Direct and indirect effects of the glyphosate

formulation Glifosato Atanor® on

freshwater microbial communities

María Solange Vera, Eugenia Di Fiori,

Leonardo Lagomarsino, Rodrigo
Sinistro, Roberto Escaray, María
Mercedes Iummato, Angela Juárez, et al.
Ecotoxicology

ISSN 0963-9292

Ecotoxicology
DOI 10.1007/s10646-012-0915-2

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 Author's personal copy
Ecotoxicology
DOI 10.1007/s10646-012-0915-2
Direct and indirect effects of the glyphosate formulation Glifosato
AtanorÒ on freshwater microbial communities
Marı́a Solange Vera • Eugenia Di Fiori • Leonardo Lagomarsino •
Rodrigo Sinistro • Roberto Escaray • Marı́a Mercedes Iummato •
Angela Juárez • Marı́a del Carmen Rı́os de Molina • Guillermo Tell
Haydée Pizarro
Accepted: 11 April 2012
Ó Springer Science+Business Media, LLC 2012
Abstract Glyphosate-based formulations are among the
most widely used herbicides in the world. The effect of the
formulation Glifosato AtanorÒ on freshwater microbial
communities (phytoplankton, bacterioplankton, periphyton
and zooplankton) was assessed through a manipulative
experiment using six small outdoor microcosms of small
volume. Three of the microcosms were added with
3.5 mg l-1 of glyphosate whereas the other three were left
as controls without the herbicide. The treated microcosms
M. S. Vera
E. Di Fiori
R. Sinistro
G. Tell
H. Pizarro (&)
Laboratorio de Limnologı́a, Departamento de Ecologı́a,
Genética y Evolución, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Ciudad Universitaria,
C1428EHA Ciudad de Buenos Aires, Argentina
e-mail: haydeepizarro@gmail.com; hay@ege.fcen.uba.ar
M. S. Vera
E. Di Fiori
L. Lagomarsino
R. Sinistro

R. Escaray
M. M. Iummato
M. d. C. Rı́os de Molina

G. Tell
H. Pizarro
Consejo Nacional de Investigaciones Cientı́ficas y Técnicas
(CONICET), Buenos Aires, Argentina
L. Lagomarsino
R. Escaray
Instituto de Investigaciones Biotecnológicas, Instituto
Tecnológico de Chascomús (IIB-INTECH), CC 164, 7130,
Chascomús, Buenos Aires, Argentina
M. M. Iummato
A. Juárez
M. d. C. Rı́os de Molina
Laboratorio de Enzimologı́a, Estrés Oxidativo y Metabolismo,
Departamento de Quı́mica Biológica, Facultad de Ciencias
Exactas y Naturales, Universidad de Buenos Aires, Ciudad
Universitaria, C1428EHA Ciudad de Buenos Aires, Argentina
A. Juárez
Departamento de Biodiversidad y Biologı́a Experimental,
Facultad de Ciencias Exactas y Naturales, Universidad de
Buenos Aires, Ciudad Universitaria, C1428EHA Ciudad de
Buenos Aires, Argentina
•
showed a significant increase in total phosphorus, not fully
explained by the glyphosate present in the Glifosato Ata-
norÒ. Therefore, part of the phosphorus should have come
from the surfactants of the formulation. The results showed
significant direct and indirect effects of Glifosato AtanorÒ
on the microbial communities. A single application of the
herbicide caused a fast increase both in the abundance of
bacterioplankton and planktonic picocyanobacteria and in
chlorophyll a concentration in the water column. Although
metabolic alterations related to oxidative stress were
induced in the periphyton community, the herbicide
favored its development, with a large contribution of fila-
mentous algae typical of nutrient-rich systems, with shal-
low and calm waters. An indirect effect of the herbicide on
the zooplankton was observed due to the increase in the
abundance of the rotifer Lecane spp. as a consequence of
the improved food availability given by picocyanobacteria
and bacteria. The formulation affected directly a fraction of
copepods as a target. It was concluded that the Glifosato
AtanorÒ accelerates the deterioration of the water quality,
especially when considering small-volume water systems.
Keywords Glyphosate formulation
Microcosms

