Bulletin of Environmental Contamination and Toxicology (2019) 102:1–6

https://doi.org/10.1007/s00128-018-2508-0

Roundup® Herbicide Decreases Quality Parameters of Spermatozoa

of Silversides Odontesthes Humensis
Tony Silveira1,2 · Antonio Sergio Varela Junior2,3 · Carine Dahl Corcini3 · William B. Domingues1 · Mariana Remião4 ·
Lucas Santos1 · Bruna Barreto1 · Ingrid Lessa1 · Diego Martins3 · Robert T. Boyle2 · Patrícia Gomes Costa2 ·
Adalto Bianchini2 · Ricardo B. Robaldo5 · Vinicius Farias Campos1 

Received: 30 July 2018 / Accepted: 29 November 2018 / Published online: 3 December 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract
The silverside (Odontesthes humensis) is a very interesting model for toxicological studies due its high sensitivity and need for
good water quality. The aim of this study was to evaluate the effects of Roundup on spermatozoa of O. humensis, after acute
exposure. The fish were exposed to 0 and 7.8 mg L−1 (a.e.) of glyphosate, respectively. Through computer-assisted sperm
analysis, a significant decrease in concentration, total and progressive motility, average path distance, straight line distance,
path average velocity, curved line velocity, straight line velocity linearity, wobble, amplitude of lateral head displacement,
cross beat frequency, and motility period of silverside spermatozoa exposed to Roundup was observed. Also, increase in
membrane fluidity, ROS production and lipid peroxidation and a decrease in the mitochondrial functionality was observed
in spermatozoa of Roundup exposed silversides. It was demonstrated that Roundup exposure in a concentration that can be
achieve in natural water bodies soon after its application in fields is able to cause losses in several sperm quality parameters,
consequently decreasing the fertilization potential of O. humensis spermatozoa.

Keywords  Glyphosate · Sperm · Fish · Toxicity · South America

Currently, herbicides with glyphosate as active ingredi- et al. 2010; Ibrahim 2015). Roundup has a high solubility
ent are the most used worldwide, being Roundup the most in water (12 g L−1) and reaches the water bodies mainly
used (not only in Brazil) but in the whole world (Guilherme through soil surface run-off (Amarante Junior et al. 2002;
Guilherme et al. 2010). The concentrations in environmen-
tal water depends upon several factors, such as how close
* Vinicius Farias Campos to the water the crops were planted, the local rainfall, the
[email protected]
soil composition, the quantity of product applied, among
1
Laboratório de Genômica Estrutural, Programa others (Vereecken 2005). Glyphosate residues have been
de Pós‑Graduação em Biotecnologia, Centro de found in natural waters around the world in concentrations
Desenvolvimento Tecnológico, Universidade Federal de varying between around 0.00013 and 1.7 mg L−1, several
Pelotas, Pelotas, RS, Brazil
days after the herbicide achieves the water (Goldsborough
2
Instituto de Ciências Biológicas, Universidade Federal and Brown 1993; Peruzzo et  al. 2008; Guilherme et  al.
do Rio Grande, Rio Grande, RS, Brazil
2010; Degenhardt et al. 2012; Ruiz-Toledo et al. 2014). It
3
Laboratório de Reprodução Animal Comparada, Programa has already been reported that concentrations of glyphosate
de Pós‑Graduação em Biologia de Ambientes Aquáticos
Continentais, Universidade Federal do Rio Grande, up to 10 mg L−1 can be considered environmentally realis-
Rio Grande, RS, Brazil tic (Langiano and Martinez 2008; Topal et al. 2015). Thus,
4
Laboratório de Biotecnologia do Câncer, Programa higher concentrations can be present in water bodies a few
de Pós‑Graduação em Biotecnologia, Centro de hours after the herbicide application. At the extreme south-
Desenvolvimento Tecnológico, Universidade Federal de ern part of Brazil, Roundup is largely used on rice and soy-
Pelotas, Pelotas, RS, Brazil bean monocultures in fields near the Lagoa Mirim, the larg-
5
Laboratório de Fisiologia, Programa de Pós‑Graduação em est lagoon of Brazil, and other neighboring smaller lagoons.
Biologia Animal, Universidade Federal de Pelotas, Pelotas, In this area, some farmers use overdoses of Roundup thus
RS, Brazil

