Computational Water, Energy, and Environmental Engineering, 2013, 2, 20-25
doi:10.4236/cweee.2013.22B004 Published Online April 2013 (
Time-Effect Relationship of Toxicity Induced by
Roundup® and Its Main Constituents in Liver of
Carassius Auratus
Jinyu Fan, Jinju Geng, Hongqiang Ren, Xiaorong Wang
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, China
Received 2013
In order to evaluate the eco-toxicological effects of Roundup® on Carassius auratus (C. auratus), fish were exposed to
32 μg/L Roundup®, isopropylamine salt of glyphosate (G.I.S) and polyoxyethylene amine (POEA) over different peri-
ods (0.5, 1, 3, 7 and 14 d). Hydroxyl radical (·OH), malondialdehyde (MDA) and acetylcholinesterase (AChE) in liver
were detected in this study. Results showed that the generation of ·OH increased before 7 d, but without significantly
difference. ·OH was induced at 1 d for POEA group, 3 d for Roundup® group and 7 d for G.I.S group. At 14 d, ·OH
generation returned to normal levels. MDA contents all increased significantly (p 0.01) during 7 days and then
reached a normal level at 14 d. AChE activity in all group tests revealed a significant inhibition (p 0.01) after 7
days exposure and then rebounded a little, but remained below the control after 14 days exposure. The rate of AChE
inhibition range from 13% - 42% in Roundup®, 6% - 40% in G.I.S, and 21% - 54% in POEA, suggesting that POEA
was more toxic compared to Roundup® and G.I.S. 32 μg/L Roundup® exposure led to the change of physiological and
biochemical indexes in C. auratus, which was a reversible process in the long run.
Keywords: Carassius Auratus; Roundup®; Hydroxyl Radical (·OH); Toxicity
1. Introduction
Roundup®, the main glyphosate formulations, is already
mixtures of glyphosate and various adjuvants at different
concentrations [1]. The original formulation of Roundup®
contains isopropylamine salt of glyphosate (G.I.S) as the
active ingredient and polyoxyethylene amine (POEA) as
the surfactant agent [2]. Since Roundup® can easily
reach the aquatic systems by runoff, drainage, leaching
or inadvertent aerial overspray, the herbicide represents a
dangerous and widely spread group of environmental
contaminants [3]. However, the knowledge on the time–
effect of toxicity induced by environmental concentration
of Roundup® and its main constituents to fish is still
Researches suggest that reactive oxygen species (ROS),
can be induced in organisms exposed to some environ-
mental contaminants [4-6]. Recent study suggested that
Roundup® might induce oxidative stress in aquatic or-
ganisms through the increased levels of tissue lipid hy-
droperoxides [7]. Few direct evidences can prove ROS
generation and oxidative stress in aquatic organisms ex-
posed to Roundup® and its main constituents.
The measurement of acetylcholinesterase (AChE) ac-
tivity in different fish tissues has also proved to be a sen-
sitive method for detecting the presence of several herbi-
cides [8,9]. The inhibition of AChE causes an accumula-
tion of acetylcholine in the synapse, which therefore
AChE cannot function in a normal way [10]. Glusczak et
al. [11] reported that Rhamdia quelen showed significant
reduction in AChE activity after exposed to Roundup®.
In this study, Carassius auratus (C. auratus), com-
monly found in China, is chosen as the testing aquatic
organism. The aim of the study is to investigate the time-
effect of environmental concentration glyphosate herbi-
cide on oxidative stress and AChE activity of native
freshwater fishes, and to compare the toxicity difference
of Roundup® and its main constituents (G.I.S and POEA)
to C. auratus.
2. Experimental Methods
2.1. Chemicals
Roundup® solution (41% purity, containing 41% G.I.S
and 18% POEA), was obtained from Monsanto Company
(St. Louis, MO, USA). G.I.S (41% purity) was purchased
from Sigma Chemical (St. Louis, MO, USA). POEA was
purchased from Haian petrochemical complex (Jiangsu,
China). The other reagents used were analytically pure,
Copyright © 2013 SciRes. CWEEE
J. Y. FAN ET AL. 21
such as the spin-trapping agent, a-phenyl-N-tert-butylni-
trone (PBN), 2-thiobarbituric acid (TBA), and bovine
serum albumin (BSA), which were purchased from Sigma
2.2. Experimental Fish and Pollutants Treatment
Gold fish (C. auratus) with 9.9 ± 0.10 cm body length and
20.2 ± 0.75 g body weight were purchased from Fuzimiao
aquatic breeding base (aquaculture facility, Nanjing,
China). All fish were acclimatized to water dechlorinated
with activated carbon for two weeks before the experi-
ment. The total mortality of fish was below 3%.
