Ecotoxicology and Environmental Safety 205 (2020) 111314

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Ecological impacts of pesticides on Astyanax jacuhiensis (Characiformes:

Characidae) from the Uruguay river, Brazil
Carjone Gonçalves a, Aline Teixeira Marins a, Aline Monique Blank do Amaral a,
Mauro Eugênio Medina Nunes b, Talise Ellwanger Müller b, Eduardo Severo a, Adriane Feijó a,
Cíntia C.R. Rodrigues b, Renato Zanella c, Osmar Damian Prestes c, Barbara Clasen d,
Vania Lucia Loro a, b, *
a
Programa de Pós-Graduação Em Biodiversidade Animal, Brazil
b
Programa de Pós-Graduação Em Ciências Biológicas, Bioquímica Toxicológica, Laboratório de Toxicologia Aquática, Universidade Federal de Santa Maria (UFSM),
97105-900, Santa Maria, RS, Brazil
c
Laboratório de Análises de Resíduos de Pesticidas (LARP), Universidade Federal de Santa Maria, 97105.900, Santa Maria, RS, Brazil
d
Departmento de Ciências Ambientais, Universidade Estadual Do Rio Grande Do Sul, 98600-000, Três Passos, RS, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Brazilian freshwater ecosystems are continuously exposed to pesticides and domestic sewage. The Uruguay River
Biomarkers was chosen for this study because of its international importance, as it flows through Brazil, Argentina, and
Biomonitoring Uruguay. It receives contaminants such as pesticides and domestic residues. Thus, the aim of this study to assess
Characidae
the accumulation of pesticides in muscle of the fish Astyanax jacuhiensis, its biochemical responses, and the
Pollution
presence of pesticides in water. In total, seven pesticides were registered in water from both river sites. Eight
pesticides were detected in fish muscle. The biochemical responses showed that brain lipid peroxidation (LPO)
and protein carbonyl (PC) in A. jacuhiensis were higher in the summer. Muscle showed the highest LPO levels in
the spring and the highest PC in the summer. Liver LPO and PC levels were higher in the spring and summer. In
the gills, the PC was higher in the spring and the LPO in the spring and winter. In the brain and in the gills,
glutathione-S-transferase activity was high in the summer and autumn. Catalase activity was lower during the
winter and spring. Non-protein thiol (NPSH) levels were lower in the brain in the winter and spring. Muscle
tissue showed lower NPSH in the winter (site 1). Liver NPSH showed increased levels in liver in the spring and
winter (site 2). The biochemical results clearly is related to pesticides and/or to the presence of other contam­
inants in the water such as metals or domestic sewage. The accumulation of pesticides in fish muscle added
evidence that pesticides have been used in the area surrounding the Uruguay River. In conclusion, the bio­
markers assayed in the present study could be used in future investigations considering other sampling sites
along Uruguay River.

1. Introduction excessive nutrient enrichment, native biodiversity decline, habitat

Freshwater is vital to life, but it makes up only 0.01% of total world
water and supports at least 100,000 species, including fish. Despite its
importance to all ecosystems, freshwaters are some of the most threat­
ened ecosystems in the world (Pelicice et al., 2017; Reis et al., 2016).
Moreover, big river systems have economic importance and are
commonly used for transport, irrigation, recreation, fisheries, and to
supply drinking water. The ecological integrity of rivers has been
severely altered by human activities, resulting in flow modification,
degradation, and water/sediment pollution (Dudgeon et al., 2006; Jia
and Chen, 2013; Li et al., 2010). Pollutants as pesticides can disrupt the
physiology of many animals in aquatic environments, and thus affecting
the vital functions of all the organisms that make up the freshwater food
chain such as fish, benthic macroinvertebrates, mollusks, and amphib­
ians. However, these disturbances appear to be especially serious for fish
and other vertebrates (Amaral et al., 2020; Azevedo et al., 2013; Burkina
et al., 2018; De Souza et al., 2016; Polo-Cavia et al., 2016; Rutkoski
et al., 2020; Wolfram et al., 2012).

* Corresponding author. Departamento de Bioquímica e Biologia molecular, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil.
E-mail addresses: vania.loro@gmail.com, vania.loro@ufsm.br (V.L. Loro).

