Science of the Total Environment 804 (2022) 150177

Contents lists available at ScienceDirect

Science of the Total Environment

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

Glyphosate and glufosinate ammonium, herbicides commonly used on

genetically modified crops, and their interaction with microplastics:
Ecotoxicity in anuran tadpoles
Rafael C. Lajmanovich a,b,⁎, Andrés M. Attademo a,b, Germán Lener b,c, Ana P. Cuzziol Boccioni a,b,
Paola M. Peltzer a,b, Candela S. Martinuzzi a,b, Luisina D. Demonte b,d, María R. Repetti d
a
Laboratorio de Ecotoxicología, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Santa Fe, Argentina
b
Consejo Nacional de Investigaciones Científicas Técnicas (CONICET), Buenos Aires, Argentina
c
Instituto de Investigaciones en Físico-Química de Córdoba-CONICET, Departamento de Química Teórica y Computacional, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba,
Córdoba, Argentina
d
Programa de Investigación y Análisis de Residuos y Contaminantes Químicos, Facultad de Ingeniería Química, Universidad Nacional del Litoral, Santa Fe, Argentina

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Glyphosate, glufosinate, microplastics,

and their mixture affected tadpoles.
• Enzymatic activities were greater af-
fected by mixtures than individual
treatments.
• Herbicide-microplastic spontaneous in-
teractions lead to strong bonds forma-
tion.
• Glufosinate-microplastic mixture showed
higher ecotoxicity and binding forces.

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

Article history: The effects of glyphosate (GLY)-based and glufosinate ammonium (GA)-based herbicides (GBH and GABH, respec-
Received 25 June 2021 tively) and polyethylene microplastic particles (PEMPs) on Scinax squalirostris tadpoles were assessed. Tadpoles
Received in revised form 21 August 2021 were exposed to nominal concentrations of both herbicides (from 1.56 to 100 mg L−1) and PEMPs (60 mg L−1), ei-
Accepted 2 September 2021
ther alone or in combination, and toxicity evaluated at 48 h. Acetylcholinesterase (AChE), carboxylesterase (CbE),
Available online 8 September 2021
and glutathione-S-transferase (GST) activities were analyzed at the three lowest concentrations (1.56, 3.12 and
Editor: Daniel Wunderlin 6.25 mg L−1, survival rates >85%) of both herbicides alone and with PEMPs. Additionally, the thermochemistry of
the interactions between the herbicides and polyethylene (PE) was analyzed by Density Functional Theory (DFT).
The median-lethal concentration (LC50) was 43.53 mg L−1 for GBH, 38.56 mg L−1 for GBH + PEMPs, 7.69 for
Keywords: GABH, and 6.25 mg L−1 for GABH+PEMPs. The PEMP treatment increased GST but decreased CbE activity, whereas
Amphibian GBH and GABH treatments increased GST but decreased AChE activity. In general, the mixture of herbicides with
Emerging contaminant PEMPs increased the effect observed in the individual treatments: the highest concentration of GBH + PEMPs in-
Herbicides creased GST activity, whereas GABH+PEMP treatments decreased both AChE and CbE activities. DFT analysis re-
Polyethylene particles
vealed spontaneous interactions between the herbicides and PE, leading to the formation of bonds at the
Plastic
herbicide-PE interface, significantly stronger for GA than for GLY. The experimental and theoretical findings of our
Density Functional Theory
study indicate that these interactions may lead to an increase in toxicity when pollutants are together, meaning po-
tential environmental risk of these combinations, especially in the case of GA.
© 2021 Published by Elsevier B.V.

⁎ Corresponding author at: Laboratorio de Ecotoxicología, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral (FBCB-UNL), Casilla de Correo 242, 3000 Santa
Fe, Argentina.
E-mail address: lajmanovich@hotmail.com (R.C. Lajmanovich).

