Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e51733, enero-diciembre 2023 (Publicado May. 16, 2023)

High frequency of larval abnormalities of the toad Rhinella arenarum

(Anura: Bufonidae) in an Argentinian agroecosystem

Jesica A. Sansiñena1; https://orcid.org/0000-0003-2440-6462

Silvia E. Plaul2*; https://orcid.org/0000-0001-7318-7863

María F. Bahl1; https://orcid.org/0000-0003-0233-5157

Andrés Piccinini1; https://orcid.org/0000-0002-2655-8139

Pedro F. Andrés Laube2; https://orcid.org/0000-0002-3122-3798

Leandro Alcalde3; https://orcid.org/0000-0002-4365-2434

Guillermo S. Natale1; https://orcid.org/0000-0001-8895-448X

1. Centro de Investigaciones del Medio Ambiente (CIM) UNLP-CONICET, Departamento de Química, Facultad de

Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Buenos Aires, Argentina;

jesicasansi@quimica.unlp.edu.ar, fbahl@quimica.unlp.edu.ar, AgPiccinini@gmail.com,

gnatale@quimica.unlp.edu.ar

2. Laboratorio de Histología y Embriología Descriptiva, Experimental y Comparada (LHYEDEC), Facultad de Ciencias

Veterinarias, Universidad Nacional de La Plata, La Plata, Buenos Aires, Argentina; splaul@fcnym.unlp.edu.ar

(*Correspondence), pfandreslaube@gmail.com

3. Instituto de Limnología “Dr. Raúl A. Ringuelet”- Sección Herpetología, CONICET, La Plata, Buenos Aires,

Argentina; alcalde@ilpla.edu.ar

Received 06-VII-2022. Corrected 02-II-2023. Accepted 28-IV-2023.

ABSTRACT

Introduction: The frequent use of pesticides is currently considered a cause of environmental pollution due to

the high rate of entry of these substances into agroecosystems. This constitutes a risk for the species that inhabit

these ecosystems, in particular anurans whose characteristics make them prone to exposure to and interaction

with environmental pollutants.

Objective: To report the occurrence of abnormalities in larvae of the common toad Rhinella arenarum inhabit-

ing ponds surrounded by agroecosystems.

Methods: In two consecutive springs (2015 and 2016), reproductive events of common toads were monitored

in temporary pond systems in agricultural and non-agricultural areas, located near the city of La Plata (Buenos

Aires, Argentina). The physicochemical parameters of the ponds were measured, and the stage of each repro-

ductive event was recorded, such as the numbers of adult toads, amplexus and clutches. In the laboratory, the

larvae were measured and photographed, their stage of development was recorded, and their morphology was

examined under a stereomicroscope. Representative samples (normal and abnormal) from each pond studied

were processed for histopathological analysis.

Results: In the field studies carried out on a population of R. arenarum collected in an agroecosystem, a lower

number of reproductive adults and clutches were observed in relation to the population of a non-agricultural

pond. A total of 1 910 larvae were collected: 529 and 1 381 larvae from ponds located in non-agricultural and

agricultural areas, respectively. Larvae from the agroecosystem showed two types of abnormalities: severe tail

flexure and abdominal bloating. In addition, five degrees of severity could be determined in relation to abdomi-

nal bloating.

https://doi.org/10.15517/rev.biol.trop..v71i1.51733

CONSERVATION

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e51733, enero-diciembre 2023 (Publicado May. 16, 2023)

INTRODUCTION

Agricultural production accompanying

population growth has increased worldwide

since the beginning of the 20th century (Car-

valho, 2017). Such increasing was possible

due to monoculture practices of genetically

modified species, a technology that led to using

multiple phytosanitary products (Bindraban

et al., 2009). The frequent use of pesticides is

currently considered a cause of environmental

pollution due to the high entrance rates these

substances into agroecosystems. Although

these products were designed to have a toxic

effect at low concentrations on target organ-

isms, many of them have high toxicity on non-

target organisms, and they also have potential

for bioaccumulation and persistence in differ-

ent environmental compartments such as soil,

water and sediment (Evenson & Gollin, 2003;

Primost et al., 2017). This situation consti-

tutes a risk for the species that inhabit these

ecosystems, in particular anurans that have

characteristics in their modes of life that make

them prone to exposure and interaction with

environmental pollutants (Agostini et al., 2013;

Brodeur et al., 2011; Natale et al., 2018; Pérez-

Iglesias et al., 2015). Furthermore, the low

migratory capacity of anurans (Sinsch, 1990)

makes them good indicators of the environment

they live (Brodeur & Vera-Candioti, 2017;

Blaustein & Wake, 1995; Guzy et al., 2012).

For more than two decades studies have

detected a decrease of amphibian populations

Conclusions: This work reports the high frequency and severity of abnormalities observed in the early stages of

R. arenarum larvae living within an agroecosystem, providing evidence of the negative impact that agricultural

activities cause on aquatic ecosystems surrounded by farming areas.

Key words: anuran larvae; abdominal bloating; histopathological studies; common toad; crop areas.

RESUMEN

Alta frecuencia de anormalidades larvales del sapo Rhinella arenarum

(Anura: Bufonidae) en un agroecosistema argentino.

Introducción: El uso frecuente de plaguicidas es considerado actualmente una causa de contaminación ambien-

tal debido a las altas tasas de ingreso de estas sustancias a los agroecosistemas. Esta situación es un riesgo para

las especies que habitan en estos ecosistemas, en particular los anuros cuyas características los hacen propensos

a la exposición e interacción con contaminantes ambientales.