https://doi.org/10.1016/j.ecoenv.2020.111314
Received 25 March 2020; Received in revised form 7 September 2020; Accepted 9 September 2020
Available online 18 September 2020
0147-6513/© 2020 Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
C. Gonçalves et al.
Potentially harmful substances, such as pesticides, are often released
directly or indirectly into the aquatic environment. When considerable
amounts of pesticides or mixtures of them reach rivers, they can
generate immediate impacts as measured by increased mortality of
aquatic organisms. They can also produce a long-term impacts,
measured by chronic damage and the accumulation of those pollutants
in the aquatic organisms (Burkina et al., 2018; Clasen et al., 2018; Jia
and Chen, 2013). There are many classes of synthetic pesticides and they
can be classified according to their chemical nature. Due to the envi­
ronmental persistence of pesticides, they can be detected in air, water,
soil, in the tissues of aquatic animals such as fish (Arisekar et al., 2019;
Ernst et al., 2018; Vieira et al., 2019). Even sub-lethal concentrations,
including of environmentally relevant pesticides, found in environment
can affect the health of fish and causing oxidative damage, among other
factors (Gonçalves et al., 2018; Sandoval-Herrera et al., 2019; Severo
et al., 2020; Vignet et al., 2019).
Recent studies have reinforced the idea that pesticides impair the
immune system, increase internal levels of free radicals, damage thyroid
function, harm growth and disrupt protein synthesis (Nunes et al., 2018;
Raibeemol and Chitra, 2020; Salbego et al., 2020). A long (90-day) of
exposure to the herbicide quinclorac (90 days) caused liver oxidative
damage by increasing lipid peroxidation (LPO) and protein carbonyl
(PC) in carps at concentrations used in rice farming (Toni et al., 2013).
Exposure to carbofuran during irrigated rice cultivation (90 days) also
disrupted normal fish metabolism, altering antioxidant enzymes, and
increasing oxidative damage (Clasen et al., 2014). The insecticide pro­
poxur reduced acetylcholinesterase (AChE) activity in the brain and
muscle of Astyanax jacuhiensis (Characiformes: Characidae) and
increased PC levels in the brain of this fish (Gonçalves et al., 2018).
Numerous river health indicators, especially biological indicators and
biomarkers assayed in bioindicators such as fish, mollusks, or crusta­
ceans, have been developed to assess the condition of a river as a means
to manage freshwater ecosystems (Jia and Chen, 2013; Li et al., 2010;
Rossi et al., 2020; Shan et al., 2020). Fish are used as biological in­
dicators of environmental changes because of their characteristics: They
are usually large, relatively easy to collect and identify, and are good
indicators of short- and long-term effects of pollutants (Amaral et al.,
2020; Loro et al., 2015; Marins et al., 2020; Pelicice et al., 2017). Fish
that inhabit rice ponds or rivers near cultivation are exposed to a high
risk of contamination from pesticide mixtures. A study that examined
fish caught in a rice field system showed that commonly used pesticides
such as glyphosate, bifenthrin, azoxystrobin, and cyproconazole showed
health risks to native fish populations that inhabit rice field due to
antioxidant enzymes and AChE inhibition (Rossi et al., 2020). In bio­
monitoring studies, biomarker measurements in organism that inhabit
the environment are crucial to identify possible damage and pesticides
effects in these organisms (Catteau et al., 2020; Rodríguez-Hernández
et al., 2017; Vieira et al., 2019). Biomarkers, such as biochemical pa­
rameters, provide evidence of changes in the environment through
biological responses. Non-protein thiols levels (NPSH), catalase (CAT)
and glutathione-S-transferase (GST) activity, LPO, and PC induction can
provide evidence of stress responses in animals and they are considered
adequate indicators of xenobiotic exposure (Atli et al., 2016; Gonçalves
et al., 2018; Loro et al., 2015; Samanta et al., 2014).
The Uruguay River basin occupies 174,612 km2 of the Southern
Brazilian territory and is crucial to the country. It is well known for its
hydroelectric potential, fishing, and provision of water supplies, and it is
the boundary between Brazil and Argentina (ANA, 2012). Itaqui is a city
bordered by the Uruguay River (29◦ S 56◦ W) located in the Pampa
biome. It is about 3404.037 km2 in size and is one of the largest rice
producers in the state of Rio Grande do Sul (RS) (IRGA, 2013). More­
over, the percentage of the human population with wastewater treat­
ment in this region is exceptionally low, about 10%, which is below the
national average (30%) (Atli et al., 2016). Despite its importance to the
region, the Uruguay River basin is impacted by several anthropogenic
activities, including agricultural chemical input and untreated
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Ecotoxicology and Environmental Safety 205 (2020) 111314
wastewater discharge (Loro et al., 2015). Few studies have determined
the environmental health of this river. In line with biomonitoring
studies, this study employed a species that inhabits the Uruguay River as
a bioindicator. A. jacuhiensis, known in Brazil as “lambari do rabo
amarelo,” is widely distributed in the Uruguay River basin and in several
other Brazilian freshwater ecosystems. Astyanax spp. have been used in
ecological investigations due to their wide distribution, omnivorous
feeding habits, and small size (Alberto et al., 2005; Costa-Silva et al.,
2015; Lemos et al., 2008; Marins et al., 2020; Prado et al., 2011).
Considering the context and regional importance of Uruguay River, this
study aimed to assess the response of some biomarkers in fish that
inhabit the environment. It also evaluated the presence of pesticides in
the Uruguay River water and in the muscle of A. jacuhiensis. The main
hypothesis was that there is a relationship between pesticides in water or
muscle and biochemical alterations observed in fish.
2. Materials and methods
2.1. Water and fish muscle sampling for pesticide determination
Water samples (500 ml) were collected by hand monthly for two
years (2014–2015) (except in flood seasons) from two sampling sites in
the Uruguay River banks (1 m deep), Itaqui city, RS, Brazil (Fig. 1) to
determine the concentrations of pesticides. We use amber glass bottle to
water store. Site 1 (S1) is located downstream of the crop fields and
upstream of the urban area (29◦ 07′ 03.1′′ S 56◦ 32′ 36.8′′ W) and site 2 (S2)
is located downstream of the central urban area (29◦ 07′ 00.7′′ S
56◦ 33′ 41.1′′ W). The chromatography assay was performed as soon as
possible after sampling, avoiding the degradation of pesticides. The
water samples were analyzed to obtain a multi-residual analysis of
pesticides according to methods described by Donato et al. (2012) and
Martins et al. (2013), considering limit of detection: 0.038 μg L− 1 and
limit of quantification: 0.013 μg L− 1 (Table 1A). The fish muscles (n = 6
fish per site per season in duplicate) were collected at the same sites and
period of water samples, and 3 g of fish muscle were used to verify the
presence of pesticides in this tissue according to method described by
Munaretto et al. (2013), adapted to small fish as A. jacuhiensis. The limit
of detection used was: 0.00018 (μg/Kg− 1) and limit of quantification:
1.75 (μg/Kg− 1). Residues of pesticides expressed as (μg/kg tissue) are
showed in Table 1B.
2.2. Fish sampling and tissue preparation
To pesticide quantification and biochemical analysis 14 fish per
point (28 for each season) were caught. Six fish to estimate the pesticide
in the muscle. Eight fish for biochemical analysis. The fish were caught
in two times for each season, so the final number of fish sampled per
season was 56. The adults of both sexes specimens of A. jacuhiensis were
collected (20.89 ± 12.83 g and 10.67 ± 2.20 cm) during one year (2015)
from the two sites [S1; S2 (Fig. 1)] in the Uruguay River in different
seasons. The biometrical parameters were: summer (17.78 ± 7.30 g;
10.42 ± 1.32 cm), autumn (29.92 ± 15.7 g; 12.27 ± 2.29 cm), winter
(25.86 ± 16.28 g; 11.56 ± 2.51 cm), and spring (15.93 ± 11.37 g; 9.15
± 2.19 cm). Fish were collected by hook-and-line, and then they were
transferred to the Laboratório de Biologia at the Universidade Federal do
Pampa, UNIPAMPA, Itaqui in polyethylene boxes containing aerated
river water. Fish were euthanized by section of spinal cord behind the
opercula. Brain, gill, liver, and muscle were removed for biochemical
analyses. The tissues were first frozen in liquid nitrogen, and then
transported to the Laboratory of Aquatic Toxicology at the Universidade
Federal de Santa Maria, UFSM. They were kept frozen at (− 80 ◦ C) until
processing the samples. For the biochemical analysis, samples of liver,
gill, brain, and muscle (n = 8, per site per season in duplicate) were
homogenized in a Potter-Elvejhem glass/Teflon homogenizer with 5 vol
(w/v) of Tris-HCl buffer (50 mM, pH 7.4). After, samples were centri­
fuged at 10,000 g for 10 min at 4 ◦ C. The supernatant was stored at
C. Gonçalves et al. Ecotoxicology and Environmental Safety 205 (2020) 111314

Fig. 1. Map of sampling sites in the Uruguay River, Itaqui city, Rio Grande do Sul, Brazil. Site 1 is downstream of farmland and upstream of the urban area. Site 2 is
downstream from the central urban area. Source: Google Maps with modifications.

Table 1
A. Average concentration of pesticides (μg L− 1) detected in the water in the Uruguay River in the summer, autumn, winter, and spring for two years (2014–2015). Limit
of detection: 0.038 μg L− 1. Limit of quantification: 0.013 μg L− 1. Table 1B. Pesticides recorded in the muscle of Astyanax jacuhiensis (μg/kg− 1) at two sampling sites in
the Uruguay River for two years (2014–2015) (n = 6). Limit of detection: 0.00018 (μg/Kg− 1). Limit of quantification: 1.75 (μg/kg− 1). The values represent the mean ±
standard error.
Summer Autumn Winter Spring
− 1
A) PESTICIDES (μg/L ) SITE 1 2 1 2 1 2 1 2

Atrazine N.D N.D N.D. N.D N.D 0.13 ± 0.01 0.11 ± 0.01
Byspiribac-sodium 0.22 ± 0.01 N.D N.D. N.D N.D N.D. N.D.
Imidacloprid 0.07 ± 0.01 N.D N.D. N.D N.D N.D. N.D.
Malathion N.D N.D 0.21 ± 0.1 N.D N.D N.D N.D. N.D
Propoxur N.D N.D 0.85 ± 0.2 0.26 0.10.1 0.13 ± 0.01. N.D N.D. N.D.
Quinclorac 0.59 ± 0.2 N.D N.D N.D N.D N.D N.D. N.D.
Simazine 0.07 ± 0.01 N.D N.D N.D N.D N.D 0.07 ± 0.01 0.07 ± 0.01

