Effect of a glyphosate-based herbicide in Cyprinus carpio: Assessmentof acetylcholinesterase activity, hematological responses and serumbiochemical parameters

Seyed Jalil Gholami-Seyedkolaei a, Alireza Mirvaghefi a,n, Hamid Farahmand a,Ali Asghar Kosari b

a Department of Fisheries, Faculty of Natural Resources, University of Tehran, Karaj 31585-4314, Iranb Department of Plant Protection, Faculty of Agricultural Sciences and Engineering, University of Tehran, Karaj, Iran

a r t i c l e i n f o

Article history:Received 22 February 2013Received in revised form29 August 2013Accepted 2 September 2013

Keywords:Common carpRoundupAcute toxicityAminotransferase enzymeHematology

a b s t r a c t

The objective of this study was to investigate the toxicity effects of acute and sublethal of Roundups as aglyphosate-based herbicide on acetylcholinesterase (AChE) activity and several hematological andbiochemical parameters of Cyprinus carpio. The LC50-96 h of Roundups to C. carpio was found to be22.19 ppm. Common carp was subjected to Roundups at 0 (control), 3.5, 7 and 14 ppm for 16 days, andthe AChE activity is verified in tissues of gill, muscle, brain and liver. After 5 days, a significant decreasewas observed in the AChE activity of muscle, brain and liver tissues. Besides, a time- and dose-dependentincrease in mean cell hemoglobin (MCH) and mean cell volume (MCV) was observed. In contrast,a significant decrease was found in the quantities of hemoglobin (Hb), hematocrit (HCT) and, red (RBC)and white (WBC) blood cell count. Also, the activities of aspartate aminotransferase (AST), alanineaminotransferase (ALT) and lactate dehydrogenase (LDH) in Roundups treated groups were significantlyhigher than the controlled group at experimental periods. However, the level of alkaline phosphatase(ALP) had a significant reduction behavior during the sampling days. It seems that the changes inhematological and biochemical parameters as well as AChE activity could be used as efficient biomarkersin order to determine Roundups toxicity in aquatic environment.

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1. Introduction

Dramatic increase in growth of world population along withdevelopment and optimization of agricultural production, has ledto an increase in application of chemical components such asherbicides in agriculture and public health sectors (Ferenczy et al.,1997). Among different types of herbicides, Roundups (glyphosate48%) as a glyphosate-based formulation has been utilized as one ofthe most commonly applied herbicides around the world(Jiraungkoorskul et al., 2002). In fact, it is commonly utilized inagriculture as a non-selective herbicide to eradicate annual andperennial plants, grasses and also broad-leaved woody species(WHO, 1994). The formulation of Roundups is composed ofisopropylamine (IPA) salt of glyphosate 480 g/L, water and poly-ethoxylated amine surfactant (POEA) (Jiraungkoorskul et al.,2002), which is believed to be much more toxic to aquaticorganisms compared to the active ingredient itself (Giesy et al.,2000). This is related to the point that its water solubility and half-

life in soil are 15,700 mg/L and 30–90 days, respectively (Cox,1998). The half-life of glyphosate in aquatic environments isnormally in range of 7–14 days. It was also demonstrated thatthe value of LC50-96 h of Roundups depending on the differentfactors including fish species, test circumstances, life stage andherbicide formulation is between 2 and 55 mg/L (Giesy et al.,2000). Measurements of polluted water bodies and aquatic animalhealth biomarkers (e.g., hematological, biochemical and enzymo-logical parameters) have been broadly used as primary diagnosticapproaches (Al-Sabti and Metcalfe, 1995; Vanzella et al., 2007).Fish blood has been studied in toxicological research and environ-mental monitoring (Piner and Üner, 2012) as a possible biomarkerof physiological and pathological alterations in fishery manage-ment and disease investigations,

In this regards, diverse hematological factors including whiteblood cell count (WBC), red blood cell count (RBC), hemoglobin(Hb), and hematological indices like mean cell hemoglobin (MCH),mean cell volume (MCV) and mean cell hemoglobin concentration(MCHC), and also biochemical parameters such as plasma glucoseand protein are widely used to assess the toxic stress. Enzymeactivities as one of the other categories of sensitive indicators havebeen also employed to identify tissue damage of fish exposed todiverse group of water pollutants (Saravanan et al., 2011a,b).

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n Corresponding author. Fax: þ98 26 3224 5908.E-mail address:

avaghefi@ut.ac.ir (A. Mirvaghefi).