Objetivo: Informar la presencia de anormalidades en larvas del sapo común Rhinella arenarum que habitan en

estanques rodeados por un agroecosistema.

Métodos: En dos primaveras consecutivas (2015 y 2016), se monitorearon los eventos reproductivos del sapo

común proveniente de sistemas de estanques temporales ubicados en zonas agrícolas y no agrícolas, cerca de

la ciudad de La Plata (Buenos Aires, Argentina). Se midieron los parámetros fisicoquímicos de los estanques

y se registraron las etapas de cada evento reproductivo como el número de sapos adultos, amplexos y nidadas.

En el laboratorio, las larvas fueron medidas y fotografiadas, se registró su estado de desarrollo y se examinó la

morfología de cada una bajo microscopio estereoscópico. Se procesaron muestras representativas (normales y

anormales) de cada estanque estudiado para análisis histopatológico.

Resultados: En la población de R. arenarum que vive dentro de un agroecosistema, se observó un menor número

de adultos reproductores y puestas en relación con la del estanque en la zona no agrícola. Se recolectaron un total

de 1 910 larvas: 529 y 1 381 larvas de estanques ubicados en zonas no agrícolas y agrícolas, respectivamente. Las

larvas del agroecosistema mostraron dos tipos de anormalidades: severa flexión de la cola y distensión abdomi-

nal. Además, se pudo determinar cinco grados de gravedad en relación con la distensión abdominal.

Conclusiones: Una alta frecuencia y severidad de anormalidades en los estadios tempranos de larvas de R. are-

narum que viven dentro de un agroecosistema proporciona evidencia del impacto negativo que las actividades

agrícolas causan en los ecosistemas acuáticos rodeados por áreas de cultivo.

Palabras clave: larvas de anuro; distensión abdominal; estudios histopatológicos; sapo común; áreas de cultivo.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e51733, enero-diciembre 2023 (Publicado May. 16, 2023)

at a global level (Croteau et al., 2008; Collins

et al., 2009; Houlahan et al., 2000; Sparling et

al., 2010; Young et al., 2004). Several factors

have been proposed as possible causes for this

phenomenon, such as destruction, fragmenta-

tion, and pollution of the habitat caused mainly

by the advance of the agricultural frontier,

and by intensive agricultural practices (crops

under cover) and extensive agricultural practic-

es of monocultures (Beebee & Griffiths, 2005;

Davidson et al., 2002).

Environmental levels of pesticides can

generate sublethal effects from short-term

(acute) and long-term (chronic) exposures.

These effects can be evaluated with biomark-

ers at different levels such as biochemical,

physiological, histological, and morphological

(Agostini et al., 2013; Brodeur et al., 2011;

Lajmanovich et al., 2011; Natale et al., 2018;

Pérez-Iglesias et al., 2015; Venturino et al.,

2003). Effects that occur at higher levels usu-

ally have a greater ecological relevance (Baird

et al., 2007) as may do the occurrence of mor-

phological abnormalities, which are expressed

at an organism level, but could have popula-

tion-level effects (Johnson et al., 2001). Most

of the studies that evaluated the occurrence of

abnormalities were performed in the laboratory

because of exposure to pesticides, detecting

abnormalities especially during the embry-

onic and larval stages (Aronzon et al., 2011;

Brunelli et al., 2009; Boccioni et al., 2020).

Field studies of abnormalities are scarce and

are mainly focused on adult anurans (Hopkins

et al., 2000; Peltzer et al., 2011; Silva & Toledo,

2010). Henle and Dubois (2017) synthesize the

information on abnormalities in the external

morphology based on anecdotal observations

of a few individuals. Reeves et al. (2008),

Lunde and Johnson (2012), and Anzaldua and

Goldberg (2019) report the presence of abnor-

mal individuals in large numbers and percent-

ages, generally from areas called “hotspot”,

which, according to Lannoo (2008) correspond

to areas with a prevalence of abnormalities

equal to or greater than 5 %.

Within the framework of a long-term

monitoring project of anurans in Northeastern

Buenos Aires province (La Plata and its sur-

roundings) that started in 1996, our team

observed a high frequency of severe abnormali-

ties in the early larval stages of the common

toad Rhinella arenarum (Hensel, 1867) that

inhabited ponds located in agroecosystems. At

present, there are few histopathology reports

associated with abnormalities in anuran lar-

vae. The objective of the present work is to

report the occurrence of abnormalities in lar-

vae of the common toad in agroecosystems

by description through macroscopic and histo-

pathological analysis.

MATERIALS AND METHODS

In two consecutives springs (2015 and

2016), reproductive events of the common

toad were monitored in two temporary pond

systems located near the city of La Plata (Bue-

nos Aires, Argentina) (Fig. 1A). The first pond

(P1, Fig. 1B) is in an area without agriculture

(34°59’4.74” S, 57°51’21.98” W), dominated

by grasses and surrounded by renewals of

native Celtis tala (Gillies ex Planch, 1848)

trees and Cyperus spp. (Linnaeus, 1753). The

other pond (P2, Fig. 1C, Fig. 1D), is in an

area with intensive agriculture and floriculture

around the headwaters of Carnaval stream

(34°55’11.67” S, 58° 6’39.00” W). All of them

are in low areas that collect rainwater and can

be dried out.