Microbial communities
Water quality
Introduction
Glyphosate (N-(phosphonomethyl)glycine), which is a
broad spectrum, non-selective and post-emergent herbicide
(Franz et al. 1997), is one of the most widely used for
agricultural weed control and for domestic and industrial
weed control. In plants and several microorganisms,
glyphosate acts by inhibiting the enzyme 5-enolpyruvyl
shikimic acid 3-phosphate synthase (EPSPS), which is
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 Author's personal copy
involved in the shikimate pathway and halts the production
of chorismate (Amrhein et al. 1980). As a result, it disrupts
the biosynthesis of aromatic amino acids, thus reducing
protein synthesis and growth, and eventually causing cel-
lular disruption and death (Salisbury and Ross 1994).
Another mechanism of action described for glyphosate is
the inhibition of the d-aminolevulinate synthase (ALAS),
an enzyme from the heme biosynthetic pathway (Duke
1988). It is noteworthy that algae, plants and animals share
the first seven steps of this pathway, related to the synthesis
of hemoproteins and chlorophylls.
In practice, what is actually used are formulations of
glyphosate (e.g. AccordÒ, Glifosato AtanorÒ, RodeoÒ,
RondoÒ, RoundupÒ, TouchdownÒ, among over a hundred
other formulations currently marketed in the world). These
commercial formulations generally include one or more
surfactants, which enhance the ability of glyphosate to
penetrate through the cuticular waxes on target plants
(Giesy et al. 2000). With the exception of RoundupÒ, in
which the surfactant is known to be polyethyloxylane
amine (Franz et al. 1997), the complete chemical compo-
sition of the formulations is generally not clearly men-
tioned by the manufacturers. It is thus difficult to know the
real mechanism of action of these formulations on the
environment considering that the chemical composition of
the mixtures is unknown.
Herbicides may reach freshwater bodies either by acci-
dental or wind-driven drift of the herbicide spray, or
through transport in surface runoff of suspended particulate
matter (Feng et al. 1990). In the case of glyphosate, once in
the aquatic system, it may affect microalgae due to their
physiological similarity with plants. Although previous
studies have provided evidence of glyphosate toxicity on
freshwater micro-photoautotrophs, most have been carried
out in single-species laboratory experiments (e.g. Forlani
et al. 2008; Lipok et al. 2010). Although this monospecific
tests are necessary, larger scale experiments are required to
extrapolate the real impact on natural communities and/or
ecosystems (Relyea and Hoverman 2006). Many experi-
mental studies have thus attempted to use complex aquatic
communities. Some authors have focused on the effects of
glyphosate on freshwater phytoplankton (Källqvist et al.
1994; Relyea et al. 2005), periphyton (Goldsborough and
Brown 1988; Relyea 2005; Relyea et al. 2005; Vera et al.
2010) and the entire microbial community (Pérez et al.
2007) in mesocosms. However, only a few studies have
evaluated the impact of this herbicide on the microbial
communities of microcosms (e.g. Pesce et al. 2009).
In Argentina, more than 170 million liters of glyphosate
were used during 2009 (*8.5 % of the total consumption
of glyphosate in the world) (CASAFE 2010) and one of the
most commonly used formulation is Glifosato AtanorÒ,
which is composed of 48 % w/v of glyphosate as
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M. S. Vera et al.
isopropylamine salt, surfactants of unknown composition
and mostly water. Thus, the objective of the present study
was to investigate the direct and indirect impacts of this
popular commercial formulation on structural features of
freshwater microbial communities (phytoplankton, bacte-
rioplankton, periphyton, and zooplankton) through a
manipulative experiment using outdoor microcosms. Since
it has been demonstrated that periphyton is a good tool to
detect glyphosate effects (Vera et al. 2010), oxidative stress
parameters (lipid damage and superoxide dismutase and
catalase activities) were studied as estimators of the met-
abolic response of the periphyton community. The exper-
iment was carried out on microcosms for analyzing the
impact of the herbicide in water bodies of small size, such
as temporary pools, widely distributed in the Pampean
region of Argentina and of great importance both as
sources of biodiversity and to human activities.
Materials and methods
Experimental design
The experiment was carried out between February and
March 2009 in six outdoor artificial small ponds (micro-
cosms). Polythene bags of *90 l were installed inside a
larger pool. Before the start of the experiment, the pool
(3,000 l) was filled with underground water, and bottom,
not rooted, aquatic plants (Ceratophyllum sp.) were added
to obtain a water body with limnological characteristics
similar to those of the neighboring shallow lakes (e.g.
Chascomús lake, Buenos Aires province, Argentina). The
periphyton assemblage was studied using artificial sub-
strates (clear polycarbonate strips of 15.7 cm2) that were
positioned in special devices (periphytometer). Once the
water column of the pool had developed into a true shallow
pond with a consolidated community of microorganisms,
eight periphytometers were placed in the pool and left for
substantial colonization for 21 days before the start of the
experiment.
On day 1 of the experiment (t0), the microcosms were
filled with water from the pool and a periphytometer with
colonized substrata was placed inside each microcosm.
Three of these microcosms were randomly selected and
treated with Glifosato AtanorÒ to obtain a nominal gly-
phosate concentration of 3.5 mg active ingredient per liter,
whereas the remaining three microcosms were used as
controls (without herbicide addition). This nominal con-
centration of glyphosate was chosen for being similar to
that recommended for aquatic and terrestrial weed control
(3.7 mg active ingredient l-1, Giesy et al. 2000). The
Glifosato AtanorÒ was bought from a commercial Argen-
tinian retailer.
 Author's personal copy
Direct and indirect effects of the glyphosate formulation Glifosato AtanorÒ
Water samples were collected from each microcosm on
five occasions. The first samples (t0) were obtained
immediately after herbicide application. The remaining
samples were collected 1, 7, 14 and 21 days after t0 (t1–t4,
respectively). In addition, a specific amount of colonized
artificial substrata was extracted on each sampling date for
the study of the periphyton assemblage.
Physical and chemical properties of the water
of the microcosms
Water temperature, pH and conductivity were measured in
situ on each sampling date with a HANNA HI 991301
portable meter, dissolved oxygen with a HANNA HI 9143
portable meter, and nephelometric turbidity with a 2100P
HachÒ portable turbidimeter.
Water samples were poured into polypropylene con-
tainers and immediately transported to the laboratory.
Water for chemical analysis was filtered immediately after
sampling through WhatmanÒ GF/F filters. Soluble reactive
phosphorus (SRP) was measured by the molybdate-ascor-
bic method. Total phosphorus (TP, from unfiltered water