https://doi.org/10.1016/j.scitotenv.2021.150177
0048-9697/© 2021 Published by Elsevier B.V.
R.C. Lajmanovich, A.M. Attademo, G. Lener et al.
1. Introduction
The incorporation of genetically modified (GM) crops resistant to
pesticides has turned the agricultural model into agrochemical-
dependent (Raman, 2017). Since the introduction of GM crops resistant
to broad-spectrum herbicides such as glyphosate (GLY) and glufosinate
ammonium (GA) in the 1990s, the land area planted and, consequently,
the ecotoxicological environmental risk and human exposure to these
herbicides have increased dramatically (Sang et al., 2021). The massive
use of herbicides reduces biodiversity and decreases the provision of
ecosystem services that maintain food security (Almeida et al., 2017).
Although this is a worldwide issue, more than 95% of GM crops in the
world are produced by only five countries (United States, Brazil,
Argentina, Canada and China). After the United States and Brazil,
Argentina is the third country in the world with the highest amounts
of GM crops, and the incorporation rate of GM technologies has been es-
timated close to saturation (Pengue, 2005). According to the ArgenBio
database (2021), there are currently 61 approved transgenic events
(evaluated and approved for agriculture by authorities), mainly for
corn, soybean, and cotton. About 60.65% of these events include toler-
ance to GLY, GA or both. Notably, GA-tolerant events have greatly
grown in the last decade. GA is a key herbicide for the management of
GLY-resistant weeds mainly because it has a broad spectrum. Currently,
there is a great variety of transgenic GA-resistant crops, including
wheat, soybean, cotton, and corn (Áy et al., 2012; Ayala et al., 2019;
Takano and Dayan, 2020).
It is broadly known that the indiscriminate use of pesticides affects
aquatic organisms, and it is not surprising that one of the main hypoth-
eses for the global decline in amphibian populations is that it is due to
the effects of these chemicals (Egea-Serrano et al., 2012). In particular,
many of the adverse effects of the use of GLY-based herbicides (GBHs)
have been observed in amphibians (Berger et al., 2018). Certainly, am-
phibian tadpoles, which are an emblematic group within vertebrates,
have been widely used to study the effects of GBHs (e.g., Mann and
Bidwell, 1999; Relyea and Jones, 2009; Howe et al., 2004; Lajmanovich
et al., 2003, 2011; Wagner et al., 2013; Bach et al., 2016; Mikó et al.,
2017; Riaño et al., 2020; Herek et al., 2021, among others). Results of
these investigations have shown several adverse effects of GBHs, includ-
ing damage in gill morphology, loss of osmotic stability or mortality, ter-
atogenic and histological effects, inhibition of B-esterases and
detoxification enzymes, erythrocyte nuclear alterations, DNA damage,
and thyroid hormone disruptions. Larval amphibians have also been
used to investigate the effects of GA-based herbicides (GABHs), which
also include inhibition of B-esterases, erythrocyte nuclear abnormali-
ties, embryotoxicity and thyroid disruption (Peltzer et al., 2013;
Lajmanovich et al., 2014; Babalola et al., 2020a, 2020b).
Microplastics (MPs), i.e. plastic particles with diameters smaller
than 5 mm, threaten biodiversity and global environmental health
worldwide (Akdogan and Guven, 2019). Due to their small size and
low biodegradation, MPs can be ingested by wild organisms, and thus
become incorporated into the food chain and accumulated in soil and
water matrices for a long time (Cole et al., 2013; Brodhagen et al.,
2017). In agroecosystems, these compounds enter by primary manufac-
ture (e.g., medical applications, waterborne paints, electronics, adhe-
sives, coatings) or as secondary MPs generated by the breakdown of
larger plastic debris (Ng et al., 2018). One of the most mass-
manufactured hydrocarbon polymers of different plastic grades is poly-
ethylene (PE); so, PE can be found in large quantities both in aquatic and
terrestrial systems (Hüffer et al., 2019). In addition, plastic mulch is
commonly used to increase crop yields; however, it produces large
amounts of PE microplastic (PEMP) waste, which contaminates agricul-
tural soils and surrounding aquatic ecosystems (Bandopadhyay et al.,
2018). This contributes to massive plastic pollution around the world
and raises a serious environmental concern that remains unclear
(Zhang et al., 2020; Serrano-Ruiz et al., 2021). In addition, in recent
years, the use of silo bags (thick laminated extruded PE plastic about
2
Science of the Total Environment 804 (2022) 150177
230–235 μm thick) for storage of grains (wheat, soybean, corn, and sun-
flower) has been successfully adopted in several Latin American coun-
tries (Casini et al., 2014). In Argentina, silo bags are widely used and
have led to an increase in grain storage from 5 million tons in 2000 to
40 million tons in 2008 (Bartosik, 2012).
Despite the global decline of amphibians, the potential risks of MPs
to these organisms are still practically unknown (Boyero et al., 2020).
Hu et al. (2018) recognized MPs in the digestive tract of several anuran
species (Pelophylax nigromaculatus, Rana limnochari, Microhyla ornata,
and Bufo gargarizans), demonstrating that amphibians can ingest MPs
from their habitats (sediments and water). Also, Hu et al. (2016)
found PEMPs in the digestive tract and gills of Xenopus tropicalis tad-
poles after 1 h of exposure, and Araújo and Malafaia (2020) reported be-
havioral toxicity on Physalaemus cuvieri tadpoles after short exposure to
PEMPs. Despite the numerous experimental studies (Bhagat et al., 2021)
focused on the interactions of MPs with polycyclic aromatic hydrocar-
bons, heavy metals, organohalogens, pharmaceuticals, pesticides, and
other emerging contaminants, only a few have investigated how MPs
alter the general toxicity of other common pollutants such as herbicides
in wild fauna. NematdoostHaghi and Banaee (2017), for example, inves-
tigated the potential effects of the herbicide paraquat and MPs on blood
biochemical biomarkers in the common carp (Cyprinus carpio), and
Zocchi and Sommaruga (2019) reported that MPs modify the toxicity
of GLY in Daphnia magna. However, whether MPs can alter the toxicity
of herbicides in amphibian tadpoles remains unknown.
The environmental risk of the different chemical species that occur
in the soil or water and their potential interaction require deep study.
Thus, it is important to determine the affinity of MPs towards pesticides,
which could be linked to the functional groups of the plastic surface (i.e.
carboxyl residues) (Bergbreiter, 1994; Bao et al., 2011; Chua et al.,
2014). In addition, MPs play a role as pesticide vectors since it has
been demonstrated that MPs carry pesticides through long distances
in wetlands or after pesticide applications followed by rains
(Seidensticker et al., 2018; Hüffer et al., 2019; Rodríguez-Seijo et al.,
2019; Wanner, 2021).
The affinity between two or more chemical species and their
physico-chemical molecular interaction can be determined by the com-
putational chemistry technique named Density Functional Theory
(DFT) (Lener et al., 2016). This technique allows the evaluation of ther-
modynamic aspects, such as bonding energy, electronic density and
structure, of this type on interaction. Stathi et al. (2006), for example,
studied the affinity and adsorptive properties of thiuram disulfate pesti-
cides on Al-montmorillonite clay surface, Belzunces et al. (2019) deter-
mined the interaction between pesticides and fungicides in Ca-
montmorillonite interfaces, and Ahmed et al. (2018) explored the inter-
action between the functional group of GLY and goethite surfaces.
The objectives of the current study were to evaluate the toxicity of a
GBH, a GABH and PEMPs, either alone or in combination, on biochemical
biomarkers of striped snouted tree frog (Scinax squalirostris) tadpoles. In
addition, computational chemistry models were simulated to obtain the
bond energies and electronic structure of the interactions between the
herbicides and PEMPs. Tadpole mortality and enzyme activities were
expected to be greater in the combined presence of herbicides and
PEMPs due to the formation of new chemical structures and compounds
which can increase their toxicity.
2. Materials and methods
2.1. Toxicity assays
2.1.1. Test substances
For the experiments with GLY and GA, we used commercial formu-
lations (based on both active ingredients), as this is the form in which
they are introduced into the environment (Relyea and Jones, 2009).
The commercial formulations chosen were: the GBH Mifos®,
Chemotecnica Co., Argentina (48% active ingredient [a.i.] GLY, N-
R.C. Lajmanovich, A.M. Attademo, G. Lener et al.
phosphonomethyl glycine) and the GABH Liberty®, Basf Co., Argentina
(20% a.i. GA, ammonium-dl-homoalanin-4-yl (methyl) phosphinate). Lit-
tle is known about other compounds integrating formulations apart from
a.i. (defined as “co-formulants”), as the chemical composition of formula-
tions is considered confidential business information. For this reason, the
safety classification of formulations is defined by their a.i. despite co-
formulants can enhance or modify a.i. toxicity (Milesi et al., 2021). Avail-
able technical data of both herbicides are summarized in Addendum
Table 1. PEMPs were purchased from Sigma-Aldrich (CAS number
9002-88-4, 40–48 μm particle size, density 0.9215–0.9255 g/mL; purity
>99%).
2.1.2. Organisms
The model test organisms were Scinax squalirostris (Anura: Hylidae)
tadpoles. This frog has an extensive distribution in Neotropical regions,
including midwestern, southern, and southeastern Brazil, in addition to
southern Uruguay, Paraguay, northeastern Argentina and eastern
Bolivia, and is cataloged as “not threatened” in the categorization of am-
phibians of Argentina (Vaira et al., 2012). This species occurs in forests,
grasslands and agricultural ecosystems, and uses ponds surrounded by
soybean and other crops (Peltzer et al., 2006).
Premetamorphic tadpoles (Gosner stages 26–30; Gosner, 1960)
were collected from an unpolluted site without agricultural activities,
situated in a natural floodplain of the Paraná River (31°36′ 50.6″ S, 60°
35′ 52.9″ W), Santa Fe, Argentina, with collection permission of the Min-
istry of Environment of Santa Fe Province (EXP. N° 02101-0018518-1).
The tadpoles were acclimated under laboratory conditions for 48 h
under a 12-h light/dark cycle in flasks with dechlorinated tap water
(DTW) (pH: 7.4 ± 0.05; conductivity: 175 ± 20 μmhos cm−1; dissolved
oxygen concentration: 6.5 ± 1.5 mg L−1; hardness: 55.5 mg L−1 of
CaCO3), at 24 ± 2 °C. Boiled lettuce was given ad libitum as food. The
mean total length of tadpoles at the start of experiment was 9.66 ±
0.24 mm, whereas their weight was 0.09 ± 0.02 g. The tadpoles used
in the bioassays were treated in conformity with the standardized ex-
perimental laboratory procedure of the American Society of Ichthyolo-
gists and Herpetologists (2004) and ASTM (2007), with minimum
adjustments for wild species, as previously described (Attademo et al.,
2016). Tadpoles were euthanized following the Animal Euthanasia
Guide proposed by the Bioethics Committee and Institutional Animal
Care and Use Committee of the Facultad de Bioquímica y Ciencias
Biológicas, Universidad Nacional del Litoral, Santa Fe, Argentina (Res.
CD N: 388/06).
2.1.3. Experimental design
To examine the combined effect between the two herbicide formu-
lations and PEMPs, an acute short-term toxicity assay was performed.
The nominal concentrations of GBH and GABH used to test single toxic-
ities were: 1.56, 3.12, 6.25, 12.5, 25, 50 and 100 mg L−1 for ecotoxicolog-
