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: enrique@bg.fcen.uba.ar

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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