The determination of the physicochemi-

cal parameters (temperature, conductivity, dis-

solved oxygen, and pH) was carried out in

situ at the moment in which the tadpoles were

found with a WA-2017SDu multiparametric.

Diurnal and nocturnal visual encounter

studies (Heyer, 1994) of R. arenarum were con-

ducted to verify presence and reproduction of

the species at each site. The reproductive sites

were located by auditory censuses during the

surveys of the study areas. Once the reproduc-

tive sites were detected, stage of each reproduc-

tive event was recorded, such as the numbers

of adult toads, amplexus and clutches. Larvae

were anesthetized with MS 222, fixed in 10 %

formaldehyde, and carried out to the laboratory.

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Subsequently, each larva was photographed

and the body length in mm was recorded, using

version 1.46r of ImageJ® program (Rasband,

1997), and the stage of development was

determinate according to Gosner (1960). In

addition, they were examined macroscopically

to record possible anomalies using the atlas of

abnormalities proposed by Bantle (1991). All

procedures were performed under an Olympus

CX31 stereomicroscope.

In addition, representative specimens (both

normal and abnormal) of each studied pond

were processed for histopathological analysis.

The fixed larvae were routinely processed

and embedded in paraffin wax (Plaul et al.,

2017). Histological sections were obtained

using a sledge microtome, prepared according

to standard protocols, and then stained with

hematoxylin and eosin (H&E) and Masson’s

trichrome techniques. The stained slides were

observed and photographed under a Leica

DM500 microscope with an integrated Leica

camera (model ICC50 W).

Statistical analyses were performed

according to Zar (2013) using version 3.1.1 of

R software (R Development Core Team, 2014).

Physicochemical parameters were evaluated

using descriptive statistics and compared using

ANOVA. Descriptive statistics were applied

for the evaluated variables (length of the body,

stage of larval development and abnormalities).

Comparisons the body length the tadpoles

between sites were made using a t-test for

mean differences. Normality and homoscedas-

ticity were corroborated by Shapiro-Wilk and

Bartlett tests. In the cases where the assump-

tions were not fulfilled and for the larval

stage of development, the comparisons were

Fig. 1. Sampling sites for Rhinella arenarum population. A. Location of both temporary ponds near of La Plata city (Buenos

Aires, Argentina), P1: non-agriculture area, P2: agriculture area. B. Photography of pond without agricultural activity. C.-D.

Photography’s of ponds surrounded by cultivated areas.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e51733, enero-diciembre 2023 (Publicado May. 16, 2023)

made using a non-parametric Kruskal-Wallis

test. The level of significance was set at 0.05

for all tests.

RESULTS

The physicochemical parameters of both

studied sites are in Table 1. In the two years

sampled, the water temperature and conduc-

tivity of P2 were significantly higher (F [12,

13.52] = 20.989, P < 0.001, F [3, 8] = 52.384,

P = 0.00001), and dissolved oxygen was sig-

nificantly lower in P2 only in the second year

of sampling (F [3, 8] = 25.652, P = 0.00019).

In P1 we registered 28 adult toads and

seven amplexus in the first year, and 50 adult

toads and 17 amplexus in the second year.

Larvae from this site have shown normal

swimming behavior like as they distributed in

dense groups of active swimming and moved

browsing in the bottom and between rocks and

plants. In P2 a smaller number of adults (14-

20) and amplexus (4-5) were observed during

the first and second year of study respectively.

Larvae shown abnormal swimming behavior,

they were scattered, floating on the surface of

the water and did not move or sink.

In spring of 2015 and 2016 a total of 529

larvae were collected from P1 and 1 381 from

P2. Larvae from P2 presented a size (t [1908]

= 29.56, P < 0.00) and staged (H [1, 1910] =

1295.87, P < 0.001) significantly lower than

larvae from P1 (Table 2).

Larvae collected in P1 did not present any

kind of abnormality (Fig. 2A, Fig. 3A) whilst

672 of the 1381 (48.7 %) larvae collected in

P2 have presented abnormalities at both mac-

roscopic and microscopic levels.

Macroscopic abnormalities observed were

mainly of two types: severe tail flexure (Fig.

2B) and abdominal bloating (Fig. 2C). The

analysis of these abnormalities showed that

29.7 % of the abnormal larvae had severe tail

flexure in 2015 and 29.6 % in 2016; and in the

case of the abdominal bloating, the percentage

was 64.8 % in 2015 and 4.19 % in 2016.

The observation under stereoscopic micro-

scope of the individuals with abdominal bloat-

ing allowed identification several degrees of

severity which, through histopathological stud-

ies, were classified into 5 categories (Fig. 3B

to Fig. 3F):

Grade 0 - Normal individuals. Spiralized intes-

tine, size, and arrangement normal. In the

microscopic analysis the organs did not

show morphological alterations. (Fig. 3A,

Fig. 4A).

Table 1

Physicochemical parameters measured in situ of the temporary ponds P1 (non-agricultural) and P2 (agroecosystem) during

the sampling years (2015-2016).

Temporary ponds Years Temperature °C pH ConductivityμSiemens/m OD mg/L

P1 2015 21.5 ± 0.44 7.43 ± 0.15 0.670 ± 0.05 6.47 ± 0.15

2016 22.5 ± 0.55 7.20 ± 0.20 0.638 ± 0.11 5.73 ± 0.35

P2 2015 23.3 ± 1.06 6.89 ± 0.78 0.879 ± 0.04 7.06 ± 0.40

2016 24.9 ± 0.10 7.43 ± 0.21 0.877 ± 0.02 4.63 ± 0.45

Table 2

Body length (mean ± SD) and larval development stage (mean ± SD) of R. arenarum collected in 2015 and 2016 in the

temporary ponds P1 and P2.

