Effect of glyphosate (Roundup Activo®) on liver of tadpoles of the Colombian endemic frog Dendropsophus molitor (Amphibia: Anura)

Journal Pre-proof Effect of glyphosate (Roundup Activo®) on liver of tadpoles of the Colombian endemic frog Dendropsophus molitor (Amphibia: Anura)

Riaño C, M. Ortiz-Ruiz, Pinto-Sánchez N. R, E. Gómez-Ramírez PII:

S0045-6535(20)30480-X

DOI:

https://doi.org/10.1016/j.chemosphere.2020.126287

Reference:

CHEM 126287

To appear in:

Chemosphere

Received Date:

25 November 2019

Accepted Date:

19 February 2020

Please cite this article as: Riaño C, M. Ortiz-Ruiz, Pinto-Sánchez N. R, E. Gómez-Ramírez, Effect of glyphosate (Roundup Activo®) on liver of tadpoles of the Colombian endemic frog Dendropsophus molitor (Amphibia: Anura), Chemosphere (2020), https://doi.org/10.1016/j. chemosphere.2020.126287

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

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Effect of glyphosate (Roundup Activo®) on liver of tadpoles of the Colombian endemic frog Dendropsophus molitor (Amphibia: Anura)

Riaño C1,2, Ortiz-Ruiz M1, Pinto-Sánchez N. R1, Gómez-Ramírez E1.

1Grupo

de ecotoxicología, Evolución, Medio ambiente y Conservación. Facultad de

Ciencias básicas y Aplicadas. Universidad Militar Nueva Granada.

2 Corresponding

Author

e-mail: [email protected] Camilo Andrés Riaño Quintero, Facultad de Ciencias Básicas y Aplicadas, Universidad Militar Nueva Granada, Cajicá, Cundinamarca, Colombia. Telephone: 57-(1)-6500000 ext. 3278

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1. ABSTRACT Glyphosate-based herbicide (GBH) using is increasing on a global scale. Few studies have investigated the sub-lethal effects of GBH in endemic amphibian species. The present work tested the GBH Active Roundup® on the tadpoles of Dendropsophus molitor. The exposure was in a range of plausible environmental concentrations (0-0.75 µg/L) during a month. D. molitor is an endemic tropical frog of South America. The exposure from 325 µg/L caused histological alterations in the liver. The high-resolution optical microscopy (HROM) detected sinusoidal dilatation and cytoplasmic vacuolization. The transmission electron microscopy (TEM) showed disorganization of the endoplasmic reticulum. Since the liver is essential for detoxification, these results suggest choric effects. Exposure to another GBH has caused histological alterations in liver tadpoles liver in a previous study, but, this study tested another endemic South-American frog for only 96h. The present work applied HROM to observe lipid alterations since it does not use organic solvents; and TEM for the ultrastructural observation of hepatocytes. Environmental risk of GBH can improve by including sub-lethal effects in endemic species.

KEYWORDS Herbicide, Lipid vesicles, Ultrastructure, Tropical frog, Histopathological alterations, Sublethal effects.

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2. INTRODUCTION

Glyphosate-based herbicides (GBH) have had a globalized use based on their effectiveness in weed eradication. The agricultural industry has developed genetically modified plants to resist the toxic effects of glyphosate, thus, increasing agricultural production (Gaines et al., 2010), but using vast amounts of GBH. This increase in the use of GBH has generated soil and water contamination, producing harmful effects in animals and humans distributed within and around fumigation zones (Tarazona et al., 2017; Tarone, 2018).

To date, the use of glyphosate has been banned in several countries, including Sri Lanka, France, El Salvador, Netherlands, and Bermuda (Malkanthi et al., 2019). However, in Colombia, the herbicide is widely used in agriculture activities related to weed control in pre and post-emergency in consumer crops and the eradication of illicit crops (Solomon et al., 2005; UNODC, 2014). Glyphosate is the most commercialized agrochemical in Colombia, with 31.25% of the total of the agricultural products, with 38 commercial formulas available being Roundup Activo®, the most used formulation (Bolaños, 2016). Roundup Activo® is composed of 446 g/L potassium salt N- (phosphonomethyl) –glycine (MONSANTO, Saint Louis, MO, EE.UU), and a substantial proportion of the surfactant, the last one is necessary for the penetration of the product in the plant (de Ruiter et al., 2004).

Several authors have described the toxic effects of the surfactant in different species of amphibians (Defarge et al., 2016; Rissoli et al., 2016; Bonfanti et al., 2018). Some of the negative effects that have been reported included alterations at different stages of the 3

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embryonic development and organs of such as liver, epidermal epithelium, or brain (Lanctôt et al., 2014; Rissoli et al., 2016; Bach et al., 2018; Bonfanti et al., 2018). For example, individuals of Leptodactylus latrans (Leptodactylidae) exposed to a concentrations between 0.37 and 5.25 mg a.e./L of Roundup Ultramax® show presence of macrophages, melanomacrophagic centers, vascular congestion, and lipidosis (Bach et al., 2018). In the epidermal epithelium of Lithobates catesbeianus tadpoles (Ranidae), Rissoli et al. (2016) reported hypertrophy, epithelial hyperplasia, and chromatid rupture to a concentration of 1 mg a.e./L of Roundup Original ® and Roundup Transorb R ®. Furthermore, decreased expression of genes encoding thyroid hormone, glucocorticoid receptor, among other effects were reported for brain samples of Lithobates sylvaticus (Ranidae) exposed to 0.21 mg a.e./L of Roundup WeatherMax® (Lanctôt et al., 2014). Tissue alterations can also include teratogenic effects and irregularity in craniofacial/ocular development, such as reported by Bonfanti et al. (2018) in Xenopus laevis (Pipidae) exposed to 20 mg or acid equivalent/L of Roundup Power 2.0 ®. At present, the state of conservation of amphibians is critical due to loss of habitat, climate change, and environmental pollution, the last one related with the excessive use of agrochemicals (Brühl et al., 2013). In the center-east of Colombia, glyphosate is often used in crops like potato, pea, and other leguminous crops. These crops are usually located close to the high Andean forests, habitat of endemic amphibians (DANE, 2016). One of them is Dendropsophus molitor, also called savanna frog. This species is endemic to the eastern cordillera of the Colombian Andes. The savanna frog is distributed between 2000 and 3600 meters above sea level and reproduces in lentic bodies of water (Méndez-Narváez, 2014; Guarnizo et al., 2015). Currently, it is listed by the IUCN as less concern (IUCN red list 2019). However, the decline in amphibian populations may be caused by contamination of 4