In the Canadian Water Quality Guideline, the safety
exposure concentration of glyphosate was 65 μg/L con-
sidered protective of aquatic life [12]. So in this experi-
ment, we set half of 65 μg/L as the sole concentration.
After acclimatization, fish were randomly divided into
sixteen groups and kept in glass aquaria. One group was
designated for control and the other groups were em-
ployed as experimental groups that received concentra-
tions 32 μg/L of Roundup® (containing 41% G.I.S and
18% POEA), G.I.S (41% purity), and 18% POEA (equal
to the concentration in Roundup®), respectively, for 0.5,
1, 3, 7 and 14 d. During the experiment, 50% water was
replaced daily by adding fresh Roundup®, G.I.S and
POEA solution to minimize contamination from meta-
bolic waste. Artificial dry food was provided once a day.
Fish were sampled after exposure. Then the fish were
dissected to obtain some fresh livers for the determina-
tion of hydroxyl radical. The rest of the livers were ho-
mogenized at 4˚C for other experiments. During the ex-
periment, the water conditions were as follows: Keep the
dissolved oxygen levels at 5 mg/L by continuous aeration,
temperature at 20˚C ± 1˚C, pH 7.0 ± 0.3.
2.3. PBN Adduct Extraction and Electron
Paramagnetic Resonance (EPR) Analysis
ROS production in livers of C. auratus were measured
using PBN as the spin-trapping agent [13]. After being
rinsed with ice-cold physiological salt water, 0.1g of an
fish liver sample was removed and homogenized quickly
in 1.0 mL 50 mmol/L PBN (dissolved in dimethylsul-
foxide (DMSO)) using a Teflon pestle in a potter ho-
mogenizer. 0.1mL supernatants was transferred to a cap-
illary tube with a diameter of 0.9 mm, and placed in liq-
uid nitrogen for EPR meaurements. The whole operation
was carried out in an incubation system with continuous
N2 puring. The EPR spectra were recorded on a Bruker
EMX 10/12 X-band spectrometer (Bruker, Germany) at
room temperature (25˚C), with the operation conditions
of magnetic field center 3470 G, scan range 200 G,
modulation frequency 100 kHz; modulation amplitude
0.5 G, microwave frequency 9.751 GHz, incident mi-
crowave power 20 mW, and sweep time 84 s for 5 scans.
2.4. Degree of Lipid Peroxidation Determined
Using Malondialdehyde (MDA)
MDA content was measured by previous published thio-
barbituric acid assay of Miller and Aust [14] with some
modification. The reaction mixture containing 0.2 mL of
tissue homogenate, 0.2 mL 8.1% sodium dodecyl sulfate
(SDS), 1.5 mL 20% acetic acid buffer (pH 3.5), 1.5 mL
1% TBA, and 1 mL distilled water was heated at 90˚C
for 90 min, then cooled at room temperature and centri-
fuged for 15 min at 3,000 rpm/min. The absorbance of
the supernatant was determined at 532 nm using a UV-
220 spectrophotometer (Shimadzu, Japan). The amount
of MDA formed was calculated by measuring the ab-
sorbance at 532 nm using a molar extinction coefficient
of 1.56 × 105 M–1·cm–1.
The total protein content from enzyme extraction was
measured using BSA as a standard [15]. All the determi-
nation assays were performed in triplicate, at a minimum.
2.5. AChE Activity Assay
Fish liver samples were weighed and homogenized in
normal saline. The homogenate was centrifuged for 10
min at 4˚C at 3,500 rpm and the supernatant was used as
the enzyme source. AChE activity was determined with a
AChE Detection kit from Nanjing Jian Cheng Biology
Company (Nanjing, China).