Years Total of individuals Temporary ponds Body length (mm) Stage development

2015 200 P1 4.84 ± 0.26 26 ± 0.26

1 000 P2 3.30 ± 0.11 24 ± 0.10

2016 329 P1 4.55 ± 0.48 25 ± 0.48

381 P2 4.40 ± 0.43 25 ± 0.43

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Grade 1 - Individuals with mild abdominal

bloating. The intestine shown a normal spi-

ral structure but with thickened intestinal

loops. Histologically, a slight displacement

of the intestine into one side was observed

with a slight bloating of the bowel and

stomach (Fig. 3B).

Grade 2 - Individuals with moderate abdo-

minal bloating. Intestine with digestive

content, displaced to the right side of the

abdominal region, a slight deformation of

the spiral structure and thickening of the

loops were observed. In the histological

samples, a hypertrophy of the wall of

the digestive tract was observed with an

intense hemorrhage in the stomach and

in some intestinal loops. Furthermore, the

loops were displaced, resulting in an oval

shape of the body in cross section (Fig.

3C, Fig. 4B).

Grade 3 - Individuals with moderate abdomi-

nal bloating and loss tissue architecture.

Although the observation of the organs in

terms of their location and appearance was

very similar to the previous grade differen-

ces were found at histological level. Sto-

mach and intestine were not differentiated

from each other, due to the thinning of

the digestive tract wall, which results in

the loss of the distinctive features of each

organ, such as villi and folds. The entire

digestive tract was filled with food (Fig.

3D, Fig. 4C).

Grade 4 - Individuals with severe abdominal

bloating. The intestine lost the typical

spiral structure and presented thickened

and displaced loops. Histologically, as in

grade 3, the wall of the digestive tract lost

its characteristic architecture and thins,

in addition, edema between the intestinal

loops and the body wall was observed (Fig.

3E, Fig. 4C).

Grade 5 - Individuals with severe abdomi-

nal bloating and compact intestine. Under

stereoscopic microscope, the tegument at

level abdominal region was shown extre-

mely thin. Due to transparency, reduction

or compaction of the intestine and a partial

loss of its spiral shape were observed. Free

digestive content could also be seen within

the abdominal cavity. Histological samples

corroborated the rupture of the walls of

the digestive tract with great cell desqua-

mation and hemorrhage (Fig. 3F, Fig. 4D).

Fig. 2. Abnormalities detected in larvae of Rhinella arenarum. A. Dorsal and ventral views of a normal larva without

morphologic abnormalities. B. Severe tail flexure (arrow). C. dorsolateral and ventrolateral views of the abdominal bloating

(arrows).

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e51733, enero-diciembre 2023 (Publicado May. 16, 2023)

Fig. 3. Rhinella arenarum larvae, observation under stereoscopic microscope and longitudinal and transverse histological

sections. A. Grade 0, normal larva. B. Grade 1, mild abdominal bloating. C.-D. Grade 2/3, moderate abdominal bloating,

respectively. E. Grade 4, severe abdominal bloating. F. Grade 5, severe abdominal bloating, and compact intestine. H&E and

Masson’s trichrome techniques. Scale bar: 500 μm.

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The percentages presented by each degree

(G) of abdominal bloating can be seen in

Table 3.

Table 3

Percentages of different degrees (G) of abdominal bloating

for 2015 and 2016 samples from P2.

G 0 G 1 G 2 G 3 G 4 G 5

2015 35.20 13.7 4.3 11.2 16.1 19.5

2016 95.82 1.57 ─ 0.26 1.83 0.52

DISCUSSION

The wetlands of the Pampean plain cover

a great part of the Buenos Aires province and

sit on soils of high productive potential that

experiences frequent annual and interannual

cycles of flooding and drainage. The inten-

sive agriculture in the pampean plain brings

an increase in the trophic state of the lagoons

that putatively could lead to a simplification

of biotic communities and a significant loss

of biodiversity (Quirós et al., 2002). In the

surroundings of the La Plata city, farming,

floriculture, and intensive and extensive agri-

culture are practiced. In recent years, studies

have determined presence of environmental

pollutants in the area, both in water and in

sediments (Agostini et al., 2013; Camilión et

al., 2003; Demetrio, 2012; Mac Loughlin et

al., 2017; Natale, 2006; Peluso, 2011; Peluso

et al., 2011; Sansiñena et al., 2018). Published

analyses performed in our both sampling sites

that surround La Plata city showed that water

from P1 has very low concentrations of heavy

metals, mainly iron and manganese, which are

not detectable or close to the detection limit

with the 3050 digestion method of EPA SW

Fig. 4. Histology of the intestinal wall in larvae of Rhinella arenarum. A. Normal intestine. B. Bowel with grade 2

abnormality, there is intense bleeding (arrows) in the digestive tract. C. Bowel with grade 3/4 abnormality, note thinning

of the bowel wall (arrow). D. Bowel with grade 5 abnormality, digestive tract wall rupture, loss of tissue architecture, cell

peeling, and bleeding. Masson’s trichrome technique. Scale bar: 50 μm.