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water sources in ecosystems with high anthropic activity, specially the pesticides used in agriculture (Relyea, 2005). Amphibians are particularly sensitive to changes in the environment, due to their high skin permeability; eggs without shell and some species have biphasic life cycles with aquatic and terrestrial life stages (Wagner et al., 2015). For this reason, these animals are key in diagnosing the state of an ecosystem (Simon et al., 2011). Consequently, the use of different histopathological approaches in vital organs allows determining the sublethal effects that xenobiotics can have on these species (Bach et al., 2018). The liver is the most important organ in detoxification processes of the organism exposed to chemical substances, particularly focusing on the analysis of hepatocytes and melanomacrophagic centers (Steinel and Bolnick, 2017). The aim was determine the effects of a commercial presentation of glyphosate (Roundup Activo®) on the liver of the tadpole stage of the tropical endemic D. molitor frog. In nature, glyphosate does not reach lethal concentrations because its levels vary with bacterial decomposition on site, interaction with soil elements, and even the changes in environmental conditions/seasons (Toretta et al., 2018). Therefore, we use glyphosate at sublethal concentrations allows us to have a more realistic approximation to the effects it has on tissues and the consequences of its use in live subjects.

3. MATERIALS AND METHODS 3.1 Animals We collected 54 tadpoles of D. molitor from an unpolluted stream at Universidad Militar Nueva Granada, Cajicá, Colombia (4°56’ 37’’ N - 74° 00’ 35’’W, 2560 m.a.s.l) using a net. Tadpoles were collected with the permission Res No 1198 given by the Environmental

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Ministry of Colombia to Universidad Militar Nueva Granada and transported to the laboratory. The specimens were acclimatized during one week according to the time used in other neotropical amphibian (Pérez-Iglesias, 2016). The tadpoles used in this study were in 24 Gosner developmental stage (Gosner, 1960) with initial length of 14.70  0.06 mm and final length of 2.45  0.17 mm (mean  SD). We maintain the physico-chemical parameters controlled (pH 7.2, temperature 16 °C, total ammoniac nitrogen (TAN) 0mg/L, nitrite 0 mg/L y dissolved oxygen (DO) >5 mg/L). The individuals were fed with commercial food tetra color type ® (47.5% protein) adjusted to 2% of biomass.

3.2 Chemicals and experimental design We evaluated the effects of glyphosate on amphibian tissues using the commercial formulation glyphosate Roundup Activo® (Bayer Cropscience). This formulation composition is 363 g/L of glyphosate acid (equivalent to 446 g/L of potassium salt of N(phosphonomethyl) –glycine), and surfactant, however, the company does not report the name of the surfactant used. The product is classified at Colombia as Toxicological category III (Slightly dangerous). We used two different concentrations of glyphosate 325 and 750 µg glyphosate acid equivalent (a.e.) /L and the control with 0 µg a.e./L, each one with three replicates prepared from Roundup Activo® (Henao et al., 2015). A total of 6 individuals per treatment were analyzed (N=54). Test subjects were maintained in semistatic systems with a total volume of 2 liters of tap water per experimental unit for 30 days. The water quality (temperature, pH, dissolved oxygen, nitrite, total ammonia nitrogen) was monitored daily with a multiparameter water quality meter® Hanna and Merck Spectroquant® test kit. Tadpoles were euthanized with benzocaine (0.5 g/L). This study

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was carried out according to the guidelines for the ethical management of fish and amphibians (AVMA, 2013).

3.3 Sample processing for histological and image analyses The tissues were fixed in 2.5% Glutaraldehyde for three days, then washed with phosphate buffer saline and post-fixed with 2% osmium tetroxide (Piloni et al., 2016). Dehydration processes were carried out with increasing concentrations of ethanol (50%, 70%, 90%, 100%). The samples were embedded with homogeneous mixtures of Poly/Bed 812® resin. Finally, the samples were polymerized in an incubator at 70°C (Piloni et al., 2016). Semithin sections 1 μm thick were obtained by high-resolution optical microscopy (HROM) using a rotatory microtome (SLEE 4060®) and stained with toluidine blue (Piloni et al., 2016). The sections were observed and photographed by an optical microscope (ZEISS®) equipped with an Axiocam digital camera (ZEISS®). We obtained measurements of the area, and lipid vesicles using Image J 1.48v software (http: //imagej.nih.gov/ij/, 2013) (Gómez, 2013). For ultrastructure analysis, we made 130 nm ultrathin tissue sample sections using ultramicrotome Leica® EM UC6. The slides were contrasted with lead citrate/uranyl acetate. The samples were analyzed in an electron microscope of transmission Jeol® JEM 1400 Plus 120 Kv. Photographs and morphometric measurements were carried out with a GATAN® camera attached to this equipment and GATAN® Digital Micrograph 1.80.70 program. 3.4 Statistical analyses

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Data were transformed using Log10 in order to satisfy the normality assumption for analysis. Differences in variables; number, area of lipid vesicles between treatments were assessed by one-way ANOVA and Tukey-Kramer test as a pos hoc (P <0.05 error type I) (Zar, 2010). All statistical analyzes were performed using the JMP version 9.0.1 (SAS Institute).