B) PESTICIDES fish muscle (μg/kg-1) SITE Summer Autumn Winter Spring

1 2 1 2 1 2 1 2

Atrazine 0.17 ± 0.2 0.28 ± 0.1 N.D 0.27 ± 0.2 0.0.25 ± 0.2 0.35 ± 0.2 0.15 ± 0.1 0.22 ± 0.3
Byspiribac-sodium N.D 0.29 ± 0.2 N.D N.D N.D. N.D. N.D
Imidacloprid 0.11 ± 0.1 0.17 ± 0.1 0.0.22 ± 0.2 0.17 ± 0.1 0.0.15 ± 0.1 0.25 ± 0.2 0.11 ± 0.0 0.27 ± 0.3
Malathion 0.18 ± 0.1 0.11 ± 0.01 N.D 0.22 ± 0.2 N.D 0.33 ± 0.3 N.D N.D
Propoxur 0.35 ± 0.3 0.27 ± 0.3 0.17 ± 0.1 0.34 ± 0.4 N.D N.D N.D
Quinclorac 0.39 ± 0.4 1.17 ± 0.5 N.D N.D N.D N.D N.D
Simazine 0.09 ± 0.1 0.28 ± 0.1 0.23 ± 0.3 0.17 ± 0.2 0.18 ± 0.1 0.25 ± 0.1 0.23 ± 0.4 0.37 ± 0.07

− 80 ◦ C until analysis. This study was approved by the Ethics Committee
on Animal Use of Universidade Federal do Pampa, UNIPAMPA (protocol
nº 004–2014). Fish sampling was authorized by the Instituto Chico
Mendes de Conservação da Biodiversidade - ICMBio (protocol nº 42972-
2).
2.3. Biomarkers assays
All biochemical analyses were determined in brain, liver, muscle,
and gill and adapted to microplate (except catalase). LPO was estimated
by a thiobarbituric acid reactive substances (TBARS) assay through a
malondialdehyde (MDA) reaction with 2-thiobarbituric acid (TBA),
which was spectrophotometrically measured according to Buege and
Aust (1978) expressed as nmol MDA/mg protein. PC content was
assayed using 2,4-Dinitrophenylhydrazine (DNPH) to bond with protein
carbonyl according to the method of Parvez and Raisuddin (2005) with
modifications described in Müller et al. (2017). The carbonyl contents
were expressed as nmol carbonyl/mg protein. Levels of NPSH were

3
C. Gonçalves et al.
determined according to Ellman (1959) and expressed as μmol NPSH/g
of tissue. GST activity was measured using 1-chloro-2,4-dinitrobenzene
(CDNB) as a substrate (Habig et al., 1974). The activity was expressed as
μmol GS-DNB/min/mg protein. Catalase activity was determined in
liver by the ultraviolet spectrophotometry method, using H2O2 as sub­
strate. CAT activity was expressed as μmol/min/mg protein (Nelson and
Kiesow, 1972). Total protein content was determined using bovine
serum albumin as standard. Absorbance of samples was measured at
595 nm according to Bradford (1976).
2.4. Statistical analyses
Normality was determined using the Shapiro-Wilk and Kolmogorov-
Smirnov tests. Comparisons between groups (sampling sites and sea­
sons) were by two-way ANOVA followed by Tukey’s test. Statistical
analyses performed with the GraphPad Prism software version 6.01.
Results of water and pesticides residues in fish muscle are presented as
means ± standard error of the mean (S.E.M).
3. Results
Seven pesticides were detected in the Uruguay River water at the two
sampling sites, during the different seasons (Table 1A). All seven pes­
ticides were registered at S1, but only three (atrazine, propoxur, and
simazine) were registered at S2. The highest pesticide concentration in
water was recorded at S1 (0.85 μg L− 1 propoxur). S1 is located next to
rural areas characterized by monoculture practice, with intensive use of
pesticides throughout the year. This fact coincide with the presence of
pesticides in Uruguay river water along the seasons. In relation to the
season, the diversity of pesticides was greatest in the summer (bispyr­
ibac-sodium, imidacloprid, quinclorac, and simazine), and these pesti­
cides were detected in the water and muscle at S1. After chemical
analysis of fish muscle, some pesticides residues found in fish at S1 and
S2 during the summer coincided with some pesticides recorded in water.
In fact, fish muscle presented greater pesticide diversity compared with
water sampled. Fish muscle showed the presence of herbicides (atrazine,
bispyribac-sodium, quinclorac, and simazine) with high concentrations
Fig. 2. Thiobarbituric acid reactive substances (TBARS) (nmol malondialdehyde [MDA]/mg protein) levels in brain (A), liver (B), gills (C), and muscle (D) from
Astyanax jacuhiensis (n = 8) collected in the Uruguay River in the summer (sum), autumn (aut), winter (win), and spring (spr). Values are expressed as mean ±
standard deviation. Different letters indicate significant statistical differences among the seasons in the same site. Asterisks indicates a significant statistical difference
between the same season at different sites (p < 0.05).
4
Ecotoxicology and Environmental Safety 205 (2020) 111314
in the summer at both sites. The neonicotinoid imidacloprid was
recorded during all four seasons and at both sites. Malathion, an orga­
nothiophosphate, was detected during the summer at S1 and S2. Finally,
the carbamate non-systemic propoxur was detected in summer and
autumn at both sites (Tables 1A and 1B).
With regard to oxidative damage, there was seasonal variation in all
analyzed tissues. The same pattern did not occur between the sampling
sites. S1 and S2 presented higher TBARS values in the brain in the
summer (Fig. 2A). TBARS levels were higher at S1 compared with S2 in
the spring. Liver (S1) and muscle (S1 and S2) had the highest levels of
TBARS in the spring compared with the other seasons (Fig. 2B and D).
The TBARS liver content was higher in the spring at S1 compared with
S2. The highest TBARS values in the gills were recorded in the winter
and in the spring at S1 (Fig. 2C).
In addition, the highest PC levels were recorded in the summer in the
brain (S1) (Fig. 3A), muscle (S1) (Fig. 3D), and in the liver (S2) (Fig. 3B).
In the brain, the PC content was higher in the summer and lower in the
spring (S1) compared with the other seasons (Fig. 3A). PC levels in the
brain were higher at S1 than S2 in the summer and autumn. In the liver,
PC content was higher in the summer and spring (S1) and in the summer
(S2) compared with the other seasons (Fig. 3B). In the muscle, there was
higher PC content in the summer (S1) (Fig. 3D). PC content in the gills
was higher in the spring (S1) (Fig. 3C). S1 differed from S2 in the
summer, winter, and spring with regard to PC content (Figs. 2 and 3). In
the brain, there were lower NPSH levels in the winter (S1). There was no
difference among seasons at S2 in brain NPSH levels. NPSH content was
not different in the gills among the seasons or between sites. In the liver,
the highest NPSH content was recorded in the winter and spring at S2
(Table 2). At S1, the highest brain GST activity was recorded in the
summer and the lowest in the spring. At S2, summer and autumn pre­
sented the highest values for brain GST activity. GST activity in the gills
at both sites was lower in the winter and in the spring as compared with
summer and autumn. GST activity in the liver was lower in the spring at
S1. At S2, the highest GST activity was recorded in the autumn and in the
winter (Table 2). GST liver activity differed between S1 and S2 in the
summer, autumn, and winter. At S1, the lowest muscle GST activity was
recorded in the winter. By contrast, at S2, the highest muscle GST
C. Gonçalves et al. Ecotoxicology and Environmental Safety 205 (2020) 111314

Fig. 3. Protein carbonyl levels (nmol carbonyl/mg protein) in brain (A), liver (B), gills (C), and muscle (D) from Astyanax jacuhiensis (n = 8) collected in the Uruguay
River in the summer (sum), autumn (aut), winter (win), and spring (spr). Values are expressed as mean ± standard deviation. Different letters indicate significant
statistical differences among the seasons in the same site. Asterisks indicate a significant statistical difference between the same season at different sites (p < 0.05).