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846. In addition, no traces of pesticides in the

water or in the sediment were found at this site

(Demetrio, 2012; Natale, 2006; Peluso, 2011;

Peluso et al., 2011). In contrast, in P2 a high

concentration of lead and zinc were detected in

the sediment samples (Camilión et al., 2003)

as well as pesticides (Agostini et al., 2013;

Mac Loughlin et al., 2017; Sansiñena et al.,

2018). In addition, it was corroborated that 13

types of pesticides were applied in the P2 dur-

ing the study period (pers. com.). Based on all

this information we are in position to suspect

that the high frequency of larvae abnormali-

ties observed in P2 is surely a consequence of

intensive agricultural activity that takes place

in this site, which causes deterioration in the

quality of environment due to contamination of

water and sediments with phytosanitary prod-

ucts (mainly pesticides).

In the two reproductive events studied

(2015 and 2016) we found, in P2 a lower

number of amplexus and clutches than P1,

a question that denotes that population of R.

arenarum toads are possibly lower in ponds

surrounded by cultivated areas.

In 2015 the larvae from P2 presented small-

er size and development than those observed in

P1. Although it may be due to population dif-

ferences in the development and growth rates,

we know that effects of climate are the same for

both sites studied, since there are distanced by

no more than 20 km in a straight line. Thus, we

must consider that the high amounts of abnor-

mal larvae and the high level of mortality we

observed in P2 are due to extrinsic factors. The

detected abnormalities had an evident effect

on the behavior of the larvae which presented

several difficulties to swim, fed, and finally

caused his death. Therefore, the reduced fit-

ness may be the reason why the larvae present-

ed less growth and development (Denoël et al.,

2012). The total percentage of abnormal larvae

observed by these authors far exceeds 5 %.

This number is, according to Read (1997), the

basal level of natural occurrence of abnormali-

ties in anuran larvae. Severtsova et al. (2012)

reported a higher percentage of larval abnor-

malities, 18 % in Rana temporaria (Linnaeus,

1758) and 16.5 % in Rana arvalis (Nilsson,

1842). These authors associated these high

levels of abnormalities with urban impacts.

Furthermore, Anzaldua and Goldberg (2019)

described a high concentration of Osteopilus

septentrionalis (Duméril & Bibron, 1841) lar-

vae with morphological abnormalities living

in drainage ditches that had been treated with

insecticide. Some of the abnormalities these

authors report were severe tail flexure and

spinal deviations or severe spinal curvatures

that clearly affect the motility of the larvae and

consequently their survival (Wilbur & Sem-

litsch, 1990). This abnormality was reported in

several laboratory works, and it was linked to

the effect caused by pesticides (Agostini et al.,

2010; Brunelli et al., 2009; Svartz et al., 2016)

and by heavy metals (Haywood et al., 2004;

Natale, 2006; Pérez-Coll et al., 1988). In field

work, the observation of this type of abnormal-

ity is scarce, and is generally associated with

different types of contamination, such as waste

from coal combustion (Hopkins et al., 2000),

wastewater (Ruiz et al., 2010), chlorides and

metals (Severtsova et al., 2012), and pesticides

(Anzaldua & Goldberg, 2019). According to

Bantle (1991), severe abnormalities in the tail

are associated with defects during the forma-

tion of the notochord but with no effects in

other body parts and organs. In the case of the

abnormal larvae studied by us they presented

tail flexure associated to abdominal bloating.

Lajmanovich et al. (2012) reported larvae

of Dendropsophus nanus (Boulenger, 1889)

with abdominal edema and inverted swimming

(belly up) coinciding with our observations in

R. arenarum. A similar case was registered by

Natale et al. (2018) in larvae of Boana pul-

chella (Duméril & Bibron, 1841) exposed to

the pesticide Aficida®. Toxicological tests per-

formed by Sansiñena et al. (2018) also showed

subcutaneous edema in larvae of B. pulchella

exposed to agroecosystem sediments. This

overview suggests that the abdominal bloating

observed in larvae of different species is related

to exposure to agrochemicals.

The analysis carried out in this work pro-

vides evidence on the occurrence of a series of

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deformities, such as alterations in swimming

caused by abnormalities in the tail, abdominal

bloating, or both. This abdominal bloating

would initially manifest itself as a thicken-

ing of the intestinal loops, generating a space

between the organs of the abdominal cavity

and the tegument, and in severe cases could

induce the rupture of the digestive tract wall.

All these events would lead to an inability to

digest and absorb food, with the consequent

accumulation and decomposition of food in the

digestive tract. This would cause, on the one

hand, a slower growth of the individual and, on

the other, a dysfunction of the whole organism,

which would trigger the death of the individual.

In conclusion, this work reports of an event

of singular relevance: the high frequency and

severity of abnormalities observed in the early

stages of R. arenarum larvae living within an

agroecosystem. The work achieves a detailed

description of the abnormalities observed at

both anatomical and histological level and

proposes a distinction of the several degrees

that the larvae suffer through during abdominal

bloating, and that could culminate in death.

