effects of glyphosate and polyoxyethylenamine on growth ...science.widener.edu/~vatnick/frontera...
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Effects of Glyphosate and Polyoxyethylenamine on Growthand Energetic Reserves in the Freshwater Crayfish Cheraxquadricarinatus (Decapoda, Parastacidae)
Jimena L. Frontera • Itzick Vatnick •
Anouk Chaulet • Enrique M. Rodrıguez
Received: 9 September 2010 / Accepted: 17 February 2011 / Published online: 19 March 2011
� Springer Science+Business Media, LLC 2011
Abstract Freshwater crayfish Cherax quadricarinatus
have a high commercial value and are cultured in farms
where they are potentially exposed to pesticides. Therefore,
we examined the sublethal effects of a 50-day exposure to
glyphosate acid and polyoxyethylenamine (POEA), both
alone and in a 3:1 mixture, on the growth and energetic
reserves in muscle, hepatopancreas and hemolymph of
growing juvenile crayfish. Exposure to two different gly-
phosate and POEA mixtures caused lower somatic growth
and decreased muscle protein levels. These effects, caused
by both compounds interacting in the mixture, could also
be synergistic because they were expressed even at the
lowest concentration. The decrease in protein levels could
be related to the greater use of other energy reserves. This
hypothesis is supported by the decrease in muscle glycogen
stores due to glyphosate exposure and the decrease in lipid
reserves associated with exposure to POEA.
Cherax quadricarinatus is a freshwater species of crayfish,
commonly known as ‘‘red claw crayfish.’’ that is native to
northern Australia. C. quadricarinatus has many advanta-
ges for farming due to several characteristics, such as rapid
growth, great potential to adapt to different climates, a
simple life cycle (direct development of eggs), and a body
weight that can reach 350 g (Jones 1997). At present, this
species is cultured intensively or semi-intensively in sev-
eral countries of Central and South America, such as
Mexico, Cuba, Ecuador (Palafox et al. 1999), and more
recently, Argentina. The grow-out of this species is usually
performed in external ponds dug in the ground. Because
most Cherax farms are in areas where several crops are
raised, the likelihood of contamination of these ponds with
herbicides, used to optimize agricultural production, is
particularly high. The biological response of aquatic
organisms to exposure to contaminants involves changes in
biochemical and cellular levels, which in turn cause
changes in the structure and function of cells and tissues
and, ultimately, changes in the physiology and behavior of
organisms (Mayer et al. 1992). Therefore, the potential
impact of exposure to pesticides is important in the
assessment of C. quadricarinatus as a commercial product.
Glyphosate-based herbicides are widely used in agri-
culture, in ornamental gardens, and for aquatic weed con-
trol in aquaculture (Giesy et al. 2000). In agriculture, their
use continues to spread to different species of plants that
are genetically modified to tolerate treatment with this
herbicide. Glyphosate, since its introduction in the late
1970s, has been regarded as one of the pesticides with the
highest rate of increase in their production and use (Giesy
et al. 2000; National Pesticide Use Database 2004). Gly-
phosate [N-(phosphonomethyl) glycine] is a nonselective
systemic herbicide that can efficiently inhibit the growth of
aquatic and terrestrial plants by acting as competitive
inhibitor of the enzyme 5-enolpyruvylshikimate-3-phos-
phate synthase, an enzyme involved in the synthesis
pathway of aromatic amino acids present in plants and
microorganisms but not in animals (Carlisle and Trevors
1988; Lydon and Duke 1989). One of the main commercial
formulations of this herbicide is Roundup (Monsanto,
Creve Coeur, Missouri), which, in addition to the active
J. L. Frontera � A. Chaulet � E. M. Rodrıguez (&)
Department of Biodiversity and Experimental Biology,
FCEyN–University of Buenos Aires, Buenos Aires, Argentina
e-mail: [email protected]
I. Vatnick
Department of Biology, Widener University, Chester,
PA 19809, USA
123
Arch Environ Contam Toxicol (2011) 61:590–598
DOI 10.1007/s00244-011-9661-3
ingredient (glyphosate), contains POEA (polyoxyethylene
amine) as a surfactant. POEA helps the emulsion of the
active ingredient, thereby increasing its absorption in plants
and other organisms (Giesy et al. 2000).