4. RESULTS Water quality The parameters pH (7.25 – 7.44), temperature (16.61 ºC – 16.35 ºC), dissolved oxygen (5.74 ppm – 5.67 ppm), ammoniacal total nitrogen (0.04 ppm – 0.13 ppm) and nitrite (0 ppm) were maintained during the 30 days of exposure to Roundup® Activo within the environmental ranges reported at Dendropsophus molitor tadpoles collection site (4°56’ 37’’ N - 74° 00’ 35’’W, 2560 m.a.s.l).

Table 1. Water quality parameters during the 30 days of exposure de D. molitor to Roundup Activo®. Data are expressed as mean ±SEM. Roundup Activo® (mg a.e./L) 0

325

750

pH

7.300 ± 0.074

7.250 ± 0.074

7.440 ± 0.074

Temperature (°C)

16.530± .140

16.350 ± 0.074

16.610 ± 0.074

DO (mg/L)

5.700 ± 0.085

5.670 ± 0.085

5.740 ± 0.085

TAN (mg/L)

0.047 ± 0.019

0.100 ± 0.019

0.130 ± 0.019

NO2- (mg/L)

0.005 ± 0.001

0.007 ± 0.001

0.003 ± 0.001

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In the Dendropsophus molitor tadpole liver of the control treatment (0 mg/L) observed hepatocytes with poligonal shape, with a prominent nucleus and one or two nucleoli. Hepatocytes arrangements in sheet separated by sinusoids with few homogeneously dispersed blood vessels. We observed few lipid vesicles and cytoplasmatic vacuoles (Fig. 1a). Ultrastructurally the rough endoplasmatic reticulum did not show structural alterations (Fig. 2a).

4.2 Histopathological effects We observed higher number and area of lipid vesicles in the treatments 325 and 750 µg a.e /L (Table 2, Fig. 3). We found lipid vesicles and sinusoidal dilation using HROM in the three treatments, being more abundant in the treatment with 750 µg a.e /L of glyphosate. In all treatments, there was absence of mortality (Table 2, Fig. 1).

Table 2. Histopathologies alterations in the liver for D. molitor exposed to Roundup Activo®. Values are expressed as a percentage. Roundup Activo® (µg a.e./L) Histopathologies findings

0

325

750

Non-homogeneous parenchyma

0

0

0

Melanomacrophage centers

0

0

0

Sinusoidal dilatation

0

20

80

Cytoplasmatic vacuolization

20

80

80

Pycnotic nuclei

0

0

0

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Lipid vesicles

20

60

80

Alteration of the rough endoplasmic reticulum

0

20

60

Glycogen granules

0

0

0

* *

H

*

*

*

Fig.1. Cross-sections of the D. molitor liver. A. Liver section control treatment (0 mg / L). B. Liver section treatment with 325 µg a.e. / L. C. Liver section treatment with 750 µg a.e. / L. The symbols are as follows lipid vesicles (red asterisks), sinusoidal dilation (black 10

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asterisks), vacuoles (arrows), hepatocytes (H), blood vessels (V). HROM technique. Bar = 20 µm. Using TEM, we observed disorganization of the rough endoplasmatic reticulum, cytoplasmic vacuoles, and some lipid vesicles (Fig. 2).

A

*

H

B

H

C

Fig. 2. Dendropsophus molitor liver showing some ultrastructural changes A) Liver section control treatment (0 mg / L). B) Liver section treatment with 325 µg a.e./ L. C) Liver section treatment with 750 µg a.e./ L. Hepatocytes (H), disorganization of 11

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the rough endoplasmic reticulum (black arrows) and cytoplasmic vacuoles (red arrows), Lipid vesicles (asterisk). TEM technique. Bar = 5 µm. The variables related to the lipid vesicles after the Log10 transformations were normally distributed as follows, the average occupied area (µm 2 ) of lipid vesicles (W= 0.95, P=0.41), and the number of lipid vesicles (W=0.93, P= 0.20). We found significant differences in the average occupied area (µm2) of lipid vesicles (R2 = 0.81, F = 33.25, p ≤ 0.001),and number of lipid vesicles (R2 = 0.78, F = 26.24, P = <0.00) in both concentrations of glyphosate with respect to the control treatment (Fig. 3). The comparisons between the two concentration of glyphosate 325 and 750 µg a.e. / L and the control have significant differences (Fig. 3). The differences between the treatments and the control are as follows control and 750 µg a.e. / L (difference of 1.29, p ≤ 0.001), followed by the difference of 325 and 750 µg a.e. / L (0.71, P = 0.00), and finally the difference of control and 325 µg a.e. / L (0.57, p ≤ 0.01).

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Fig. 3. Number of lipid vesicles (LV) and filled occupied area of LV (µm2), present in the liver of D. molitor exposed to different glyphosate concentrations in Roundup Activo®. Data are expressed as mean ± SD.

DISCUSSION

The Dendropsophus molitor tadpole liver of the control treatment (0 mg /L) presented polygonal hepatocytes, with a prominent nucleus and one or two nucleoli. Hepatocytes arrangements in sheet separated by sinusoids with little homogeneously dispersed blood vessels (Fig. 1a, 2a). Those liver structure arrangement were similar to that described for other amphibian species, which have different grade of phylogenetical proximity with Dendropsophus molitor (Jetz and Pyron, 2018), such as Rhinella arenarum (Svartz et al., 2019), Leptodactylus latrans (Bach et al., 2018), Bufo gargarizans (Wu et al., 2017), Triturus carnifex (Capaldo et al., 2016), Leptodactylus latinasus (Pérez-Iglesias, 2016), Ambystoma mexicanum (Ortiz-Ordoñez et al., 2016) and Lissotriton italicus (Bernabo et al., 2014).

