Chemosphere 250 (2020) 126287

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Effect of glyphosate (Roundup Active®) on liver of tadpoles of the

colombian endemic frog Dendropsophus molitor (amphibia: Anura)
Camilo Rian~ o a, *, Mo
 nica Ortiz-Ruiz a, Nelsy Rocío Pinto-Sa
nchez a,
Edwin Go mez-Ramírez a
a n, Medio ambiente y Conservacio
Grupo de ecotoxicología, Evolucio sicas y Aplicadas, Universidad Militar Nueva Granada,
n, Facultad de Ciencias ba
Colombia

h i g h l i g h t s

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

Sinusoidal dilatation, cytoplasmic vacuolization are concentration-dependent glyphosates

Disorganization of the endoplasmic reticulum at high glyphosate concentration.

The high-resolution optical microscopy permits observation lipid alterations.

a r t i c l e i n f o a b s t r a c t

Article history: Glyphosate-based herbicide (GBH) using is increasing on a global scale. Few studies have investigated the
Received 25 November 2019 sub-lethal effects of GBH in endemic amphibian species. The present work tested the GBH Roundup
Received in revised form Active® on the tadpoles of Dendropsophus molitor. The exposure was in a range of plausible environ-
13 February 2020
mental concentrations (0e0.75 mg a.e./L) during a month. D. molitor is an endemic tropical frog of South
Accepted 19 February 2020
Available online 21 February 2020
America. The exposure from 325 mg a.e./L caused histological alterations in the liver. The high-resolution
optical microscopy (HROM) detected sinusoidal dilatation and cytoplasmic vacuolization. The trans-
Handling Editor: David Volz mission 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
Keywords: histological alterations in liver tadpoles liver in a previous study, but, this study tested another endemic
Herbicide South-American frog for only 96h. The present work applied HROM to observe lipid alterations since it
Lipid vesicles does not use organic solvents; and TEM for the ultrastructural observation of hepatocytes. Environmental
Ultrastructure risk of GBH can improve by including sub-lethal effects in endemic species.
Tropical frog
© 2020 Elsevier Ltd. All rights reserved.
Histopathological alterations
Sublethal effects

1. Introduction producing harmful effects in animals and humans distributed

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,
* Corresponding author. Camilo Andre s Rian
~ o Quintero, Facultad de Ciencias
sicas y Aplicadas, Universidad Militar Nueva Granada, Cajic
Ba a, Cundinamarca,
Colombia.
~ o).
E-mail address: u0500743@unimilitar.edu.co (C. Rian
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 her-
bicide is widely used in agriculture activities related to weed con-
trol 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 Active®, the most
~ os, 2016). Roundup Active® is composed of
used formulation (Bolan
446 g/L potassium salt N- (phosphonomethyl) eglycine