The half-life of glyphosate is 7–70 days, whereas that of
POEA is from 21 to 28 days depending on environmental
conditions (Giesy et al. 2000). Environmental water con-
centrations of glyphosate in the United States have reached
values B2.7 mg acid equivalents (ae)/l (Solomon and
Thompson 2003) and 2.8 mg ae/l (Giesy et al. 2000). Up to
7.6 mg ae/l have been reported in Australia (Mann and
Bidwell 1999). Because most of the components of gly-
phosate-based herbicides bind to sediment and degrade
quickly, it was generally assumed that contamination of
aquatic and terrestrial habitats with this herbicide represent
low risk for nontarget organisms (Relyea 2005). Moreover,
the acute toxicity of glyphosate is considered to be low by
the World Health Organization (WHO 1994). However,
recent studies indicate that continuous exposure to gly-
phosate could adversely affect both terrestrial and aquatic
habitats as well as nontarget organisms, such as amphibians
(Howe et al. 2004; Relyea 2004, 2005). Numerous studies
have investigated the effects of glyphosate formulations on
larvae of amphibians, and their results indicate that the
surfactants, moreso than the active ingredient, may be
responsible for the observed mortality (Bidwell and Gorrie
1995; Howe et al. 2004; Mann and Bidwell 1999; Relyea
2004, 2005; Relyea et al. 2005). Nonionic surfactants, such
as POEA, may exert their adverse effects through disrup-
tion of the respiratory surfaces of aquatic organisms
(Lindgren et al. 1996).
Although numerous studies have determined the acute
lethal toxicity of glyphosate and its commercial formula-
tions, little is known about the effects of exposure to sub-
lethal concentrations of these toxins. Exposure to sublethal
concentrations of glyphosate, corresponding to\2% of the
96-h LC50, caused ultrastructural damage in the liver of the
fish C. capio (Szarek et al. 2000). Alterations in liver,
kidney, and gills of Nile tilapia (Oreochromis niloticus)
after exposure to acute and chronic sublethal concentra-
tions of Roundup also have been demonstrated (Jiraung-
koorskul et al. 2002, 2003). Other previous studies have
reported that exposure of fish to the commercial herbicide
Roundup produced alterations on several enzymatic and
metabolic parameters (Glusczak et al. 2007).
The objective of this study was to examine the effects of
exposure to sublethal doses of an experimental formulation
of glyphosate on growth, energy use, and reserve levels in
juvenile freshwater crayfish C. quadricarinatus. We also
determined the relative contribution to the toxicity of the
active ingredient glyphosate and the POEA emulsifier
present in this formulation.
Materials and Methods
Water
All bioassays were conducted in semistatic conditions
according to standard procedures recommended by the
American Public Heath Association (1995). Water used
was prepared from tap water (hardness 80 mg/l as CaCO3
equivalents) purified through a filter system (Hidroquil)
composed of a series of three filters with replaceable car-
tridges to retain sediment, organic matter (by activated
charcoal), and cations (using a cationic resin). The water
was dechlorinated by holding it for at least 48 h in a
storage tank, and the pH was maintained at 8.0 ± 0.5 with
the addition of 0.1 N NaOH or HCl 0.1 N when necessary.
Dissolved oxygen was always [5 mg/l.