Table 2
Non-protein thiol (NPSH) levels (μmol NPSH/g tissue) and glutathione S-transferase (GST) (μmol GS-DNB min/mg protein) activity in brain, liver, gill, and muscle and
catalase (CAT) (μmol/min/mg protein) activity in liver of Astyanax jacuhiensis (n = 8) collected from the Uruguay River in the summer (sum), autumn (aut), winter
(win), and spring (spr). Values are expressed as mean ± standard deviation. Different letters indicate significant statistical differences among the seasons in the same
site (p < 0.05).
Site 1 Site 2

Summer Autumn Winter Spring Summer Autumn Winter Spring

NPSH Brain 2.85 ± 0.67a 2.55 ± 0.16a,b 1.91 ± 0.13c 2.21 ± 0.30b,c 2.64 ± 0.10 2.47 ± 0.38 2.13 ± 0.18 2.26 ± 0.28
Gills 2.85 ± 0.67 2.55 ± 0.16 2.71 ± 0.1 2.36 ± 0.29 2.64 ± 0.10 2.47 ± 0.38 2.45 ± 0.09 2.25 ± 0.50
Liver 2.41 ± 0.35 2.36 ± 0.16 2.74 ± 0.55 2.56 ± 0.22 2.28 ± 0.19a 2.25 ± 0.22a 3.22 ± 0.30b 2.73 ± 0.16b
Muscle 0.52 ± 0.04a 0.52 ± 0.05a 0.59 ± 0.04a,b 0.66 ± 0.04 b 0.61 ± 0.05 0.59 ± 0.03 0.60 ± 0.02 0.54 ± 0.05
GST Brain 0.16 ± 0.04a 0.10 ± 0.01b 0.07 ± 0.00b,c 0.03 ± 0.007c 0.12 ± 0.02a 0.15 ± 0.04a 0.06 ± 0.00b 0.03 ± 0.00b
Gills 0.08 ± 0.01a 0.08 ± 0.01a 0.04 ± 0.01b 0.04 ± 0.01b 0.08 ± 0.01a 0.08 ± 0.01a 0.04 ± 0.01b 0.04 ± 0.01b
Liver 0.09 ± 0.02a 0.08 ± 0.02a 0.07 ± 0.00a 0.08 ± 0.01a 0.05 ± 0.00a 0.12 ± 0.02b 0.13 ± 0.00b 0.04 ± 0.01a
Muscle 0.03 ± 0.01a 0.04 ± 0.00a 0.02 ± 0.00b 0.03 ± 0.00ab 0.02 ± 0.00a 0.02 ± 0.01a 0.02 ± 0.00a 0.03 ± 0.00a
CAT Liver 0.50 ± 0.16 0.54 ± 0.09 0.58 ± 0.16 0.54 ± 0.08 0.34 ± 0.07a 0.37 ± 0.12a 0.70 ± 0.16b 0.52 ± 0.14b

activity was in the autumn (Table 2). CAT activity did not differ among
seasons at S1, but at S2, the highest liver CAT activity was recorded in
the winter follow by spring (Table 2).
4. Discussion
There are scarced studies to evaluate the presence and accumulation
of pesticides in fish collected in the Uruguay River and its relationship
with biochemical parameters. The results of this study highlight the
possible impacts of pesticides on aquatic biota, represented here by the
fish species A. jacuhiensis. We registered pesticides at both sampling sites
of the Uruguay River (up- and downstream of the city) in all seasons.
Summer was the season with the greatest diversity of pesticides: sima­
zine, bispyribac-sodium, quinclorac (herbicides), and imidacloprid
(insecticide). In the state of Rio Grande do Sul, the purchase of simazine
and imidacloprid was estimated to be about 151.41 and 687.77 tons per
year, respectively (IBAMA, 2016). The purchase of atrazine was even
higher, at an estimated 1074.45 tons per year. The high amounts of
pesticides in the summer coincide with regional crop management,
which begins in the spring when simazine and atrazine were detected.
Simazine and atrazine are herbicides that are widely used on crops in
Southern Brazil. In a previous study, atrazine was detected in the water
of the Passo Real reservoir (Southern Brazil) in all seasons at six sampled
sites (Amaral et al., 2020). High atrazine levels in water were detected in
the Uruguay River, near the city of Uruguaiana, this factor is concerning
for the Pampa biome (Loro et al., 2015). Nevertheless, Brazilian law
stipulates the maximum concentration of 2 μg L− 1 of atrazine and
simazine in potable water (CONAMA, 2005). Atrazine has been associ­
ated with locomotor, genotoxic, and oxidative disorders in fish
(Adeyemi et al., 2015; Liu et al., 2016). Marins et al. (2018) verified the
occurrence of oxidative damage and bioaccumulation of atrazine in carp
even when safe concentrations stipulated by Brazilian law were tested.
An unbalanced antioxidant system could decrease the fitness of the fish
to cope with the adversities and challenges of the environment
throughout the seasons.
For example, quinclorac, an herbicide registered at S1 in the sum­
mer, also caused oxidative damage in Cyprinus carpio (Menezes et al.,
2012; Toni et al., 2013). In the autumn, the insecticides malathion and
propoxur were detected, and propoxur was also detected at S1 in the
winter. Propoxur is a household carbamate insecticide that has already
been found in the Uruguay River (Loro et al., 2015), and it has been
associated with disrupted biochemical and hematological parameters in

