Enviro Toxic and Chemistry - 2023 - Darmani - Concentrations of The Glyphosate Based Herbicide Roundup Causes

Homa Darmani ORCID iD: 0000-0001-7905-0294
Concentrations of the glyphosate-based herbicide Roundup® causes developmental
defects in Artemia salina

Accepted Article
Running Title: Developmental toxicity of Roundup in Artemia salina
Homa Darmani,* Dua’a Riyad Husain Al-Saleh

Department of Biotechnology and Genetic Engineering, Faculty of Science and Arts,
Jordan University of Science and Technology, Jordan
Acknowledgment:
The authors declare no conflict of interest.
The authors would like to thank the Deanship of Research at Jordan University of
Science and Technology for financial support (Grant number 20210440).
The authors would like to thank Haifa Hammad for her help in taking the fluorescent
photographs of the Artemia salina nauplii.
Data Availability:
Raw data can be found in the Supporting Information.
Author Contribution statement:
Conceptualization, Homa Darmani; Data curation, Dua’a Al-Saleh; Formal analysis,
Homa Darmani; Funding acquisition, Homa Darmani; Methodology, Homa Darmani and
Dua’a Al-Saleh; Project administration, Homa Darmani; Supervision, Homa Darmani;
Visualization, Homa Darmani; Writing – original draft, Homa Darmani and Dua’a Al-
Saleh; Writing – review & editing, Homa Darmani.
*Corresponding Author: Professor Homa Darmani, Department of Biotechnology and
Genetic Engineering, Faculty of Science and Arts, Jordan University of Science &
Technology, P.O. Box 3030, Irbid 22110, Jordan. e-mail: darmani@just.edu.jo Telephone
Number: +962797495043
Abstract
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may lead to differences between this version and the Version of Record. Please cite this
article as doi: 10.1002/etc.5639.
This article is protected by copyright. All rights reserved.

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Roundup® is the most used glyphosate-based herbicide. During agricultural use it may
directly contaminate existing aquatic ecosystems, posing severe concerns for the safety of
non-target terrestrial and aquatic organisms. We investigated the outcome of exposure to

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different concentrations of glyphosate in Roundup® on cyst hatchability, toxicity, and
teratogenic effects in the aquatic crustacean Artemia salina (A. salina) that inhabits
diverse types of salt waters and, as a filter feeder, carries a greater risk of being exposed
to pollutants. We found that exposure to 144 and 288 μg/mL glyphosate in Roundup®
resulted in cysts unable to complete diapause, and hatchability was completely inhibited
during all exposure times tested (17 h- 48 h). Glyphosate concentrations of 288 μg/mL in
Roundup® was lethal to A. salina nauplii and the lower concentrations (9, 18, 36, 72
μg/mL) had no significant effects on viability. In addition, sublethal and environmentally
safe concentrations of glyphosate (0.72 μg/mL) in Roundup® also affected the early
development of A. salina nauplii, with significantly decreased body lengths and reduced
widths of the tail, abdomen and head. The increased level of catalase activity observed in
nauplii exposed to 0.72 μg/mL glyphosate for 24 h and those exposed to 7.2 and 72
μg/mL glyphosate for 48 h may be linked to excessive ROS levels that had been induced
by Roundup®. In conclusion, Roundup® containing greater than 72 μg/mL glyphosate
totally inhibited hatching of cysts and exerted toxic effects on A. salina nauplii. The
increased prevalence of developmental defects in the nauplii observed at 0.72 μg/mL
glyphosate, signifies possible teratogenicity of Roundup® exposure even at
concentrations of glyphosate, possibly due to disturbance of the antioxidant defenses,
which needs further investigation.