Animals
Advanced juvenile C. quadricarinatus (N = 50)were pur-
chased from a commercial hatchery (Pinzas Rojas S. R. L,
Tucuman, Argentina) and had an initial average body
weight of 4.72 ± 0.61 g. Once in the laboratory, the
crayfish were placed in glass aquaria containing 15 l of
water and were acclimated for 4 weeks at the same water
quality and other environmental conditions subsequently
used in the experiment. The animals were fed, ad libitum, a
balanced fish pellet (TetraColor) and Elodea sp. The ani-
mals were held in a room maintained at a light-to-dark
cycle of 14–10 h at 27�C ± 1�C.
To avoid cannibalism during the experiment, crayfish
were placed in wide-mouth plastic containers filled with
400 ml water to allow for ample gas exchange. The med-
ium was changed twice a week. The animals were fed,
ad libitum, every 2 days with balanced pellets (TetraPond
40% and TetraColor 60%, w/w).
Treatments
The experiment lasted 50 days. At this time, a small
sample of hemolymph from the base of the appendages of
each animal was extracted using a 29G needle and a
tuberculin syringe. Then, all animals were killed, and tissue
samples were stored at -70�C for subsequent analysis.
Animals were randomly assigned to five groups of ten
individuals each as follows:
Group C (control) = water without addition of
chemicals.
Group G = glyphosate at a concentration of 22.5 mg/l.
Group P = POEA (polyoxyethylene amine) at a con-
centration of 7.5 mg/l.
Arch Environ Contam Toxicol (2011) 61:590–598 591
123
Group LCM (low-concentration mixture) = a mixture
of 15 mg/l (3.75 mg/l POEA and 11.25 mg/l glyphosate).
Group HCM (high-concentration mixture) = a mixture
of 30 mg/l (7.5 mg/l POEA and 22.5 mg glyphosate).
All stock solutions of glyphosate (as acid) and POEA
(99.8% purity; Sigma, St. Louis, Missouri) were prepared
weekly by dissolving the appropriate amount of the
chemicals in distilled water. Sublethal concentrations
assayed were chosen that were \96-h LC50 values esti-
mated for juveniles of C. quadricarinatus ([400 mg/l for
glyphosate and 82.26 mg/l for POEA; Frontera 2010).
Because glyphosate could be unstable under certain
conditions (Giesy et al. 2000), a separate trial was con-
ducted by checking the nominal concentrations of gly-
phosate in a wide range of concentrations, always mixing
glyphosate with POEA in the same proportion used for
exposing juvenile crayfish. Duplicate test solutions were
aged for 24 h, and glyphosate concentration was deter-
mined by means of ionic chromatography: DIONEX
(Sunnyvale, CA) DX-100 chromatograph with a conduc-
tivity detector and a 25-ll sample loop using a DIONEX
AS-4 as analytical chromatographic column and a mixture
of NaOH/Na2CO3 4 mM/9 mM as eluent with a flow rate
of 2 ml/min.
Growth
Weight gain was determined using the weight on days 1
and 50 of the study according to the following algorithm
(Eq. 1):
Weight gain ¼ FW� IWð Þ=IW� 100; ð1Þ
where IW is the initial weight and FW is the final weight of
juveniles.
Molts
The containers were examined daily for molts, and the
exoskeleton was kept for 48 h in the container (because
molted animals often eat it), and then the uneaten remains
were discarded.
Oxygen Consumption
At days 45 and 46, oxygen consumption of each animal was
measured in a closed constant volume respirometer that was
completely filled with dechlorinated water and fitted with an
oxygen electrode (Lutron, Taipei, Taiwan, Mod: DO-5510)
connected to a computer by means of an analog-digital
interface. Inside the chamber, a magnetic stirrer was placed
inside a plastic cage, which was glued to the bottom to
prevent injury to the crayfish and to achieve continuous
water circulation. Once the animal was placed in the
chamber, oxygen levels (mg O2/l) were recorded every 5 s
for 15 min. The data obtained were stored for later analysis
using linear regression.