ical comparison, as previously indicated in Lajmanovich et al. (2011,
2014). These concentrations were prepared with DTW, and with and
without 60 mg L−1 of PEMPs. A treatment with PEMPs alone
(60 mg L−1) was also tested. The chosen concentration of PEMPs has
been indicated as a realistic pollution condition for amphibian tadpoles
(Araújo et al., 2020; Anbumani and Kakkar, 2018). A negative control
treatment with DTW was also added.
Both the control and test concentrations were evaluated in triplicate.
Glass flasks (13.5 cm high; 12.5 cm in diameter) containing 1 L of test
solution and ten tadpoles per assay (N = 30 tadpoles per treatment)
were used. Tests were made at 24 °C ± 2 °C and under a 12-h light/
dark cycle. Tadpole mortality was monitored and dead animals were re-
moved every 24 h. Median lethal concentration (LC50) and the no- and
lowest-observed-effect concentrations (NOEC and LOEC, respectively)
were calculated. Both control and test solutions were made in triplicate.
The cumulative mortality rates for each treatment were calculated at 24
and 48 h of exposure. To obtain a homogeneous distribution of PEMPs
during the bioassay, the solution in each glass flask was continuously
3
Science of the Total Environment 804 (2022) 150177
aerated to maintain the dispersion of the particles in water and prevent
PEMP aggregation (Yifeng et al., 2016).
At the end of the assay (48 h), seven tadpoles from the control and
PEMP treatments, as well as from the treatments with the three lowest
concentrations of both herbicides with and without PEMPs, which
displayed survival rates higher than 85% (Lajmanovich et al., 2010;
Attademo et al., 2021), were used for biochemical determination of ace-
tylcholinesterase (AChE), carboxylesterase (CbE), and glutathione-S-
transferase (GST) activities.
2.2. Enzymatic activities
Tadpoles (n = 7 per treatment) were weighed (g), and then homog-
enized (1:10, w/v) in ice-cold 25 mM sucrose and 20 mM Tris-HCl
buffer (pH 7.4) containing 1 mM EDTA, by using a polytron tissue
grinder. The homogenates were centrifuged at 10,000g for 15 min at
4 ± 1 °C, and stored at −80 °C until measurements. Total protein con-
centration was determined by the Biuret method (Kingsley, 1942) to
calculate enzymatic activity levels.
Enzymatic activities were measured at 25 °C, using a JENWAY 6405
UV-VIS spectrophotometer, following the specifications of the methods
to measure AChE, CbE and GST in amphibians, routinely used in our lab-
oratory (Attademo et al., 2021; Lajmanovich et al., 2019; Peltzer et al.,
2019).
AChE activity was determined colorimetrically following Ellman
et al. (1961), and expressed as nmol min−1 mg−1 protein (molar extinc-
tion coefficient (MEC) = 13.6 × 103 M−1 cm−1).
CbE activity was measured by the method of Bunyan and Jennings
(1968), using α naphthyl acetate (α-NA) (1.04 mg mL−1 in acetone)
as substrate. Hydrolysis of α-NA was expressed as nmol of hydrolyzed
substrate min−1 mg−1 protein (MEC = 3.225 × 103 M−1 cm−1).
GST activity was measured spectrophotometrically according to
Habig et al. (1974) and modifications of Habdous et al. (2002) for mam-
mal serum GST activity. Whole GST activity was expressed as nmol
min−1 mg−1 protein (MEC = 9.61 × 103 M−1 cm−1).
2.3. Chemical determination of the herbicides
To confirm the exposure concentrations of the commercial formula-
tions used, the concentrations of GLY plus its metabolite
aminomethylphosphonic acid (AMPA) and GA were determined as de-
scribed by Demonte et al. (2018). The concentrations of the two herbi-
cides showed low variations compared to the predicted nominal
concentrations (Addendum Table 2).
2.4. Computational quantum chemistry of the interaction between the
herbicides and PE
The interactions between the two herbicides and PEMPs were com-
putationally characterized considering the herbicide active ingredients
(GLY and GA) and the PE molecule. It is important to point out that sim-
ulating the interaction of all the components of a real system is as com-
plex as not feasible, since there are many not-informed substances
(surfactants, micelles, metals, etc.) of commercial herbicide formula-
tions that are under industrial secret. In addition, since the state of art
of the pesticide formulation is dynamic (Jibrin et al., 2001; Liu et al.,
2021), the assignation of chemical combination would require a com-
plete characterization for every formulation, which is forbidden by pat-
ent laws.
The generalized gradient approximation in the Perdew–Burke–
Ernzerhof version (Perdew et al., 1996) was used to perform the DFT
calculations and provide information regarding thermochemical and
electronic structure properties, as implemented in the SIESTA code
(Soler et al., 2002). Indeed, the Troullier–Martins norm-conserving
pseudopotentials (Troullier and Martins, 1991) were used to represent
the nucleus and core electrons of the species considered. The basis set
R.C. Lajmanovich, A.M. Attademo, G. Lener et al.
used for the expansion of the Kohn–Sham eigenstates is composed of a set
of numerical atomic orbitals including polarization orbitals. An energy
shift of 50 meV was chosen as a compromise between accuracy and com-
putational efficiency. We took an energy cut-off of 200 Ry, and a double-f
plus polarization orbital basis set. We used the 6 6 6 k-point and 6 1 6 k-
point for the Brillouin zone for bulk and slab respectively. The coordinates
were optimized by conjugate gradients, and all geometries were opti-
mized using the conjugate gradient method until the force on each
atom was lower than 0.01 eV/Å. In real environments, PE has been ob-
served as fiber as a consequence of bio- and photo-degradation (Cole
et al., 2016; Lambert and Wagner, 2016; Song et al., 2019). Here, PE
frames were modeled as a 1D fiber, built from the ethylene monomer.
In addition, as a consequence of natural surface degradation and residues
that remain after their chemical synthesis, plastics have functional groups
on their surface (Bergbreiter, 1994; Hakkarainen and Albertsson, 2004).
To describe these surface properties and evaluate the interaction of PE
functional groups with GLY and GA molecules, the carboxyl group
(PECOOH) was proposed as adsorbent material of GLY and GA molecules.
To study the herbicides-PE interaction, calculations were run as fol-
lows.
First, each herbicide structure and PE fiber were optimized as iso-
lated frames. Then, herbicides-PE interfaces were studied by placing
pesticides on the PE slabs facing the functional groups, followed by
quantum dynamics using a thermostat at 300 K for 1 ps. Finally, the ge-
ometries obtained were optimized by relaxation using the conjugate
gradient algorithm.
The adsorption energies of the herbicides-PE interactions were ob-
tained by the following thermodynamic equation:
EB ¼ EðPE−Herbicide− Þ − ðEPE þ EHerbicide Þ
where EB is the adsorption energy of the herbicide-PE system, EPE is the
energy of the PE slab, and EHerbicide is the energy of the pesticide
molecule isolated.
The pseudo-charge density difference Δn was defined as follows
(Lener et al., 2016):
Δn ¼ ΔnðPE−HerbicideÞ − ðΔnPE þ ΔnHerbicide Þ
where Δn is the pseudo-charge density of the system, Δn(PE-Herbicide) is
the pseudo-charge density of the final PE/herbicide system, ΔnPE is the
pseudo-charge density of the PE slab, and Δn Herbicide is the pseudo-
charge density of the pesticide. The molecular graphics were created
with the XCRYSDEN package (Kokalj, 2003).
2.5. Toxicity data analysis
The LC50 values and their respective 95% confidence intervals were
calculated via the Trimmed Spearman-Karber method (Hamilton et al.,
1977). Significant differences (p ≤ 0.05) between LC50 values were
determined by the criterion of non-overlapping 95% confidence intervals
(APHA, 1989; Wheeler et al., 2006). The data of biochemical parameters
(enzymatic activity) were expressed as mean ± standard error (SE).
Kolmogorov-Smirnov test and Levene test were used to confirm normality
Table 1
Comparison of the median lethal concentration (LC50 (95% confidence intervals)), lowest observed effect concentration (LOEC), and no observed effect concentration (NOEC) values
(mg L−1) in Scinax squalirostris tadpoles exposed to a glyphosate-based herbicide (GBH) and a glufosinate-ammonium-based herbicide (GABH) with and without polyethylene
microplastics (PEMPs).
24 h
LC50 LOEC
GBH 61.56 (55.66–73.36) 50
GBH + PEMPs 47.54(35.77–63.17) 50
GABH 10.15 (8.52–12.01) 12.5
GABH+PEMPs 6.25 (5.02–7.68) 6.25
Values in bold indicate LC50 significantly different between GABH and GABH+PEMPs (no overlapping of 95% confidence intervals).
Science of the Total Environment 804 (2022) 150177
and homogeneity of variances, respectively. The differences in enzymatic
activity of tadpoles from control and treatments were analyzed with
one-way ANOVA and Dunnett's test for post-hoc comparisons. The results
were statistically analyzed using GraphPad InStad©. Values were consid-
ered significant at p < 0.05. Since no significant differences were found
among replicates (p > 0.05), replicates from each treatment were pooled.
3. Results
3.1. Toxicity
The results of the acute toxicity bioassay for LC50, LOEC and NOEC are
summarized in Table 1. No mortality was observed in the control or PEMP
treatment (60 mg L−1). At 48 h, the LC50 values for GBH ranged from
43.53 (35.61–53.21) mg L−1 for the herbicide alone to 38.56
(30.08–49.41) mg L−1 for the herbicide in mixture with PEMPs
(GBH + PEMP treatment), whereas the LC50 values for GABH were 7.69
(6.45–9.17) mg L−1 for the herbicide alone and 6.25 (5.02–7.68) mg
L−1 for the herbicide in mixture with PEMPs (GABH+PEMP treatment).
Differences between GABH and GABH+PEMP LC50 were statistically
significant at 24 h. LOEC and NOEC values were the same at 24 and 48 h
for GBH and GABH+PEMP treatments (50–25 and 6.25–3.12, respec-
tively). For GBH + PEMP and GABH treatments, the LOEC and NOEC
values at 24 h (50–25 and 12.5–6.25 mg L−1 respectively for each treat-
ment) decreased by 50% at 48 h (25–12.5 and 6.25–3.12 mg L−1).
3.2. Enzyme biomarkers
At the end of the assay (48 h), the mean value of AChE activity in
ð1Þ control tadpoles was 43.97 ± 6.77 nmol min−1 mg−1, whereas that in
treated tadpoles was significantly altered (F = 16.889, p < 0.05;
Fig. 1). The three GBH concentrations highly decreased AChE activity
(by 45% and 36.5% at 1.65 and 6.25 mg L−1, respectively), whereas
6.25 mg L−1 GBH + PEMPs reduced it by 25%. The highest GABH con-
centration (6.25 mg L−1) also significantly decreased AChE activity
(by 45%), whereas the GABH+PEMPs treatment decreased it from
24.45 to 50%.
ð2Þ CbE activity, which in control tadpoles was 2.34 ±
0.35 nmol min−1 mg−1, was significantly reduced by 33% in PEMP-
treated tadpoles and in those treated with 6.24 mg L −1 GABH
+PEMPs (F = 4.92, p < 0.05; Fig. 2).
Regarding GST activity, control tadpoles recorded a mean of
106.74 ± 30.47 min−1 mg−1, whereas PEMP-treated tadpoles showed
a significant increase (by 40%; F = 21.546; p < 0.05; Fig. 3). GST activity
was also increased at the highest GBH concentration (6.25 mg L−1) with
and without PEMPs (by 110% and 64%, respectively), at 6.25 mg L−1
GABH (96%), and at 1.56 mg L−1 GABH+PEMPs (37%).
3.3. Computational quantum chemistry of the interaction between the
herbicides and PE
The optimized geometries, as well as the differential electronic den-
sity, for the herbicide-PECOOH interaction are shown in Fig. 4. In both
cases, the binding energy was exothermic (−67.6 kJ mol−1 and
48 h
NOEC LC50 LOEC NOEC
25 43.53 (35.61–53.21) 50 25
25 38.56 (30.08–49.41) 25 12.5
6.25 7.69 (6.45–9.17) 6.25 3.12
3.12 6.25 (5.02–7.68) 6.25 3.12
4
R.C. Lajmanovich, A.M. Attademo, G. Lener et al. Science of the Total Environment 804 (2022) 150177