Furthermore, it gives evidence on the nega-

tive impact that agricultural activity causes on

aquatic ecosystems that are surrounded by crop

areas, on the population dynamics, among other

population parameters, causing a population

decline. In addition, the finding in the field of

abnormal larvae is an event of great relevance

on the biology and ecology of the species, due

to its severity, and in the ecotoxicological con-

text raised, it is an indicator of the existence of

stress on the ecosystem, highlighting the role

of amphibians as indicators of the health of the

environment. Given the percentages of occur-

rence of malformations observed in the present

study, P2 could be a “hotspot” according to

criteria of Lanoo (2008), for which it is neces-

sary to continue with frequent systematic and

continuous monitoring at this site to achieve

an early detection and a complete record of

this type of events. Finally, it is necessary to be

aware of these events, which undoubtedly occur

and are not easy to detect unless prolonged and

detailed sampling is carried out in the field.

Ethical statement: the authors declare

that they all agree with this publication and

made significant contributions; that there is no

conflict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are

fully and clearly stated in the acknowledge-

ments section. A signed document has been

filed in the journal archives.

ACKNOWLEDGMENTS

This research was funded by PICT 2015-

3137 of ANPCyT. Sansiñena J. A. and Bahl

M. F. received scholarships from the Consejo

Nacional de Investigaciones Científicas y Téc-

nicas (CONICET). We are deeply grateful

to José Marinelli for allowing us to conduct

samplings in his field. We also express our

gratitude to Marina Scrocchi, who performed

the translation and revision of the manuscript.

REFERENCES

Agostini, M. G., Kacoliris, F., Demetrio, P., Natale, G. S.,

Bonetto, C., & Ronco, A. E. (2013). Abnormalities

in amphibian populations inhabiting agroecosystems

in northeastern Buenos Aires Province, Argentina.

Diseases of Aquatic Organisms, 104(2), 163–171.

Agostini, M. G., Natale, G. S., & Ronco, A. (2010).

Lethal and sublethal effects of cypermethrin to Hyp-

siboas pulchellus tadpoles. Ecotoxicology, 19(8),

1545–1550.

Anzaldua, S. P., & Goldberg, J. (2019). Hotspot of tadpole

abnormality in suburban south-west Florida. Herpe-

tological Journal, 29(2), 115–124.

Aronzon, C. M., Sandoval, M. T., Herkovits, J., & Pérez-

Coll, C. S. (2011). Stage-dependent toxicity of

2,4-dichlorophenoxyacetic on the embryonic develo-

pment of a South American toad, Rhinella arenarum.

Environmental Toxicology, 26(4), 373–381.

Baird, D. J., Brown, S. S., Lagadic, L., Liess, M., Maltby,

L., Moreira-Santos, M., & Scott, G. I. (2007). In situ-

based effects measures: Determining the ecological

relevance of measured responses. Environmental

Assessment and Management: An International Jour-

nal, 3(2), 259–267.

Bantle, J. A. (1991). Atlas of Abnormalities: A Guide for the

Performance of FETAX. Oklahoma State University,

Printing Services.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e51733, enero-diciembre 2023 (Publicado May. 16, 2023)

Beebee, T. J. & Griffiths, R. A. (2005). The amphibian

decline crisis: a watershed for conservation biology?

Biological Conservation, 125(3), 271–285.

Bindraban, P. S., Bulte, E. H., & Conijn, S. G. (2009). Can

large-scale biofuels production be sustainable by

2020? Agricultural Systems, 101(3), 197–199.

Blaustein, A. R., & Wake, D. B. (1995). The puzzle of

declining amphibian populations. Scientific Ameri-

can, 272(4), 52–57.

Boccioni, A. P. C., Lajmanovich, R. C., Peltzer, P. M., Atta-

demo, A. M., & Martinuzzi, C. S. (2020). Toxicity

assessment at different experimental scenarios with

glyphosate, chlorpyrifos and antibiotics in Rhinella

arenarum (Anura: Bufonidae) tadpoles. Chemosphe-

re, 273, 128475.

Brunelli, E., Bernabò, I., Berg, C., Lundstedt-Enkel, K.,

Bonacci, A., & Tripepi, S. (2009). Environmentally

relevant concentrations of endosulfan impair deve-

lopment, metamorphosis and behaviour in Bufo bufo

tadpoles. Aquatic Toxicology, 91(2), 135–142.

Brodeur, J. C., Suarez, R. P., Natale, G. S., Ronco, A. E.,

& Zaccagnini, M. E. (2011). Reduced body condi-

tion and enzymatic alterations in frogs inhabiting

intensive crop production areas. Ecotoxicology and

Environmental Safety, 74(5), 1370–1380.

Brodeur, J. C., & Vera-Candioti, J. (2017). Impacts of agri-

cult ure and pesticides on amphibian terrestrial life

stages: Potential biomonitor/bioindicator species for

the Pampa region of Argentina. In M. L. Larramendy

(Ed.), Ecotoxicology and Genotoxicology: Non-tra-

ditional Terrestrial Models Issues in Toxicology No.

32. (pp. 163–194). The Royal Society of Chemistry.

Camilión, M. C., Manassero, M. J., Hurtado, M. A., &

Ronco, A. E. (2003). Copper, lead and zinc distribu-

tion in soils and sediments of the south-western coast

of the Río de la Plata estuary. Journal of Soils and

Sediments, 3(3), 213–220.

Carvalho, F. P. (2017). Mining industry and sustainable

development: time for change. Food and Energy

Security, 6(2), 61–77.

Collins, J. P., Crump, M. L., & Lovejoy III, T. E. (2009).

Extinction in our Times: Global Amphibian Decline.

Oxford University Press.

Croteau, M. C., Davidson, M. A., Lean, D., & Trudeau, V.