Energy Reserves
At the end of the experiment, levels of glycogen, protein,
and lipids were assessed in three tissues (muscle, hepato-
pancreas, and hemolymph). Glycogen was extracted by the
method of Van Handel (1965) and quantified as glucose
using a glucose oxidase and peroxidase assay (kit from
Wiener Laboratories, Buenos Aires, Argentina) after per-
forming acid hydrolysis with HCl and subsequent neu-
tralization with Na2CO3. Total lipids were extracted using
the method of Folch et al. (1957) and quantified by the
method of Fring and Dunn (1970) by measuring absor-
bance of the sulphatephosphovainillin complex at wave-
length of 530 nm using olive oil as standard. Protein
extraction was performed by the addition of 30% KOH to
each sample followed by a 2-h incubation at 100�C. Protein
concentration was assessed according to Lowry et al.
(1951) using bovine albumin as a standard and measuring
absorbance at 650 nm.
Data Analysis
To test significant differences between means, considering
the whole set of variables, multivariate analysis of variance
(MANOVA) was used. For univariate comparisons, one-
way ANOVA, followed by Tukey’s multiple-comparisons
test (Sokal and Rohlf 1981), was used. Data normality and
homogeneity of variances were always confirmed. A 5%
confidence level was considered in all cases.
Results
No mortality was observed in any of the experimental
groups during the experiment. Only one animal molted
during the experiment, so that the frequency of molting
was not a variable considered for analysis. Figure 1 shows
the measured glyphosate concentrations plotted against
nominal concentrations. A high correlation coefficient
(R2 = 0.9773) was found.
By means of MANOVA, a significant correlation among
dependent variables was verified (Wilks k44,63 = 0.011,
p \ 0.001). Animals exposed to either glyphosate or POEA
grew slower in terms of weight gain (1.497 ± 0.616 and
1.177 ± 0.709 g, respectively) compared with controls
(2.950 ± 0.660 and 0.058 ± 0.013 g, respectively). How-
ever, only in animals exposed to both concentrations of the
592 Arch Environ Contam Toxicol (2011) 61:590–598
123
glyphosate and POEA mixture was weight gain (LCM =
-0.609 ± 0.984 and HCM = -1.740 ± 2.026 g) signifi-
cantly different from that of the control group (Fig. 2).
The average rate of oxygen consumption of animals
exposed to glyphosate (0.030 ± 0.003 mg O2/s/g tissue),
the surfactant POEA (0.026 ± 0.002 O2/s/g tissue), and the
lower concentration of the mixture (0.029 ± 0.001 mg O2/
s/g tissue) was significantly lower (p \ 0.05) than that of
the control group (0.039 ± 0.004 mg O2 /s/g tissue;
Fig. 3). However, the HCM group did not differ signifi-
cantly (p [ 0.05) from the control or the rest of the
experimental groups.
The concentration of glycogen in abdominal muscle was
significantly decreased (p \ 0.001) in both the glyphosate
group (0.723 ± 0.047 mg/g tissue) and the two mixture
concentration groups (HCM = 0.777 ± 0.063 and
LCM = 0.989 ± 0.078 mg/g tissue). Only the POEA
group had muscle glycogen levels (1.504 ± 0.078 mg/g
tissue) similar to those of the control group
(1.497 ± 0.167 mg/g tissue; Fig. 4a). Significantly, the
glycogen concentration of crayfish exposed to both gly-
phosate and the highest mixture concentration was
approximately half that of the control group.
In muscle, total protein levels were significantly
decreased (p \ 0.05) by both glyphosate and POEA mix-
tures (HCM = 58.529 ± 1.97 and LCM = 58.482 ±
4.527 mg/g tissue) with respect to both the control group
(71.576 ± 3.940 mg/g tissue) and the group exposed
to glyphosate (72.966 ± 3.373 mg/g tissue; Fig. 4b).