The liver is an organ with different metabolic functions, and the exposure to xenobiotics can result in changes in its histological structure (Cakici, 2015). In the present study, we observed histopathological alterations after one-month exposure to Roundup Active® in a range of 350-750 µg a.e. / L. This chronic exposure resulted in sinusoidal dilation, cytoplasmic vacuolation, and accumulation of lipid vesicles (Fig. 1, Fig. 3). Additionally, 13

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by TEM the disorganization of the rough endoplasmic reticulum were observed (Fig. 2). These alterations were concentration-dependent. The hepatic sinusoidal dilation in vertebrates is caused by obstruction of hepatic venous flow, which leads to vascular stasis and parenchymal congestion (Brancatelli et al., 2018). This alteration can generate osmoregulatory problems in nutrient transport and metabolism of waste products (Sparling et al., 2010). The sinusoidal dilation observed was similar to that reported for adults of Pelophylax ridibundus after exposure to two insecticides (Paunescu et al., 2010, 2012) and also to a fungicide (Paunescu et al., 2018). Likewise, this alteration has been reported in adults of the frog Bufotes variabilis induced by the insecticide Carbaryl (Cakici, 2015) an in adults of Triturus carnifex after daily exposure to heavy metal concentrations, such as cadmium (Capaldo et al., 2016). These reports suggest that sinusoidal dilatation seems to be a general response to pesticide intoxication in adult amphibians, and in our study is the first report in tadpoles under herbicide exposure. The acute effects caused by GHBs in the liver of rats are related to increased lipid peroxidation, which can lead to alterations in the liver activity and abnormalities in hepatic tissues over longer periods of exposure, in this case, the accumulation of lipid vesicles (Pandey et al., 2019). We observed vesicles located in the cytoplasm of hepatocyte exhibited a small size (less than 1 um in diameter) and nucleus centrally located. This alteration indicates a microvesicular steatosis which is caused by mitochondrial β-oxidation of fatty acid in the process of oxidative stress (Pandey et al., 2019). The microvesicular steatosis could be induced by several factors including exposure to drugs and toxins (Fromenty et al., 1997).On the other hand, Sinhorin et al., (2014) observed an increase in thiobarbituric acid reactive substances (TBARS) in the liver of Pseudoplatystoma sp, after 14

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exposure to Roundup Original® at concentrations of 2.25, 4.5, 7.5 and 15 mg / L during 96h, the same was found by Menezes et al., (2011) in Rhamdia quelen exposed to 0.45 and 0.9 mg / L of Roundup® for 48 hours and Glusczak et al., (2011) in Leporinus obtusidens exposed to 3, 6, 10 and 20 mg / L for 96 hours. This suggest that GBHs are related to alteration of lipid metabolism. In amphibians, an increase in lipid peroxidation caused by GBH has also been reported in short periods of exposure, in the tadpole’s liver of Lithobates catesbeianus exposed to 36, 72, and 144 μg / L of Roundup Original® for 7 days, in all treatments with GBH increase in the concentration of TBARS was observed. This occurs due to high lipid peroxidation, which is related to the loss of integrity of the cell membrane, disorders in glycolipid metabolism and high-energy demand, which can lead to cell damage, lipid accumulation, and apoptosis (Dornelles and Oliveira, 2013). High levels of lipid peroxidation cause cytoplasmic vacuolization in the liver (OrtizOrdoñez et al., 2016), and also the accumulation and displacement of triglycerides and phospholipids to the periphery of the hepatocyte (Bach et al., 2018). The cytoplasmic vacuolization observed seems to be a consequence of the earliest responses to Roundup Activo® exposure, such as oxidative stress and lipid disorder (El-Shenawy, 2009; Modesto C., 2010; Milic et al., 2018). Moreover, the Roundup Activo® exposure increased the number of deposits of lipid vesicles on the periphery of hepatocytes (Fig. 1), which was related to lipid oxidation and the disorganization of the rough endoplasmic reticulum (Bernabo et al., 2014). This anomaly has been reported in several amphibian species exposed to different xenobiotics. For example, in tadpoles of Bufo gargarizans exposed to mercury (Shi et al., 2018), in adult specimens of Lissotriton italicus exposed 15

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to nonylphenol ethoxylate (Bernabo et al., 2014). The increased number of lipid vesicles can generate alterations in protein synthesis, specifically lipoproteins necessary for the transport and release of lipids from hepatocytes (Rondón-Barragán et al., 2012). The registered alterations seem to be a consequence of a physiological stress process induced by chemical intoxication.

In acute exposure, is common found melanomacrophagic centers and the eosinophils accumulation are among the most common alterations after exposure to xenobiotics. The Roundup ® Ultramax and pure glyphosate caused melanomacrophagic centers and the eosinophils accumulation in the frog Leptodactylus latrans after 96 h exposure (Bach et al., 2018). These accumulations did not occur in the present work with the frog Dendropsophus molitor. Although both species are endemic, Dendropsophus molitor is a tropical species, and Leptodactylus latrans is a subtropical species. Bach et al., (2018) exposed tadpoles to Roundup ® Ultramax for 96 h; meanwhile, tadpoles were exposed to Active Roundup® for one month in the present study. The accumulation of melanomacrophagic centers and eosinophils was also observed in tadpoles of the bullfrog Lithobates catesbeianus exposed to the herbicide Clomazone for 96 h (De Oliveira et al., 2016). This study suggests that the alteration in melanomacrophagic centers and eosinophils numbers is related to the exposure duration of chemical stress and early response to liver intoxication that results in the alteration observed in the present study.