Keywords: Aquatic invertebrates, aquatic toxicology, developmental toxicity,
ecotoxicology, herbicide
Introduction
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In 1974 the Monsanto Company introduced the herbicidal agent glyphosate (N-
(phosphonomethyl) glycine) for use in agriculture under the proprietary name Roundup®.
Farmers rapidly accepted glyphosate for agricultural weed control and after the
introduction of glyphosate resistant genetically modified crops the rate of use of these
herbicides increased further and now it stands as the most used agricultural, domestic,
urban, and rural herbicide (Mesnage & Antoniou, 2017; Tang et al., 2021). Indeed, there has
been a 100-fold increase in use of glyphosate-based herbicides globally in 2016, and it
has been estimated that in 2023 this will increase to about 1000 million kg (Mesnage &
Antoniou, 2017; Mertens et al., 2018).
Glyphosate inhibits the activity of 5-enolpyruvylshikimate-3-phosphate synthase
(EPSPS), the central enzyme required by plants and various microorganisms for the
biosynthesis of aromatic amino acids (Cerdeira & Duke, 2006; Gill et al., 2017). By
preventing the synthesis of 5-enolpyruvylshikimate-3-phosphate (EPSP), glyphosate
inhibits the shikimate pathway which operates only in plants and some microorganisms
(Gill et al., 2017; Lozano et al., 2018). Since this pathway does not exist in humans and
animals, glyphosate containing herbicides were thought to have low potential toxicity and
this encouraged their widespread global use (Sinhorin et al., 2014; Tizhe et al., 2014; van
Bruggen et al., 2018). In 2015, glyphosate was classified as a potential carcinogen
(IARC, 2017) and since then there has been global concern due to the constant and
recurrent human exposure, through the widespread usage of herbicides that contain

glyphosate as the active agent in agronomy and in domestic, urban, and rural
environments, and consequently, the presence of residues in foods and water (Myers et
al., 2016). During agricultural use, glyphosate may directly contaminate existing aquatic
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ecosystems through runoff and soil leaching after rainfall, resulting in severe concerns for
safety of non-target terrestrial as well as aquatic organisms (Abrantes et al., 2009).
Adding to the problem, the resistance of glyphosate to degradation means that this
potential carcinogen will accumulate in soil, surface water, groundwater, and sediments,
and thus in all forms of life that exist in these environments (Gasnier et al., 2009;
Mesnage et al., 2015; van Bruggen et al., 2018; de Melo et al., 2020). In fact, glyphosate
levels of up to 10.3 mg acid equivalent/L have been reported in surface freshwater
(Ronco et al., 2008; Córdova López et al., 2019; Parlapiano et al., 2021). Furthermore,
levels of 1690 ng/L have been found in saltwater in the Western Pacific (Wang et al.,
2016) and 13-1377 μg/L have been reported in the Baltic Sea (Skeff et al., 2015;
(Parlapiano et al., 2021).
Many studies have investigated the consequences of exposure of different species of
organisms to glyphosate and/or glyphosate-based herbicides (such as Roundup®) in fresh
water ecosystems. These studies have reported reproductive and developmental toxicity
in numerous freshwater species with exposure to glyphosate/herbicide inducing delays in
the age of first reproduction in Cladocerans (Reno et al., 2014; Lares et al., 2022), as well
as abnormal embryonic development in Daphnia, estuarine crabs (Avigliano et al., 2014),
amphibians (Howe et al., 2004; Paganelli et al., 2010; Flach et al., 2022), and fish
(Sulukan et al., 2017; Zebral et al., 2018; Zhang et al., 2017; Lanzarin et al., 2019, 2020;
Panetto et al., 2019). However, there still remains a gap in the knowledge about the

effects of glyphosate in marine ecosystems, since there are still very few studies
regarding the effects on marine organisms (Zaller et al., 2015). Artemia salina (A. salina)
is an aquatic crustacean that inhabits diverse types of saltwaters like saline lakes, as well
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as oceans, and, as a filter feeder, carries a greater risk of being exposed to pollutants in
comparison to those species that are not filter feeders (Ates et al., 2013, 2015). The
suitability of A. salina as a model system to investigate the ecotoxicology of pollutants
like glyphosate, stems from the ready availability, and multiple life stages of this
organism (Caldwell et al., 2003; Nunes et al., 2006; Libralato, 2014; Zhu et al., 2017).
Furthermore, depending on the developmental stages, A. salina nauplii at the instar I, II
and III stages have differing levels of susceptibility to pollutants (Barahona & Sánchez-
Fortún, 1996; Caldwell et al., 2003). An acute toxicity study of Roundup® on A. salina has
reported significant increases in mortality rate, with 48-h LC50 values being 14.19 mg of
glyphosate (acid equivalent/L) but the study did not go into detail regarding the effects of
sublethal concentrations. In this study we used A. salina cysts and nauplii, to investigate
the outcome of exposure to Roundup® herbicide on highly saline aquatic environments
found in or near the ocean.
Materials and Methods
Roundup®
Roundup® Weed & Grass Killer (Monsanto Company, Marysville, OH, USA) was
purchased from the local market and was used as the glyphosate formulation. The
Roundup® formulation contained the active ingredient glyphosate isopropylamine salt (2
%) and exposure levels to this herbicide in the current study is described as the
concentration of glyphosate isopropylamine in the formulation.