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carps (Gül et al., 2012). Propoxur also led to the decline of AChE activity
in Astyanax sp. at concentrations found in the Uruguay River (15 and 30
μg L− 1) (Gonçalves et al., 2018). Malathion is extensively used in agri­
cultural practices. It was detected in the Uruguay River in levels mark­
edly above (20 times) those established by the Brazilian law (0.01 μg
L− 1). Malathion is recognized as a potent endocrine disruptor that im­
pairs fish growth (Lal et al., 2013). The presence of higher pesticides
concentrations at S1 can be explained by their proximity to crops, while
S2 is located near an urban center. Simazine, atrazine, and propoxur
were registered at both sites, data that confirm the use of pesticides in
addition to domestic discharge could be the cause of the effects observed
in the A. jacuhiensis sampled in this region. Given that these pesticides
were detected simultaneously in the environment, they could present
different effects: additive, synergistic, or antagonistic. In fact, pesticide
and metals affect fish health and usually causes oxidative damage. A
study considering fish kept in two subtropical agro-ecosystems for 120
days revealed the impact of chemicals on fish health. The results showed
that the investigated sites exhibited different levels of metals and pes­
ticides. Fish confined in agricultural sites accumulated higher levels of
endosulfan and copper. Integrated biomarker analysis revealed a
decrease in the health status on the fish over time. Fish confined shown
oxidative damage, DNA damage and histological alterations (Vieira
et al., 2019).
The results of pesticide residue analysis in fish muscle showed the
presence of a large diversity of pesticides even though they were not
present in the water at all the sampling sites. This finding can be
explained in part by the free movement of fish along the river. In this
way, these animals can retain different types of pollutants throughout
their lives. Some authors have reported similar results where pesticides
founded in water were different from those recorded at fish muscle
(Ernst et al., 2018; Pérez-Parada et al., 2018). Another important
observation is the potential risk to humans who consume fish from this
river. When Cyprinus carpio were reared for 100 days and exposed to the
pesticides applied to irrigated rice culture, lambda-cyhalothrin (18
μg/kg− 1) and tebuconazole (39.9 μg/kg− 1) were detected in the muscle
(Clasen et al., 2018). Pesticide exposure in fish generally increases the
susceptibility to diseases, due to a reduced capacity of the fish immune
and antioxidant system. Some evidences suggest that pesticides induce
oxidative damage due to free radical generation, and promote changes
in the defense mechanisms of the organism, leading to oxidative damage
in different tissues (Clasen et al., 2014; Lal et al., 2013; Oropesa et al.,
2009; Rossi et al., 2020; Samanta et al., 2014; Toni et al., 2013; Vieira
et al., 2019). LPO is one of the most used biomarkers for quantifying
oxidative stress in lipids, expressed in this study as TBARS levels. In the
present work, the brain had the highest TBARS values in the summer,
which coincides with the greater presence of pesticides in the river. In
the spring, the herbicides atrazine and simazine were detected at both
sites, and liver and muscle tissues presented the highest TBARS levels.
Simazine and atrazine have been suggested as LPO promoters of lipid
peroxidation in fish and other animals (Adeyemi et al., 2015; Marins
et al., 2018; Oropesa et al., 2009), along with quinclorac (Menezes et al.,
2012; Toni et al., 2013), and imidacloprid (Wang et al., 2016; Vignet
et al., 2019), which were detected in the summer. Moreover, the highest
PC and TBARS levels in the liver were registered at S1, which was most
contaminated by pesticides in the spring. In the summer, PC levels in the
brain were higher at both sites. Consistently, the increase in GST activity
observed in the brain at S1 in the summer could be a detoxification
response against pesticide exposure. A similar response was recorded in
Astyanax sp. in a river also used for public supply (Bueno-Krawczyk
et al., 2015). Studies have suggested a role for increased GST activity in
the presence of toxic chemicals (Azevedo et al., 2013; Loro et al., 2015;
Nunes et al., 2018). In the brain and gills, GST activity followed the same
pattern at both sites; the highest activity was recorded in the summer
and autumn. In the autumn, propoxur occurred at both sampling sites.
Propoxur is a highly toxic carbamate insecticide used for domestic
purposes. According to the World Health Organization (WHO) for
6
Ecotoxicology and Environmental Safety 205 (2020) 111314
pesticide classification, propoxur, imidacloprid, and bentazone are all
class II toxins (WHO, 2009). When the same fish species used in the
present study was exposed to propoxur at concentrations founded in
Uruguay River, oxidative damage was recorded with increasing in lipid
peroxidation and protein oxidation in brain and muscle (Gonçalves
et al., 2018).
CAT, another important scavenger enzyme, catalyzes H2O2 produced
during various metabolic processes into less toxic molecules, namely
water and oxygen (Van der Oost et al., 2003). Liver CAT activity
increased only at S2, with highest activity during the winter follow by
spring. In the S1 did not have any change in the CAT activity. So, the
increased activity recorded in the S2 could be related to the presence of
pesticides in the fish muscle, due to absence of recorded pesticides in the
winter at water. Non-protein thiols levels (NPSH) are an indicator of the
antioxidant capacity of the organism. In this work, NPSH levels in the
liver did not increase. We observed a significant increase in muscle
NPSH at S1 in the spring. Increased levels of NPSH in fish collected at the
Uruguay River has been reported (Loro et al., 2015). However, we also
observed a decrease in the levels of brain NPSH in the winter. NPSH
depletion has been reported in similar study (Costa-Silva et al., 2015),
and may be related to the presence of propoxur, the only pesticide
detected in the winter. Propoxur acts by inhibiting AChE activity,
although it is a less potent AChE inhibitor than other carbamates, it is
highly toxic to fish. It also is a potential contaminant of the aquatic
environment due to its high solubility in water, as has been shown by
other studies (Gonçalves et al., 2018; Schmuck and Mihail, 2004; Wang
et al., 2009).
The Uruguay River basin is continuously exposed to pesticides;
indeed, we detected pesticides in all seasons in the water and fish
muscle. In addition to exposure to pesticides commonly used in crops,
we also found propoxur, a domestic pesticide. This fact clearly attests to
the increased use of pesticides in the area. Besides pesticides, domestic
discharges and other pollutants such as metals could be the reasons of
the biochemical alterations recorded in Astyanax sp. collected in the
Uruguay River. In summary, changes in biomarkers were recorded and
could be used in future studies to understand the role of these chemical
compounds on fish’s health. Thus, considering the impact of pesticides
use in this important region, this study is a relevant contribution to
assess the environmental quality using toxicological biomarkers in fish.
5. Conclusions
The main reason for the pollution in the Uruguay River may be due to
intensive agricultural practices, unplanned urbanization, and disposal of
domestic effluents. The results of this study highlight the contamination
of the river by pesticides, especially mixtures of compounds. Summer
was the season with high concentration of different agrochemicals in
fish muscle and this fact could be related to the intense agricultural
activity in this period. Hence, there is a relationship between the pres­
ence of pesticides in the water and in the fish muscle with the different
biochemical alterations recorded. The results indicate that adequate
management and monitoring of the waters of the Uruguay River is
essential to control pollutants and save this aquatic environment.
Credit author statement
Carjone Gonçalves – Doctor thesis using part of results obtained.
Experimental procedures, fish caught, water samples, statistical analysis
and write the manuscript with help of Aline Monique Blank do Amaral
and Bárbara Clasen. Aline Teixeira Marins, Mauro Eugênio Medina
Nunes, Talise Ellwanger Müller, Adriane Feijó, Cintia Corte Real
Rodrigues, Eduardo Severo – Experimental analysis and help with sta­
tistical interpretation of the results. Renato Zanella, Osmar Damian
Prestes – Financial support and analysis of pesticides in water and fish
muscle. Barbara Clasen and Vania Lucia Loro – Financial support,
revision of the manuscript. Vania L. Loro was the adviser of Carjone
C. Gonçalves et al. Ecotoxicology and Environmental Safety 205 (2020) 111314