Please cite this article as: Gholami-Seyedkolaei, S.J., et al., Effect of a glyphosate-based herbicide in Cyprinus carpio: Assessment ofacetylcholinesterase.... Ecotoxicol. Environ. Saf. (2013), http://dx.doi.org/10.1016/j.ecoenv.2013.09.011i

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Among different types of enzymes, both alanine aminotransferase(ALT) and aspartate aminotransferase (AST) have been proved toplay an essential role in protein and carbohydrate metabolism.Thus, they appear to be useful as reliable biomarkers with whichthe tissue damage caused by the toxicants can be recognizedNemcsok and Benedeczky (1990). In addition, as a result of thechemical stress and anaerobic capability of tissue, another type ofenzyme called the lactate dehydrogenase (LDH) has been identi-fied to be used as an alternative bioindicator. Alkaline phosphatase(ALP) is also an enzyme found in certain body tissues such as liver,which is produced by the cells lining the small bile ducts (Speitand Hartmann, 1999).

It was reported that acetylcholinesterase (AChE) enzyme has akey role in the cholinergic transmission of the nervous systems(Ferenczy et al., 1997). Since the activity of this enzyme changeswith different contaminant concentrations, it could be an appeal-ing biomarker for measurement of the neurotoxicity changes(Ferenczy et al., 1997; Sturm et al., 1999; Miron et al., 2005).Many researchers previously proved that the AChE enzyme activ-ity can inhibit by several pesticides such as organophosphate,carbamate and glyphosate (Glusczak et al., 2007; Yogesh et al.,2009; Modesto and Martinez, 2010a,b; Cattaneo et al., 2011). Thisinhibition was negatively affected on the growth, survival, orienta-tion to food and reproductive behavior of fishes exposed todifferent pollutants (Dutta and Arends, 2003).

Previous investigations suggested that the regular tissuesaffected by inhibition activity of AChE are mostly brain and muscletissues (Cattaneo et al., 2011; Glusczak et al., 2007; Moraes et al.,2011), and only a few numbers of studies have been conducted ongill and liver tissues (Oruç and Usta, 2007; Piner and Üner, 2012;Xing et al., 2012).

Due to having similar biochemical responses to those found inmammals, teleost fish including common carp might be consequentlya good indicator of contamination caused by a wide range ofpollutants. The common carp (Cyprinus carpio L.) is a fish speciesthat is cultivated and consumed widely throughout the world.

Therefore, the objectives of this study were to determine the96 h-LC50 value of the common carp subjected to glyphosate stressand the effects of its sub-lethal concentrations on the AChEactivity of different tissues (brain, muscle, gill and liver), hemato-logical and biochemical parameters.

2. Materials and methods

2.1. Experimental fish specimen and chemicals

The common carp (C. carpio L.) with almost same sizes (Length 10.1272.01 cm,Weight 41.0370.15 g) were collected from a local fish farm (Cilver carp, Rasht, Iran)and used for the experiment. Fish specimens were subjected to a prophylactictreatment by bathing twice in 0.05% potassium permanganate (Merck Chemical Co.,Darmstadt, Germany) for 2 min to avoid any dermal infections. Fish was adapted tolaboratory conditions under natural photoperiod (12 h:12 h L/D) for at least 25 daysbefore exposure. The fishes were kept in continuously aerated water at lowdensities in three glass tanks (1000 L). The quality characteristics of used waterwere as follows: temperature 2071 1C, pH 7.0270.07, dissolved oxygen6.5070.12 mg/l, water hardness 16375 mg/L CaCO3 and daily water exchangerate 5%. Fish was fed twice a day with commercial carp pellets at the manufac-turer's recommended rate (2% of their body weight twice a day). Also, the fishhealth was evaluated based on behavioral changes during direct observations.

A commercial formulation of glyphosate (N-(phosphonomethyl) glycine),Roundups, (Monsanto company, St. Louis, Missouri USA) containing isopropylam-monium salt of glyphosate at 480 g/l as the active ingredient (equivalent to 360 gglyphosate per liter) and POEA as surfactant was used in this study. All the otherchemicals were purchased from Merck Chemical Co. (Darmstadt, Germany).