A. salina cysts and nauplii density
A. salina encysted embryos used in this study were purchased from Carolina Biological
Supply Company (Burlington, NC 27215-3398, USA). 0.5 g of A. salina cysts were

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hydrated in cold deionized water at 5 °C for 3 h, and the cysts that had sunk down (90 %
had sunk down) were collected and placed in a container with artificial salt water ((25 g
NaCl/L distilled water) and left to acclimatize at 28 °C for 2 h before the hatching
procedure.
Hatching assay
The effect of exposure to different concentrations of Roundup® in artificial salt water on
the hatching rate of A. salina cysts was investigated. The hatching assay was performed
in 24-well microplates. Different concentrations of glyphosate in the Roundup® (0, 9, 18,
36, 72, 144, 288 μg/mL) were prepared in artificial salt water and 1 mL aliquots
transferred to the wells of the microtiter plates. Hydrated and acclimatized A. salina cysts
(22 cysts per well) were then added and the plates incubated at 28 °C under continuous
illumination with shaking in an orbital shaking incubator at 60 rpm. Hatchability was
examined using a stereomicroscope at 7, 17, 24, 41 and 48 h. The experiment was
repeated on three separate occasions, with triplicate samples for each concentration. The
hatching percentage was calculated according to the following equation: where n is the
number of hatched out cysts and c is the number of decapsulated cysts (Madhav et al.,
2017).
𝑛
h% = [ ] × 100
𝑛+𝐶
Acute toxicity test and mortality A. salina nauplii

A. salina cysts (0.5 g) were hydrated, as described above, and the sunken cysts (90 %)
were collected and placed in a container with 250mL of artificial salt water and left to
acclimatize at 28 °C for 2 h before the hatching procedure. The beaker was continuously
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illuminated and kept for 24 h at 28 ± 1 °C in an orbital shaking incubator at 60 rpm.
Following hatching of the cysts, A. salina nauplii (15-20 nauplii) were added to 500 µl of
Roundup® solutions containing glyphosate (0, 9, 18, 36, 72, 144, 288 μg/mL) in 24-well
plates. The nauplii were incubated at 28 °C for 24 h and were not fed during the exposure
period. The number of dead nauplii (entirely motionless) was counted using a
stereomicroscope after exposure for 24 h, and the percentage mortality was calculated.
The experiment was repeated on three separate occasions, with triplicate samples for each
concentration.
Morphological analysis- acute toxicity testing

A. salina cysts (0.5 g) were added to vessels containing 250 mL solutions of different
concentrations of glyphosate in Roundup® (0, 0.72, 7.2 and 72 μg/mL) prepared in
artificial salt water. They were hatched, as previously described in an orbital shaking
incubator (60 rpm) at 28 °C. After hatching, nauplii were separated into two groups. One
group was exposed for 24 h and the other for 48 h to glyphosate in Roundup®. The
highest concentration used was 72 μg/mL since this exposure level resulted in minimal
mortality and the nauplii did not show any visible signs of stress. The nauplii were
removed from the culture (for each concentration of glyphosate in the Roundup®),
euthanized in 10 % buffered formalin and examined using a Kern microscope.
Photographs of the nauplii were taken and the body size (length from the head to the
anus) and the width of the head, abdomen and tail were measured using the microscope
VIS software image analysis program. The experiment was repeated on three separate

occasions, with triplicate samples for each concentration and a total of 20 larvae were
photographed each time for each concentration.
Catalase enzyme activity

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A. salina cysts were added to vessels containing 100 mL solutions of different
concentrations of glyphosate in Roundup® (0, 0.72, 7.2 and 72 μg/mL) prepared in
artificial salt water. They were hatched, as previously described in an orbital shaking
incubator (60 rpm) at 28 °C. After hatching, nauplii were separated into two groups. One
group was exposed for 24 h and the other for 48 h to glyphosate in Roundup®. A catalase
activity assay kit (ab83464 Abcam, USA) was used to determine the levels of this
antioxidant enzyme in the nauplii following the relevant exposure time to glyphosate.
Briefly, the nauplii were harvested and washed in cold phosphate buffered saline,
weighed, frozen at -80 °C. Then, 200 µL of ice-cold assay buffer was added (for every
100 mg nauplii weight using quadruplicate samples) and the nauplii homogenized with a
homogenizer on ice, with 10-15 passes then centrifuged at 7370 RCF for 15 minutes at 4
°C to remove any insoluble material. Finally, the supernatant was collected and
transferred into a new tube and was kept on ice for the assay. Catalase activity was
determined according to the manufacturer’s instructions and the absorbance was
measured at 570 nm using a microplate reader. The assay included both positive controls
(wells that contained catalase) and negative controls (wells without catalase).
Apoptosis analysis using Acridine Orange (AO) staining.
The degree of apoptosis in A. salina nauplii was determined after acute exposure (24 h
and 48 h) to different concentrations of glyphosate (0.72, 7.2 and 72 μg/mL) in
Roundup®, as described above, using acridine orange staining. Following exposure to