Gonçalves. All co-authors contribute for the experimental procedures the Brazilian Pampa biome. Environ. Sci. Pollut. Res. 22, 15526–15535. https://doi.
org/10.1007/s11356-015-4737-7.
and manuscript building.
De Souza, R.V., Garbossa, L.H.P., Campos, C.J.A., Vianna, L.F.de N., Vanz, A., Rupp, G.S.,
2016. Metals and pesticides in comercial bivalve mollusc production áreas in the
Notrh and South Bays, Santa Catarina (Brazil). Marine Pollution 105, 377–384.
Declaration of competing interest https://doi.org/10.1016/j.marpolbul.2016.02.024.
Donato, F., Kemmerich, M., de Facco, J.F., do Frigi, C.A., Prestes, O., Adaime, M.B.,
2012. Simultaneous determination of pesticide and antibiotic residues at trace levels
The authors declare that they have no known competing financial in water samples by SPE and LC-MS/MS. Brazilian Journal of Anal. Chem. 2,
interests or personal relationships that could have appeared to influence 331–340.
the work reported in this paper. Dudgeon, D., Arthington, A.H., Gessner, Kawabata, Z., Knowler, D.J., Lévêque, C.,
Naiman, R.J., Prieur-Richard, A., Soto, D., Stiassny, M.L.J., Sullivanm, C.A., 2006.
Freshwater biodiversity: importance, threats, status and conservation challenges.
Acknowledgments Biol. Rev. 81, 163–182. https://doi.org/10.1017/S1464793105006950.
Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77.
https://doi.org/10.1016/0003-9861(59)90090-6.
This study was financed in part by the Coordenação de Aperfeiçoa­ Ernst, F., Alonso, B., Colazzo, M., Pareja, L., Cesio, V., Pereira, A., Márquez, A., Errico, E.,
mento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. Segura, A.M., Heinzen, H., Pérez-Parada, A., 2018. Occurrence of pesticide residues
in fish from South American rainfed agroecosystems. Sci. Total Environ. 631–632,
Vania Lucia Loro received research fellowship from CNPq under process
169–179.
number: 309314/2017-8. We would like to thank the Universidade Gonçalves, C.R., Marins, A.T., Amaral, A.M.B., Leitemperger, J., Severo, E.S., Moraes, B.
Federal de Santa Maria (UFSM) and Universidade Federal do Pampa S., Zanella, R., Loro, V.L., 2018. Biochemical responses in freshwater fish exposed to
(UNIPAMPA) for financial support. insecticide propoxur. Bull. Environ. Contam. Toxicol. 100, 524–528. https://doi.
org/10.1007/s00128-018-2285-9.
Gül, A., Benli, A.Ç.K., Ayhan, A., Memmi, B.K., Selvi, M., Sepici-Dinçel, A.,
References Cakiroğullari, G.Ç., Erkoç, F., 2012. Sublethal propoxur toxicity to juvenile common
carp (Cyprinus carpio L., 1758): biochemical, hematological, histopathological, and
genotoxicity effects. Environ. Toxicol. Chem. 31, 2085–2092. https://doi.org/
Adeyemi, J.A., Martins-Junior, A.C., Barbosa, F., 2015. Teratogenicity, genotoxicity and
10.1002/etc.1924.
oxidative stress in zebrafish embryos (Danio rerio) co-exposed to arsenic and
Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione-S-transferases, the first
atrazine. Comp. Biochem. Physiol. 172–173, 7–12. https://doi.org/10.1016/j.
enzimatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139.
cbpc.2015.04.001.
Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA),
Agência Nacional de Águas (ANA), 2012. Panorama da Qualidade das Águas Superficiais
2016. Boletim Anual de Produção Importação, Exportação e Vendas de Agrotóxicos
do Brasil. ANA, Brasília, pp. 182–189.
no Brasil. https://www.ibama.gov.br/agrotoxicos/relatorios-de-comercializac
Alberto, A., Camargo, A.F.M., Verani, J.R., Costa, O.F., Fernandes, M.N., 2005. Health
ao-de-agrotoxicos.
variables and gill morphology in the tropical fish Astyanax fasciatus from a sewage-
Instituto Rio Grandense do Arroz (IRGA), 2013. Censo da lavoura de arroz irrigado do
contaminated river. Ecotoxicol. Environ. Saf. 61, 247–255. https://doi.org/10.1016/
Rio Grande do Sul – safra 2012/2013. Porto Alegre. IRGA.
j.ecoenv.2004.08.009.
Jia, Y.T., Chen, Y.F., 2013. Ecological Indicators River health assessment in a large river:
Amaral, A.M.B., Moura, L.K., Pellegrin, D., Guerra, L.J., Cerezer, F.O., Saibt, N.,
bioindicators of fish population. Ecol. Indicat. 26, 24–32.
Prestes, O.D., Zanella, R., Loro, V.L., Clasen, B., 2020. Seasonal factors driving
Lal, B., Sarang, M.K., Kumar, P., 2013. Malathion exposure induces the endocrine
biochemical biomarkers in two fish species from a subtropical reservoir in southern
disruption and growth retardation in the catfish, Clarias batrachus (Linn.). Gen.
Brazil: an integrated approach. Environ. Pollut. 266, 115168. https://doi.org/
Comp. Endocrinol. 181, 139–145. https://doi.org/10.1016/j.ygcen.2012.11.004.
10.1016/j.envpol.2020.115168.
Lemos, C.T., Iranço, F.A., Oliveira, N.C.D., de Souza, G.D., Fschel, J.M., 2008.
Arisekar, U., Shakila, R.J., Jeyasekaran, G., Shalini, R., Kumar, P., Malani, A.H., Rani, V.,
Biomonitoring of genotoxicity using micronuclei assay in native population of
2019. Accumulation of organochlorine and pyrethroid pesticide residues in fish,
Astyanax jacuhiensis (Characiformes: Characidae) at sites under petrochemical
water, and sediments in the Thamirabarani river system of southern peninsular
influence. Sci. Total Environ. 406, 337–343. https://doi.org/10.1016/j.
India. Environ. Nanotechnol. Monit. Manag. 11, 100194. https://doi.org/10.1016/j.
scitotenv.2008.07.006.
enmm.2018.11.003.
Li, L., Zheng, B., Liu, L., 2010. Biomonitoring and bioindicators used for river
Atli, G., Canli, E.G., Eroglu, A., Canli, M., 2016. Characterization of antioxidant system