Fig. 1. AChE activity (nmol min−1 mg−1 of protein) in Scinax squalirostris tadpoles exposed to a glyphosate-based herbicide (GBH) and a glufosinate-ammonium-based herbicide (GABH)
with and without polyethylene microplastics (PEMPs). Data are expressed as mean ± SE. Significantly different from control (CO): * p < 0.05, ** p < 0.01; Dunnett's test).

−100.6 kJ mol−1 for GLY and GA, respectively), being stronger in GA
than in GLY. The differential electronic density (Fig. 4) showed a red
lobe in the herbicide-PE interfaces, specifically between the PECOOH
frame and the phosphonic groups of both herbicides.
4. Discussion
The herbicide market has grown worldwide, being responsible for
an increase of 50% in the total pesticide consumption, reaching an an-
nual rate of 17% during 1990 and 2005 (Zhang et al., 2011). In
Argentina, the two herbicides here evaluated (GLY and GA) increased
by approximately 250% from 2000 to 2015 (Scursoni et al., 2019). Re-
garding MPs, Blettler et al. (2017) conducted pioneering studies on con-
tamination by macro-, meso- and MPs in freshwater ecosystems of the
alluvial plain of the Paraná River and found an average of 4654 MP frag-
ments/m2 (ranging from 131 to 12,687 MPs/m2) in sediments of the
wetlands on its shore (Blettler et al., 2019). In the same way, by using
spectrometry, Mitchell et al. (2021) verified the plastic nature of the
MPs present in these ecosystems, which showed characteristics of
PEMPs. These wetlands are one of the regions of Argentina with the

Fig. 2. CbE activity (nmol min−1 mg−1 of protein) in Scinax squalirostris tadpoles exposed to a glyphosate-based herbicide (GBH) and a glufosinate-ammonium-based herbicide (GABH)
with and without polyethylene microplastics (PEMPs). Data are expressed as mean ± SE. Significantly different from control (CO): * p < 0.05 Dunnett's test).

Fig. 3. GST activity (nmol min−1 mg−1 of protein) in Scinax squalirostris tadpoles exposed to a glyphosate-based herbicide (GBH) and a glufosinate-ammonium-based herbicide (GABH)
with and without polyethylene microplastics (PEMPs). Data are expressed as mean ± SE. Significantly different from control (CO): * p < 0.05, ** p < 0.01; Dunnett's test).