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mitigating losses by volatization or drifting to non-target groups of this study. Also, no difference between length
species, and also, many do not respect grace periods. Due (p = 0.93) or mass (p = 0.90) of silversides of both con-
to these facts, this herbicide has great relevance for the eco- trol and exposed groups were detected. The experimen-
toxicology of aquatic environments and creates concerns tal setup consisted in a control and a Roundup treatment
(Lushchak et al. 2009; Salbego et al. 2010). both in duplicate totaling in four tanks with five fish each.
Odontesthes humensis de Buen, 1953, also known as sil- The control group was exposed to the water in absence of
verside. In nature, this species occur in coastal lagoons from Roundup herbicide. The Roundup group was submitted to
southern Brazil, Uruguay and Argentina (Bemvenuti 2006). acute exposure of Roundup herbicide on the nominal con-
Currently, some fishes from these species occupy freshwa- centration of 10 mg L−1 (a.e.) of glyphosate for 24 h. After
ter environments, despite all Odontesthes spp. have a recent the exposure, the fish were anesthetized with 50 mg L −1
common marine origin (Campanella et al. 2015). Thus, the of benzocaine and euthanized by decapitation. The tes-
freshwater silversides have an interesting ability to tolerate tes were dissected and stored in the extender Beltsville
salinity variations. This euryhaline characteristic has drawn Thawing Solution (Minitüb, Germany) at 4°C for 1 h until
attention of researchers to its application in aquaculture in the sperm analysis. The real concentration of glyphosate
estuarine regions, where there are continuous alterations in was measured in water samples as described previously
water salinity. However, even though the use of Roundup in (Zebral et al. 2018). A Shimadzu 2010 Plus equipped with
Brazil is intensive, there is a lack of investigations using Bra- a flame photometric detector (FPD) and a DB-17 fused
zilian aquatic native species to investigate the effects of this silica capillary column (30 m × 0.25 mm I.D., 0.25 µm film
herbicide (Albinati et al. 2009) and O. humensis is among thickness) (J&W, USA) were used to perform the analy-
the native species exposed to Roundup. Studies focused on ses. The optimized column oven temperature program was
fish reproduction have shown that pure glyphosate at 5 and from 125 to 200°C at 15°C min−1. The total run time was
10 mg ­L−1 and the Roundup Original® formulation at 0.7 5 min. Helium was used as the carrier gas, maintained
and 1.3 mg L−1 of glyphosate can cause negative effects at a constant flow-rate of 1.5  mL  min −1. A calibration
on sperm cell quality of the zebrafish Danio rerio and of curve (0.15–10 mg L−1 glyphosate) was built and used for
the guppy Poecilia vivipara, respectively (Harayashiki et al. glyphosate concentration determination in water samples.
2013; Lopes et al. 2014). All injections were performed with 1 µL in the split flow
Thus, the aim of this study was to evaluate the effects of (10:1) mode at 260°C. The limit of detection and quanti-
Roundup herbicide in silverside O. humensis sperm cells, fication of the method was 0.05 mg L−1 and 0.15 mg L−1,
following acute exposure in a realistic concentration. respectively. Recoveries for spiked matrices were higher
than 80%. The method showed adequate linearity for the
calibration curve ­(r2 = 0.999). Measured concentrations of
Materials and Methods glyphosate in the media after the experimental period was
7.8 ± 1.46 mg L−1 (a.i.) of glyphosate (n = 6).
The silversides Odontesthes humensis used in this study Spermatozoa kinetic parameters were determined using a
came from eggs collected from wild broodstock in Lagoa computer-assisted sperm analysis (CASA) system (AndroVi-
Mirim and hatched in tanks. The fish were 1.5 years old sion 3.5, Minitub, Germany) combined with an Axio Scope.
and have mean length of 15.2 ± 2.2 cm and mean mass of A1® optical microscope (Zeiss, Germany). For evaluations,
25.7 ± 11.2 g at experimental time. The silversides were five activations per sample were prepared and one to two
sustained in 1000 L cylindrical plastic tanks, being fed fields per activation were analyzed up to 10 s. Each sample
three times a day with commercial feed (Supra, 38% crude was investigated to include at least 500 cells.
protein) until satiety. The sides of tanks were opaque to To perform the flow-cytometry analysis, the Attune®
reduce visual stress. The animals were kept since hatching Acoustic Focusing Flow Cytometer (Applied Biosystems,
in environmental water collected from Arroio Chasque- USA) was used. Each sample was exposed to 2 mM of
iro, less than 30 km from the collection site. The water Hoechst 33342–H33342 (Sigma-Aldrich, USA) for 5 min
parameters were pH 7.0 ± 0.7, temperature 13.3 ± 1.8°C, before each analysis. Events were detected by fluoro-
dissolved oxygen 9.7 ± 0.6 mg L−1, total ammonia levels chrome with violet laser (UV 405 nm) and photomulti-
0.4 ± 0.2 mg L−1, and salinity 3.25 ± 0.67 ppt since the plier (PMT) VL1 (450/40 nm) filter (Silva et al. 2017).
beginning of acclimation until the end of experimental The acquisition rate was 200 events s −1 totaling 20,000
period. The acclimation period was four weeks and experi- events per sample. All assays were performed in dupli-
mental period was 24 h. The use and handling of the ani- cate. The non-sperm events were eliminated from analysis
mals was approved by the Ethics Committee on Animal by scatter plots of FSC × SSC (Petrunkina et al. 2005) and
Experimentation of the Federal University of Pelotas. No negative fluorescence of H33342 events (debris). Plasma
fish mortality was observed in any of the experimental membrane integrity of spermatozoa were evaluated using