samples) and total dissolved phosphorous (TDP, from GF/F
filtered water) were converted to SRP after acid digestion
with potassium persulfate. Nitrate (NO3-) and nitrite
(NO2-) were measured by Cd reduction followed by dia-
zotation and NH4 by the indophenol blue method. Organic
nitrogen (ON) was determined by the Kjeldahl method
(APHA (American Publication Health Association) 2005).
Total nitrogen (TN) was considered as the sum of nitrates,
nitrites, and organic nitrogen. The methodology taken from
APHA (American Publication Health Association) (2005)
was applied.
For alkalinity measurements the titration with H2SO4
0.1 N method (APHA (American Publication Health
Association) 2005) was followed.
Total suspended solids (TSS) were estimated after fil-
tration of a water sample through pre-rinsed and pre-
combusted (530 °C, 2 h) GF/F filters, dried until constant
weight at 103–105 °C. Organic matter (OM) was deter-
mined as the mass difference after combustion at 530 °C
for 3 h of GF/F dry filters following APHA (American
Publication Health Association) (2005). Both TSS and OM
were not determined at t1 and t2 due to the large water
volume required for their analyses.
Glyphosate analysis
Glyphosate concentrations in the water samples were
determined with ion chromatography using a DIONEX
DX-100 chromatograph with a conductivity detector and a
25 ll sample loop (Pessagno et al. 2008). A DIONEX
AS-4 was used as analytical chromatographic column. A
mixture of NaOH/Na2CO3 4 mM/9 mM was chosen as
eluent with a flow rate of 2 ml min-1. The experimental
error was below 5 %. The glyphosate dissipation rate
(k) and half-life in water were estimated using Ct =
C0 e(-kt), where Ct is concentration at time t and C0 is the
initial concentration.
Periphyton
On each sampling date, the periphyton obtained from
21 days of colonization was removed from substrata by
means of a fine brush, for the analyses of the following
variables: chlorophyll a and b concentrations (P-Chl a and
P-Chl b), dry weight (DW), ash-free dry weight (AFDW),
taxonomical composition and density of the algal fraction.
For chlorophyll determinations, the periphyton from one
substratum of each microcosm was scraped and centrifu-
gated (10 min at 7,0009g). Cells were broken in 80 %
(v/v) acetone by sonication using a Cole Parmer CP600
4710 Ultrasonic Homogenizer. After 1 h of incubation at
4 °C, the extracts were clarified by centrifugation for
10 min at 7,0009g and absorbance was read at 663.2 and
646.8 nm in a Shimadzu UV/vis spectrophotometer.
Chlorophyll a and b concentrations were calculated using
Lichtenthaler (1987)’s equations. Other substratum from
each microcosms was used for the rest of mass variables.
DW was estimated from samples filtered through What-
manÒ GF/C filters pre-combusted to 440 °C for 2 h prior to
use and later weighing the material dried at 60 °C on a
stove up to constant weight. AFDW was determined as the
mass difference after 3 h of calcination (440 °C) of dry
samples (APHA (American Publication Health Associa-
tion) 2005). The autotrophic index (AI) was estimated as
the AFDW : chlorophyll a ratio. An AI value higher than
200 indicates a high proportion of heterotrophic, non-
chlorophyllous organisms or organic detritus (APHA
(American Publication Health Association) 2005).
Samples for algae identification were obtained from one
substratum from each microcosms and the material was
fixed with 2 % formalin and analyzed using an optical
microscope at 91,000 magnification. For algae quantifi-
cation, samples were obtained for one substratum of each
microcosms and fixed with 1 % acidified Lugol’s iodine
solution. Counts were performed using the inverted
microscope technique (Utermöhl 1958) at 9400 magnifi-
cation with a counting error \15 %, estimated according to
Venrick (1978).
To estimate all the oxidative stress parameters, cells
were harvested from one substratum of each microcosms
by scraping and then centrifuged at 3,0009g for 20 min,
washed with 0.134 M (pH 6.5) potassium phosphate buffer,
and resuspended in the same buffer containing protease
inhibitors (0.2 mM benzamidine and 0.5 mM phenyl
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methyl sulfonyl fluoride). Cells were then disrupted by
sonication using a Cole Parmer CP600 4710 Ultrasonic
Homogenizer. The homogenates were centrifuged at
11,0009g for 30 min, and the supernatant was used as
enzyme and lipid peroxide source. All procedures were
carried out at 4 °C.
Lipid peroxides were quantified through dosage of
thiobarbituric acid (TBA) reactive substances (TBARs),
according to Buege and Aust (1978). An aliquot of 175 ll
of the 11,0009g supernatant was mixed with 1 ml of
reagent containing TBA, after incubation in a boiling water
bath for 30 min. The absorbance of cleared supernatant
was measured at 535 nm on a spectrophotometer Shimadzu
UV/vis. The TBARs content was estimated as equivalent of
malondialdehyde (MDA), since for the calculations we
used the molar extinction coefficient (e = 156 mM-1
cm-1) for the MDA-TBA complex. The results were
expressed as nmol MDA per g of AFDW.
Superoxide dismutase (SOD) activity was determined
according to Beauchamp and Fridovich (1971). This
method is based on the inhibition of the photochemical
reduction of nitro blue tretrazolium (NBT). The reaction
was carried out in 50 mM potassium phosphate buffer (pH
7.8), and the absorbance was measured at 560 nm. The
results were expressed as units of SOD per mg of protein
(one unit of SOD: the amount of enzyme necessary to
inhibit the NBT reduction rate by 50 %).
Catalase (CAT) activity was measured using H2O2 as
substrate and calculated using an extinction coefficient of
40 M-1 cm-1 (Aebi 1984). The decay of peroxide was
monitored for 30 s at 240 nm, in a reaction mixture con-
taining 50 mM potassium phosphate buffer (pH 7.4) and
10 mM H2O2. The results were expressed as CAT units per
mg of proteins (one CAT unit: the enzyme amount that
transforms 1 mmol of H2O2 per min).
All periphytic variables were expressed by triplicate on
an area basis.
Phytoplankton and bacterioplankton
The abundance of the different size fractions of phyto-
plankton and bacterioplankton was estimated on all sampling
dates from water samples of each microcosm. Micro-
([20 lm) and nanophytoplankton (2–20 lm) were quanti-
fied as described for periphyton. Picophytoplankton
(0.2–2 lm) and bacterioplankton samples were preserved
with 2 % ice-cold glutaraldehyde. Picophytoplankton and
bacterioplankton samples were filtered through 0.2-lm pore
size black polycarbonate filters (Isopore GTPB 02500; Iso-
pore, Billerica, Massachusetts, USA) stained with DAPI
(Porter and Feig 1980). Then, each filter was mounted on a
microscope slide with a drop of immersion oil (Immersol
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M. S. Vera et al.
Zeiss 518 F; Zeiss, Jena Germany) and algae and bacteria
were quantified using an epifluorescence microscope
(Olympus BX40). To estimate the picophytoplankton and
bacterioplankton abundance, a minimum of 20 fields and 400
individuals were counted on each slide (error \15 %).
Phytoplankton chlorophyll a concentrations (Chl a) were
measured after extraction with methanol (Lopretto and Tell
1995).
Zooplankton