5
R.C. Lajmanovich, A.M. Attademo, G. Lener et al.
Fig. 4. Optimized geometries and differential pseudo-electronic density for (A) the glyphosate (GLY) and (B) glufosinate-ammonium (GA) interaction with polyethylene (PE). Red lobes
indicate accumulation of electronic density; blue lobes indicate depletion of electronic density. Density of charge at the isosurface 1.10−4 e/Bohr3.
highest amphibian biodiversity where tadpoles inhabit (Peltzer and
Lajmanovich, 2007).
Anuran amphibians often breed in water bodies adjacent to crop
fields (Bishop et al., 1999; Peltzer et al., 2006). For this reason, labora-
tory and field researches have highlighted the strong relation between
herbicides and amphibian exposure, reinforcing the ecotoxicological
risk of pesticides for these vertebrates (EFSA, 2018). The GLY field
concentrations that may be particularly relevant to amphibian tad-
poles are not only those observed in the environment, but also
those directly related to amphibian habitats (temporal small
ponds). Mann and Bidwell (1999) measured a worst-case scenario
for wild tadpoles after direct over-spraying with GBH in a small su-
perficial water body (5–10 cm in depth) at the maximum authorized
application rate and recorded values of ≈21 mg L−1 of GBH. Further-
more, considering that GBH and GABH are applied at similar rates
(approximately 600–980 g a.e. ha −1; Costa et al., 2018) and that
they have similar potential to contaminate surface water (0.031
and 0.064‰ of the active ingredient applied, respectively;
Screpanti and Accinelli, 2005), the worst-case exposure ranges in
temporal ponds for tadpoles would be similar.
The present study is the first to simultaneously examine the effects
of exposure to GBH and GABH and their interaction with PEMPs on am-
phibian tadpoles. The results based on LC50 show that GABH is five-fold
more toxic than GBH. The first research on GBH toxicity carried out by
Mann and Bidwell in 1999 in several amphibian tadpole species re-
ported a wide range of LC50 values between 8.1 and 48.7 mg L−1 for
6
Science of the Total Environment 804 (2022) 150177
the GBH Roundup® and up to 911 mg L−1 for the GBH Roundup
Bioactive®. Regarding this, it has been reported that the LC50 value of
GBH for tadpoles fundamentally depends on the chemical structure of
the surfactants used in each trademark, which may have an intrinsic
toxicity (Mann et al., 2009; Vincent and Davidson, 2015). In the present
study, the LC50 value obtained for the GBH used (Mifos®) in treated
S. squalirostris tadpoles at 48 h was 43.53 mg L−1, similar to that previ-
ously found by us for the GBH Roundup Ultra-Max® in treated Rhinella
arenarum tadpoles at the same developmental stages (45.95 mg L−1)
(Lajmanovich et al., 2019). In contrast, regarding GABH, few data are
available on its acute toxicity in amphibians. In the present study, the
LC50 obtained for the GABH used (Liberty®) in treated S. squalirostris
tadpoles was 7.69 mg L−1 at 48 h, which is similar to values reported
in other tadpole species exposed to the same herbicide. Dinehart et al.
(2010), for example, determined LC50 values for the GABH Ignite®
(Bayer Crop Science) of 3.55 and 5.55 mg L−1 for treated Spea
bombifrons and S. multiplicata tadpoles, respectively, at 48 h. Neverthe-
less, in the sympatric hylid frog Boana pulchella, also exposed to the
GABH Liberty®, the LC50 found was considerably higher
(21.47 mg L−1 at 48 h) (Peltzer et al., 2013). A similar value (LC50 =
24.9 mg L−1 at 96 h) was reported by Babalola and van Wyk (2018)
for Xenopus laevis tadpoles exposed to the GABH Basta® (Bayer Crop
Science). The intra- or interspecific toxic variability of herbicides is a
well-known phenomenon observed in amphibians (e.g., Holcombe
et al., 1987; Howe et al., 2004; Jones et al., 2009; Lajmanovich et al.,
2010).
R.C. Lajmanovich, A.M. Attademo, G. Lener et al.
The synergisms between MPs and pesticides represent an amazing
threat for aquatic organisms worldwide, because they may potentially
exacerbate the toxicity of several pollutants in surface waters (Zocchi
and Sommaruga, 2019). Agathokleous et al. (2021) highlighted that
the study of pollutant interactions may contribute to better understand-
ing and predicting the effects of MPs, taking into account that organisms
are exposed to multiple stressors in the environment. MPs could inter-
act with other environmental stressors (pesticides, heavy metals, and
several toxic elements) and exacerbate amphibian decline (Boyero
et al., 2020). It is important to note that the PEMP concentration tested
in the present study (60 mg L−1) is not lethal for Scinax acuminatus tad-
poles after short exposure (48 h). Araújo and Malafaia (2020) also used
a PEMP concentration of 60 mg L−1 to simulate a realistic contamination
condition for Neotropical amphibian tadpoles (Physalaemus cuvieri) at
7 days of experimental assay, and reported that, at the end of PEMP ex-
posure, treated tadpoles showed locomotion alterations, anxiety-like
behaviors and anti-predatory response deficits. In our case, based on
our LC50 results, both herbicide formulations in mixture with PEMPs
were more ecotoxic than individually and more toxic at 24 than at
48 h. In fact, at 24 h, the LC50 values of the GABH+PEMP mixture
were statistically significant respect to those of the GABH treatment.
For this mixture, the LOEC and NOEC decreased to 50% at 24 h compared
with the GABH treatment. Several authors have reported that MPs, es-
pecially PE particles, increase the toxicity of pesticides (e.g., Garrido
et al., 2019; Bellas and Gil, 2020), which may explain the lower LC50
values obtained in this study for the mixtures. Furthermore, Zocchi
and Sommaruga (2019) revealed that MPs modify the toxicity of GLY,
increasing the mortality of Daphnia magna exposed to Roundup® for-
mulation and two different MPs (microbeads and PE-terephthalate or
polyamide fibers). According to these authors, the GBH toxicity caused
by MPs may be related to the different sorption properties of GLY-
based chemical formulations.
Regarding sublethal effects, it is important to evaluate the organism
response to low concentrations of pesticides, since they can induce del-
eterious effects on individual fitness and alter the survival of amphibian
tadpoles and adults (Svartz et al., 2016). Despite the differences in tox-
icity based on LC50, the sublethal effects based on enzymatic activities
were quite similar in the treatments with the individual herbicides.
The two herbicides reduced tadpoles AChE activity, being pronounced
in all GBH concentrations and only at the highest GABH concentration,
indicating a greater response to the effect of GBH. Both GBH and
GABH exposure increased GST activity only at the highest concentra-
tions evaluated. In accordance, several researches have reported similar
results in anuran larvae and other organisms exposed to the same her-
bicides (e.g., Lajmanovich et al., 2011, 2019; Takano and Dayan, 2020).
In pure chemical terms, GLY and GA are organophosphorous herbi-
cides that contain carbon and phosphorus, indicating that they could in-
hibit cholinesterases. Based on this, in 2011, both our group
(Lajmanovich et al., 2011) and that of Ruamthum et al. (2011) were
the first to document the effects of GBH on AChE activity in amphibian
tadpoles. In addition, we reported that sublethal concentrations of
GABH inhibited AChE activity in B. pulchella tadpoles (Peltzer et al.,
2013). Such inhibition of AChE activity by GBH and GABH exposures re-
inforces the potential toxicity of such herbicides to amphibian popula-
tions and other communities of ponds surrounding crops where these
herbicides are massively used (Lajmanovich et al., 2014). Overall, our
present results showed that the GABH+PEMP mixture decreased both
AChE and CbE activities compared to the treatments with the herbicide
alone. Since CbE and other several hydrolases are known to degrade
synthetic polymers (e.g., PE, polypropylene, polystyrene, polyvinyl
chloride, and others) (Kawai et al., 2019), this probably explains the ev-
ident decrease in CbE activity in the PEMP treatment (60 mg L−1). How-
ever, CbE activity was inhibited only at the highest GABH concentration,
while AChE activity was significantly reduced at all GABH+PEMP mix-
ture concentrations. In previous studies in GBH-treated R. arenarum tad-
poles, we found that both AChE and CbE activities were inhibited
7
Science of the Total Environment 804 (2022) 150177
respect to controls (Lajmanovich et al., 2011). To our knowledge,
there are no data to support these results and explain how these en-
zymes detoxify both herbicides. In this sense, the levels of CbE activity
measured can be explained in terms of false positive results since no
data exist to assure that herbicides or MPs are potent CbE inhibitors.
Further research is necessary to demonstrate the way in which CbE con-
tributes to the detoxification of GLY and GA individually or in mixture
with MPs, taking into consideration their chemical interaction.
Regarding GST activity, GST is an important enzyme of phase II of
biotransformation, a process that protects cells from xenobiotics
(e.g., pesticides) by catalyzing the conjugation of a wide variety of elec-
trophilic substrates (van der Oost et al., 2003). In the present study, GST
activity was increased by both GBH and GABH exposure, but only at the
highest concentration evaluated. Therefore, the increase in GST activity
indicates a good defense of tadpoles against the toxic effects of the two
herbicides (Lajmanovich et al., 2011; Cuzziol-Boccioni et al., 2021). The
induction of GST activity in the PEMP treatment showed an accurate re-
sponse of the biotransformation process. This represents a key point in
the fitness of amphibian tadpoles in their natural environment due to
the crucial role of GST in the detoxification process of xenobiotics and
protection against oxidative stress (e.g. Freitas et al., 2017; Pal et al.,
2018; Carvalho et al., 2020). Our results show that the effect on GST ac-
tivity was enhanced by exposure to the GBH + PEMP mixture since it
was higher at the maximum concentration of the mixture than at the
same concentration of GBH alone. These results suggest that MPs inter-
act with GBH, increasing the antioxidant system of tadpoles. In general,
exposure to pesticides and other contaminant mixtures increases GST
activity under oxidative stress conditions (Güngördü et al., 2016). For
example, in the clam Scrobicularia plana, GST activity increases after ex-
posure to PEMPs with several adsorbed chemical contaminants
(O'Donovan et al., 2018). By contrast, at the lowest concentrations
here studied, the GBH + PEMP mixture slightly diminished the effect
of GBH alone, regarding both GST and AChE activities. This could indi-
cate that the interaction of both compounds at low concentrations alters
the processes in which both enzymes are involved and affects their ad-
equate response.
The results of our bioassay demonstrated a differential ecotoxicolog-
ical response of S. squalirostris tadpoles to the two herbicides evaluated
(GLY and GA) when they were mixed with PEMPs, suggesting a great in-
teraction between these compounds. The computational chemistry