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Bulletin of Environmental Contamination and Toxicology (2019) 102:1–6 3

LIVE/DEAD Sperm Viability Kit (Invitrogen, USA) Results and Discussion

(SYBR-14/ propidium iodide (PI) dye). The rate was
obtained through the equation: This is the first study that evaluated the effects of water con-
(
Not damage spermatozoa and with functional membrane
) taminated with Roundup herbicide in spermatozoa of a wild
Spermatozoa positive to H33342
× 100 silverside species. The present study also provided sperm
characteristics not yet reported for O. humensis. The knowl-
Sperm plasma membrane fluidity was evaluated using edge of spermatozoa toxicology is important not only for an
two fluorescent probes to allow differentiation between individual reproductive sphere but also for biomonitoring,
low and high fluidity cells, merocyanine 540 (M540) and since these cells, instead of whole animals, can be used as
YO-PRO-1. The rate of membrane fluidity was obtained an alternative to detect environmental contaminants (Lor-
through the equation: enzetti et al. 2011). Furthermore, the detection of negative
( )
Spermatozoa with low fluidity
× 100
Spermatozoa with low fluidity + Spermatozoa with high fluidity

The mitochondrial functionality of spermatozoa was impact of contaminants in reproductive cells may signify a
evaluated using rhodamine 123 (Rh123) (Sigma-Aldrich, more profound impact in future generations. This situation
USA) and PI probes (Varela Junior et al. 2012). Mito- may indicate a population decline and/or an extinction risk
chondria functionality rate was calculated through the (Purvis et al. 2000).
equation: In the present study, the exposure to Roundup at con-
( )
Spermatozoa with high mitochondrial membrane potential
× 100
Spermatozoa with high mitochondrial membrane potential + Spermatozoa with low mitochondrial membrane potential