Although protein levels of animals treated with POEA
(63.054 ± 2.999 mg/g tissue) were significantly (p \ 0.05)
lower than levels of the G group, they were not significantly
different (p [ 0.05) from the control. Figure 4c shows that
the group exposed to the highest concentration of the mixture
(HCM) had significantly (p \ 0.05) lower lipid levels
(0.059 ± 0,005 mg/g tissue) than the control group
(0.085 ± 0.006 mg/g tissue). In contrast, animals exposed
to the lower concentration of the mixture (0.112 ±
0.006 mg/g tissue) had increased lipid levels significantly
different from both the control (0.085 ± 0.006 mg/g tissue)
and the group treated with surfactant (0.079 ± 0.011 mg/ g
tissue).
The groups exposed to glyphosate and the HCM mix-
ture, both with the same concentration of glyphosate, had
Fig. 1 Nominal and measured concentrations of glyphosate (ae)
mixed with POEA in the same ratio used for exposing juvenile C.quadricarinatus and aged for 24 h. Ionic chromatography was used.
Replicates are indicated by black or white dots, and corresponding
mean values are referred on the Y-axis
Fig. 2 Average values ± SE of weight gain (a) and specific growth
rate (b) of juvenile C. quadricarinatus. C control G glyphosate
(22.5 mg ea/l), P POEA (7.5 mg/l), LCM glyphosate ? POEA
(15 mg /l), and HCM glyphosate ? POEA (30 mg/l). N = 10 in
each treatment. Different letters indicate statistically significant
differences (p \ 0.05)
Fig. 3 Average ± SE oxygen consumption rate (mg O2/s/g) in C.quadricarinatus juveniles. C control, G glyphosate (22.5 mg ea/l),
P POEA (7.5 mg/l), LCM glyphosate ? POEA (15 mg /l), and HCMglyphosate ? POEA (30 mg/l). N = 10 in each treatment. Differentletters indicate statistically significant differences (p \ 0.05)
Arch Environ Contam Toxicol (2011) 61:590–598 593
123
increased levels of hepatopancreatic glycogen (3.244 ±
0.462 and 2.937 ± 0.422 mg/g tissue, respectively), but
this was not significantly different (p [ 0.05) compared
with the control (2.303 ± 0.409 mg/g tissue; Fig. 5a).
However, group G showed significant differences (p \0.05) with respect to LCM (1.545 ± 0.356 mg/g tissue).
For total protein levels in the hepatopancreas, group
HCM (36.484 ± 2.807 mg/g tissue) was the only group that
exhibited a significant decrease (p \ 0.05) compared with
control (45.693 ± 3.311 mg/g tissue; Fig. 5b). However,
groups P (39.899 ± 2.994 mg/g tissue), LCM (41.811 ±
2.505 mg/g tissue), and HCM (36.484 ± 2.807 mg/g tis-
sue) showed significantly lower protein levels (p \ 0.05)
than the group treated with glyphosate (50.578 ±
3.158 mg/g tissue).
The group exposed to glyphosate showed decreased
levels of total lipids in hepatopancreas (0.595 ±
0.066 mg/g tissue), whereas the group exposed to POEA
Fig. 4 Average levels (mg/g ± SE) of glycogen (a), proteins (b), and
total lipids (c) in muscle of C. quadricarinatus juveniles. C control,
G glyphosate (22.5 mg ea/l), P POEA (7.5 mg/l), LCM glyphos-
ate ? POEA (15 mg /l), and HCM glyphosate ? POEA (30 mg/l).
N = 10 in each treatment. Different letters indicate statistically
significant differences (p \ 0.05)
Fig. 5 Average levels (mg/g ± SE) of glycogen (a), proteins (b), and
total lipids (c) in hepatopancreas of C. quadricarinatus juveniles.