https://doi.org/10.1016/j.chemosphere.2020.126287
0045-6535/© 2020 Elsevier Ltd. All rights reserved.
2 ~ o et al. / Chemosphere 250 (2020) 126287
C. Rian
(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 em-
bryonic development and organs of such as liver, epidermal
epithelium, or brain (Lancto ^t et al., 2014; Rissoli et al., 2016; Bach
et al., 2018; Bonfanti et al., 2018). For example, individuals of Lep-
todactylus 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 re-
ceptor, among other effects were reported for brain samples of
Lithobates sylvaticus (Ranidae) exposed to 0.21 mg a.e./L of Roundup
WeatherMax® (Lancto ^t 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 pollu-
tion, 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 m above sea
level and reproduces in lentic bodies of water (Me ndez-Narva ez,
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 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 he-
patocytes and melanomacrophagic centers (Steinel and Bolnick,
2017).
The aim was determine the effects of a commercial presenta-
tion of glyphosate (Roundup Active®) 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
(Torretta 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.
2. Materials and methods
2.1. Animals
We collected 54 tadpoles of D. molitor from an unpolluted
stream at Universidad Militar Nueva Granada, Cajica , Colombia
(4 560 3700 N - 74 000 3500 W, 2560 m.a.s.l) using a net. Tadpoles
were collected with the permission Res No 1198 given by the
Environmental 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 (Pe rez-Iglesias et al., 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) 0 mg/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.
2.2. Chemicals and experimental design
We evaluated the effects of glyphosate on amphibian tissues
using the commercial formulation glyphosate Roundup Active®
(Bayer Cropscience). This formulation composition is 363 g/L of
glyphosate acid (equivalent to 446 g/L of potassium salt of N-
(phosphonomethyl) eglycine), and surfactant, however, the com-
pany 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 mg glyphosate acid equivalent (a.e.)/L and the control
with 0 mg a.e./L, each one with three replicates prepared from
Roundup Active® (Henao et al., 2015). A total of 6 individuals per
treatment were analyzed (N ¼ 54). Test subjects were maintained
in semi-static systems with a total volume of 2 L 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 was carried out according to the
guidelines for the ethical management of fish and amphibians
(AVMA, 2013).
2.3. 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 con-
centrations 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 mm 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 micro-
scope (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) (Go  mez,
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
 ~ o et al. / Chemosphere 250 (2020) 126287
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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.
2.4. Statistical analyses
Data were transformed using Log10 in order to satisfy the
normality assumption for analysis. Differences in variables; num-
ber, 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).
3. Results
3.1. Water quality
The parameters pH (7.25e7.44), temperature (16.61  C e
16.35  C), dissolved oxygen (5.74 ppme5.67 ppm), ammoniacal
total nitrogen (0.04 ppme0.13 ppm) and nitrite (0 ppm) were
maintained during the 30 days of exposure to Roundup Active®
within the environmental ranges reported at Dendropsophus moli-
tor tadpoles collection site (4 560 3700 N - 74 000 3500 W,
2560 m.a.s.l) (see Table 1 )
In the Dendropsophus molitor tadpole liver of the control treat-
ment (0 mg/L) observed hepatocytes with poligonal shape, with a
prominent nucleus and one or two nucleoli. Hepatocytes arrange-
ments in sheet separated by sinusoids with few homogeneously
dispersed blood vessels. We observed few lipid vesicles and cyto-
plasmatic vacuoles (Fig. 1a). Ultrastructurally the rough endo-
plasmatic reticulum did not show structural alterations (Fig. 2a).
3.2. Histopathological effects
We observed higher number and area of lipid vesicles in the
treatments 325 and 750 mg a.e/L (Table 2, Fig. 3). We found lipid
vesicles and sinusoidal dilation using HROM in the three treat-
ments, being more abundant in the treatment with 750 mg a.e/L of
glyphosate. In all treatments, there was absence of mortality
(Table 2, Fig. 1).
Using TEM, we observed disorganization of the rough endo-
plasmatic reticulum, cytoplasmic vacuoles, and some lipid vesicles
(Fig. 2).
The variables related to the lipid vesicles after the Log10
transformations were normally distributed as follows, the average
occupied area (mm 2) of lipid vesicles (W ¼ 0.95, P ¼ 0.41), and the
number of lipid vesicles (W ¼ 0.93, P ¼ 0.20).
We found significant differences in the average occupied area
(mm2) of lipid vesicles (R2 ¼ 0.81, F ¼ 33.25, p  0.001), and number
of lipid vesicles (R2 ¼ 0.78, F ¼ 26.24, P ¼ <0.00) in both concen-
trations of glyphosate with respect to the control treatment (Fig. 3).
Table 1
Water quality parameters maintainedduring the 30 days of exposure of D. molitor to
three concentrations of Roundup Active® (0, 325 and 725 mg a.e./L). Data are
expressed as mean ± SEM.
Water quality parameters Roundup Active ® (mg a.e./L)
0 325 750
pH 7.300 ± 0.074 7.250 ± 0.074 7.440 ± 0.074
Temperature ( C) 16.530 ± 0.140 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
3
The comparisons between the two concentration of glyphosate 325
and 750 mg 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 mg a.e./L (difference of 1.29, p  0.001),
followed by the difference of 325 and 750 mg a.e./L (0.71, P ¼ 0.00),
and finally the difference of control and 325 mg a.e./L (0.57,
p  0.01).
4. Discussion
The Dendropsophus molitor tadpole liver of the control treat-
ment (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 (Figs. 1a and 2a). Those liver structure arrangement
were similar to that described for other amphibian species, which
have different grade of phylogenetical proximity with Dendrop-
sophus 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 (Pe rez-Iglesias et al., 2016), Ambystoma
mexicanum (Ortiz-Ordon ~ ez 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 histo-
pathological alterations after one-month exposure to Roundup
Active® in a range of 350e750 mg a.e./L. This chronic exposure
resulted in sinusoidal dilation, cytoplasmic vacuolation, and accu-
mulation of lipid vesicles (Figs. 1 and 3). Additionally, 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 alter-
ation can generate osmoregulatory problems in nutrient transport
and metabolism of waste products (Sparling et al., 2010). The si-
nusoidal 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 re-
ports 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 pe-
riods 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 mm in diameter)
and nucleus centrally located. This alteration indicates a micro-
vesicular steatosis which is caused by mitochondrial b-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 thi-
obarbituric acid reactive substances (TBARS) in the liver of Pseu-
doplatystoma sp, after exposure to Roundup Original® at
concentrations of 2.25, 4.5, 7.5 and 15 mg/L during 96h, the same
was found by De Menezes et al., (2011) in Rhamdia quelen exposed
to 0.45 and 0.9 mg/L of Roundup® for 48 h and Glusczak et al.
4 ~ o et al. / Chemosphere 250 (2020) 126287
C. Rian
Fig. 1. Cross-sections of the D. molitor liver. A. Liver section control treatment (0 mg/L). B. Liver section treatment with 325 mg a.e./L. C. Liver section treatment with 750 mg a.e./L.
The symbols are as follows lipid vesicles (red asterisks), sinusoidal dilation (black asterisks), vacuoles (arrows), hepatocytes (H), blood vessels (V). HROM technique. Bar ¼ 20 mm.
(2011) in Leporinus obtusidens exposed to 3, 6, 10 and 20 mg/L for
96 h. 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 mg/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 accumula-
tion, and apoptosis (Dornelles and Oliveira, 2013).
High levels of lipid peroxidation cause cytoplasmic vacuoliza-
tion in the liver (Ortiz-Ordon ~ ez et al., 2016), and also the accu-
mulation 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 Active® exposure, such as oxidative stress
and lipid disorder (El-Shenawy, 2009; Modesto and Martinez,
2010; Mili c et al., 2018). Moreover, the Roundup Active® expo-
sure increased the number of deposits of lipid vesicles on the pe-
riphery 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 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 (Rondo  n-Barraga
n et al., 2012). The registered alter-
ations seem to be a consequence of a physiological stress process
induced by chemical intoxication.
In acute exposure, is common found melanomacrophagic cen-
ters and the eosinophils accumulation are among the most com-
mon 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 Roundup Active® for one
month in the present study. The accumulation of melanomacro-
phagic centers and eosinophils was also observed in tadpoles of the
bullfrog Lithobates catesbeianus exposed to the herbicide Cloma-
zone 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 histopathol-
ogies related to glyphosate-based herbicides (GBH).Howe et al.
(2004) exposed tadpoles of Rana pipiens for 42 days to two GBHs;
Roundup Original®, Roundup Transorb® and POEA surfactant,
chronic exposure caused a decrease in snout e vent length, gonadal
histological abnormalities, tail damage and increased meta-
morphosis time. The exposure for 20 days of tadpoles from Rana
pipiens, Bufo americanus and Hyla versicolor to Roundup® in
 ~ o et al. / Chemosphere 250 (2020) 126287
C. Rian 5