Zooplankton abundance was estimated at t0 from the water
of the pool and at t4 from the water of each microcosm. At
the end of the experiment, the entire content of the
enclosures was filtered through a 55 lm pore mesh.
Microzooplankton (\200 lm) samples were analyzed in
1 ml Sedgwick–Rafter chambers, counting organisms
under a binocular microscope, and subsamples were
obtained using a Hensen-Stempel pipette. Mesozooplank-
ton ([200 lm) was counted in 5 ml Bogorov chambers
under a stereomicroscope, and subsamples were taken with
a Russell device. The larval stages were recorded and the
number of aliquots counted (at least three) was calculated
with a maximum error of 10 %.
Statistical analyses
For all variables, differences between treatments were
assayed using repeated-measures analysis of variance (RM
ANOVA), with two treatments (control and herbicide
treatment) and five sampling times (t0–t4). RM ANOVA
analyses were followed by multiple comparisons tests (post
hoc testing), using Tukey’s test (p \ 0.05). Only for zoo-
plankton variables, differences between treatments at t4
(21 days) were assayed using one-way analysis of variance
(one-way ANOVA). Prior to each analysis, Kolmogorov–
Smirnov tests (with Lilliefors’ correction), Levene median
tests and Mauchley Sphericity tests were run in order to test
data for normality, homoscedasticity and sphericity,
respectively. Whenever the data did not conform, the val-
ues were transformed (square-root or log) as necessary.
Results
Physical and chemical properties of the water
of the microcosms
At t0, the water was alkaline, with high values of pH
(8.97 ± 0.01) and conductivity (4.05 ± 0.02 mS cm-1 at
21 °C). Mean water temperature was 27.22 ± 0.16 °C,
dissolved oxygen 4.5 mg l-1 and turbidity 2.75 NTU.
 Author's personal copy
Direct and indirect effects of the glyphosate formulation Glifosato AtanorÒ
Initial total phosphorus (TP) was 76 lg l-1 and total
nitrogen (TN) 4,308.99 lg l-1.
Water temperature ranged from 21.3 to 27.4 °C and
conductivity from 3.05 to 4.44 mS cm-1 throughout the
experiment. Dissolved oxygen concentration varied
between 4.5 and 11.2 mg l-1. Significant differences were
observed between treatments, times and the treatment-by-
time interaction for dissolved oxygen (RM ANOVA,
p \ 0.05). Mean comparisons revealed significantly higher
values in treated microcosms as from t1. Nephelometric
turbidity fluctuated between 1.17 and 3.23 NTU in the
controls and between 1.39 and 7.38 NTU in the micro-
cosms treated with Glifosato AtanorÒ. Although turbidity
did not vary significantly between treatments (RM
ANOVA, p [ 0.05), values were smaller in controls
throughout the experiment. pH increased in all the micro-
cosms during the experimental period, and significant dif-
ferences were observed between treatments, times and the
treatment-by-time interaction (RM ANOVA, p \ 0.05).
Mean comparisons showed significantly higher values in
treated microcosms in the last two sampling dates (t3 and t4).
From t0 onwards, TP concentration ranged between 32
and 90 lg P l-1 in control microcosms and between 922
and 1,564 lg P l-1 in the microcosms added with herbi-
cide. Significant differences between treatments, times and
treatment-by-time interaction were observed for TP con-
centration (RM ANOVA, p \ 0.05), showing a significant
increase after herbicide addition, with values between 10
and 30 times higher than those in controls. Soluble reactive
phosphorus (SRP) ranged between 3 and 40 lg P l-1 along
the experiment. SRP concentrations showed only signifi-
cant differences between times (RM ANOVA, p \ 0.05),
with a remarkable increase until the first week of exposure
and a decrease after that date.
Total and organic nitrogen concentration (TN and ON)
averaged 2,881.3 ± 1,082.1 lg N l-1 and 2,387.8 ± 978.2
lg N l-1 (mean ± SD), respectively. The RM ANOVA
showed significant differences between times (p \ 0.05) but
not between treatments (p [ 0.05) for both variables. TN
and ON decreased significantly during the study period.
Ammonium concentrations ranged between 0.0 and
11.2 lg N l-1 and between 0.0 and 4.5 in lg N l-1 in con-
trol and treated microcosms, respectively. The concentration
of ammonia showed significant differences due to the treat-
ment-by-time interaction (RM ANOVA, p \ 0.05). Mean
comparisons revealed lower values at t1 and higher values at
t4 for controls with respect to the microcosms enriched with
Glifosato AtanorÒ.
Nitrite concentrations varied between 1.4 and 34.2
lg N l-1 and between 23.1 and 36.5 lg N l-1 and nitrate
concentrations fluctuated between 2.2 and 1,131.9 lg
N l-1 and 233.1 and 904.8 lg N l-1 in the microcosms
with and without the herbicide, respectively. Significant
differences were observed between times and treatments,
as well as in the treatment-by-time interaction for these two
variables (RM ANOVA, p \ 0.05). For nitrite values,
mean comparisons revealed significant differences between
treatments at t4, with values 20 times higher in controls
than in treated microcosms. The concentration of N–NO2-
did not show differences between times in control micro-
cosms. In contrast, treated microcosms showed a signifi-
cant decrease at t4. Tukey post hoc comparisons for nitrate
concentrations showed higher values in the controls than in
the treated microcosms at t3 and t4. Both treatments
showed a similar pattern over time.
Total suspended solids (TSS) ranged from 2.4 to
7.8 mg l-1 and from 4.1 to 15.3 mg l-1 and organic matter
(OM) ranged from 1.8 to 6.4 mg l-1 and from 3.3 to
13.0 mg l-1 for control and treated microcosms, respec-
tively. Although mean TSS and OM were always higher in
microcosms with Glifosato AtanorÒ than in controls, no
significant differences between treatments, times or treat-
ment-by-time interaction were observed for these variables
(RM ANOVA, p [ 0.05).
Glyphosate analysis
Mean glyphosate concentration after the herbicide appli-
cation was 3.45 ± 0.36 mg l-1 (mean ± SD). Treated
microcosms presented an estimate glyphosate dissipation
rate (k) of 0.04 day-1 (±0.02 standard error, SE) with a
half-life of 16.02 days.
Periphyton assemblage
The periphyton assemblage was dominated by Chlorophyta
(53 %) in both treatments during the whole experiment,
followed by Cyanobacteria and Bacillariophyta (Fig. 1).
Abundance of live Chlorophyta ranged from 39,285 to
248,205 algae cm-2, and significant differences were
observed due to times (RM ANOVA, p = 0.000) and the
time-by-treatment interaction (RM ANOVA, p = 0.000).
Mean comparisons showed a significant increase in control
at t1 and higher values in treated microcosms as from t3
(Fig. 1a). With respect to diatoms, the abundance of living
organisms varied between 5,841 and 48,988 algae cm-2,
both extreme values recorded at t4. The RM ANOVA
revealed significant differences due to times (p = 0.000),
treatments (p = 0.028) and the interaction between treat-
ments and time (p = 0.000); in controls, the abundance of
living diatoms decreased over time, whereas in the treated
microcosms the values remained constant until the last
sampling date, on which the abundance increased signifi-
cantly as compared to earlier times (Fig. 1a). Cyanobacteria
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M. S. Vera et al.