In chronic exposure, some authors have reported histopathologies related to glyphosatebased herbicides (GBH). Howe et al., (2004) exposed tadpoles of Rana pipiens for 42 days 16

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to two GBHs; Roundup Original®, Roundup Transorb® and POEA surfactant, chronic exposure caused a decrease in snout – vent length, gonadal histological abnormalities, tail damage and increased metamorphosis time. The exposure for 20 days of tadpoles from Rana pipiens, Bufo americanus and Hyla versicolor to Roundup® in microcosm conditions showed a significant increase in mortality in the three species, independent of the type of soil where the GBH was applied (Relyea, 2005). King and Wagner (2010) exposed six species of amphibians tadpoles; Ambystoma gracile, Ambystoma macrodactylum, Anaxyrus boreas, Pseudacris regilla, Rana cascadae, and Rana luteiventris, for 16 days. They found that the amphibian species evaluated respond differentially to the GBH, having a survival time of 1 day for tadpoles of Pseudacris regilla and up to 16 days for Ambystoma gracile in concentrations between 1 and 5 mg/L, concluding that chronic toxicological effects have a high variation among amphibian species. This study is the first report of chronic effects of GBH in the tadpole liver of the South-American endemic frogs of a tropical region. Alteration in the number of lipid vesicles and disorganization of the endoplasmic reticulum were observed. The registered alterations can affect liver functions (Sparling et al., 2010), and they were caused by exposure to sublethal concentration of Roundup Activo ®. These results indicate that environmental risk assessment of GBH should consider endemic amphibian species of tropical regions such as the frog Dendropsophus molitor. Consequently, the environmental risk assessment of GBH should be locally performed considering endemic species of tropical regions. At lower level of vertebrates that depend of water, distributed at Bogotá we found amphibians such as Edwards’ Rocket Frog Hyloxalus edwardsi and the Orphan salamander Bolitoglossa capitana listed as critically endangered, the Elegant Robber Frog Pristimantis elegans listed as vulnerable, and Bogota 17

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Tree frog Hyloscirtus bogotensis, Peters’ mushroomtongue salamander Bolitoglossa adspersa listed as near threatened. The Bogota Rocket Frog Hyloxalus subpunctatus and the Bogota Robber Frog Pristimantis bogotensis are listed as least concern. At Fish level, the fish captain of the Savannah Trichomycterus venulosus listed as critically endangered, and Guapucha Grundulus bogotensis as least concern (IUCN, 2019). As presented before, some of the species mentioned previously have been formally listed as endangered and could become extinct, unless action is taken for their protection.

Funding This study was supported by the vice-rectory of investigations of the Universidad Militar Nueva Granada in the project CIAS 2550.

Acknowledgments We are in debt with Miguel Banoy who collected the tadpoles. Lina Maria helped with the English revision of the manuscript. We thank to Dr. Martha P. Ramírez Pinilla, and three anonymous authors who helped improve the written document.

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1

References

2

American Veterinary Medical Association (AVMA). Guidelines for the euthanasia of

3

animals: (2013), Edition. Schaumburg, IL: American Veterinary Medical Association.

4 5

Bach N.C., Marino D.J.G., Natale G., and Somoza G.M. Effects of Glyphosate and its

6

commercial formulation, Roundup ® Ultramax, on liver histology of tadpoles of the

7

neotropical frog, Leptodactylus latrans (amphibia: Anura). Chemosphere, 202 (2018), 289 -

8

297. https://doi.org/10.1016/j.chemosphere.2018.03.110.

9 10

Bernabo I., Biasone P., Macirella R., Tripepi S., and Brunelli E. Liver histology and

11

ultrastructure of the Italian newt (Lissotriton italicus): Normal structure and modifications

12

after acute exposure to nonylphenol ethoxylates. Experimental and Toxicologic Pathology,

13

66 (2014), 455 - 468. https://doi.org/10.1016/j.etp.2014.09.002.

14 15

Bolaños E. La “suspensión” del Glifosato en la erradicación de cultivos ilícitos en

16

Colombia: ¿una solución humanitaria o un cambio para que todo siga igual? Trabajo de

17

Grado. Universidad Católica de Colombia. Facultad de Derecho. Bogotá, Colombia. 2016.

18

https://repository.ucatolica.edu.co/bitstream/10983/4372/4/Suspensi%C3%B3n%20del%20

19

glifosato%20en%20Colombia%20pdf.pdf

20 21

Bonfanti P., Saibene M., Bacchetta R., Mantecca P., and Colombo A. A glyphosate

22

micro-emulsion formulation displays teratogenicity in Xenopus laevis. Aquatic Toxicology

23

195 (2018), 103 - 113. https://doi.org/10.1016/j.aquatox.2017.12.007.

24 19

Journal Pre-proof

25

Brancatelli G, Furlan A, Calandra A, and Dioguardi Burgio M. Dilatación sinusoidal

26

hepática. Abdominal Radiology, 43 (8) (2018), 2011 - 2022.

27

https://doi.org/10.1007/s00261-018-1465-8.

28 29

Brühl C., Schmidt T., Pieper S., and Alscher A. Terrestrial pesticide exposure of

30

amphibians: An underestimated cause of global decline?. Scientific Reports, 3 (2013),

31

1135-1138. doi:10.1038/srep01135.

32 33

Cakici, O. Histopathologic changes in liver and kidney tissues induced by carbaryl in

34

Bufotes variabilis (Anura: bufonidae). Experimental and Toxicologic Pathology, 67 (2015),

35

237 - 243. https://doi.org/10.1016/j.etp.2014.12.003.

36 37

Capaldo A., Gay L., Scudiero R., Trinchella F., Caputo I., Lepretti M., Marabotti A.,

38

Esposito C., and Laforgia V. Histological changes, apoptosis and metallothionein levels in

39

Triturus carnifex (Amphibia, Urodela) exposed to Environmental cadmium concentrations.