2.6. Statistical Analysis
Statistical analyses were performed using SPSS statisti-
cal package version 16.0. Data were expressed as mean
values±standard deviation (SD). The differences between
experimental groups and the control were compared by a
one-way analysis of variance (ANOVA), and signifi-
cantly different treatments were identified by Dunnett’s
test. The level of statistical significance was set at sig-
nificantly different from control p
0.05 (*) and highly
significantly different from control p0.01 (**).
3. Results and Discussion
3.1. Free Radical Production by Induction of
Roundup®, G.I.S and POEA
Signals of PBN adducts of fish hepatic after Roundup®,
G.I.S and POEA exposure could be detected using EPR
(Figure 1). A six line spectrum of three groups with two
hyperfine coupling splitting peaks was observed. Ac-
cording to previous literature [4,5,13,16], the trapped
ROS was likely to be the hydroxyl radical (·OH) and the
levels could be expressed with the second couplet inten-
sity of the triplets in the EPR spectra.
Figure 2 showed the kinetics of ·OH generation over
Copyright © 2013 SciRes. CWEEE
Figure 1. EPR was used to detect PBN-radical adducts in
the liver of C.auratus with Roundup®, G.I.S and POEA.
Figure 2. ·OH signal intensity in fish liver after exposure to
32 μg/L Roundup® (blank bars), G.I.S (grey bars) and POEA
(black bars), for different experimental periods.
different periods (0.5, 1, 3, 7 and 14 d) of 32 μg/L
Roundup®, G.I.S and POEA exposure. ·OH generation
increased first and then decreased nearly with the control
group, but without significantly difference. ·OH was in-
duced at 1 d for POEA group, 3 d for Roundup® group
and 7 d for G.I.S group. ·OH accumulation was earlier
under POEA group conditions than under Roundup®
group and G.I.S group, indicating that the speed of oxi-
dant stress of POEA is faster than Roundup® and G.I.S in
C. auratus. The maximum accumulation of ·OH genera-
tion was 118% (of the control) in Roundup®, 132% (of
the control) in G.I.S and 135% (of the control) in POEA.
Considering the speed and the maximum of ·OH accu-
mulation, POEA is supposed to be more toxic than the
other two substances. Howe et al. [17] found that for
Rana clamitans, acute toxicity values in order of de-
creasing toxicity were POEA Roundup®.
It was reported that the ·OH can be significantly in-
duced by phenanthrene, pentachlorophenol, pyrene and
2-Chlorophenol in fish liver [18-21]. In the present study,
the ·OH generation induced by Roundup®, G.I.S and
POEA, but without obviously accumulated. It might be
explained that the activities of antioxidant enzymes were
activated and induced to remove ·OH to protect organ-
isms from oxidative stress. After 14 days, the ·OH signal
intensity returned to normal levels. This could be ex-
plained as follows: First, the ·OH had been reduced by
antioxidant defense systems, or metabolized to less harm-
ful radicals. Second, the fish had adjusted itself to com-
bat the oxidative stress. Third, glyphosate formulations
does not accumulation in vivo [22] and protect the fish
from many harmful effects.
3.2. Changes in MDA
One of the most damaging effects ROS and their prod-
ucts in cells is the peroxidation of membrane lipids,
which can be indicated by MDA detection [16]. MDA
contents in fish livers after exposure to Roundup®, G.I.S
and POEA were illustrated in Figure 3. Roundup®, G.I.S
and POEA group elevated (p 0.01) the MDA con-
tents during 7 days and returned to normal levels at 14 d.
The MDA contents increased at 0.5 d after Roundup®,
G.I.S and POEA exposure, and they kept increasing until
a maximum level were reached at 3 d in Roundup®
(148% of the control) and G.I.S (170% of the control), at
1 d in POEA (180% of the control). The speed of lipid
peroxidation may be further confirmed that POEA was
more toxic than the other two pollutants. Obviously,
Roundup® and its main constituents exposure resulted in
an accumulation of lipid peroxidation in C. auratus dur-
ing 7 days. But at 14 d, MDA contents were the same as
the control group, indicating that exposure of low con-
centration of Roundup® to fish is a reversible process.
This maybe explained by an adaptive response takes
place in the cells or might be due to the activities of
various damage removal and repair enzymes, to mini-
mize the concentration of ·OH to the basal level and
blocked lipid peroxidation in the cell.