Abundance algae living groups (algae cm ) a microcosms. Significant differences were observed due to
-2

4.0 e
+5 times (RM ANOVA, p = 0.000), treatments (RM
Cyanobacteria ANOVA, p = 0.001) and the time-by-treatment interaction
Chlorophyta
Bacillariophyta
(RM ANOVA, p = 0.000). In the treated microcosms,
+5
3.0 e there was a progressive and significant increase in dead
diatoms with respect to controls from t1 onwards (Fig. 1b).
+5
After t2, assemblages of metaphyton appeared on the
2.0 e
artificial substrata in the herbicide-treated microcosms, and
remained there throughout the rest of the experimental
+5
period. These metaphyton were composed mainly of
1.0 e
filamentous Chlorophyta Oedogonium spp. and Mougeotia
sp. The abundance of filamentous Chlorophyta ranged
0.0 from 1,360 to 26,378 algae cm-2 and from 1,526 to
C T C T C T C T C T 4,582 algae cm-2 in the microcosms with and without
Glifosato AtanorÒ, respectively. Significant differences
b were observed between treatments (p = 0.004), times
Abundance algae dead groups (algae cm )
-2

7.0 e
+4
(p = 0.000) and the interaction (p = 0.000). Mean com-
Chlorophyta parisons revealed that the abundance of filamentous green
6.0 e
+4 Bacillariophyta
algae increased significantly over time in the microcosms
5.0 e
+4 treated with the herbicide but not in the controls.
Dry weight (DW) and ash-free dry weight (AFDW) ran-
ged from 46.4 to 334.0 lg cm-2 and from 28.5 to
+4
4.0 e

+4 259.1 lg cm-2, respectively. Periphytic chlorophyll a var-

3.0 e
2.0 e
+4
+4
1.0 e
0.0
C T C T C T C T
0 1 7 14
Time (d)
Fig. 1 Mean abundance of a living and b dead algae for control
(C) and Glifosato AtanorÒ-treated (T) microcosms throughout the
study period. n = 3
abundance averaged 73,299 ± 31,698 ind. cm-2, and sig-
nificant differences were observed due to times (p = 0.000),
treatments (p = 0.004) and the time-by-treatment interac-
tion (p = 0.000). Mean comparisons showed higher values
in treated microcosms from t3 onwards (Fig. 1a). No dead
organisms were detected for Cyanobacteria.
The abundance of dead Chlorophyta averaged
2,709 ± 2,464 and 4,490 ± 2,621 algae cm-2 in control
and treated microcosms, respectively. The RM ANOVA
showed significant differences due to times (p = 0.000),
treatments (p = 0.018) and the time-by-treatment interac-
tion (p = 0.003), and mean comparisons revealed that the
abundance of dead Chlorophyta increased significantly
over time in the treated microcosms and only at t3 and t4 in
control microcosms, always with higher values in treated
microcosms than in controls (Fig. 1b). The abundance of
dead diatoms ranged from 1,244 to 24,200 ind. cm-2 in
controls and from 1,575 to 59,690 ind. cm-2 in the treated
ied between 0.03 and 0.4 lg cm-2 and between 0.15 and
0.7 lg cm-2 while chlorophyll b concentration between
0.02 and 0.16 lg cm-2 and between 0.08 and 0.16 lg cm-2
in control and treated microcosms, respectively. Significant
differences between treatments, times and the treatment-by-
C T
21 time interaction were observed for these variables (RM
ANOVA, p \ 0.05). Mean comparisons revealed significant
higher values in the treated microcosms than in controls, in t3
and t4 for DW and AFDW (Fig. 2a, b) and as from t2 for
P-Chl a and P-Chl b (Fig. 2c, d).
The autotrophic index (AI) averaged 487.4 ± 269.9 for
the whole experiment and ranged from 187.9 to 1,001.0
and from 223.3 to 1,264.4 in the microcosms with and
without Glifosato AtanorÒ, respectively. Statistically sig-
nificant differences due to times (RM ANOVA, p = 0.000)
and the time-by-treatment interaction (RM ANOVA,
p = 0.003) were observed for the AI. Mean comparisons
revealed AI values approximately three times higher in
controls than in the treated microcosms at t4. The AI ten-
ded to increase significantly in both microcosms as from t3,
but there was a significant decrease in the AI of the treated
microcosms between t3 and t4.
Damage to lipids evaluated according to the content of
TBARs (as MDA equivalents) varied between 0.4 and
1.6 nmol MDA/g AFDW and between 0.5 and 3.1 nmol
MDA/g AFDW in the control and treated microcosms
respectively. The RM ANOVA revealed significant dif-
ferences between times (p = 0.006) and the interaction
between treatments and time (p = 0.015) for this variable.
TBARs concentration remained without significant

123
 Author's personal copy
Direct and indirect effects of the glyphosate formulation Glifosato AtanorÒ

a 400 b 400
Control Control

Ash-free dry weight (µg cm )

-2
Treatment ** Treatment
Dry weight (µg cm ) * *
-2
300 300
*

200 200

100 100

0 0
0 1 7 14 21 0 1 7 14 21

c 0.8 d 0.8
* Control
Control
P-Chlorophyll a (µg cm )
-2

Treatment Treatment

P-Chlorophyll b (µg cm )
-2
0.6 0.6

**

0.4 0.4
*

0.2 0.2 *
**
**

0.0 0.0
0 1 7 14 21 0 1 7 14 21
Time (d) Time (d)