(2008). Global increases in ultraviolet B radiation:

potential impacts on amphibian development and

metamorphosis. Physiological and Biochemical Zoo-

logy, 81(6), 743–761.

Davidson, C., Shaffer, H. B., & Jennings, M. R. (2002).

Spatial tests of the pesticide drift, habitat destruction,

UV-B, and climate-change hypotheses for California

amphibian declines. Conservation Biology, 16(6),

1588–1601.

Demetrio, P. M. (2012). Estudio de efectos biológicos de

plaguicidas utilizados en cultivos de soja RR y eva-

luación de impactos adversos en ambientes acuáticos

de agroecosistemas de la región pampeana. Univer-

sidad Nacional de La Plata.

Denoël, M., D’Hooghe, B., Ficetola, G. F., Brasseur, C.,

De Pauw, E., Thomé, J. P., & Kestemont, P. (2012).

Using sets of behavioral biomarkers to assess short-

term effects of pesticide: a study case with endosulfan

on frog tadpoles. Ecotoxicology, 21(4), 1240–1250.

Evenson, R. E., & Gollin, D. (2003). Assessing the impact

of the Green Revolution, 1960 to 2000’. Science,

300(5620), 758–762.

Gosner, K. L. (1960). A simplified table for staging anuran

embryos and larvae with notes on identification. Her-

petologica, 16(3), 183–190.

Guzy, J. C., McCoy, E. D., Deyle, A. C., Gonzalez, S. M.,

Halstead, N., & Mushinsky, H. R. (2012). Urbaniza-

tion interferes with the use of amphibians as indica-

tors of ecological integrity of wetlands. Journal of

Applied Ecology, 49(4), 941–952.

Haywood, L. K., Alexander, G. J., Byrne, M. J., &

Cukrowska, E. (2004). Xenopus laevis embryos and

tadpoles as models for testing for pollution by zinc,

copper, lead and cadmium. African Zoology, 39(2),

163–174.

Henle, K., & Dubois, A. (2017). Studies on anomalies in

natural populations of amphibians. Mertensiella, 25,

185–242.

Heyer, W. R. (1994). Variation within the Leptodactylus

podicipinus-wagneri complex of frogs (Amphibia:

Leptodactylidae). Smithsonian Contributions to Zoo-

logy, 446, 1–132.

Hopkins, W. A., Congdon, J., & Ray, J. K. (2000). Incidence

and impact of axial malformations in larval bullfrogs

(Rana catesbeiana) developing in sites polluted by a

coal-burning power plant. Environmental Toxicology

and Chemistry: An International Journal, 19(4),

862–868.

Houlahan, J. E., Findlay, C. S., Schmidt, B. R., Meyer, A.

H., & Kuzmin, S. L. (2000). Quantitative evidence

for global amphibian population declines. Nature,

404(6779), 752.

Johnson, P. T., Lunde, K. B., Ritchie, E. G., Reaser, J. K.,

& Launer, A. E. (2001). Morphological abnormality

patterns in a California amphibian community. Her-

petologica, 57(3), 336–352.

Lajmanovich, R. C., Attademo, A. M., Peltzer, P. M., Jun-

ges, C. M., & Cabagna, M. C. (2011). Toxicity of four

herbicide formulations with glyphosate on Rhinella

arenarum (Anura: Bufonidae) tadpoles: B-esterases

and glutathione S-transferase inhibitors. Archives of

12 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e51733, enero-diciembre 2023 (Publicado May. 16, 2023)

Environmental Contamination and Toxicology, 60(4),

681–689.

Lajmanovich, R. C., Peltzer, P. M., Attademo, A. M.,

Cabagna-Zenklusen, M. C., & Junges, C. M. (2012).

Los agroquímicos y su impacto en los anfibios: un

dilema de difícil solución. Química Viva, 11(3),

184–198.

Lannoo, M. J. (2008). Malformed frogs: The Collapse of

Aquatic Ecosystems. University of California Press.

Lunde, K. B., & Johnson, P. T. (2012). A practical guide for

the study of malformed amphibians and their causes.

Journal of Herpetology, 46(4), 429–442.

Mac Loughlin, T. M., Peluso, M. L., & Marino, D. J. G.

(2017). Pesticide impact study in the peri-urban hor-

ticultural area of Gran La Plata, Argentina. Science of

the Total Environment, 598, 572–580.

Natale, G. S. (2006). Análisis ecotoxicológico de una

comunidad de anuros de la Región Pampeana. Facul-

tad de Ciencias Naturales y Museo.

Natale, G. S., Vera-Candioti, J., Ruiz de Arcaute, C., Solo-

neski, S., Larramendy, M. L., & Ronco, A. E. (2018).

Lethal and sublethal effects of the pirimicarb-based

formulation Aficida® on Boana pulchella (Duméril

and Bibron, 1841) tadpoles (Anura, Hylidae). Eco-

toxicology and Environmental Safety, 147, 471–479.

Peluso, M. L. (2011). Evaluación de efectos biológicos y

biodisponibilidad de contaminantes en sedimentos

del Río de la Plata y afluentes. Facultad de Ciencias

Exactas.

Peluso, M. L., Giusto, A., Rossini, G. D. B., Ferrari, L.,

Salibián, A., & Ronco, A. E. (2011). Hyalella cur-

vispina (amphipoda) as a test organism in laboratory

toxicity testing of environmental samples. Fresenius

Environmental Bulletin, 20(2), 372–376.

