed to different concentrations of glyphosate

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Tadpoles' exposure to glyphosate

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