Fig. 2 Mean values of a dry weight, b ash-free dry weight and the same sampling date are indicated by asterisks: * p \ 0.05;
periphytic, c chlorophyll a and d chlorophyll b concentrations in ** p \ 0.01. Significant differences between consecutive times within
control and treated microcosms throughout the study period. Error a treatment are indicated by diamonds: e p \ 0.05; ee p \ 0.01, in
bars represent ?1 SD. Significant differences between treatments at grey for control and black for treated microcosms. n = 3

variations along the experiment until t4, when TBARs
content increased significantly in the treated microcosms
with respect to controls, revealing damage to lipids in the
community treated with Glifosato AtanorÒ (Fig. 3a).
Catalase activity varied from 0.1 to 1 units mg-1 of
protein and from 0.1 to 0.5 units mg-1 of protein in the
control and treated microcosms, respectively. The RM
ANOVA revealed significant differences between treat-
ments (p = 0.009), times (p = 0.000) and the treatment-
by-time interaction (p = 0.008) for this parameter. Mean
comparisons showed lower catalase activity in the treated
microcosms than in the controls, showing significant dif-
ferences from t2 onwards (Fig. 3b).
Superoxide dismutase activity varied from 66.5 to
303.6 units mg-1 of protein and from 83.9 to 386.6
units mg-1 of protein in the control and treated microcosms,
respectively. The RM ANOVA revealed significant differ-
ences between treatments (p = 0.027), times (p = 0.000)
and the treatment-by-time interaction (p = 0.013) for this
parameter. Mean comparisons revealed significant higher
values in the treated microcosms than in controls at t1 and t2,
showing levels similar to those of the control microcosms in
the remaining sampling dates (Fig. 3c).
Phytoplankton and bacterioplankton
The micro- and nanophytoplankton community was dom-
inated by Chlorophyta (804–158,438 ind. ml-1) in both
treatments during the whole experiment, followed by Ba-
cillariophyta (0–918 ind. ml-1) and Cyanobacteria (0–759
ind. ml-1). Micro- and nanophytoplankton abundance
ranged from 1.1 9 103 to 1.6 9 105 ind. ml-1 and from
1.8 9 103 to 6.3 9 104 ind. ml-1 in microcosms with and
without herbicide, respectively. The RM ANOVA revealed
significant differences due to times (p = 0.000), treatment
(p = 0.007) and the interaction between treatment and time
(p = 0.001) for the abundance of this algal community.
Mean comparisons showed that this abundance increased
significantly more than two-fold at t2 in the treated
microcosms with respect to controls and decreased signif-
icantly at t3 for both treatments.
The values of phytoplankton chlorophyll a varied between 0.9
and 12.6 lg l-1 for controls and between 4.5 and 39.2 lg l-1 for
treated microcosms, and significant differences for the time-by-
treatment interaction (RM ANOVA, p = 0.025) were observed
for Chl a. Chlorophyll a concentration of the treated phyto-
plankton community showed a small significant decrease at t1

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M. S. Vera et al.

a 3.5
a +5
3.0 e
TBARs (nanomoles MDA g AFDW )
-1

Control Control
3.0 Treatment ** +5 Treatment **
2.5 e
**
2.5

Pcy (cells ml )
+5

-1
2.0 e
2.0
+5
*
1.5 e
1.5
+5
1.0 e
1.0
+4
5.0 e
0.5

0.0 0.0
0 1 7 14 21 0 1 7 14 21

b 1.2 b +7
Control 1.0 e
** Control
Treatment Treatment *
1.0
+6 *
CAT (units mg protein )

8.0 e
-1

Bacteria (cells ml )
*

-1
0.8
+6 *
6.0 e
0.6

+6
4.0 e
0.4
**
+6
0.2 * 2.0 e

0.0
0.0
0 1 7 14 21 0 1 7 14 21
c Time (d)
50
0
Control
Treatment Fig. 4 Mean abundance of a picocyanobacteria (Pcy) and b bacterio-
plankton during the experiment in control and treated microcosms.
SOD (units mg protein )

40
0
-1

** * Error bars represent ?1 SD. Significant differences between treat-

ments at the same sampling date are indicated by asterisks: * p \ 0.05;
30
0 ** p \ 0.01. Significant differences between consecutive times within a
treatment are indicated by diamonds: e p \ 0.05; ee p \ 0.01, in
grey for control and black for treated microcosms. n = 3
20
0

10
0 Picocyanobacteria (Pcy) abundance ranged from
0
0 1 7 14
Time (d)
Fig. 3 Mean values of periphytic a thiobarbituric acid reactive
substances (TBARs) levels as malondialdehyde (MDA) equivalents,
b catalase (CAT) activity and c superoxide dismutase (SOD) activity
in control and treated microcosms throughout the study period. Error
bars represent ?1 SD. Significant differences between treatments at
the same sampling date are indicated by asterisks: * p \ 0.05;
** p \ 0.01. Significant differences between consecutive times within
a treatment are indicated by diamonds: e p \ 0.05; ee p \ 0.01, in
grey for control and black for treated microcosms. n = 3
and a significant increase at t2 with respect to controls. This
increase in Chl a was observed until the end of the experiment but
without significant differences between treatments.
5.1 9 104 to 2.7 9 105 cells ml-1 during the whole
experiment, and significant differences were observed due
21 to times (p = 0.000), treatments (p = 0.000) and time-by-
treatment interaction (p = 0.000). Mean comparisons
showed that Pcy abundance was significantly higher in the
treated microcosms than in controls as from t2 (Fig. 4a).
The other group of autotrophic picoplankton, the pic-
oeukaryotes, were almost absent from the water samples of
both treatments. Heterotrophic bacterioplankton abundance
averaged 4.2 9 106 ± 6.8 9 105 cells ml-1 in the controls
and 5.96 9 106 ± 1.68 9 106 cells ml-1 in the treated
microcosms. Statistically significant differences in the
bacteria abundance were observed between treatments
(RM ANOVA, p = 0.001), times (RM ANOVA,
p = 0.007) and the interaction between time and treatment
(RM ANOVA, p = 0.000). Bacteria abundances were