40

Aquatic Toxicology, 173 (2016), 63 - 73. https://doi.org/10.1016/j.aquatox.2016.01.009.

41 42

Defarge N., Takács E., Lozano V. L., Mesnage R., Vendômois J.S., Seralini, G. E., and

43

Székács A. Co-formulants in glyphosate-based herbicides disrupt aromatase activity in

44

human cells below toxic levels. International Journal of Environmental Research and Public

45

Health, 13, (2016), 1 - 17. https://doi.org/10.3390/ijerph13030264.

46 47

De Oliveira C., Fernandes L., Miglioranza G., Salla R., Camargo F., Jones M., and Silva –

48

Zacarin E. Hepatic effects of the clomazone herbicide in both its free form and associated 20

Journal Pre-proof

49

with chitosan-alginate nanoparticles in bullfrog tadpoles. Chemosphere, 149 (2016), 304 -

50

313. https://doi.org/10.1016/j.chemosphere.2016.01.076.

51 52

Departamento Administrativo Nacional de Estadistica (DANE). Encuesta Nacional

53

Agropecuaria ENA. Boletin técnico comunicación informativa. (2016). Bogotá, Colombia,

54

1 - 31.

55

https://www.dane.gov.co/files/investigaciones/agropecuario/enda/ena/2017/boletin_ena_20

56

17.pdf

57 58

De Menezes C., da Fonseca M., Loro V., Santi A., Cattaneo R., Clasen B., Pretto A., and

59

Morsch V. Roundup effects on oxidative stress parameters and recovery pattern of

60

Rhamdia quelen. Archives of Environmental Contamination and Toxicology, 60(4) (2011),

61

665 - 71. doi: 10.1007/s00244-010-9574-6.

62 63

de Ruiter H., Kempenaar C., and Blom G. Foliar Absorption of Crop Protection Agents:

64

Influence of Cpa Properties, Formulation and Plant Species. (2004). A Literature Study for

65

the Dutch Research Programme Pesticides and the Environment (DWK-359) Theme B-2.

66

http://edepot.wur.nl/24422.

67 68

Dornelles M., and Oliveira G. Effect of Atrazine, Glyphosate and Quinclorac on

69

Biochemical Parameters, Lipid Peroxidation and Survival in Bullfrog Tadpoles (Lithobates

70

catesbeianus). Archives of Environmental Contamination and Toxicology, 66(3) (2013),

71

415 - 429. doi:10.1007/s00244-013-9967-4.

72 21

Journal Pre-proof

73

El-Shenawy N. Oxidative stress responses of rats exposed to Roundup and its active

74

ingredient glyphosate. Environmental Toxicology and Pharmacology, 28 (2009), 379 - 385.

75

https://doi.org/10.1016/j.etap.2009.06.001

76 77

Fromenty B., Berson A., and Pessayre D. Microvesicular steatosis and steatohepatitis: role

78

of mitochondrial dysfunction and lipid peroxidation. Journal of Hepatology, 26 Suppl

79

(1997), 13 - 22.

80 81

Gaines TA., Zhang W., Wang D., Bukun B., Chisholm S.T., Shaner DL., Nissen S.J.,

82

Patzoldt W.L., Tranel P.J., Culpepper A.S., Grey T.L., Webster T.M., Vencill W.K.,

83

Sammons R.D., Jiang J., Preston C., Leach J.E., and Westra P. Gene amplification confers

84

glyphosate resistance in Amaranthus palmeri. Proceedings of the National Academy of

85

Sciences of the United States of America, 107 (2010), 1029 - 1034.

86

https://doi.org/10.1073/pnas.0906649107.

87 88

Glusczak L., Loro V., Pretto A., Moraes B., Raabe A., Duarte M., da Fonseca M., de

89

Menezes C., and Valladão D. Acute exposure to glyphosate herbicide affects oxidative

90

parameters in piava (Leporinus obtusidens). Archives of Environmental Contamination and

91

Toxicology, 61(4) (2011), 624 - 630. doi: 10.1007/s00244-011-9652-4.

92 93

Gómez E. Efecto de una presentación comercial de glifosato en alevinos de Cachama

94

blanca (Piaractus brachypomus). Universidad de los Llanos. (2013), 1 - 92.

95

http://repositorio.unimagdalena.edu.co/jspui/handle/123456789/3123. 22

Journal Pre-proof

96 97

Gosner KL. A simplified table for staging anuran embryos and larvae with notes on

98

identification. Herpetologica, 16 (1960), 183 - 190.

99 100

Guarnizo CE., Paz A., Muñoz-Ortiz A., Flechas S.V., Méndez-Narváez J., and Crawford

101

A.J. DNA barcoding survey of anurans across the Eastern Cordillera of Colombia and the

102

impact of the Andes on cryptic diversity. PLoS ONE, 10 (2015), e0127312.

103

https://doi.org/10.1371/journal.pone.0127312

104 105

Henao L.M., Montes CM., and Bernal M.H. Acute toxicity and sublethal effects of the

106

mixture glyphosate (Roundup® Active) and Cosmo-Flux® 411F to anuran embryos and

107

tadpoles of four Colombian species. Revista de Biología Tropical, 63(1) (2015), 223 - 233.

108

DOI 10.15517/RBT.V63I1.12893

109 110

Jetz, W. and Pyron, R.A. The interplay of past diversification and evolutionary isolation

111

with present imperilment across the amphibian tree of life. Nature Ecology and Evolution,

112

2(2018), 850 - 858. https://doi.org/10.1038/s41559-018-0515-5.