Roundup®, A. salina nauplii were washed with PBS and transferred to 24 well plates and
exposed to 100 𝜇L of acridine orange (5 𝜇g/mL) for 20 min at room temperature and then
washed with PBS three times. The nauplii were examined using a fluorescence
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microscope (Optika, Italy) for the presence of small green spots (indicator of apoptosis)
and photographed. The experiment was repeated on three separate occasions, with
triplicate samples for each concentration.
Statistical Analysis
Results were expressed as means ± standard deviation. Percentage data were first arcsine
transformed before statistical analysis. Either a two-way or one-way ANOVA (Analysis
of Variance) was performed to analyze the data with Minitab 17 statistical software
package. Any significant differences between the controls and the glyphosate exposed
groups were analyzed using Tukey’s pairwise comparisons. Statistical significance was
taken as p < 0.05. Normal probability plots showed that the data were normally
distributed.
Results
The results showed that exposure to different concentrations of glyphosate (0, 9, 18, 36,
72, 144, 288 μg/mL) in Roundup® for 17 h, 24 h, 41 h and 48 h significantly affected the
percentage hatchability of A. salina cysts. Indeed, two way ANOVA revealed highly
significant effects of concentration, time and also the interaction between concentration
and time on percentage hatchability (p < 0.001). Follow-up by one way ANOVA
revealed that hatchability was not significantly affected by the different times of exposure
alone (p > 0.05). However, one way ANOVA for each time interval revealed significant
differences in hatchability when cysts had been exposed to different concentrations of

glyphosate in Roundup® (Figure 1; p < 0.001). Tukey’s pairwise comparison revealed
that glyphosate at concentrations of 144 and 288 μg/mL completely inhibited hatching
during all exposure times and these inhibitory effects were significant at all the different
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concentrations, except for cysts exposed to 36 μg/mL of glyphosate at the 17 h exposure
time.
We also found significant decreases in the viability of A. salina nauplii that had been
exposed for 24 h at concentrations of 144 and 288 μg/mL glyphosate in Roundup®
(Figure 2), in comparison with all the other concentrations tested (0-72 μg/mL; p < 0.001,
ANOVA; Tukey’s pairwise comparison). The LC50 values were 80 μg/mL glyphosate in
Roundup®.
Figure 3 shows the actual body measurements of A. salina nauplii that were taken to
investigate the effects of exposure to glyphosate (0.72, 7.2, and 72 μg/mL) in Roundup®
on the development of A. salina nauplii when cysts had been exposed and hatching
initiated at these concentrations. These measurements included the length of the body,
and the width of the head, tail and abdomen (Figure 4). A two-way ANOVA was
performed to analyze the effect of concentration and time of exposure to glyphosate in
Roundup® on these morphological measurements. The results showed statistically
significant interactions between the effects of concentration (p < 0.001; 3 DF; F = 42.33),
and time of exposure (p < 0.001; 1 DF; F = 58.08), as well as interaction between
concentration and exposure time (p < 0.05; 3 DF; F = 3.11) on the width of the head of A.
salina nauplii. For the abdomen width, there were significant interactions between the
effects of concentration (p < 0.001; 3 DF; F = 14.77) and time of exposure (p < 0.001; 1
DF; F = 526.62). A. salina tail lengths were also significantly affected by concentration