123
 Author's personal copy
Direct and indirect effects of the glyphosate formulation Glifosato AtanorÒ
Fig. 5 Mean zooplankton
abundance at 0 (t0) and 21 (t4)
days after the beginning of the
experiment in control (C) and
treated (T) microcosms.
Significant differences between
treatments at t4 are indicated by
asterisks: ** p \ 0.01. n = 3
significantly higher in the treated microcosms than in
controls as from t1 (Fig. 4b).
Zooplankton
Total abundance of zooplankton at t0 was 346 ind. l-1 with
a high dominance of copepods, represented with over 98 %
by calanoid nauplii (Fig. 5). No significant differences
between treatments were observed for total zooplankton
abundance at t4 (one-way ANOVA, p = 0.984), with
average values of 328 ± 10 and 328 ± 55 ind l-1 in the
microcosms with and without herbicide, respectively. Two
orders of copepods, Calanoida and Cyclopoida, were
identified and adult organisms of nauplii were distin-
guished (Fig. 5). No significant differences between treat-
ments at t4 were detected for cyclopoid adults or nauplii
(one-way ANOVA, p [ 0.05). However, a significant
increase in calanoid adult abundance at t4 with respect to t0
was detected in both treatments, with density values sig-
nificantly higher in controls at t4 (one-way ANOVA,
p = 0.000, Fig. 5). With respect to the calanoid nauplii,
significantly lower densities were obtained in both treat-
ments at t4 than at t0, but with no significant differences
between treatments at t4 (one-way ANOVA, p = 0.081,
Fig. 5).
Total abundance of Cladocerans was 3 ind. l-1 at t0 and
138 ± 20 ind. l-1 at t4 in controls and 134 ± 43 ind. l-1
in treated microcosms. Two genera of Cladocerans were
identified, Alona sp. and Moina sp., and only a significant
increase in Moina sp. was detected at t4 as compared to t0,
but without differences between treatments (one-way
ANOVA, p [ 0.05, Fig. 5). The total abundance of rotifers
at t0 was 38 ind. l-1. With respect to the rotifers identified,
Lecane was the only genus that showed significant differ-
ences between treatments at t4 (one-way ANOVA,
p = 0.003, Fig. 5), with abundance values 20 times greater
in the treated microcosms.
Discussion
In this study, it was observed that a single application of
Glifosato AtanorÒ caused water turbidity as well as chan-
ges in other physical and chemical variables, which led to
rapid impoverishment of the water quality of the micro-
cosms studied. The worsening conditions of the micro-
cosms were faster than those previously observed in
mesocosms (*30,000 l) using RoundupÒ (Vera et al.
2010). The disparity in the responses between micro- and
mesocosms could be due to the effect of the resource
confinement and concentration due to morphometric dif-
ferences (Pesce et al. 2009), which results in a better
availability of resources by organisms at lower volumes,
with an overall acceleration of the system functional pro-
cesses. On the other hand, the input of high amounts of
phosphorus may also promote microbial development. The
concentration of Glifosato AtanorÒ added to the micro-
cosms (i.e. 3.5 mg glyphosate l-1) contributed with
490 lg phosphorus l-1, being 56 % of the TP measured in
the water at t1. As a consequence, the rest of the phos-
phorus should have come from the surfactants presented in
the formulation, which would enhance the potential tox-
icity of the formulation, considering that phosphorus pro-
motes eutrophication in water bodies. In previous studies
conducted in mesocosms (Pérez et al. 2007; Vera et al.
2010), a marked increase in TP was also recorded in treated
mesocosms but fully explained by the phosphorus from the
glyphosate of the RoundupÒ added.
Immediately after the application of Glifosato AtanorÒ,
both turbidity and total suspended solids showed a trend to
increase, and the organic fraction was always dominant in
the presence of the herbicide. This fact was clearly asso-
ciated with the increase in the planktonic chlorophyll a and
the density of planktonic bacteria and picocyanobacteria. A
high development of picocyanobacteria was observed by
Pérez et al. (2007) in the presence of RoundupÒ, showing
the clear resistance of some cyanobacteria to glyphosate, as
123
 Author's personal copy
stated by Forlani et al. (2008) and Lipok et al. (2010).
Moreover, the abrupt and rapid increase in bacterial density
in the treated microcosms can be explained by the fact that
bacteria can degrade the glyphosate molecule. Amorós
et al. (2007) showed a significant augment in Aeromonas
spp. and Stenotrophomonas maltophilia from the first day
of exposure to RoundupÒ in water samples from a shallow
lake in Spain. However, negative effects of glyphosate
have been reported in Vibrio fischeri in culture (Bonnet
et al. 2007), and in aquatic bacteria in a mesocosm study
(Chan and Leung 1986). Clearly, it would be important to
further study the effect of glyphosate and its formulations
on bacteria structure.
The sudden increase in bacterioplankton after the addi-
tion of the herbicide was quickly followed by an
enhancement in nano- and microphytoplankton, probably
favored by the improved nutrient loading. Later, and after
the decrease in net phytoplankton, the picocyanobacteria
proliferated, probably favored by the presence of the her-
bicide and the competitive advantage given by the decrease
in larger phytoplankton organisms. At 2 weeks, picocy-
anobacteria became the dominant autotrophic fraction in
the water column of the microcosms exposed to Glifosato
AtanorÒ. The growth of picocyanobacteria and periphytic
algae (see below), associated with an increase in photo-
synthesis, seems to be the cause of the higher values of
dissolved oxygen concentration and pH of the water in the
presence of the herbicide.
It was also observed that the herbicide caused a clear
positive effect on micro- and nanophytoplankton, although
differences between treatments were significant only at t2.
Unlike that observed in mesocosms (Pérez et al. 2007),
these autotrophic fractions proliferated in smaller devices
with Glifosato AtanorÒ, as it was also evident in chloro-
phyll a concentrations in the water column. The clear
decrease in nano-and microphytoplankton abundance at t4
recorded in both treatments could be due to a high grazing
pressure by Moina sp., a cladoceran that multiplied in both
treatments. However, the possible sedimentation of algae
in the quiet water of the microcosms as well as the sig-
nificant increase in periphyton, which could compete for
resources with the larger phytoplankton, should not be
disregarded.
An indirect effect of the herbicide on the zooplankton
was observed due to the increase in the abundance of the
rotifer Lecane spp. at t4 as a consequence of the improved
food availability given by picocyanobacteria and bacteria,
which increased in density in the microcosms treated with