ecosystems: definitions, approaches and trends. Procedia Environ. Sci. 2,
parameters in four freshwater fish species. Ecotoxicol. Environ. Saf. 126, 30–37.
1510–1524. https://doi.org/10.1016/j.proenv.2010.10.164.
https://doi.org/10.1016/j.ecoenv.2015.12.012.
Liu, Z., Wang, Y., Zhu, Z., Yang, E., Feng, X., Fu, Z., Jin, Y., 2016. Atrazine and its main
Azevedo, J.S., Braga, E.S., Assis, H.C.S., Ribeiro, O.C.A., 2013. Biochemical changes in
metabolites alter the locomotor activity of larval zebrafish (Danio rerio).
the liver and gill of Cathorops spixii collected seasonally in two Brazilian estuaries
Chemosphere 148, 163–170. https://doi.org/10.1016/j.chemosphere.2016.01.007.
under varying influences of anthropogenic activities. Ecotoxicol. Environ. Saf. 96,
Loro, V.L., Murussi, C., Menezes, C., Leitemperger, J., Severo, E., Guerra, L., Costa, M.,
220–230. https://doi.org/10.1016/j.ecoenv.2013.06.021.
Perazzo, G.X., Zanella, R., 2015. Spatial and temporal biomarkers responses of
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram
Astyanax jacuhiensis (Cope, 1894) (Characiformes: Characidae) from the middle rio
quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.
Uruguai, Brazil. Neotrop. Ichthyol. 13, 569–578. https://doi.org/10.1590/1982-
72, 248–254. https://doi.org/10.1016/0003-2697(76)90527-3.
0224-20140146.
Buege, J.A., Aust, S.D., 1978. Microssomal lipid peroxidation. Methods Enzymol. 52,
Marins, A.T., Rodrigues, C.C.R., de Menezes, C.C., Gomes, L.C.J., Costa, M.D., Nunes, M.
302–309.
E.M., Vieira, S.V., Donato, F.F., Zanella, R., Silva, L.P., Loro, V.L., 2018. Integrated
Bueno-Krawczyk, A.C.D., Guiloski, I.C., Piancini, L.D.S., Azevedo, J.C., Ramsdorf, W.A.,
biomarkers response confirm the antioxidant role of diphenyl diselenide against
Ide, A.H., Guimarães, A.T., Cestari, M.M., Assis, S.H.C., 2015. Multibiomarker in fish
atrazine. Ecotoxicol. Environ. Saf. 151, 191–198. https://doi.org/10.1016/j.
to evaluate a river used to water public supply. Chemosphere 135, 257–264. https://
ecoenv.2018.01.021.
doi.org/10.1016/j.chemosphere.2015.04.064.
Marins, A.T., Severo, E.S., Leitemperger, J.W., Cerezer, C., Muller, T.E., Costa, M.D.,
Burkina, V., Zamaratskaia, G., Sakalli, S., Giang, P.T., Kodes, V., Grabic, R., Velisek, J.,
Weimer, G.H., Bandeira, N.M.G., Prestes, O.D., Zanella, R., Loro, V.L., 2020.
Turek, J., Kolarova, J., Zlabek, V., Randak, T., 2018. Complex effects of pollution on
Assessment of river water quality in an agricultural region of Brazil using biomarkers
fish in major rivers in the Czech Republic. Ecotoxicol. Environ. Saf. 164, 92–99.
in a native neotropical fish, Astyanax spp. (Characidae). Bull. Environ. Contam.
https://doi.org/10.1016/j.ecoenv.2018.07.109.
Toxicol. 104, 575–581. https://doi.org/10.1007/s00128-020-02821-0.
Clasen, B., Leitemperger, J., Murussi, C., Pretto, A., Menezes, C., Dalabona, F.,
Martins, M.L., Donato, F.F., Prestes, O.D., Adaime, M.B., Zanella, R., 2013.
Marchezan, E., Adaime, M.B., Zanella, R., Loro, V.L., 2014. Carbofuran promotes
Determination of pesticide residues and related compounds in water and industrial
biochemical changes in carp exposed to rice field and laboratory conditions.
effluent by solid-phase extraction and gas chromatography coupled to triple
Ecotoxicol. Environ. Saf. 101, 77–82. https://doi.org/10.1016/j.
quadrupole mass spectrometry. Anal. Bioanal. Chem. 405, 7697–7709. https://doi.
ecoenv.2013.12.012.
org/10.1007/s00216-013-7235-0.
Clasen, B., Loro, V.L., Murussi, C.R., Pretto, A., Menezes, C., Dalabona, F., Marchezan, E.,
Menezes, C.C., Leitemperger, J., Santi, A., Lópes, T., Veiverberg, C.A., Peixoto, S.,
Adaime, M.B., Zanella, R., Loro, V.L., 2018. Bioaccumulation and oxidative stress
Adaime, B.M., Zanella, R., Barbosa, N., Loro, V.L., 2012. The effects of diphenyl
caused by pesticides in Cyprinus carpio reared in a rice-fish system. Sci. Total
diselenide on oxidative stress biomarkers in Cyprinus carpio exposed to herbicide
Environ. 626, 737–743. https://doi.org/10.1016/j.scitotenv.2018.01.154.
quinclorac (Facet®). Ecotoxicol. Environ. Saf. 8, 91–97. https://doi.org/10.1016/j.
Catteau, A., Bado-Nilles, A., Beaudouin, R., Joachim, S., Palluel, O., Turies, C., Galet, C.,
ecoenv.2012.04.022.
Geffard, A., Jean-Marc Porcher, J.-M., 2020. An active biomonitoring approach
Munaretto, J.S., Ferronato, G., Ribeiro, L.C., Martins, M.L., Adaime, M.B., Zanella, R.,
using three-spined stickleback (Gasterosteus aculeatus, L.) to assess the efficiency of a
2013. Development of a multiresidue method for the determination of endocrine
constructed wetland as tertiary treatment of wastewater. Ecol. Indicat. 114, 106238.
disrupters in fish fillet using gas chromatography-triple quadrupole tandem mass
https://doi.org/10.1016/j.ecolind.2020.106238.
spectrometry. Talanta 116, 827–834. https://doi.org/10.1016/j.
Conselho Nacional do Meio Ambiente (CONAMA), 2005. Resolução n.◦ 357, de 17 de
talanta.2013.07.047.
março de 2005 publicada no DOU n.◦ de 18/03, pp. 58–63.
Müller, T.E., Nunes, M.E., Menezes, C.C., Marins, A.T., Leitemperger, J., Gressler, A.C.L.,
Costa-Silva, D.G., Nunes, M.E.M., Wallau, G.L., Martins, I.K., Zemolin, A.P.P., Cruz, L.C.,
Carvalho, F.B., Freitas, C.M., Quadros, V.A., Fachinetto, R., Rosemberger, D.B.,
Rodrigues, N.R., Lopes, A.R., Posser, T., Franco, J.L., 2015. Oxidative stress markers
in fish (Astyanax sp. and Danio rerio) exposed to urban and agricultural effluents in