113 114

Lanctôt C., Navarro-Martín L., Robertson C., Park B., Jackman P., Pauli B.D., and Trudeau

115

V.L. Effects of glyphosate-based herbicides on survival, development, growth and sex

116

ratios of wood frog (Lithobates sylvaticus) tadpoles. II: agriculturally relevant exposures to

117

Roundup Weathe Max® and vision® under laboratory conditions. Aquatic Toxicology, 154

118

(2014), 291 - 303. https://doi.org/10.1016/j.aquatox.2014.05.025.

119 23

Journal Pre-proof

120

Malkanthi S., Sandareka U., Wijerathne A., and Sivashankar P. Banning of Glyphosate and

121

its Impact on Paddy Cultivation: A study in Ratnapura District in Sri Lanka. The Journal of

122

Agricultural Sciences - Sri Lanka, 14 (2019), 129 - 44.

123

http://dx.doi.org/10.4038/jas.v14i2.8515.

124 125

Méndez-Narváez J. Diversidad de anfibios y reptiles en hábitats altoandinos y paramunos

126

en la cuenca del río Fúquene, Cundinamarca, Colombia. Biota Colombiana, 15(1) (2014),

127

94 - 103. http://revistas.humboldt.org.co/index.php/biota/article/view/310.

128 129

Milić M., Zunec S., Micek V., Kasuba V., Mikolic A., Lavakovic B.L., Zivkovic T.,

130

Pavicic I., Marjanovic A.M., Pizent A., Vrdoljak A.L., Valencia-Quintana R., Sanchez-

131

Alarcon J. and Zeljezic D. Oxidative stress, cholinesterase activity, and DNA damage in the

132

liver, whole blood, and plasma of Wistar rats following a 28-day exposure to glyphosate.

133

Arhiv Za Higijenu Rada I Toksikologiju (2018), 154 - 168. DOI: 10.2478/aiht-2018-69-

134

3114.

135 136

Modesto K., and Martínez C. Roundup causes oxidative stress in liver and inhibits

137

acetylcholinesterase in muscle and brain of the fish Prochilodus lineatus, Chemosphere, 78

138

(2010) 294 - 299. https://doi.org/10.1016/j.chemosphere.2009.10.047

139 140

Ortiz-Ordoñez E., López-López E., Sedeño-Díaz J., Uría E., Morales I., Pérez M., and

141

Shibayama M. Liver histological changes and lipid peroxidation in the amphibian

142

Ambystoma mexicanum induced by sediment elutriates from the Lake Xochimilco.

24

Journal Pre-proof

143

International Journal of Environmental Science and Technology, 46 (2016), 156 - 164.

144

https://doi.org/10.1016/j.jes.2015.06.020.

145 146

Pandey A., Dhabade P., and Kumarasamy A. Inflammatory effects of subacute exposure of

147

Roundup in rat liver and adipose tissue. Dose-Response, 17 (2019), 1 - 11. DOI:

148

10.1177/1559325819843380

149 150

Paunescu A., Ponepal C.M., Drghici O., and Marinescu A.G. Liver Histopathologic

151

alterations in the frog Rana (Pelophylax) Ridibunda induce by the action of rendan 40EC

152

insecticide. Analele Universitatii din Oradea Fascicula Biologie, 17 (2010), 166 - 169.

153 154

Paunescu A., Ponepal C.M., Grigorean V.T., and Popescu M. Histopathological changes in

155

the liver and kidney tissue of marsh frog (Pelophylax ridibundus) induced by the action of

156

Talstar 10EC insecticide. Analele Universitatii din Oradea Fascicula Biologie, XIX (1)

157

(2012), 5 -10.

158 159

Paunescu A., Soare L.C., Fierascu R.C., Fierascu I., and Ponepal M.C. The influence of six

160

pesticides on physiological indices of Pelophylax ridibundus (Pallas, 1771). Bulletin of

161

Environmental Contamination and Toxicology, 100 (3) (2018), 376 - 383.

162

https://doi.org/10.1007/s00128-018-2277-9.

163 164

Pérez-Iglesias J.M., Franco-Belussi L., Moreno L., Tripole S., De Oliveira C., and Natale

165

G.S. Effects of Glyphosate on hepatic tissue evaluating melanomacrophages and

25

Journal Pre-proof

166

erythrocytes responses in anuran Leptodactylus latinasus. Environmental Science and

167

Pollution Research, 10 (2016), 9852 - 9861. https://doi.org/10.1007/s11356-016-6153-z.

168 169

Piloni N., Perazzo J., Fernandez V., Videla L., and Puntarulo S. Sub-chronic iron overload

170

triggers oxidative stress development in rat brain: implications for cell protection.

171

BioMetals, 29 (2016), 119 - 130. https://doi.org/10.1007/s10534-015-9902-4.

172 173

Relyea R. The lethal impact of roundup on aquatic and terrestrial amphibians. Ecol Appl,

174

(2005); 19 (1), 276-276.

175 176

Rissoli R.Z., Abdalla F.C., Costa M.J., Rantin F.T., McKenzie D.J., and Kalinin A.L.

177

Effects of glyphosate and the glyphosate based herbicides Roundup Original® and

178

Roundup Transorb® on respiratory morphophysiology of bullfrog tadpoles. Chemosphere,

179

156 (2016), 37 - 44. https://doi.org/10.1016/j.chemosphere.2016.04.083.

180 181

Rondón-Barragán I. S., Marin-Mendez G., Chacón-Novoa., Naranjo-Suarez L., Pardo-

182

Hernández D., and Eslava-Mocha P.R. El glifosato (Roundup®) y Cosmoflux® 411F

183

inducen estrés oxidativo en cachama blanca (Piaractus brachypomus). Orinoquia, 16 (2)

184

(2012), 162 - 166. https://www.redalyc.org/pdf/896/89659212002.pdf.