analysis confirmed the interaction of the active ingredients of the herbi-
cides with PE. According to the DFT analysis, the high solubility of the
herbicides-PE combination may lead to an interaction effect on the tox-
icity mechanism that indicates a possible interaction. Likewise, it has
been reported that MPs can absorb and concentrate different pollutants
(e.g., pesticides) present in the environment (Antunes et al., 2013) and,
therefore, increase their toxicity (e.g., in an agroecosystem scenario).
Bellas and Gil (2020), for example, found that PEMPs increased the tox-
icity of the pesticide chlorpyrifos in copepods. The fact that the two mix-
tures studied in the present work have exothermic binding energy
shows a favored electronic interaction between the interfaces, which
made possible the generation of a new chemical compound. Further-
more, the stronger interaction obtained for GA-PECOOH respect to the
moderate one for GLY-PECOOH may be related to the statistically
significant increased toxicity detected at 24 h for GABH+PEMP. To our
knowledge, this is the first approach to analyze herbicide-MP interac-
tions. It is important to point out that, in our study, we used the term
“interaction” because we made no quantitative evaluation. Conse-
quently, further studies are needed to elucidate the way in which
these compounds interact (synergism, antagonism, other).
Finally, the differential electronic density analysis for both mixtures
evaluated evidenced the accumulation of electronic density between
the PECOOH frame and the phosphonic groups of pesticides. These
results indicate the formation of a chemical bond at the interface and
suggest that PE systems can adsorb these herbicides, generating hot-
spots of concentration of pesticides which might enter an organism as
R.C. Lajmanovich, A.M. Attademo, G. Lener et al.
a new pollutant. Moreover, the higher desorption energies observed for
GA-PE respect to GLY-PE indicates that, once formed, it is more difficult
to slow down the PE bond with GA than that with GLY. Thus, the forma-
tion of a new pollutant from the interactions between pesticides and
plastics, which seems to enhance the individual toxicological effects,
represents a great and new environmental risk, especially in the case
of GA.
5. Conclusion
Overall, the increase in ecotoxicity and the alterations in the bio-
chemical parameters in S. squalirostris tadpoles exposed to GBH and
GABH in mixture with PEMPs reinforced our expectation that the co-
occurrence of MPs and herbicides in water bodies represents an ecotox-
icological risk for amphibian tadpoles. DFT calculations made evident
that the main tendency of GA to bind to PE, which allows the pesticide
concentration to enter the organism, drastically increases the toxicity,
as observed in the experiments of GABH+PEMP mixture. Therefore,
the MPs present in agricultural ponds and polluted water bodies can in-
crease the toxicity and bioavailability of pesticides. Our results demon-
strated the higher electronic affinity and toxicity of GA-PE compared
to those of GLY-PE. Due to the exponential increase in evidence of the
negative effects on wildlife of herbicides such as GLY and GA, which
are frequently used in GM crops, and the ecocide agricultural models
dispersing worldwide, it is imperative to apply higher regulatory re-
quirements and precaution principles on the use of GBHs and GABHs.
We also recommend the urgent decrease in the use of plastic materials
in agriculture (e.g., silo bags) and the implementation of more efficient
and mandatory recycling systems for the mitigation of these products
(potential source of MP pollution) through the use of innovative policies
and environmental awareness strategies among farmers. Finally, we
highlight the importance of addressing a change in the production
model through socioeconomic compensation. There is an urgent need
to transform the current agro-industrial model based on the use of her-
bicides such as GLY and GA for transgenic crops, which generates thou-
sands of tons of grains that are accumulated due to financial speculation
in plastic silo-bags, into an agroecological model that could safeguard
the health of ecosystems.
CRediT authorship contribution statement
Rafael C. Lajmanovich: Methodology, Investigation, Formal analy-
ses, Writing - original draft. Andrés M. Attademo: Formal analysis, In-
vestigation, Writing - review & editing. German Lener: Methodology,
Formal analysis, Writing. Ana P. Cuzziol Boccioni: Investigation, Formal
analyses, Writing - original draft. Paola M. Peltzer: Formal analysis, In-
vestigation, Writing - review & editing. Candela S. Martinuzzi: Method-
ology. Luisina D. Demonte: Chemical Analyses. Marìa R. Repetti:
Chemicals Analyses, Writing - review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
This study was supported in part by the National Agency for Promo-
tion of Science and Technology (ANPCyT FONCyT PICT, 2018 No. 3293;
and 1069), and the Course of Action for Research and Science Promotion
(CAI + D-UNL, PIC No. 50620190100036LI), Argentina. This work used
the Eulogia Cluster from CCAD-UNC, which is a part of SNCAD-
MinCyT, Argentina. We are especially grateful to Carlos Gálvez for self-
lessly providing us the commercial formulations of herbicides used in
this study. We also thank María Victoria Gonzalez Eusevi (Scientific
8
Science of the Total Environment 804 (2022) 150177
English Service) for proofreading the manuscript and correcting gram-
mar and spelling mistakes.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2021.150177.
References
Agathokleous, E., Iavicoli, I., Barceló, D., Calabrese, E.J., 2021. Micro/nanoplastics effects on
organisms: a review focusing on “dose”. J. Hazard. Mater. 12, 6084. https://doi.org/10.
1016/j.jhazmat.2021.126084.
Ahmed, A.A., Leinweber, P., Kühn, O., 2018. Unravelling the nature of glyphosate binding
to goethite surfaces by: ab initio molecular dynamics simulations. Phys. Chem. Chem.
Phys. 20, 1531–1539. https://doi.org/10.1039/c7cp06245a.
Akdogan, Z., Guven, B., 2019. Microplastics in the environment: a critical review of cur-
rent understanding and identification of future research needs. Environ. Pollut. 254,
113011. https://doi.org/10.1016/j.envpol.2019.113011.
Almeida, V.E.S., Friedrich, K., Tygel, A.F., Melgarejo, L., Carneiro, F.F., 2017. Use of geneti-
cally modified crops and pesticides in Brazil: growing hazards. Cien. Saude Colet.
22 (10), 3333–3339. https://doi.org/10.1590/1413-812320172210.17112017.
Anbumani, S., Kakkar, P., 2018. Ecotoxicological effects of microplastics on biota: a review.
Environ. Sci. Pollut. Res. Int. 25 (15), 14373–14396. https://doi.org/10.1007/s11356-
018-1999-x.
Antunes, J.C., Frias, J.G.L., Micaelo, A.C., Sobral, P., 2013. Resin pellets from beaches of the
Portuguese coast and adsorbed persistent organic pollutants. Estuar. Coast. Shelf Sci.
130, 62e69. https://doi.org/10.1016/j.ecss.2013.06.016.
APHA, 1989. Standard Methods for the Examination of Water and Wastewater. 17th edn.
American PublicHealthAssociation, Washington DC.
Araújo, A.P.D.C., Malafaia, G., 2020. Can short exposure to polyethylene microplastics
change tadpoles' behavior? A study conducted with neotropical tadpole species be-
longing to order anura (Physalaemus cuvieri). J. Hazard. Mater. 391, 122214.
https://doi.org/10.1016/j.jhazmat.2020.122214.
Araújo, A.P.D.C., de Melo, N.F.S., de Oliveira Jr., A.G., Rodrigues, F.P., Fernandes, T., de
Andrade-Vieira, J.E., Rocha, T.L., Malafaia, G., 2020. How much are microplastics
harmful to the health of amphibians? A study with pristine polyethylene
microplastics and Physalaemus cuvieri. J. Hazard. Mater. 382, 121066. https://doi.
org/10.1016/j.jhazmat.2019.121066.
ArgenBio©, 2021. Consejo Argentino para la Información y el Desarrollo de la
Biotecnología. Cultivos transgénicos aprobados en Argentina. Available at http://
www.argenbio.org/cultivos-transgenicos (Accessed 03 March 2021).
ASIH, 2004. American Society of Ichthyologists and Herpetologists. Guidelines for Use of
Live Amphibians and Reptiles in Field and Laboratory Research. second ed. Herpeto-
logical Animal Care and Use Committee (HACC) of the American Society of Ichthyol-
ogists and Herpetologists, Washington D.C.
ASTM, 2007. Standard guide for conducting acute toxicity tests with fishes, macroinverte-
brates, and amphibians. Biological Effects and Environmental Fate, ASTM E,
Pensilvania.
Attademo, A.M., Lajmanovich, R.C., Peltzer, P.M., Junges, C.M., 2016. Acute toxicity of met-
aldehyde in the invasive rice snail pomace acanaliculata and sublethal effects on tad-
poles of a non-target species (Rhinella arenarum). Water Air Soil Pollut. 227 (11),
400. https://doi.org/10.1007/s11270-016-3083-9.
Attademo, A.M., Lajmanovich, R.C., Peltzer, P., Cuzziol Boccioni, A.P., Martinuzzi, C.,
Simonielo, F., Repetti, M.R., 2021. Effects of the emulsifiable herbicide Dicamba on
amphibian tadpoles: an underestimated toxicity risk? Environ. Sci. Pollut. Res.
https://doi.org/10.1007/s11356-021-13000-x.
Áy, Z., Mihály, R., Cserháti, M., Kótai, É., Pauk, J., 2012. The effect of high concentrations of
glufosinate ammonium on the yield components of transgenic spring wheat
(Triticuma estivum L.) constitutively expressing the bar gene. Sci. World J. 657945.
https://doi.org/10.1100/2012/657945 (2012).
Ayala, F., Fedrigo, G.V., Burachik, M., Miranda, P.V., 2019. Compositional equivalence of
event IND-ØØ412-7 to non-transgenic wheat. Transgenic Res. 28 (2), 165–176.
https://doi.org/10.1007/s11248-019-00111-y.
Babalola, O.O., van Wyk, J.H., 2018. Comparative early life stage toxicity of african clawed
frog, X. laevis following exposure to selected herbicide formulations applied to erad-
icate alien plants in SouthAfrica. Arch. Environ. Contam. Toxicol. 75 (2015), 8–16.
https://doi.org/10.1007/S00244-017-0463-0.
Babalola, O.O., Truter, J.C., Van Wyk, J.H., 2020a. Lethal and teratogenic impacts of
imazapyr, diquat dibromide, and glufosinate ammonium herbicide formulations
using frog embryo teratogenesis assay-xenopus (FETAX). Arch. Environ. Contam.
Toxicol. 80 (4), 708–716. https://doi.org/10.1007/s00244-020-00756-5.
Babalola, O.O., Truter, J.C., Archer, E., van Wyk, J.H., 2020b. Exposure impacts of environ-
mentally relevant concentrations of a glufosinate ammonium herbicide formulation
on larval development and thyroid histology of Xenopus laevis. Arch. Environ.
Contam. Toxicol. https://doi.org/10.1007/s00244-020-00758-3.
Bach, N.C., Natale, G.S., Somoza, G.M., Ronco, A.E., 2016. Effect on the growth and develop-
ment and induction of abnormalities by a glyphosate commercial formulation and its
active ingredient during two developmental stages of the south-american creole frog,
Leptodactylus latrans. Environ. Sci. Pollut. Res. Int. 23, 23959–23971. https://doi.org/
10.1007/s11356-016-7631-z.
R.C. Lajmanovich, A.M. Attademo, G. Lener et al.
Bandopadhyay, S., Martin-Closas, L., Pelacho, A.M., DeBruyn, J.M., 2018. Biodegradable
plastic mulch films: impacts on soil microbial communities and ecosystem functions.
Front. Microbiol. 9, 819. https://doi.org/10.3389/fmicb.2018.00819.
Bao, L.J., You, J., Zeng, E.Y., 2011. Sorption of PBDE in low-density polyethylene film: im-