C control, G glyphosate (22.5 mg ea/l), P POEA (7.5 mg/l), LCMglyphosate ? POEA (15 mg /l), and HCM glyphosate ? POEA
(30 mg/l). N = 10 in each treatment. Different letters indicate
statistically significant differences (p \ 0.05)
594 Arch Environ Contam Toxicol (2011) 61:590–598
123
(0.943 ± 0.074 mg/g tissue) had higher levels compared
with the control group (0.785 ± 0.059 mg/g tissue;
Fig. 5c); however, the differences were not statistically
significant (p [ 0.05). In contrast, hepaopancreas lipid
level of the glyphosate group (0.595 ± 0.066 mg/g tissue)
was significantly (p \ 0.05) lower than that of the POEA
group and even lower that the level of the HCM group
(0.830 ± 0.070 mg/g tissue; Fig. 5c).
The hemolymph glucose level was not affected signifi-
cantly (p [ 0.05) in animals exposed to the different
treatments (Fig. 6a). Total hemolymph protein levels were
also not significantly affected (p [ 0.05). Hemolymph
total lipid levels were significantly (p \ 0.05) decreased
with exposure to POEA (0.046 ± 0.004 mg/g tissue).
Although a decrease in the hemolymph lipid levels in the
two groups treated with the mixture of glyphosate and
POEA was seen (LCM = 0.072 ± 0.006 and HCM =
0.063 ± 0.008 mg/g tissue), the only significant difference
(p \ 0.05) was between group HCM and the control
(0.085 ± 0.009 mg/g tissue; Fig. 6c).
Discussion
Glyphosate concentrations tested in the current study were
clearly greater than the worst-case concentrations previ-
ously reported in nature (Solomon and Thompson 2003;
Giesy et al. 2000; Mann and Bidwell 1999). However
because C. quadricarinatus crayfish are commonly grown
in earthen ponds located in fields mainly dedicated to soy
farming, glyphosate is likely to be found at peak concen-
trations in these ponds due to air spray dispersion after
fumigation or by water run-off from the neighboring crops.
Unfortunately, no data are currently available in Argentina
about the actual associated glyphosate concentration.
In this study we found that weight gain was inhibited by
both concentrations of the glyphosate and POEA mixture
assayed. Moreover, because almost all animals did not molt
during the experiment, the inhibition of growth in terms of
body weight was exerted during the relatively long inter-
molt period. The increment of organic content and ener-
getic reserves in most crustacean tissues certainly occur
during the late postmolt and intermolt periods (Nelson
1991). The decreased growth seen in the current study
could be explained by the depletion of energy reserves in
one or more tissues as discussed in later text.
The oxygen consumption rate is commonly used to esti-
mate of the metabolic rate of organisms; in addition, the rate
of oxygen consumption is a good indicator of metabolic
damage to organisms exposed to pollutants (Mayer et al.
1992). In the current study, C. quadricarinatus exhibited
lower oxygen consumption (relative to control) with expo-
sure to glyphosate, POEA, and the lowest concentration of
the mixture. It has been proposed that the surfactant POEA
acts synergistically with glyphosate at the mitochondrial
level, thereby depressing the activity of several mitochon-
drial complexes and affecting oxidative phosphorylation
(Peixoto 2005). Therefore, the decrease in oxygen con-
sumption observed in this experiment may be related to
some form of metabolic arrest (enzyme inhibition) caused
Fig. 6 Average levels (mg/g ± SE) of glucose (a), proteins (b), and
total lipids (c) in hemolymph of C. quadricarinatus juveniles.