the herbicide. On the other hand, a direct effect of the
herbicide on zooplankton may have occurred, affecting
directly a fraction of copepods as a target. Although a clear
decrease in density of calanoid nauplii was observed in
both treatments, this fraction showed lesser abundance in
123
M. S. Vera et al.
the microcosms treated at t4. Moreover, the density of
calanoid adults was significantly lower in the treatment
with Glifosato AtanorÒ, which would be reflecting the
effect of the herbicide on larval development. It was ruled
out the possible reduction of adult calanoid copepods by
interspecific competition with rotifers of similar eating
habits. Rotifers of the genus Lecane appear to be abundant
and, like calanoid copepods, feed on picoplankton and
bacteria. Since the food (bacteria and picocyanobacteria)
was not a limiting factor in the microcosms with the her-
bicide, the smaller development of calanoid nauplii in this
treatment does not appear to be due to competition, but a
direct toxicological effect of the herbicide. There is a
current profound debate on the mode of action of gly-
phosate on non-target organisms, and its potential effect as
an endocrine disruptor in animals has not been ruled out
(Paganelli et al. 2010; Walsh et al. 2000). All the nauplii
stages of the copepod Cyclops vernalis have been found to
be sensitive to four different herbicides (Robertson and
Bunting 1976), which suggests that glyphosate or any of its
formulations, such as Glifosato AtanorÒ, could have
caused a direct effect on these sensitive stages in the
present experiment. In natural systems, these impacts at the
zooplankton level could certainly have an effect on higher
trophic levels, including fish that consume this kind of
organisms.
In agreement with that reported by Peterson et al. (1994)
and Tsui and Chu (2003), diatoms appeared as the peri-
phytic algae most sensitive to the herbicide. Moreover,
according to Pérez et al. (2007) and Vera et al. (2010) for
RoundupÒ in mesocosms, cyanobacteria were the auto-
trophic component of the periphyton most favored in the
presence of the glyphosate formulation. Although they
were not dominant, there was a marked temporary increase
in filamentous green algae typical of metaphyton in the
herbicide treatment. This indicates that the system could
tend towards the ‘‘Sheltered State’’ dominated by algae,
such as Oedogoniales and/or Zygnematales (Goldsborough
and Robinson 1996) in environments with high amounts of
nutrients, stability of the water column and high light
irradiance, pH and conductivity values. All these features
were present in the microcosms with herbicide.
Unlike the general behavior of the periphyton in mes-
ocosms with RoundupÒ (Pérez et al. 2007; Vera et al.
2010), in the present experiment there was a significant
increase in DW and AFDW, probably due to the
enhancement in bacteria, organic detritus and heterotrophic
organisms, which is consistent with phenomena of organic
pollution (Mudge and Seguel 1999). The increase in peri-
phytic chlorophyll a could be attributable to the presence of
high amounts of filamentous green algae which is also
revealed by the increase in chlorophyll b concentrations in
microcosms treated with Glifosato AtanorÒ. The increase
 Author's personal copy
Direct and indirect effects of the glyphosate formulation Glifosato AtanorÒ
in periphyton biomass recorded here is consistent with a
previous study conducted in mesocosms using RoundupÒ
at a similar concentration of the active ingredient
(3.8 mg l-1, Relyea 2005).
Oxidative stress has been proposed as a mechanism of
action for different xenobiotics (Bagchi et al. 1995).
Besides, oxidative stress has been observed to be induced
by glyphosate formulations in one species of microalgae
and other aquatic organisms (Contardo-Jara et al. 2009;
Costa et al. 2008; Lushchak et al. 2009; Romero et al.
2011). Despite the increase in periphytic biomass found in
the microcosms with the glyphosate formulation studied in
this work, the damage to lipids as evidenced by the higher
concentration of TBARs in the treated microcosms sug-
gests that the herbicide would be inducing oxidative stress
in the entire community, i.e. in all the autotrophic and
heterotrophic components. SOD activity showed similar
results: the higher levels at t1 and t2 in the treated
microcosms showed an antioxidant response that seems to
have prevented lipid oxidative damage under this exposure
time. A subsequent decrease in its activity occurred at t4,
thus resulting in the increase in the TBARs level at this
time. The results of CAT activity indicate that this anti-
oxidant response of the periphyton community is being
damaged by glyphosate. Since CAT is a hemoprotein, its
lower level in the presence of Glifosato AtanorÒ could be
explained by the inhibition of the enzyme ALAS, the
secondary mechanism of action reported for glyphosate
(Duke 1988). For algae, the use of indicators of oxidative
stress can complement the information obtained by clas-
sical evaluation criteria such as mortality, and can be
successfully used to assess oxidative damage by herbicides
(Qian et al. 2009; Ross et al. 2006). Romero et al. (2011)
found increased levels of MDA and SOD activity in
Chlorella kessleri exposed to Glifosato AtanorÒ. These
previous studies were performed using monospecific algae
cultures. Bonnineau et al. (2011) and Guasch et al. (2010)
analyzed the impact on periphyton of some herbicides
different from glyphosate by means of oxidative stress
parameters. In this study, periphyton community exposed
to Glifosato AtanorÒ also showed alterations in these
parameters, which would indicate the induction of meta-
bolic alterations related to oxidative stress.
In conclusion, a single application of Glifosato AtanorÒ
caused a fast stimulation of bacterioplankton and planktonic
picocyanobacteria as well as of chlorophyll a concentration
in the water column, accelerating the eutrophication of the
system. In this scenario, intensive and repeated applications
of herbicides such as Glifosato AtanorÒ, quickly promote
profound changes in temporary water bodies, so widely
scattered in Argentina as well as in other countries around the
world.
Acknowledgments We thank our colleagues at the Laboratorio de
Ecologı́a y Fotobiologı́a Acuática (IIB-INTECH) and Laura Sánchez
and Griselda Chaparro for field assistance. We also thank Dr. Marı́a
dos Santos Afonso (Universidad de Buenos Aires) for laboratory
assistance and two anonymous reviewers for their helpful suggestions.
This work was supported by ANPCyT (PICT 01104 and PICT 0908)
and UBACyT 01/W550 and UBACyTX187. The authors declare that
they have no conflict of interest.
Conflict of Interest The authors declare that they have no conflict
of interest.
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