plications for bioavailability of BDE-209. Environ. Toxicol. Chem. 30, 1731–1738.
https://doi.org/10.1002/etc.564.
Bartosik, R., 2012. An inside look at the silo-bag system. In: Navarro, S., Banks, H.J., Jayas,
D.S., Bell, C.H., Noyes, R.T., Ferizli, A.G., Emekci, M., Isikber, A.A., Alagusundaram, K.
(Eds.), Proc 9th. Int. Conf. on Controlled Atmosphere and Fumigation in Stored Prod-
ucts, Antalya, Turkey, pp. 117–128.
Bellas, J., Gil, I., 2020. Polyethylene microplastics increase the toxicity of chlorpyrifos to
the marine copepod Acartia tonsa. Environ. Pollut. 260, 114059. https://doi.org/10.
1016/j.envpol.2020.114059.
Belzunces, B., Hoyau, S., Bessac, F., 2019. Interaction of metamitron and fenhexamid with
ca 2-montmorillonite clay surfaces: a density functional theory molecular dynamics
study. J. Comput. Chem. 40, 1449–1462. https://doi.org/10.1002/jcc.25799.
Bergbreiter, D.E., 1994. Polyethylene surface chemistry. Prog. Polym. Sci. 19, 529–560.
https://doi.org/10.1016/0079-6700(94)90004-3.
Berger, G., Graef, F., Pallut, B., Hoffmann, J., Brühl, C.A., Wagner, N., 2018. How does chang-
ing pesticide usage over time affect migrating amphibians: a case study on the use of
glyphosate-based herbicides in German agriculture over 20 years. Front. Environ. Sci.
6, 6.
Bhagat, J., Nishimura, N., Shimada, Y., 2021. Toxicological interactions of microplastics/
nanoplastics and environmental contaminants: current knowledge and future per-
spectives. J. Hazard. Mater. 5 (405), 123913. https://doi.org/10.1016/j.jhazmat.2020.
123913.
Bishop, C.A., Mahony, N.A., Struger, J., Ng, P., Pettit, K.E., 1999. Anuran development, den-
sity and diversity in relation to agricultural activity in the Holland River watershed,
Ontario, Canada (1990–1992). Environ. Monit. Assess. 57, 21–43.
Blettler, M.C.M., Ulla, M.A., Rabuffetti, A.P., Garello, N., 2017. Plastic pollution in freshwater
ecosystems: macro-, meso-, and microplastic debris in a floodplain lake. Environ.
Monit. Assess. 189 (11), 581. https://doi.org/10.1007/s10661-017-6305-8.
Blettler, M.C.M., Garello, N., Ginon, L., Abrial, E., Espinola, L.A., Wantzen, K.M., 2019. Mas-
sive plastic pollution in a mega-river of a developing country: sediment deposition
and ingestion by fish (Prochilodus lineatus). Environ. Pollut. 255 (Pt 3), 113348.
https://doi.org/10.1016/j.envpol.2019.113348.
Boyero, L., López-Rojo, N., Bosch, J., Alonso, A., Correa-Araneda, F., Pérez, J., 2020.
Microplastics impair amphibian survival, body condition and function. Chemosphere
244, 125500. https://doi.org/10.1016/j.chemosphere.2019.125500.
Brodhagen, M., Goldberger, J.R., Hayes, D.G., Inglis, D.A., Marsh, T.L., Miles, C., 2017. Policy
considerations for limiting unintended residual plastic in agricultural soils. Environ.
Sci. Pol. 69, 81–84. https://doi.org/10.1016/j.envsci.2016.12.014.
Bunyan, P.J., Jennings, D.M., 1968. Organophosphorus poisoning; some properties ofavian
esterase. J. Agric. Food Chem. 16, 326–331.
Carvalho, C.D.S., Moraes-Utsunomiya, H.S., Pasquoto-Stigliani, T., Costa, M.J., Fernandes,
M.N., 2020. Biomarkers of the oxidative stress and neurotoxicity in tissues of the bull-
frog, Lithobates catesbeianus to assess exposure to metals. Ecotoxicol. Environ. Saf.
2020 (196), 110560. https://doi.org/10.1016/j.ecoenv.2020.110560.
Casini, C., Santa Juliana, M., Audicio, S., 2014. Estudio del almacenamiento de granos en
bolsas plásticas Para pequeños agricultores. en: almacenamiento de granos en silo
bolsa. Resultados Investig. 2009–2013, 329–340.
Chua, E.M., Shimeta, J., Nugegoda, D., Morrison, P.D., Clarke, B.O., 2014. Assimilation of
polybrominated diphenyl ethers from microplastics by the marine amphipod,
allorchestescompressa. Environ. Sci. Technol. 48, 8127–8134. https://doi.org/10.
1021/es405717z.
Cole, M., Lindeque, P., Fileman, E., Halsband, C., Goodhead, R., Moger, J., Galloway, T.S.,
2013. Microplastic ingestion by zooplankton. Environ. Sci. Technol. 47, 6646–6655.
Cole, M., Lindeque, P.K., Fileman, E., Clark, J., Lewis, C., Halsband, C., Galloway, T.S., 2016.
Microplastics Alter the properties and sinking rates of zooplankton faecal pellets. En-
viron. Sci. Technol. 50, 3239–3246. https://doi.org/10.1021/acs.est.5b05905.
Costa, F.R., Rech, R., Duke, S.O., Carvalho, L.B., 2018. Lack of effects of glyphosate and
glufosinate on growth, mineral content, and yield of glyphosate- and glufosinate-
resistant maize. GM Crops Food 9 (4), 189–198. https://doi.org/10.1080/21645698.
2018.1511204.
Cuzziol-Boccioni, A.P., Lajmanovich, R.C., Peltzer, P.M., Attademo, A.M., Martinuzzi, C.S.,
2021. Toxicity assessment at different experimental scenarios with glyphosate, chlor-
pyrifos and antibiotics in Rhinella arenarum (Anura: Bufonidae) tadpoles.
Chemosphere 273, 128475. https://doi.org/10.1016/j.chemosphere.2020.128475.
Demonte, L.D., Michlig, N., Gaggiotti, M., Adam, C.G., Beldoménico, H.R., Repetti, M.R.,
2018. Determination of glyphosate, AMPA and glufosinate in dairy farm water from
Argentina using a simplified UHPLC-MS/MS method. Sci. Total Environ. 645, 34–43.
https://doi.org/10.1016/j.scitotenv.2018.06.340].
Dinehart, S.K., Smith, L.M., McMurry, S.T., Smith, P.N., Anderson, T.A., Haukos, D.A., 2010.
Acute and chronic toxicity of roundup weather max and ignite 280 SL to larval Spea
multiplicata and S. bombifrons from the southern High Plains USA. Environ. Pollut.
158, 2610–2617. https://doi.org/10.1016/j.envpol.2010.05.006.
Efsa, 2018. Scientific opinion on the state of the science on pesticide risk assessment for
amphibians and reptiles. EFSA J. 16, 5125. https://doi.org/10.2903/j.efsa.2018.5125.
Egea-Serrano, A., Relyea, R.A., Tejedo, M., Torralva, M., 2012. Understanding the impact of
chemicals on amphibians: a meta-analytic review. Ecol. Evol. 2 (7), 1382–1397.
Ellman, G.L., Courtney, K.D., Andres Jr., V., Featherstone, R.M., 1961. A new and rapid col-
orimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7 (2),
88–95. https://doi.org/10.1016/0006-2952(61)90145-9.
Freitas, J.S., Felício, A.A., Teresa, F.B., Alves de Almeida, E., 2017. Combined effects of tem-
perature and clomazone (Gamit®) on oxidative stress responses and B-esterase ac-
tivity of Physalaemus nattereri (Leiuperidae) and Rhinella schneideri (Bufonidae)
9
Science of the Total Environment 804 (2022) 150177
tadpoles. Chemosphere 185, 548–562. https://doi.org/10.1016/j.chemosphere.2017.
07.061.
Garrido, S., Linares, M., Campillo, J.A., Albentosa, M., 2019. Effect of microplastics on the
toxicity of chlorpyrifos to the microalgae Isochrysis galbana, clone t-ISO. Ecotoxicol.
Environ. Saf. 173, 103–109. https://doi.org/10.1016/j.ecoenv.2019.02.020.
Gosner, K.L., 1960. A simplified table for staging anuran embryos and larvae with notes on
identification. Herpetologica 16, 183–190.
Güngördü, A., Uçkun, M., Yologlu, E., 2016. Integrated assessment of biochemical markers
in premetamorphic tadpoles of three amphibian species exposed to glyphosate- and
methidathion-based pesticides in single and combination forms. Chemosphere 144,
2024–2035. https://doi.org/10.1016/j.chemosphere.2015.10.125.
Habdous, M., Vincent-Viry, M., Visvikis, S., Siest, G., 2002. Rapid spectrophotometric
method for serum glutathione S-transferases activity. Clin. Chim. Acta 326 (1–2),
131–142. https://doi.org/10.1016/S0009-8981(02)00329-7.
Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases the first enzymatic
step in mercapturic acid formation. Int. J. Biol. Chem. 249 (22), 7130–7139.
Hakkarainen, M., Albertsson, A.C., 2004. Environmental degradation of polyethylene. Adv.
Polym. Sci. 169, 177–199. https://doi.org/10.1007/b13523.
Hamilton, M.A., Russo, R.C., Thurston, R.V., 1977. Trimmed spearman-karber method for
estimating median lethal concentrations in toxicity bioassays. Environ. Sci. Technol.
11 (7), 1–719. https://doi.org/10.1021/es60130a004.
Herek, J.S., Vargas, L., RinasTrindade, S.A., Rutkoski, C.F., Macagnan, N., Hartmann, P.A.,
Hartmann, M.T., 2021. Genotoxic effects of glyphosate on physalaemus tadpoles. En-
viron. Toxicol. Pharmacol. 81, 103516. https://doi.org/10.1016/j.etap.2020.103516.
Holcombe, G.W., Phipps, G.L., Sulaiman, A.H., Hoffman, A.D., 1987. Simultaneous multiple
species testing: acute toxicity of 13 chemicals to 12 diverse freshwater amphibian,
fish, and invertebrate families. Arch. Environ. Contam. Toxicol. 16 (6), 697–710.
https://doi.org/10.1007/BF01055420.
Howe, C.M., Berrill, M., Pauli, B.D., Helbing, C.C., Werry, K., Veldhoen, N., 2004. Toxicity of
glyphosate-based pesticides to four north american frog species. Environ. Toxicol.
Chem. 23 (8), 1928–1938. https://doi.org/10.1897/03-71.
Hu, L., Su, L., Xue, Y., Mu, J., Zhu, J., Xu, J., Shi, H., 2016. Uptake, accumulation and elimina-
tion of polystyrene microspheres in tadpoles of Xenopus tropicalis. Chemosphere
164, 611–617. https://doi.org/10.1016/j.chemosphere.2016.09.002.
Hu, L., Chernick, M., Hinton, D.E., Shi, H., 2018. Microplastics in small water bodies and
tadpoles from Yangtze river Delta, China. Environ. Sci. Technol. 52 (15), 8885–8893.
Hüffer, T., Metzelder, F., Sigmund, G., Slawek, S., Schmidt, T.C., Hofmann, T., 2019. Polyeth-
ylene microplastics influence the transport of organic contaminants in soil. Sci. Total
Environ. 657, 242–247. https://doi.org/10.1016/j.scitotenv.2018.12.047.
Jibrin, M.O., Liu, Q., Jones, J.B., Zhang, S., 2001. Surfactants in plant disease management: a
brief review and case studies. Plant Pathol. 70, 495–510. https://doi.org/10.1111/ppa.
13318.
Jones, D.K., Hammond, J.I., Relyea, R.A., 2009. Very highly toxic effects of endosulfan across
nine species of tadpoles: lag effects and family-level sensitivity. Environ. Toxicol.
Chem. 28, 1939–1945. https://doi.org/10.1897/09-033.1.
Kawai, F., Kawabata, T., Oda, M., 2019. Current knowledge on enzymatic PET degradation
and its possible application to waste stream management and other fields. Appl.
Microbiol. Biotechnol. 103 (11), 4253–4268. https://doi.org/10.1007/s00253-019-
09717-y.
Kingsley, G.R., 1942. The direct biuret method for the determination of serum proteins as
applied to photoelectric and visual colorimetry. J. Lab. Clin. Med. 27, 840–845.
Kokalj, A., 2003. Computer graphics and graphical user interfaces as tools in simulations
of matter at the atomic scale. Comput. Mater. Sci. 28, 155–168. https://doi.org/10.
1016/S0927-0256(03)00104-6.
Lajmanovich, R.C., Sandoval, M.T., Peltzer, P.M., 2003. Induction of mortality and malfor-
mation in Scinax nasicus tadpoles exposed to glyphosate formulations. Bull. Environ.
Contam. Toxicol. 70 (3), 612–618. https://doi.org/10.1007/s00128- 003-0029-x.
Lajmanovich, R.C., Peltzer, P.M., Junges, C.M., Attademo, A.M., Sanchez, L.C., Basso, A.,
2010. Activity levels of B-esterases in the tadpoles of 11 species of frogs in the middle
Paraná River floodplain: implication for ecological risk assessment of soybean crops.
Ecotoxicol. Environ. Saf. 73, 1517–1524. https://doi.org/10.1016/j.ecoenv. 2010.07.
047.
Lajmanovich, R.C., Attademo, A.M., Peltzer, P.M., Junges, C.M., Cabagna, M.C., 2011. Toxic-
ity of four herbicide formulations with glyphosate on Rhinella arenarum (Anura:
Bufonidae) tadpoles: b-esterases and glutathione S-transferase inhibitors. Arch. Envi-