(p < 0.001; 4 DF; F = 33.09) and exposure time (p < 0.001; 1 DF; F = 284.21). Lastly,
significant interactions were observed between the body lengths and concentration (p <
0.001; 3 DF; F = 10.75), time of exposure (p < 0.001; 1 DF; F = 2999.75), as well as
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interaction between concentration and time of exposure (p < 0.01; 3 DF; F = 4.13).
Further analysis by one-way ANOVA (followed by Tukey’s pairwise comparison) was
performed for each measurement group at each time of exposure (24 h or 48 h). Larvae
exposed for 24 h, had significantly reduced body lengths at all concentrations (p < 0.001;
3 DF; F = 16.5; ANOVA and Tukey’s pairwise comparison) whereas those exposed for
48 h showed reduced body lengths at the higher concentrations of 7.2 and 72 μg/mL
glyphosate in Roundup® (p < 0.005; 3 DF; F = 4.86). Tail widths were also reduced
significantly in nauplii exposed to all concentrations of glyphosate for both exposure
times, in comparison to controls (p < 0.001; 24 h exposure 3DF and F = 20.68; 48 h
exposure 3DF and F = 23.51). The abdomen of the nauplii showed significant reductions
in width when exposed to 7.2, and 72 μg/mL glyphosate for 24 h and to all the
concentrations for 48 h, in comparison to the controls (p < 0.001; 24 h exposure 3DF and
F = 8.92; 48 h exposure 3DF and F = 7.02). Furthermore, the measurements of abdomen
width were much more reduced when the nauplii had been exposed for 48 h, in
comparison to 24 h, at all concentrations tested. In addition, we observed that the head
widths of the nauplii were significantly decreased at all concentrations, and at both
exposure times, in comparison to the controls (p < 0.001; 3DF and F = 12.22 for both
exposure times).
Figure 5 shows the level of catalase activity in A. salina nauplii when cysts were hatched
in Roundup® and the newly hatched nauplii incubated in the same concentrations of

Roundup® for 24 h and 48 h. Two-way ANOVA showed significant interactions
between catalase activity and concentration of glyphosate in Roundup® (p < 0.001; 3 DF;
F = 24.71), time of exposure (p < 0.001; 1 DF; F = 205.72), as well as interaction

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between concentration and time of exposure (p < 0.01; 3 DF; F = 8.71). Further analysis
by one way ANOVA and Tukey’s pairwise comparison for each time interval showed
that for the 24 h exposure group, at the highest concentration used (72 μg/mL) the level
of catalase activity was significantly decreased, in comparison with the control group and
the group exposed to 0.72 μg/mL glyphosate in Roundup®. Interestingly the level of
catalase activity was increased at the lowest concentration used (0.72 μg/mL), in
comparison with the controls and the higher concentrations used. When the level of
catalase activity was examined for the 48 h exposure group, significantly increased levels
of activity were observed, in comparison with the controls. Furthermore, when A. salina
nauplii were exposed to glyphosate for 48 h, the level of catalase activity was reduced at
all the concentrations tested, when compared to nauplii that had been exposed for 24 h.
Acridine orange staining of A. salina nauplii that had been incubated for 24 h and 48 h in
Roundup® containing different concentrations of glyphosate was carried out to determine
if exposure to low levels of glyphosate would lead to increased apoptosis in the living
nauplii. Figures 6a and 6b show that exposure to glyphosate resulted in increased levels
of apoptotic cells as shown by the punctated green fluorescence in the fluorescent images,
in comparison to the control counterparts.
Discussion
The use of herbicides based on glyphosate has continued to increase, especially with the
production of genetically modified crops that are resilient to glyphosate. The maximum

levels of aquatic glyphosate in freshwater ecosystems in different countries globally has
been estimated to reach 105 μg/mL which represents a moderate to considerable risk in
95 % of the countries investigated. Unfortunately, 90 % of these countries worldwide do

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not have any kind of legislation to reduce the exposure of aquatic ecosystems to
glyphosate concentrations. This means that there is a moderate to substantial risk of
toxicity to aquatic organisms. Although some studies have investigated the effects of
glyphosate exposure in freshwater ecosystems, there are still very few studies regarding
the effects on marine organisms.
A. salina has been approved by the US Environmental Protection Agency as a model
system to determine acute toxicity of compounds. They have been used in many
ecotoxicological assays that have investigated several types of manufactured products
that pollute the ocean such as microplastics (Albendín et al., 2021; Ñañez Pacheco et al.,
2021) which often travel up the food chain, as well as materials used in nanotechnology
(Ates et al., 2015, 2020; Madhav et al., 2017). Since studies on the effects of Roundup®
are limited, we investigated the outcome of exposure to Roundup® using the aquatic
crustacean A. salina that inhabits diverse types of salt waters like saline lakes, as well as
oceans, and, as a filter feeder, carries a greater risk of being exposed to pollutants. We
found that glyphosate in Roundup® completely inhibited hatching of A. salina cysts at
concentrations of 144 and 288 μg/mL during all exposure times tested (17 h – 48 h).
Exposure to these levels of Roundup® prevented termination of this state of diapause
where growth of the organism has halted, with diminished metabolic processes and
heightened tolerance of stress (Robbins et al., 2010; MacRae, 2016; Drinkwater & Clegg,
2018). Grave adverse consequences of exposure to Roundup® were also reported in the