185 186

Shi Q., Sun N., Kou H., Wang H., and Zhao H. Chronic effects of mercury on Bufo

187

gargarizans larvae: Thyroid disruption, liver damage, oxidative stress and lipid metabolism

188

disorder. Ecotoxicology and Environmental Safety, 164 (2018), 500 - 509.

189

https://doi.org/10.1016/j.ecoenv.2018.08.058. 26

Journal Pre-proof

190 191

Simon M., Puky M., Braun M., and Tóthmérész B. Frogs and toads as biological indicators

192

in environmental assessment. Murray JL (ed.), Frogs: Biology, Ecology and Uses, (2011),

193

Hauppauge, Nueva York: Nova Science Publishers Inc.

194 195

Sinhorin V., Sinhorin A., Teixeira J., Miléski K., Hansen P., Moreira P., Kawashita N.,

196

Baviera A., and Loro V. Effects of the acute exposition to glyphosate-based herbicide on

197

oxidative stress parameters and antioxidant responses in a hybrid Amazon fish surubim

198

(Pseudoplatystoma sp). Ecotoxicology and Environmental Safety, 106 (2014), 181 - 187.

199

doi: 10.1016/j.ecoenv.2014.04.040.

200 201

Steinel N., and Bolnick D. Melanomacrophage Centers As a Histological Indicator of

202

Immune Function in Fish and Other Poikilotherms. Frontiers in Immunology, 8 (2017),

203

827. https://doi.org/10.3389/fimmu.2017.00827.

204 205

Solomon K., Anadon A., Cerdeira A., Marshall J., and Sanin L. Estudio de los efectos del

206

Programa de Erradicación de Cultivos Ilícitos mediante la aspersión aérea con el herbicida

207

Glifosato (PECIG) y de los cultivos ilícitos en la salud humana y en el medio ambiente.

208

(2005). Informe preparado para la Comisión Interamericana para el Control del Abuso de

209

Drogas (CICAD), División de la Organización de los Estados Americanos (OEA)

210

Washington, D.C., Estados Unidos de América. http://scm.oas.org/pdfs/2007/CP17420-

211

S.pdf.

212

27

Journal Pre-proof

213

Sparling D., Linder G., Bishop C., and Krest S. Ecotoxicology of amphibians and reptiles.

214

CRC Press. (2010). SETAC Books, Boca Raton, Florida. Second Edition. 916.

215

Svartz G., Sandoval M.T., Gosatti M., Pérez S., and Pérez C. Lethality, neurotoxicity,

216

morphological, Histological and celular alterations of Ni-Al nanoceramics on the embryo-

217

larval development of Rhinella arenarum. Environmental Toxicology and Pharmacology,

218

69 (2019), 36 - 43. https://doi.org/10.1016/j.etap.2019.03.020.

219 220

Tarazona J.V., Court-Marques D., Tiramani M., Reich H., Pfeil R., Istace F., and

221

Crivellente F. Glyphosate toxicity and carcinogenicity: A review of the scientifc basis of

222

the European Union assessment and its differences with IARC. Archives of Toxicology, 91

223

(2017), 2723 - 2743. https://doi.org/10.1007/s00204-017-1962-5.

224 225

Tarone R.E. On the International Agency for Research on Cancer classification of

226

glyphosate as a probable human carcinogen. European Journal of Cancer Prevention, 27

227

(2018), 82 - 87. https://doi.org/10.1097/CEJ.0000000000000289.

228 229

The IUCN Red List of Threatened Species. Version 2019-1 (IUCN, 2019);

230

http://www.iucnredlist.org/. (Consulted at 11/08/2019).

231 232

Torretta V., Katsoyiannis I.A.,Viotti P., and Rada E.C. Critical Review of the Effects of

233

Glyphosate Exposure to the Environment and Humans through the Food Supply Chain.

234

Sustainability, 10 (2018), 950.

235

28

Journal Pre-proof

236

United Nations Office on Drugs and Crime (UNODC). Colombia Monitoreo de cultivos de

237

Coca. Bogota, Gobierno de Colombia (2014). https://www.unodc.org/documents/crop-

238

monitoring/Colombia/Colombia_Monitoreo_de_Cultivos_de_Coca_2014_web.pdf.

239 240

Wagner N., Lötters S., Veith M., and Viertel B. Acute Toxic Effects of the Herbicide

241

Formulation Focus® Ultra on Embryos and Larvae of the Moroccan Painted Frog,

242

Discoglossus scovazzi. Archives of Environmental Contamination and Toxicology, 69 (4)

243

(2015), 535 - 544. https://doi.org/10.1007/s00244-015-0176-1.

244 245

Wu C., Zhang Y., Chai L., and Wang H. Histological changes, lipid metabolism and

246

oxidative stress in the liver of Bufo gargarizans exposed to cadmium concentrations.

247

Chemosphere, 179 (2017), 337 - 346. https://doi.org/10.1016/j.chemosphere.2017.03.131.

248 249

Zar J. Biostatistical Analysis. Prentince Hall, (2010), 5. New Jersey. USA. ISBN: 97.

29

Journal Pre-proof

CRediT autor statement Camilo Riaño: Methodology, Formal Analysis, Investigation, Writing –Original Draft, Visualization Mónica Ortiz: Methodology, Investigation, Writing –Original Draft Nelsy R Pinto-Sánchez: Conceptualization, Methodology, Resources, Writing – Review & Editing, , Visualization, Supervision, Funding Acquisition Edwin Gómez: Conceptualization, Methodology, Resources, Writing – Review & Editing, Supervision, Project Administration, Funding Acquisition

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

Effects of glyphosate-based herbicides in South-American endemic tadpole frogs

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Sinusoidal dilatation, cytoplasmic vacuolization are concentration-dependent glyphosates



Disorganization of the endoplasmic reticulum at high glyphosate concentration

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The high-resolution optical microscopy permits observation lipid alterations