Pérez-Coll, C. S., Herkovits, J., & Salibián, A. (1988).

Embryotoxicity of lead on Bufo arenarum. Bulletin

of Environmental Contamination and Toxicology,

41(2), 247–252.

Pérez-Iglesias, J., Soloneski, S., Nikoloff, N., Natale, G.

S., & Larramendy, M. (2015). Toxic and genotoxic

effects of the imazethapyr-based herbicide formula-

tion Pivot H® on montevideo tree frog Hypsiboas

pulchellus tadpoles (Anura, Hylidae). Ecotoxicology

and Environmental Safety, 119, 15–24.

Peltzer, P. M., Lajmanovich, R. C., Sanchez, L. C., Attade-

mo, A. M., Junges, C. M., Bionda, C. L., Martino, A.,

& Basso, A. (2011). Morphological abnormalities in

amphibian populations. Herpetological Conservation

and Biology, 6(3), 432–442.

Plaul, S. E., Andrés Laube, P. F., Pacheco Marino, S. G.,

Santamaría Martín, C. J., Moyano, D. A., & Barbei-

to, C. G. (2017). Morphological techniques used in

ichthyopathological diagnosis. In A. Méndez-Vilas

(Ed.), Microscopy and imaging science approaches

to applied research and education (pp. 269–280).

Badajoz.

Primost, J. E., Marino, D. J., Aparicio, V. C., Costa, J.

L., & Carriquiriborde, P. (2017). Glyphosate and

AMPA,“pseudo-persistent” pollutants under real-

world agricultural management practices in the

Mesopotamic Pampas agroecosystem, Argentina.

Environmental Pollution, 229, 771–779.

Quirós, R., Rosso, J. J., Rennella, A., Sosnovsky, A., &

Boveri, M. (2002). Análisis del estado trófico de las

lagunas pampeanas (Argentina). Interciencia, 27(11),

584–591.

R Development Core Team. (2014). R: A language and

environment for statistical computing. Vienna, Aus-

tria: R Foundation for Statistical Computing. Availa-

ble in: http://www.R-project.org/

Rasband, W. (1997). ImageJ. US National Institutes of

Health, Bethesda, MD, USA.

Read, J. (1997). Comparative abnormality rates of the

trilling frog at Olympic Dam. Herpetofauna-Sydney,

27, 23–27.

Reeves, M. K., Dolph, C. L., Zimmer, H., Tjeerdema, R. S.,

& Trust, K. A. (2008). Road proximity increases risk

of skeletal abnormalities in wood frogs from National

Wildlife Refuges in Alaska. Environmental Health

Perspectives, 116(8), 1009–1014.

Ruiz, A. M., Maerz, J. C., Davis, A. K., Keel, M. K., Ferrei-

ra, A. R., Conroy, M. J., Morris, L. A., & Fisk, A. T.

(2010). Patterns of development and abnormalities

among tadpoles in a constructed wetland receiving

treated wastewater. Environmental Science & Techno-

logy, 44(13), 4862–4868.

Sansiñena, J. A., Peluso, M. L., Costa, C. S., Demetrio, P.

M., Mac Loughlin, T. M., Marino, D. J., Alcalde, L.,

& Natale, G. S. (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. Ecological Indicators, 93,

100–110.

Severtsova, E. A., Aguillon-Gutierrez, D. R., & Severts-

ov, A. S. (2012). Frequent anomalies in larvae of

common and moor frogs in Moscow area and in the

Suburbs of Moscow, Russia. Russian Journal of Her-

petology, 19(4), 337–348.

Silva, N., & Toledo, L. (2010). Bokermannohyla saxicola

(NCN), Scinax curicica (Lanceback Treefrog), Sci-

nax squalirostris (Snouted Treefrog), Trachycephalus

mesophaeus (Golden-eyed Treefrog), and Elachis-

tocleis sp. (Oval Frog). Morphology Herpetological

Review, 41(3), 333–334.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e51733, enero-diciembre 2023 (Publicado May. 16, 2023)

Sinsch, U. (1990). Migration and orientation in anuran

amphibians. Ethology Ecology & Evolution, 2(1),

65–79.

Sparling, D. W., Linder, G., Bishop, C. A., & Krest, S.

(2010). Ecotoxicology of Amphibians and Reptiles.

CRC Press.

Svartz, G., Aronzon, C., & Coll, C. P. (2016). Comparative

sensitivity among early life stages of the South Ame-

rican toad to cypermethrin-based pesticide. Envi-

ronmental Science and Pollution Research, 23(3),

2906–2913.

Venturino, A., Rosenbaum, E., Caballero de Castro, A.,

Anguiano, O. L., Gauna, L., Fonovich de Schroeder,

T., & Pechen de D’Angelo, A. M. (2003). Biomarkers

of effect in toads and frogs. Biomarkers: biochemical

indicators of exposure, response, and susceptibility to

chemicals, 8(3–4), 167–186.

Wilbur, H. M., & Semlitsch, R. D. (1990). Ecological

consequences of tail injury in Rana tadpoles. Copeia,

1, 18–24.

Young, B. E., Stuart, S. N., Chanson, J. S., Cox, N. A., &

Boucher, T. M. (2004). Joyas que están desapare-

ciendo: El estado de los anfibios en el Nuevo Mundo.

NatureServe.

Zar, J. H. (2013). Biostatistical analysis. Pearson.