non-target planktonic crustacean Daphnia, where an increased incidence of aborted eggs
and thus failure of embryonic development was observed (Suppa et al., 2020). Similarly
female estuarine crabs that were exposed to another formulation of Roundup®

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(RoundUp®UltraMax) had significantly lowered numbers of hatched larvae (Avigliano et
al., 2014). Other studies also found that exposure of zebra fish to RoundUp®UltraMax at
lower concentrations of 8.5 μg/mL caused decrease in the hatching rate compared to
controls (Lanzarin et al., 2019). Furthermore, decreased hatch rates were also observed in
Oryzias javanicus exposed to glyphosate at concentrations of 100 μg/mL. On the other
hand, other studies have reported higher hatching rates in zebrafish embryos exposed to
pure glyphosate (Zhang et al., 2017). The latter has been attributed to the glyphosate-
induced reduction in the elasticity of the chorion and increased embryonic locomotor
activity resulting in increased hatching rates. In addition, other studies on zebrafish have
also found premature hatching in zebrafish in response to glyphosate-based herbicides
such as Roundup® (de Brito Rodrigues et al., 2017). Interestingly, glyphosate and the
commercial formulation Roundup Express® did not adversely affect the embryo-larval
development of either the Pacific oyster Crassostrea gigas (upto 200 μg L−1) nor the sea
urchin Paracentrotus lividus (Akcha et al., 2012; His et al., 1999).
The majority of the investigations on glyphosate and glyphosate-based herbicides have
been carried out on zebrafish and few have examined the effects on Artemia. Different
studies report different lethal concentrations of glyphosate in Roundup® for A. salina
ranging from of 14.19 μg/mL to 500ppm for Round-up®Platinum (500 μg/mL) in the
case of A. franciscana following 48 h exposure (de Brito Rodrigues et al., 2017;
Parlapiano et al., 2021). We found that Roundup® containing glyphosate concentrations

of 288 μg/mL in Roundup® was lethal at 24 h of exposure. At the lower concentrations
(9, 18, 36, 72 μg/mL), no significant effects on viability were observed.
We examined the effects of Roundup® on morphological changes and chose

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concentrations that ranged from 0.72 - 72 μg/mL glyphosate in Roundup® since the
highest exposure level resulted in minimal mortality and the nauplii did not show any
visible signs of stress. Also, the lowest exposure level falls within the glyphosate
concentration that is considered to be environmentally safe drinking water levels in the
United States which is 0.7 μg/mL (USEPA, 2009). We found that sublethal and
environmentally safe concentrations of glyphosate in Roundup® also affected the early
development of A. salina nauplii. The developmental abnormalities observed included
significantly decreased body lengths and significantly reduced width of the tail, abdomen
and head of A. salina nauplii that had hatched from cysts incubated in Roundup® and the
nauplii exposed for 24 h and 48 h in the same concentrations of Roundup®. For example,
nauplii exposed to 0.72 μg/mL glyphosate showed decreased tail width and abdomen
width, in comparison to the controls. According to our knowledge, there are no reports of
glyphosate-based herbicides’ effects on these early developmental morphological features
of A. salina. Our results on decreased body lengths agree with another study that showed
reductions in the embryonic body length of the amphibian Xenopus laevis on exposure to
glyphosate at higher concentrations that ranged from 97-243 μg/mL (Flach et al., 2022).
Significantly shorter body lengths have also been reported in zebrafish embryos exposed
to 400 μg/mL pure glyphosate in comparison with the controls (Zhang et al., 2017). The
significantly reduced head width of A. salina nauplii that had been exposed to Roundup®
containing as low as 0.72 μg/mL glyphosate, in the current study, are in line with the