A B
H *

Fig. 2. Dendropsophus molitor liver showing some ultrastructural changes A) Liver section control treatment (0 mg/L). B) Liver section treatment with 325 mg a.e./L. C) Liver section
treatment with 750 mg a.e./L. Hepatocytes (H), disorganization of the rough endoplasmic reticulum (black arrows) and cytoplasmic vacuoles (red arrows), Lipid vesicles (asterisk).
TEM technique. Bar ¼ 5 mm.

Table 2 microcosm conditions showed a significant increase in mortality in

Histopathologies alterations in the liver for D. molitor exposed to Roundup Active®. the three species, independent of the type of soil where the GBH
Values are expressed as a percentage.
was applied (Relyea, 2005). King and Wagner, (2010) exposed six
Roundup Active® (mg species of amphibians tadpoles; Ambystoma gracile, Ambystoma
a.e./L) macrodactylum, Anaxyrus boreas, Pseudacris regilla, Rana cascadae,
Histopathologies findings 0 325 750
and Rana luteiventris, for 16 days. They found that the amphibian
Non-homogeneous parenchyma 0 0 0 species evaluated respond differentially to the GBH, having a sur-
Melanomacrophage centers 0 0 0
vival time of 1 day for tadpoles of Pseudacris regilla and up to 16
Sinusoidal dilatation 0 20 80
Cytoplasmatic vacuolization 20 80 80 days for Ambystoma gracile in concentrations between 1 and 5 mg/
Pycnotic nuclei 0 0 0 L, concluding that chronic toxicological effects have a high variation
Lipid vesicles 20 60 80 among amphibian species. This study is the first report of chronic
Alteration of the rough endoplasmic reticulum 0 20 60 effects of GBH in the tadpole liver of the South-American endemic
Glycogen granules 0 0 0
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 concentra-
tion of Roundup Active®. These results indicate that environmental
risk assessment of GBH should consider endemic amphibian spe-
cies 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 re-
gions. At lower level of vertebrates that depend of water, distrib-
uted at Bogota we found amphibians such as Edwards’ Rocket Frog
Hyloxalus edwardsi and the Orphan salamander Bolitoglossa cap-
itana listed as critically endangered, the Elegant Robber Frog Pris-
Fig. 3. Number of lipid vesicles (LV) and filled occupied area of LV (mm2), present in the timantis elegans listed as vulnerable, and Bogota Tree frog
liver of D. molitor exposed to different glyphosate concentrations in Roundup Active®.
Data are expressed as mean ± SD.
Hyloscirtus bogotensis, Peters’ mushroomtongue salamander Boli-
toglossa 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
6 ~ o et al. / Chemosphere 250 (2020) 126287
C. Rian
of the Savannah Trichomycterus venulosus listed as critically en-
dangered, 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-president for research of
the Universidad Militar Nueva Granada in the project CIAS 2550.
CRediT authorship contribution statement
~ o: Methodology, Formal analysis, Investigation,
Camilo Rian
Writing - original draft, Visualization. Mo nica Ortiz-Ruiz: Meth-
odology, Investigation, Writing - original draft. Nelsy Rocío Pinto-
nchez: Conceptualization, Methodology, Resources, Writing -
Sa
review & editing, Visualization, Supervision, Funding acquisition.
Edwin Go  mez-Ramírez: Conceptualization, Methodology, Re-
sources, Writing - review & editing, Supervision, Project adminis-
tration, Funding acquisition.
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 au-
thors who helped improve the written document.
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