ron. Contam. Toxicol. 60 (4), 681–689. https://doi.org/10.1007/s00244-010-9578-2.
Lajmanovich, R.C., Cabagna-Zenklusen, M.C., Attademo, A.M., Junges, C.M., Peltzer, P.M.,
Bassó, A., Lorenzatti, E., 2014. Induction of micronuclei and nuclear abnormalities in
tadpoles of the common toad (Rhinella arenarum) treated with the herbicides
Liberty® and glufosinate-ammonium. Mutat. Res. Genet. Toxicol. Environ. Mutagen.
769, 7–12. https://doi.org/10.1016/j.mrgentox.2014.04.009.
Lajmanovich, R.C., Peltzer, P.M., Attademo, A.M., Martinuzzi, C.S., Simoniello, M.F., Colussi,
C.L., Cuzziol-Boccioni, A.P., Sigrist, M., 2019. First evaluation of novel potential syner-
gistic effects of glyphosate and arsenic mixture on Rhinella arenarum (Anura:
Bufonidae) tadpoles. Heliyon 5 (10), e02601. https://doi.org/10.1016/j.heliyon.2019.
e02601.
Lambert, S., Wagner, M., 2016. Formation of microscopic particles during the degradation
of different polymers. Chemosphere 161, 510–517. https://doi.org/10.1016/j.
chemosphere.2016.07.042.
Lener, G., Vélez, P., Leiva, E.P.M., Moyano, E.L., Carbonio, R.E., 2016. Chemical adsorption of
phenacyl-1,2,3-benzotriazole over AMoO4 (010) scheelite surfaces. structure and
electronic properties. Comput. Theor. Chem. 1090, 120–128. https://doi.org/10.
1016/j.comptc.2016.06.007.
Liu, B., Fan, Y., Zhao, H., Luo, S., Wang, H., Guan, B., Li, Q., Yue, J., Dong, Z., Wang, Y., Jiang, L.,
2021. Control the entire journey of pesticide application on superhydrophobic plant
R.C. Lajmanovich, A.M. Attademo, G. Lener et al.
surface by dynamic covalent trimeric surfactant coacervation. Adv. Funct. Mater.
https://doi.org/10.1002/adfm.202006606 (IF 16.836 ).
Mann, R.M., Bidwell, J.R., 1999. The toxicity of glyphosate and several glyphosate formu-
lations to four species of south western australian frogs. Arch. Environ. Contam.
Toxicol. 36 (2), 193–199. https://doi.org/10.1007/s002449900460.
Mann, R.M., Hyne, R.V., Choung, C.B., Wilson, S.P., 2009. Amphibians and agricultural
chemicals: review of the risks in a complex environment. Environ. Pollut. 157,
2903–2927.
Mikó, Z., Ujszegi, J., Gál, Z., Hettyey, A., 2017. Effects of a glyphosate-based herbicide and
predation threat on the behaviour of agile frog tadpoles. Ecotoxicol. Environ. Saf. 140,
96–102. https://doi.org/10.1016/j.ecoenv.2017.02.032.
Milesi, M.M., Lorenz, V., Durando, M., Rossetti, M.F., Varayoud, J., 2021. Glyphosate herbi-
cide: reproductive outcomes and multigenerational effects. Front. Endocrinol. (Lau-
sanne) 12, 672532. https://doi.org/10.3389/fendo.2021.672532.
Mitchell, C., Quaglino, M.C., Posner, V.M., Arranz, S.E., Sciara, A.A., 2021. Quantification and
composition analysis of plastic pollution in riverine beaches of the lower Paraná
River, Argentina. Environ. Sci. Pollut. Res. Int. 28 (13), 16140–16151. https://doi.
org/10.1007/s11356-020-11686-z.
NematdoostHaghi, B., Banaee, M., 2017. Effects of micro-plastic particles on paraquat tox-
icity to common carp (Cyprinus carpio): biochemical changes. Int. J. Environ. Sci.
Technol. 14, 521–530. https://doi.org/10.1007/s13762-016-1171-4.
Ng, E.L., Huerta Lwanga, E., Eldridge, S.M., Johnston, P., Hu, H.W., Geissen, V., Chen, D.,
2018. An overview of microplastic and nanoplastic pollution in agroecosystems. Sci.
Total Environ. 627, 1377–1388. https://doi.org/10.1016/j.scitotenv.2018.01.341.
O'Donovan, S., Mestre, N.C., Abel, S., Fonseca, T.G., Carteny, C.C., Cormier, B., Keiter, S.H.,
Bebianno, M.J., 2018. Ecotoxicological effects of chemical contaminants adsorbed to
microplastics in the clam Scrobicularia plana. Front. Mar. Sci. 5, 143.
Pal, S., Samanta, P., Kole, D., Mukherjee, A.K., Ghosh, A.R., 2018. Acute toxicity and oxida-
tive stress responses in tadpole of skittering frog, euphlyctiscyanophlyctis (Schneider,
1799) to sodium fluoride exposure. Bull. Environ. Contam. Toxicol. 100 (2), 202–207.
https://doi.org/10.1007/s00128-017-2264-6.
Peltzer, P.M., Lajmanovich, R.C., 2007. Amphibians. In: Iriondo, M.H., Paggi, J.C., Parma, M.J.
(Eds.), The Middle Paraná River: Limnology of a Subtropical Wetland. Springer, Berlin
Heidelberg New York, pp. 327–340.
Peltzer, P.M., Lajmanovich, R.C., Attademo, A.M., Beltzer, A.H., 2006. Diversity of anurans
across agricultural ponds in Argentina. Biodiver. Conserv. 15, 3499–3513. https://
doi.org/10.1007/s10531-004-2940-9.
Peltzer, P.M., Junges, C.M., Attademo, A.M., Bassó, A., Grenón, P., Lajmanovich, R.C., 2013.
Cholinesterase activities and behavioral changes in Hypsiboas pulchellus (Anura:
Hylidae) tadpoles exposed to glufosinate ammonium herbicide. Ecotoxicology 22
(7), 1165–1173. https://doi.org/10.1007/s10646-013-1103-8.
Peltzer, P., Lajmanovich, R.C., Martinuzzi, C.S., Attademo, A.M., Curi, L., Sandoval, M., 2019.
Biotoxicity of diclofenac on two larval amphibians: assessment of development,
growth, cardiac function and rhythm, behavior and antioxidant system. Sci. Total En-
viron. 683, 624–637. https://doi.org/10.1016/j.scitotenv.2019.05.275.
Pengue, W.A., 2005. Transgenic crops in Argentina: the ecological and social debt. Bull. Sci.
Technol. Soc. 25, 314–322. https://doi.org/10.1177/0270467605277290.
Perdew, J.P., Burke, K., Ernzerhof, M., 1996. Generalized gradient approximation made
simple. Phys. Rev. Lett. 77, 3865–3868. https://doi.org/10.1103/PhysRevLett.77.3865.
Raman, R., 2017. The impact of genetically modified (GM) crops in modern agriculture: a
review. GM Crops Food 8 (4), 195–208. https://doi.org/10.1080/21645698.2017.
1413522.
Relyea, R.A., Jones, D.K., 2009. The toxicity of roundup original max to 13 species of larval
amphibians. Environ. Toxicol. Chem. 28 (9), 2004–2008. https://doi.org/10.1897/09-
021.1.
Riaño, C., Ortiz-Ruiz, M., Pinto-Sánchez, N.R., Gómez-Ramírez, E., 2020. Effect of glypho-
sate (Roundup Active®) on liver of tadpoles of the colombian endemic frog
Dendropsophus molitor (amphibia: Anura). Chemosphere 250, 126287. https://doi.
org/10.1016/j.chemosphere.2020.126287.
Rodríguez-Seijo, A., Santos, B., Ferreira Da Silva, E., Cachada, A., Pereira, R., 2019. Low-
density polyethylene microplastics as a source and carriers of agrochemicals to soil
and earthworms. Environ. Chem. 16, 8–17. https://doi.org/10.1071/EN18162.
Ruamthum, W., Visetson, S., Milne, J.R., Bullangpoti, V., 2011. Effect of glyphosate-based
herbicide on acetylcholinesterase activity in tadpoles, Hoplobatrachus rugulosus.
Commun. Agric. Appl. Biol. Sci. 76 (4), 923–930.
10
Science of the Total Environment 804 (2022) 150177
Sang, Y., Mejuto, J.C., Xiao, J., Simal-Gandara, J., 2021. Assessment of glyphosate impact on
the agrofood ecosystem. Plants (Basel, Switzerland) 10 (2), 405. https://doi.org/10.
3390/plants10020405.
Screpanti, C., Accinelli, C., 2005. Glyphosate and glufosinate-ammonium runoff from a
corn-growing area in Italy. Agron. Sustain. Dev. 25, 407–412.
Scursoni, J.A., Vera, A.C.D., Oreja, F.H., Kruk, B.C., de la Fuente, E.B., 2019. Weed manage-
ment practices in Argentina crops. Weed Technol. 33, 459–463. https://doi.org/10.
1017/wet.2019.26.
Seidensticker, S., Grathwohl, P., Lamprecht, J., Zarfl, C., 2018. A combined experimental
and modeling study to evaluate pH-dependent sorption of polar and non-polar com-
pounds to polyethylene and polystyrene microplastics. Environ. Sci. Eur. 30, 1–12.
https://doi.org/10.1186/s12302-018-0155-z.
Serrano-Ruiz, H., Martin-Closas, L., Pelacho, A.M., 2021. Biodegradable plastic mulches:
impact on the agricultural biotic environment. Sci. Total Environ. 750, 141228.
https://doi.org/10.1016/j.scitotenv.2020.141228.
Soler, J.M., Artacho, E., Gale, J.D., García, A., Junquera, J., Ordejón, P., Sánchez-Portal, D.,
2002. The SIESTA method for ab initio order-N materials simulation. J. Phys. Condens.
Matter 14, 2745–2779. https://doi.org/10.1088/0953-8984/14/11/302.
Song, Y., Cao, C., Qiu, R., Hu, J., Liu, M., Lu, S., Shi, H., Raley-Susman, K.M., He, D., 2019. Up-
take and adverse effects of polyethylene terephthalate microplasticsfibers on terres-
trial snails (Achatinafulica) after soil exposure. Environ. Pollut. 250, 447–455. https://
doi.org/10.1016/j.envpol.2019.04.066.
Stathi, P., Christoforidis, K.C., Tsipis, A., Hela, D.G., Deligiannakis, Y., 2006. Effects of dis-
solved carboxylates and carbonates on the adsorption properties of thiuramdisulfate
pesticides. Environ. Sci. Technol. 40, 221–227. https://doi.org/10.1021/es051451s.
Svartz, G., Aronzon, C., Pérez Coll, C., 2016. Comparative sensitivity among early life stages
of the south american toad to cypermethrin-based pesticide. Environ. Sci. Poll. Res.
Int. 23 (3), 2906–2913. https://doi.org/10.1007/s11356-015-5547-7.
Takano, H.K., Dayan, F.E., 2020. Glufosinate-ammonium: a review of the current state of
knowledge. Pest Manag. Sci. 76 (12), 3911–3925. https://doi.org/10.1002/ps.5965.
Troullier, N., Martins, J.L., 1991. Efficient pseudopotentials for plane-wave calculations. II.
Operators for fast iterative diagonalization. Phys. Rev. B 43, 8861–8869. https://doi.
org/10.1103/PhysRevB.43.8861.
Vaira, M., Akmentins, M., Attademo, M., Baldo, D., Barrasso, D.A., Barrionuevo, S., et al.,
2012. Categorización del estado de conservación de los anfibios de la República
Argentina. Cuad. Herpetol. 26, 131–159.
Van der Oost, R., Beyer, J., Vermeulen, N.P., 2003. Fish bioaccumulation and biomarkers in
environmental risk assessment: a review. Environ. Toxicol. Pharmacol. 13 (2),
57–149. https://doi.org/10.1016/S1382-6689(02)00126-6.
Vincent, K., Davidson, C., 2015. The toxicity of glyphosate alone and glyphosate-surfactant
mixtures to western toad (Anaxyrus boreas) tadpoles. Environ. Toxicol. Chem. 34
(12), 2791–2795. https://doi.org/10.1002/etc.3118.
Wagner, N., Reichenbecher, W., Teichmann, H., Tappeser, B., Lötters, S., 2013. Questions
concerning the potential impact of glyphosate-based herbicides on amphibians. Envi-
ron. Toxicol. Chem. 32 (8), 1688–1700. https://doi.org/10.1002/etc.2268.
Wanner, P., 2021. Plastic in agricultural soils – a global risk for groundwater systems and
drinking water supplies? – a review. Chemosphere 264, 128453. https://doi.org/10.
1016/j.chemosphere.2020.128453.
Wheeler, M.W., Park, R.M., Bailer, A.J., 2006. Comparing median lethal concentration
values using confidence interval overlap or ratio tests. Environ. Toxicol. Chem. 25,
1441–1444.
Yifeng, L., Yan, Z., Yongfeng, D., Wei, J., Yanping, Z., Jinju, G., Lili, D., Hongqiang, R., 2016.
Uptake and accumulation of polystyrene microplastics in zebrafish (Danio rerio)
and toxic effects in liver. Environ. Sci. Technol. 7, 4054–4060. https://doi.org/10.
1021/acs.est.6b00183.
Zhang, W.J., Jiang, F.B., Ou, J.F., 2011. Global pesticide consumption and pollution: with
China as a focus. Proc. Int. Acad. Ecol. Environ. Sci. 1, 125–144.
Zhang, D., Ng, E.L., Hu, W., Wang, H., Galaviz, P., Yang, H., Sun, W., Li, C., Ma, X., Fu, B., Zhao,
P., Zhang, F., et al., 2020. Plastic pollution in croplands threatens long-term food secu-
rity. Glob. Chang. Biol. 26 (6), 3356–3367. https://doi.org/10.1111/gcb.15043.
Zocchi, M., Sommaruga, R., 2019. Microplastics modify the toxicity of glyphosate on
Daphnia magna. Sci. Total Environ. 697, 134194. https://doi.org/10.1016/j.scitotenv.
2019.134194.