defects observed in the craniofacial development of zebrafish embryos exposed to
glyphosate at concentrations of 50 μg/mL which had significantly decreased ceratohyal
cartilage length (Díaz-Martín et al., 2021). In addition, significantly smaller head size
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was observed in zebrafish embryos exposed to 400 μg/mL pure glyphosate in comparison
with the controls (Zhang et al., 2017). We found decreased body length and head width of
A. salina nauplii at much lower concentrations of 0.72 μg/mL of glyphosate in
Roundup® and this is related to the fact that Roundup® formulation consists of a mixture
of chemicals in addition to the active ingredients and as such exerts greater toxicity than
the active ingredient alone (Mesnage & Antoniou, 2017). It is important to emphasize that
the observed developmental defects in A. salina nauplii in this study occurred at
exceptionally low exposure levels to glyphosate (0.72 μg/mL) in Roundup® which are
environmentally relevant concentrations. Indeed, it is reported that levels of glyphosate in
aquatic environments may vary between 0.01-0.7 μg/mL and could increase to 1.7 - 5.2
μg/mL when the herbicides are applied directly or when there is accidental contamination
(Harayashiki et al., 2013; Annett et al., 2014; Zhang et al., 2017).
Toxic effects of Roundup® and other glyphosate-based herbicides are reported to occur
through various mechanisms including increased formation of reactive oxygen species
(ROS) which lead to a surge in oxidative stress, free radical production, and disruption of
antioxidant defense systems (de Moura et al., 2017). We found a significant increase in
catalase activity in A. salina nauplii exposed to 0.72 μg/mL glyphosate for 24 h and in
nauplii exposed for 48 h. The increased catalase activity may be linked to excessive ROS
levels that had been induced by Roundup® (Halliwell & Gutteridge, 1990; Lushchak et al.,
2005). On the other hand, we also observed decreased catalase activity in nauplii that had

been exposed for 24 h to 7.2 and 72 μg/mL glyphosate and the decreased levels in the
activity of catalase may result in buildup of ROS that may contribute to the
developmental abnormalities observed. However, these assumptions need to be clarified

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further by determining the total ROS formation and the effects on the activity of other
antioxidant enzymes (SOD, GPx; GPx-Se) in Artemia salina nauplii so that a definitive
conclusion can be reached regarding the observed developmental effects and the role of
Roundup® exposure on oxidative stress parameters.
In the current study, we also found increased levels of Roundup®-induced apoptosis in A.
salina nauplii and these findings are in line with another report examining glyphosate-
induced abnormal embryonic development in zebrafish. The morphological abnormalities
observed in the latter study were linked to cellular apoptosis resulting from elevated
levels of ROS (Sulukan et al., 2017). Furthermore, glyphosate has been reported to
induce cell apoptosis via the miR203-PI3K/AKT axis in carp lymphocytes (Wang et al.,
2020).
In conclusion, Roundup® containing glyphosate concentrations at levels greater than 72
μg/mL totally inhibited hatching of cysts and exerted toxic effects on A. salina nauplii. In
addition, exposure to levels as low as 0.72 μg/mL glyphosate in Roundup® caused an
increased prevalence of developmental defects in the nauplii, including reduced body
length and reduced width of the head, abdomen, and tail during the first 24-48 h of
development, signifying possible teratogenicity of Roundup® exposure even at
environmentally relevant concentrations of glyphosate. These effects may be related to
Roundup® -induced oxidative stress, disturbance of the antioxidant defenses, and
induction of apoptosis which need further investigation. Finally, longer term exposure

effects of A. salina to Roundup® at environmentally relevant concentrations are needed
to see if the early developmental abnormalities persist until adulthood.
Figure Legends
Accepted Article
Figure 1: The effect of exposure to different concentrations of glyphosate in Roundup®
for different times on hatchability of cysts of A. salina. Bars represent means ± SD.
Means that do not share a letter are significantly different from each other (One way
ANOVA; Tukey’s pairwise comparisons for each data set at each time interval).

not share a letter are significantly different from each other (One way ANOVA; Tukey’s
for 24 h on viability of nauplii of A. salina. Data represent means ± SD. Means that do

pairwise comparisons).
Accepted Article
Figure 3: Red lines indicate the different body measurements of A. salina nauplii that

were taken. The photograph shows a control nauplius.
Accepted Article
Accepted Article
for 24 h and 48 h on body lengths and width of the tail, abdomen and head of nauplii of
A. salina. Data represent means ± SD. Means that do not share a letter are significantly
different from each other (One way ANOVA; Tukey’s pairwise comparisons for each
data set at 24 h or 48 h).

Accepted Article
for 24 h and 48 h on catalase activity in nauplii of A. salina. Data represent means ± SD.
Means that do not share a letter are significantly different from each other (One way
ANOVA; Tukey’s pairwise comparisons for each data set at 24 h or 48 h).