The chloromethyl 2′,7′-dichlorodihydrofluorescein centration of 7.8 mg L−1 (a.e.) of glyphosate results in a

diacetate (CM-H2DCFDA) probe (Invitrogen, Spain), a significant decrease of the majority of kinetics parameters of
derivative of fluorescein, was used for detection of reac- the analyzed spermatozoa, which only distance curved line
tive oxygen species (ROS). This fluorescence probe was (DCL) and straightness (STR) was not affected (Table 1). In
combined with PI (Sigma-Aldrich, USA). Only intact zebrafish (D. rerio), the exposure to 5 and 10 mg L−1 of pure
cells (PI negative) were selected and classified as sperma- glyphosate for 24 and 96 h also reduced the motility period
tozoa. ROS production was obtained by the mean of the and sperm motility, but did not alter sperm cells concentra-
green fluorescence intensity. In order to evaluate plasma tion (Lopes et al. 2014). In Rio de La Plata one-sided live-
membrane lipid peroxidation (LPO), the sperm were bearer (Jenynsia multidentata), the treatment with the same
loaded with 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)- Roundup formulation tested in this study at concentration of
4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid 0.5 mg L−1 (a.e.) of glyphosate for 24 h led to a significant
­(C11-BODIPY), a lipophilic membrane probe that changes reduction in cell concentration and motility percentage of
irreversibly its fluorescence from red to green upon oxi- spermatozoa from these fish (Sánchez et al. 2017).
dation. LPO rate was obtained by the equation: Membrane integrity was the only, among the non-kinetics
( )
Median intensity of green fluorescence (peroxidated lipid)
× 100
Median intensity of green fluorescence + Median intensity of red fluorescence (lipid not peroxidated)

The normality of all results was tested using Shap- parameters, that did not present a negative response after
iro–Wilk test. The unpaired t-test was employed to com- exposure to Roundup in silversides (Fig. 1a). Additionally,
pare the length and the weight across the treated groups, the exposure of up to 16 mg L−1 (a.e.) of Roundup did not
since only these parameters presented normal distribu- affect the membrane integrity of oyster spermatozoa (Akcha
tion. In all other parameters the significant differences et al. 2012). In the other hand, in other fish species, the
among medians were evaluated by the nonparametric membrane integrity of spermatozoa was directly affected
Mann–Whitney test. Quantitative data were expressed as by the contact with several pollutants, including Roundup
mean ± standard error of the mean (SEM). and pure glyphosate (Roychoudhury et al. 2010; Harayashiki
et al. 2013; Hulak et al. 2013; Lopes et al. 2014; Acosta