C control, G glyphosate (22.5 mg ea/l), P POEA (7.5 mg/l), LCMglyphosate ? POEA (15 mg /l), and HCM = glyphosate ? POEA
(30 mg/l). N = 10 in each treatment. Different letters indicate
statistically significant differences (p \ 0.05)
Arch Environ Contam Toxicol (2011) 61:590–598 595
123
by exposure to glyphosate and POEA. The prawn Palae-
monetes pugio also exhibited a decrease in oxygen con-
sumption with chronic exposure to the herbicide
pentachlorophenol, which inhibits the activity of Krebs
cycle enzymes (Ranga Rao et al. 1979). However, a relative
increase in the oxygen consumption of C. quadricarinaus
(with respect to the other treatments except the control) was
seen at the highest concentration of the mixture. This
response could be related to a more intense, unspecific stress
caused by this later treatment, counterbalancing the inhibi-
tion mentioned previously. Similarly, a study in the estua-
rine crab Chasmagnatus granulata reported an increase in
oxygen consumption with both acute and chronic exposure
to the insecticides parathion and 2,4-D (Rodrıguez and
Monserrat 1991) but only at high concentrations. During the
resistance phase of chronic stress, an increase in oxygen
consumption is usually seen in several crustaceans and fish
as an unspecific response (Mayer et al. 1992).
Crayfish exposed to glyphosate alone and both doses of
the mixtures of glyphosate and POEA mixtures had a sig-
nificant decrease in muscle glycogen levels. Glycogen is the
main source of energy used by crustaceans (Herreid and
Full 1988), and its levels can be affected by stress. Other
aquatic organisms, such as fish, have shown negative effects
on muscle glycogen reserves with exposure to glyphosate
based herbicides, suggesting that these organisms use
muscle glycogen as an energy source to compensate for this
stress (Glusczak et al. 2006, 2007). Similar results were
obtained in crustaceans exposed to various toxicants
(Coglianese and Neff 1982; Graney and Giesy 1986). Pos-
sible causes for the decrease in muscle glycogen levels
include interference with the mechanisms of carbohydrate
absorption in the intestine, inhibition of glucose uptake (and
possibly other monosaccharides) in muscle cells, and inhi-
bition of glycogen synthesis from monosaccharides.
It is worthy to note a correlation between the decrease in
glycogen reserves and the decrease of somatic growth
(growth rates) in crayfish exposed to both concentrations of
the glyphosate–POEA mixture. Although exposure to pure
glyphosate also produced a significant decrement of gly-
cogen in muscle, neither weight gain or specific growth
rate were statistically significantly decreased. Therefore,
the contribution of POEA to the lowering of growth rate is
important. In fact, in the POEA-exposed animals, the lipid
content in muscle decreased. The decrease in lipid levels
caused by the greater concentration of the glyphosate–
POEA mixture could be therefore attributed mainly to the
POEA. This effect of POEA was also seen in hemolymph,
where POEA exposure caused a significant decrease in
lipid levels. POEA could possibly decrease absorption of
glycerol and fatty acids in the intestine. Additionally, given
the lipophilic nature of the POEA surfactant (Giesy et al.
2000; Tsui and Chu 2003), it is possible that these
substances were solubilized by POEA and were excreted in
feces along with the fraction of POEA that was not
absorbed. POEA could also inhibit hemolymphatic trans-
port of glycerol and fatty acids (inhibiting transport pro-
teins) and/or inhibit the absorption of lipids in by muscle
and other tissues. Finally, in muscle cells, POEA could
inhibit lipid synthesis from glycerol and fatty acids. Several
of these possible effects have been previously reported for
several pollutants (Mayer et al. 1992).
Glycogen and lipids are the main sources of energy in
crustaceans; however, under stressful conditions, protein
can also be mobilized and serve as a source of energy by
way of the oxidation of amino acids (Graney and Giesy
1986), especially once the glycogen and lipid reserves have
decreased. In the present work, a significant decrease in the
muscle protein content was seen in both concentrations of
the glyphosate and POEA mixtures. This decrease could be
a result of the effect of glyphosate and POEA on glycogen
and lipid reserves, respectively. However, some type of
direct inhibition of the glyphosate–POEA mixture on pro-
tein synthesis can not be excluded, although there is no
previous evidence to support this possibility. A decrease in
the level of white-muscle protein has also been reported in
freshwater fish exposed to Roundup (Glusczak et al. 2007).