Accepted Article
Figure 6: Acridine orange staining of nauplii of A. salina exposed to different
concentrations of glyphosate (A: control; B: 0.72 μg/mL; C: 7.2 μg/mL; D: 72 μg/mL) in
Roundup® for 24 h (6a) and 48 h (6b). Cells that have undergone apoptosis appear as
punctated green fluorescence.
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Enviro Toxic and Chemistry - 2023 - Darmani - Concentrations of The Glyphosate Based Herbicide Roundup Causes

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Homa Darmani ORCID iD: 0000-0001-7905-0294

Concentrations of the glyphosate-based herbicide Roundup® causes developmental

defects in Artemia salina

Homa Darmani,* Dua’a Riyad Husain Al-Saleh

This article is protected by copyright. All rights reserved.

non-target terrestrial and aquatic organisms. We investigated the outcome of exposure to

μg/mL) had no significant effects on viability. In addition, sublethal and environmentally

by Roundup®. In conclusion, Roundup® containing greater than 72 μg/mL glyphosate

increased prevalence of developmental defects in the nauplii observed at 0.72 μg/mL

glyphosate, signifies possible teratogenicity of Roundup® exposure even at

concentrations of glyphosate, possibly due to disturbance of the antioxidant defenses,

which needs further investigation.

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been a 100-fold increase in use of glyphosate-based herbicides globally in 2016, and it

Antoniou, 2017; Mertens et al., 2018).

Glyphosate inhibits the activity of 5-enolpyruvylshikimate-3-phosphate synthase

preventing the synthesis of 5-enolpyruvylshikimate-3-phosphate (EPSP), glyphosate

Bruggen et al., 2018). In 2015, glyphosate was classified as a potential carcinogen

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safety of non-target terrestrial as well as aquatic organisms (Abrantes et al., 2009).

levels of up to 10.3 mg acid equivalent/L have been reported in surface freshwater

(Parlapiano et al., 2021).

Many studies have investigated the consequences of exposure of different species of

organisms to glyphosate and/or glyphosate-based herbicides (such as Roundup®) in fresh

in numerous freshwater species with exposure to glyphosate/herbicide inducing delays in

as abnormal embryonic development in Daphnia, estuarine crabs (Avigliano et al., 2014),

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suitability of A. salina as a model system to investigate the ecotoxicology of pollutants

Furthermore, depending on the developmental stages, A. salina nauplii at the instar I, II

the outcome of exposure to Roundup® herbicide on highly saline aquatic environments

found in or near the ocean.

Materials and Methods

Roundup® formulation contained the active ingredient glyphosate isopropylamine salt (2

concentration of glyphosate isopropylamine in the formulation.

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Supply Company (Burlington, NC 27215-3398, USA). 0.5 g of A. salina cysts were

The effect of exposure to different concentrations of Roundup® in artificial salt water on

in 24-well microplates. Different concentrations of glyphosate in the Roundup® (0, 9, 18,

illumination with shaking in an orbital shaking incubator at 60 rpm. Hatchability was

examined using a stereomicroscope at 7, 17, 24, 41 and 48 h. The experiment was

Acute toxicity test and mortality A. salina nauplii

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Morphological analysis- acute toxicity testing

concentrations of glyphosate in Roundup® (0, 0.72, 7.2 and 72 μg/mL) prepared in

euthanized in 10 % buffered formalin and examined using a Kern microscope.

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photographed each time for each concentration.

Catalase enzyme activity

concentrations of glyphosate in Roundup® (0, 0.72, 7.2 and 72 μg/mL) prepared in

determined according to the manufacturer’s instructions and the absorbance was

Apoptosis analysis using Acridine Orange (AO) staining.

and 48 h) to different concentrations of glyphosate (0.72, 7.2 and 72 μg/mL) in

Roundup®, as described above, using acridine orange staining. Following exposure to

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triplicate samples for each concentration.

transformed before statistical analysis. Either a two-way or one-way ANOVA (Analysis

differences in hatchability when cysts had been exposed to different concentrations of

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exposed for 24 h at concentrations of 144 and 288 μg/mL glyphosate in Roundup®

performed to analyze the effect of concentration and time of exposure to glyphosate in

Roundup® on these morphological measurements. The results showed statistically

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Further analysis by one-way ANOVA (followed by Tukey’s pairwise comparison) was