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Table 1  Kinect parameters evaluated by CASA of spermatozoa idea that sperm toxicity on different parameters appears to
from silversides Odontesthes humensis non-exposed and exposed to be species-specific.
Roundup treatment of 7.8 mg L−1 (acid equivalent) of glyphosate for
24 h.
The present study reveals that membrane fluidity of
silverside spermatozoa was higher in the fish exposed to
Treatment groups Roundup (Fig. 1b). Besides this, there are no more studies
Control Roundup® in scientific literature that evaluated the relation between
0 mg L−1 (a.e.) of 7.8 mg L−1 (a.e.) Roundup and membrane fluidity in sperm cells. However,
glyphosate of glyphosate
the elevated level of polyethoxylated tallow amine (POEA)
Concentration (×106 mL−1) 0.071 ± 0.003a 0.057 ± 0.003b surfactant in Roundup formulation may act solubilizing
Total motility (%) 17.240 ± 0.960a 9.944 ± 0.981b the lipids from plasma membrane leading to an increase of
Progressive motility (%) 4.098 ± 0.410a 3.078 ± 0.403b membrane fluidity. This effect of surfactants has already
DAP (µm) 11.370 ± 0.262a 10,120 ± 0.236b been observed in other biological membranes (Pettitt and
DCL (µm) 17.210 ± 0.611a 15.430 ± 0.479a Buhr 1998; Glover et al. 1999).
DSL (µm) 8.924 ± 0.430a 7.733 ± 0.254b Mitochondrial function is one of the key parameters
VAP (µm s− 1) 27.170 ± 0.892a 23.020 ± 0.490b governing the fertilization (Akcha et al. 2012). Sperm cells
VCL (µm s− 1) 40.160 ± 1.329a 35.350 ± 0.982b with high mitochondrial function are more viable and prob-
VSL (µm s− 1) 20.110 ± 0.867a 16.580 ± 0.373b ably increases the period of motility in fish, which results
STR (%) 0.755 ± 0.012a 0.744 ± 0.011a in higher rates of in vitro fertilization (He and Woods III
LIN (%) 0.540 ± 0.015a 0.471 ± 0.019b 2004; Gillan et al. 2005). In O. humensis, the Roundup expo-
WOB (%) 0.727 ± 0.016a 0.619 ± 0.017b sure causes a decrease in mitochondrial function (Fig. 1c).
ALH (µm) 2.424 ± 0.090a 1.848 ± 0.115b The same pattern has already been observed in zebrafish
BCF (Hz) 26.540 ± 0.949a 23.260 ± 0.760b exposed to 10 mg L−1 (a.e.) of glyphosate for 24 and 96 h
MP (s) 544 ± 16.540a 422 ± 27.180b (Lopes et al. 2014) and in guppy exposed to Roundup at
0.7 mg L− 1 (a.e.) for 96 h (Harayashiki et al. 2013). The
Data are expressed as the mean and standard error of the mean. Dif-
reduced mitochondrial respiration may lead to a decrease in
ferent letters in a same row represent a statistical difference (p < 0.05)
ATP production and, consequently, to a lower metabolism
DAP distance average path; DCL distance curved line; DSL distance
straight line; VAP velocity average path; VCL velocity curved line; and cell motility, observed in this study by CASA analysis
VSL velocity straight line; STR (VSL/VAP) straightness; LIN (VSL/ (Christen et al. 1987; Perchec et al. 1995).
VCL) linearity; WOB (VAP/VCL) wobble; ALH amplitude of lateral The accumulation of ROS in sperm cells can result in
head displacement; BCF beat cross frequency; MP motility period cell damage and general loss of sperm quality, including
damage of membrane structure; alterations in the mito-
et al. 2016; Gerber et al. 2016; Sánchez et al. 2017). Due chondrial function; and loss of fertilization capacity (Shiva
to being the first protective barrier of the spermatozoa, its et al. 2011). The exposure to Roundup led to an increase in
cell membrane has already been reported as more sensitive ROS production levels in silverside sperm cells (Fig. 2a),
to different types of pollutants (Acosta et al. 2016). How- which can be directly related to changes in cell membrane,
ever, herbicides can also produce negative effects in internal mitochondrial functionality and spermatozoa motility, as it
sperm cell functionalities without leading to loss of mem- was observed by this study. Increases in the ROS produc-
brane integrity (Akcha et al. 2012). This corroborates to the tion has already been reported in zebrafish spermatozoa

Fig. 1  Quality parameters evaluated by flow cytometry of spermato- mitochondrial functionality (%). Data are expressed as the mean and
zoa from silversides Odontesthes humensis non-exposed and exposed standard error of the mean (n = 15). The asterisk represent a statistical
to Roundup treatment of 7.8  mg  L−1 (acid equivalent) of glypho- difference (p < 0.05)
sate for 24 h. a membrane integrity (%). b membrane fluidity (%). c

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Bulletin of Environmental Contamination and Toxicology (2019) 102:1–6 5

Fig. 2  Quality parameters evaluated by flow cytometry of spermato- oxidation (%). Data are expressed as the mean and standard error
zoa from silversides Odontesthes humensis non-exposed and exposed of the mean (n = 15). The asterisk represents a statistical difference
to Roundup treatment of 7.8 mg L−1 (acid equivalent) of glyphosate (p < 0.05)
for 24  h. a ROS production (fluorescence intensity). b Lipid per-

after cryopreservation (Hagedorn et al. 2012). Further- generate great concern and raises a critical issue about the
more, ROS levels were increased following the exposure to maintenance of future population of O. humensis.
Roundup in Rio de La Plata onesided livebearer (Sánchez
et al. 2017) and in guppy (Harayashiki et al. 2013), how- Acknowledgements  This study was supported by the Brazilian
CAPES, CNPQ (Grant No. 422292/2016-8) and FAPERGS (Grant
ever these evaluations were carried out in non-reproduc- No. 16/2551).
tive cells. This is the first study that evaluated ROS pro-
duction directly in sperm cells of a fish species.
LPO generated by accumulation of ROS can cause a
loss of membrane fluidity and integrity, affecting also the References
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