7
C. Gonçalves et al.
Loro, V.L., 2017. Sodium selenite prevents paraquat-induce neurotoxicity in
zebrafish. Mol. Neurobiol. 55 (3), 1928–1941. https://10.1007/s12035-017-0441-6.
Nelson, D.P., Kiesow, L.A., 1972. Enthalpy of decomposition of hydrogen peroxide by
catalase at 25◦ C (with molar extinction coefficients of H2O2 solution in the UV).
Anal. Biochem. 49, 474–478.
Nunes, M.E.M., Müller, T.E., Murussi, C., Amaral, A.M.B., Gomes, J.L.C., Marins, A.T.,
Leitemperger, J., Rodrigues, C.C.R., Fiuza, T.L., Costa, M.D., Severo, E.S.,
Rosemberg, D.B., Loro, V.L., 2018. Oxidative effects of the acute exposure to a
pesticide mixture of cypermethrin and chlorpyrifos on carp and zebrafish – a
comparative study. Comp. Biochem. Physiol. C 206–207, 48–53. https://doi.org/
10.1016/j.cbpc.2018.03.002.
Oropesa, A.-L., García-Cambero, J.P., Solera, F., 2009. Glutathione and malondialdehyde
levels in common carp after exposure to simazine. Environ. Toxicol. Pharmacol. 27,
30–38. https://doi.org/10.1016/j.etap.2008.08.003.
Parvez, S., Raisuddin, S., 2005. Protein carbonyls: novel biomarkers of exposure to
oxidative stress-inducing pesticides in freshwater fish Channa punctate (Bloch).
Environ. Toxicol. Pharmacol. 20, 112–117. https://doi.org/10.1016/j.
etap.2004.11.002.
Pelicice, F.M., Azevedo-Santos, V.M., Vitule, J.R.S., Orsi, M.L., Junior, D.P.L.,
Magalhães, A.L.B., Pompeu, P.S., Junior, M.P., Agostinho, A.A., 2017. Neotropical
freshwater fishes imperilled by unsustainable policies. Fish Fish. 18, 1119–1133.
https://doi.org/10.1111/faf.12228.
Polo-Cavia, N., Burraco, P., Gomez-Mestre, I., 2016. Low levels of chemical
anthropogenic pollution may threaten amphibians by impairing predator
recognition. Aquat. Toxicol. 172, 30–35. https://doi.org/10.1016/j.
aquatox.2015.12.019.
Pérez-Parada, A., Goyenola, G., de Mello, F.T., Heinzen, H., 2018. Recent advances and
open around pesticide dynamics and effects on freshwater fishes. Cur. Opin. Environ.
Sci. Health 4, 38–44. https://doi.org/10.1016/j.coesh.2018.08.004.
Prado, P.S., Souza, C.C., Bazzoli, N., Rizzo, E., 2011. Reproductive disruption in lambari
Astyanax fasciatus from a Southeastern Brazilian reservoir. Ecotoxicol. Environ. Saf.
74, 1879–1887. https://doi.org/10.1016/j.ecoenv.2011.07.017.
Raibeemol, K.P., Chitra, K.C., 2020. Induction of immunological, hormonal and
histological alterations after sublethal exposure of chlorpyrifos in the freshwater
fish, Pseudetroplus maculatus (Bloch, 1795). Fish Shellfish Immunol. 102, 1–12.
https://doi.org/10.1016/j.fsi.2020.04.005.
Reis, R.E., Albert, J.S., Di Dario, F., Mincarone, M.M., Petry, P., Rocha, L.A., 2016. Fish
biodiversity and conservation in South America. J. Fish. Biol. 89, 12–47. https://doi.
org/10.1111/jfb.13016.
Rodríguez-Hernández, A., Camacho, M., Henríquez-Hernández, L.A., Boada, L.D.,
Valerón, P.F., Zaccaroni, A., 2017. Comparative study of the intake of toxic
persistent and semi persistent pollutants through the consumption of fish and
seafood from two modes of production (wild-caught and farmed). Sci. Total Environ.
575, 919–931.
Rossi, A.S., Fantón, N., Michlig, M.P., Repetti, M.R., Cazenave, J., 2020. Fish inhabiting
rice fields: bioaccumulation, oxidative stress and neurotoxic effects after pesticides
application. Ecol. Indicat. 113, 106186. https://doi.org/10.1016/j.ecolind.2020./
106186.
Rutkoski, C.F., Macagnan, N., Folador, A., Skovronski, V.J., do Amaral, A.M.B.,
Leitemperger, J., Costa, M.D., Hartmann, P.A., Müller, C., Loro, V.L., Hartmann, M.
T., 2020. Morphological and biochemical traits and mortality in Physalaemus
gracilis (Anura: Leptodactylidae) tadpoles exposed to the insecticide chlorpyrifos.
Chemosphere 250, 126162. https://doi.org/10.1016/j.chemosphere.2020.126162.
Salbego, J., Seben, D., Sippert, L.R., Gressler, L.T., da Cunha, J.A., Zanella, R.,
Vaucher, R.A., Marchesan, E., Baldisserotto, B., Loro, V.L., Golombieski, J.I., 2020.
Ecotoxicology and Environmental Safety 205 (2020) 111314
Toxicological response of silver catfish (Rhamdia quelen) after acute exposure to a
commercial insecticide containing thiamethoxam. J. Environ. Sci. Heal. B. 55,
749–755. https://doi.org/10.1080/03601234.2020.1782115.
Samanta, P., Pal, S., Mukherjee, A.K., Ghosh, A.R., 2014. Biochemical effects of
glyphosate based herbicide, Excel Mera 71 on enzyme activities of
acetylcholinesterase (AChE), lipid peroxidation (LPO), catalase (CAT), glutathione-
S-transferase (GST) and protein content on teleostean fishes. Ecotoxicol. Environ.
Saf. 107, 120–125. https://doi.org/10.1016/j.ecoenv.2014.05.025.
Sandoval-Herrera, N., Mena, F., Espinoza, M., Romero, A., 2019. Neurotoxicity of
organophosphate pesticides could reduce the ability of fish to escape predation
under low doses of exposure. Sci. Rep. 9, 10530. https://doi.org/10.1038/s41598-
019-46804-6.
Schmuck, G., Mihail, F., 2004. Effects of the carbamates fenoxycarb, propamocarb and
propoxur on energy supply, glucose utilization and SH-groups in neurons. Arch.
Toxicol. 78, 330–337. https://doi.org/10.1007/s00204-004-0546-3.
Severo, E.S., Marins, A.T., Cerezer, C., Costa, D., Nunes, M., Prestes, O.D., Zanella, R.,
Loro, V.L., 2020. Ecological risk of pesticide contamination in a Brazilian river
located near a rural area: a study of biomarkers using zebrafish embryos. Ecotoxicol.
Environ. Saf. 190, 110071. https://doi.org/10.1016/j.ecoenv.2019.110071.
Shan, Y., Yan, J., Hong, X., Qin, J., 2020. Effect of imidacloprid on the behavior,
antioxidant system, multixenobiotic resistance, and histopathology of Asian
freshwater clams (Corbicula fluminea). Aquat. Toxicol. 218, 105333. https://doi.org/
10.1016/j.aquatox.2019.105333.
Toni, C., Menezes, C., Clasen, B., Leitemperger, J., Pretto, A., Adaime, M.B., Martins, M.,
Loro, V.L., 2013. Oxidative stress in carp exposed to quinclorac herbicide under rice
field condition. Ecotoxicol. Environ. Saf. 92, 27–31. https://doi.org/10.1016/j.
ecoenv.2013.01.028.
Van der Oost, R., Beyer, J., Vermeulen, N.P.E., 2003. Fish bioaccumulation and
biomarkers in environmental risk assessment: a review. Environ. Toxicol.
Pharmacol. 13, 57–149. https://doi.org/10.1016/S1382-6689(02)00126-6.
Vieira, C.E.D., Costa, P.G., Caldas, S.S., Tesser, M.E., Risso, W.E., Escarrone, A.L.V.,
Primel, E.G., Bianchini, A., Martinez, C.B.R., 2019. An integrated approach in
subtropical agro-ecosystems: active biomonitoring, environmental contaminants,
bioaccumulation, and multiple biomarkers in fish. Sci. Total Environ. 666, 508–524.
https://doi.org/10.1016/j.scitotenv.2019.02.209.
Vignet, C., Cappello, T., Fu, Q., Lajoie, K., De Marco, G., Clérandeau, C., Mottaz, H.,
Maisano, M., Hollender, J., Schirmer, K., Cachot, J., 2019. Imidacloprid induces
adverse effects on fish early life stages that are more severe in Japanese medaka
(Oryzias latipes) than in zebrafish (Danio rerio). Chemosphere 225, 470–478. https://
doi.org/10.1016/j.chemosphere.2019.03.002.
Wang, C., Lu, G., Cui, J., Wang, P., 2009. Sublethal effects of pesticide mixtures on
selected biomarkers of Carassius auratus. Environ. Toxicol. Pharmacol. 28, 414–419.
https://doi.org/10.1016/j.etap.2009.07.005.
Wang, J., Wang, J., Wang, G., Zhu, G., Wang, J., 2016. DNA damage and oxidative stress
induced by imidacloprid exposure in the earthworm Eisenia fetida. Chemosphere
144, 510–517. https://doi.org/10.1016/j.chemosphere.2015.09.004.
World Health Organization (WHO), 2009. The WHO Recommended Classification of
Pesticides by Hazard and Guidelines to Classification. Wissenchaftliche
Verlagsgesellschaft mb H, Stuttgart, Germany.
Wolfram, G., Höss, S., Orendt, C., Schmitt, C., Adámek, Z., Bandow, N.,
Grobschartner, M., Kukkonen, J.V.K., Leloup, V., Doval, J.C.L., Muñoz, I.,
Traunspurger, W., Tuikka, A., Liefferingr, C.V., Von der Ohe, P.C., Deckere, E., 2012.
Assessing the impact of chemical pollution on benthic invertebrates from three
different European rivers using a weight-of-evidence approach. Sci. Total Environ.
438, 498–509. https://doi.org/10.1016/j.scitotenv.2012.07.065.