Detailed studies have provided evidence that some
xenobiotics induced MDA contents following stress on
many species [5,21,24]. In the present study, ·OH gen-
eration induced without significantly difference, which
suggested the self-adjusting of fish by activating anti-
oxidant capacity to combat the cellular excess ROS gen-
eration. However, the increased MDA contents during 7
days proved that the lipid peroxidation in fish liver was
promoted and further revealed that the fish was already
Figure 3. MDA content in fish liver after exposure to 32 μg/L
Roundup® (blank bars), G.I.S (grey bars) and POEA (black
bars), for different experimental periods.
Copyright © 2013 SciRes. CWEEE
J. Y. FAN ET AL. 23
in the status of oxidative stress although the results did
not showed the accumulate of ·OH. According to Luo et
al. [20], the higher MDA level by the end of the exposure
to 2-chlorophenol in C. auratus suggested the oxidative
damage occurred although hydroxyl radical returning to
the normal level. In the study, the MDA content did not
have a time-response pattern consistent with ·OH gen-
eration at all exposure time in Roundup®, G.I.S and
POEA, which might be due to the effectiveness of the
antioxidant system in providing protection. Sun et al. [21]
found the similar change pattern of MDA in liver after
different doses of pyrene were exposed to the fish (C.
3.3. Changes in AChE Activity
AChE activity in the liver of C. auratus exposed to
Roundup®, G.I.S and POEA, for different experimental
periods was displayed in Figure 4. All tests revealed a
significant inhibition of AChE activity during 7 days
exposure, then restored to the level of control group after
14 d. The inhibition percentages in Roundup®, G.I.S and
POEA of the control fish was 13% - 42%, 6% - 40% and
21% - 54%, respectively. The degree of AChE reduction
showed the toxicity order of the chemicals was: POEA
Roundup® G.I.S. On the basis of Giesy et al. [24], the
LC50 values (mg/L) for rainbow trout were between 8.2
and 27 for Roundup®, between 0.65 and 7.4 for POEA.
Organophosphorus pesticides have several toxic prop-
erties, the most prominent effect of which is AChE inhi-
bition. AChE activity is therefore widely used in bio-
monitoring studies as a biomarker of organopho- sphorus
pesticide exposure [25]. In this study, the reduction of
AChE activity is assumed to have been resulted from the
direct action of Roundup®, G.I.S and POEA exposure on
active site of this enzyme. Glusczak et al. [26] reported
the inhibition of this enzyme in the brain of Leporinus
obtusidens exposed to 3, 6, 10 and 20 mg/L glyphosate
for 96 h. Modesto and Martinez [27] also reported a de-
creasing AChE activity in the brain of Prochilodus
Figure 4. AChE activity in fish liver after exposure to 32 μg/L
Roundup® (blank bars), G.I.S (grey bars) and POEA
(black bars), for different experimental periods.
lineatus after exposure to 1 and 5 mg/L Roundup® for 96 h.
The exposure of the three pollutants led to the maximum
inhibition of AChE in POEA by 54%. The rate of AChE
inhibition may not be considered a life-threatening situa-
tion since fish are capable of tolerating over 90% AChE
inhibition [28]. After 14 days exposed to Roundup®, G.I.S
and POEA, AChE activity rebounded a little, which were
consistant with the alteration of ·OH generation and
MDA contents, indicating that long-term exposure of
Roundup®, G.I.S and POEA with low concentration in C.
auratus is a reversible process.
4. Conclusions
The present study showed that Roundup®, G.I.S and
POEA may cause changes in the metabolic and enzy-
matic parameters of fish during 7 days, such as ·OH gen-
eration addition, lipid peroxidation and AChE inhibition,
implying that the fish was already in the status of oxida-
tive stress. At 14 d, ·OH generation, MDA contents and
AChE activity all returned to the normal level, indicating
that the exposure of Roundup®, G.I.S and POEA with 32
μg/L in C. auratus is a reversible process in a long time.
According to the toxic effects of Roundup®, G.I.S and
POEA on C. auratus, POEA may be the most toxic pol-
lutant. Roundup®, G.I.S and POEA should be distin-
guished when assessing the toxicity of this pesticide.
5. Acknowledgements
The research was funded through the National Science
Foundation of China (No. 21077051, 51278241) and the
Jiangsu Natural Science Foundation (No. BK2011057).
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