Moreover, the fact that decreased protein levels (lower
than the control) were observed even at the lowest con-
centration of the glyphosate–POEA mixture, but not with
glyphosate or POEA alone—and also taking into account
that the lower concentration of the mixture (15 mg/l) con-
tained only half the concentrations of glyphosate and POEA
tested in isolation (22.5 and 7.5 mg/l, respectively)—could
suggest a possible synergistic effect between glyphosate
and POEA. According to the 96-h LC50 values estimated by
Frontera (2010) in assays with juvenile C. quadricarinatus
exposed to either glyphosate or POEA, 15 mg/l of the
mixture (3.75 mg/l POEA and 11.25 mg/l glyphosate) is
equivalent to\0.074 toxic units (TU), defined as the actual
concentration/LC50. However, 7.5 mg/l POEA represents
0.091 TU, despite the fact that 15 mg/l of the mixture
produced a significant decrease in protein synthesis, which
was not observed with either POEA or glyphosate alone, at
twice the concentration presented in the mixture.
The hepatopancreas is one of the main organs for the
detoxification of xenobiotics in crustaceans (Vogt 2002). In
the hepatopancreas of animals exposed to glyphosate and
the highest concentration of the glyphosate–POEA mixture
(in which the concentration of glyphosate was the same as
in the first group), there was a slight tendency for crayfish
to have greater glycogen levels, but this was not statisti-
cally significant. Previous studies have reported increased
liver glycogen levels, simultaneously with decreased
muscle glycogen, of freshwater fish exposed to Roundup
(Glusczak et al. 2006, 2007). It is known that in vertebrates
596 Arch Environ Contam Toxicol (2011) 61:590–598
123
under chronic stress, glycogen stores are replenished in the
liver (analogous to the hepatopancreas of crustaceans),
whereas lipid reserves and protein are consumed in various
tissues (Mayer et al. 1992). This response to stress may
have evolved to deal with any episode of acute stress that
may occur during chronic stress. The literature on this
subject in invertebrates is scarce, and in this sense the
results of this study indicate an interesting parallel with the
observation mentioned previously in fish exposed to
Roundup, e.g., lipid levels increase in liver and decrease in
muscle. We observed a similar pattern, but only the gly-
cogen decrease in muscle was statistically significant.
Hemolymph functions as a temporary storage site for
glucose, proteins, and lipids from various tissues and cells.
It is a transit zone that reflects the balance between
resorption of reserves from storage in tissues (e.g., the
hepatopancreas) and the recruitment of these same reser-
vations by other tissues, such as muscle. There is also an
endocrine control for maintaining the hemolymphatic
homeostasis, which is represented, for example, by circu-
lating levels of the crustacean hyperglycemic hormone
(CHH). It has been noted, however, that various parame-
ters, such as molting stage, stress, activity, reproduction
stage, starvation, and acclimation temperature, can affect
glucose levels in hemolymph (Chang and O’Connor 1983).
However, the significant decrease of circulating lipid levels
in the group treated exposed to POEA alone may suggest
that intestinal lipid absorption in these animals is affected
of this toxicant as hypothesized previously.
In summary, crayfish exposed to both low and high
concentrations of glyphosate–POEA mixture (in a 3:1
proportion) had lower body-weight gain than either the
control or the crayfish exposed to either of the compounds
assayed alone. This effect was strongly correlated to a
decrease in muscle protein levels by the same treatments.
Additionally, glyphosate was able to produce a significant
decrement in muscle glycogen stores, whereas POEA
affected lipid reserves in the same tissue. The decrease of
both glycogen and lipid reserves, as observed in the mix-
ture, could have lead to lower protein levels and decreased
somatic growth in juvenile crayfish C. quadricarinatus.
Acknowledgments This study was supported by grants from AN-
PCyT (PICT 2006–01104 and PICT 2010) and UBACYT 2008-2010
program (EX241). It was also supported by Widener University
Faculty Development Grant (2009) to I. V.
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