2.2. Measurement of sub-lethal concentrations and experiment design

A total of 180 immature C. carpio were used in acute toxicity bioassays todetermine the LC50-96 h value of Roundups. This test was performed according to

the OECD guideline test 203 under static-renewal test conditions (OECD, 1992).Nominal concentrations of active ingredient tested were 0 (control), 19, 22, 25, 28and 31 mg/l and each of the concentration was set in triplicate in 300 L fiberglasstanks. 10 fish specimens in each tank were randomly exposed to each of the sixRoundups target concentrations. The water was changed daily to reduce thebuildup of metabolic wastes. In the next step, the mortality of fish after Roundups

exposure for 24, 48, 72 and 96 h was recorded. LC50-96 h value was determined bythe Probit analysis test according to the applied method by Aydın and Koprucu(2005). Based on the obtained value for LC50-96 h, three test concentrations ofRoundups including 1 (�15% of LC50¼3.5 ppm), 2 (�30% of LC50¼7 ppm) and 3(�60% of LC50¼14 ppm) were determined in order to design the sublethal tests forthe in vivo experiment. Common carps were exposed for 16 days to glyphosatebased on the half-life of the herbicide (Cattaneo et al., 2011), 50% of the water wasrenewed and additional herbicide was transferred to the tanks by the 4th day afterthe beginning of experiment to sustain the expected concentration.

The analysis of glyphosate concentration in water was carried out using anHPLC system as previously explained by Shiogiria et al. (2012). A Knauer (Berlin,Germany) HPLC system has been used which included a K-1001 HPLC pump,a K-1001 solvent organizer, an on-line degasser, a dynamic mixing chamber and ascanning HPLC fluorescence detector (Model RF-10XL) with the excitation andemission wavelengths set at 266 nm and 316 nm, respectively. The separation wasperformed on an ACEs 5C 18 reverse phase (4.6 mm�250 mm�5 μm). Themobile phase was acetonitrile:dihydrogenphosphate buffer (3:97 v/v) at a flowrate of 1.0 mL/min. The chromatographic data was collected and recorded usingEuroChrom 2000 software from Knauer, which was controlled under Windows XPplatform.

After the onset of Roundups exposure, the rate of six fishes per treatment at 1,3, 5, 9 and 16 days were removed, and immediately anesthetized with benzocaine(0.1570.05 g/L). Blood samples were collected through tail vein puncture from thecontrol and treated groups. Whole blood was used for the estimation of hemato-logical parameters. The residual blood sample were centrifuged at 4500 rpm and at4 1C for 15 min and the plasma was removed and stored at �70 1C the biochemicalanalysis. In the next step, they were killed by medullar section and the requiredtissues (gill, muscle, brain and liver) for the analysis were removed by dissection.Then, these tissues were washed in ice-cold physiological saline solution (0.59%NaCl) and stored at �80 1C for the analysis.

2.3. AChE activity assay

The gill, muscle, brain and liver tissues were thawed and homogenized in ice(5–10 volume) in potassium phosphate buffer (0.1 M, pH 7.5), and then centrifugedat 12,000 rpm and at 4 1C for 15 min. The obtained supernatant was applied foranalysis of the AChE activity. This assay was carried out according to thecolorimetric technique of Ellman et al. (1961) developed by Alves Costa et al.(2007). The final concentration of the AChE iodide substrate was used, and theEllman's color reagent (5,5′-dithiobis-(2-nitrobenzoic acid) or DTNB) were 9 and0.5 mM, respectively. An ELISA microplate reader (Beckman Colter, Fullerton, CA,USA) was used to determine absorbance amount at 415 nm. The AChE activity wasexpressed as nmol DTNB/min/mg protein. Also, the protein concentration in brainsamples was determined by the method of Lowry et al. (1951). Bovine albumin wasused as standard.

2.4. Hematological analysis

The counts of RBC and WBC, blood Hb (g/dl) content, hematocrit (HCT)percentage and differential leukocytes were determined using a hemocytometeraccording to the method applied by Klont et al. (1994). The blood indices such asMCV, MCH and MCHC were calculated according to the method applied bySaravanan et al. (2011a,b).

2.5. Serum enzyme assay

Serum ALT, AST, ALP and LDH concentrations were determined using anautomated chemical analyzer (Auto analyzer (Ependrof)) and the commercialdiagnostic kits (Pars Azmoon Co., Tehran, Iran).

2.6. Statistical analysis

All analytical experiments were performed in triplicate, and the results werepresented as a mean of the three values with the standard deviation. All the datawere tested for normality (Kolmogorov-Smirnov test). The results were subjectedto two-way analysis of variance (ANOVA) and Tukey multiple-range tests usingSPSS 15.0 (SPSS Inc., USA) software. A value of po0.05 was considered statisticallysignificant.

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3. Results

3.1. Fish behavior and acute toxicity of glyphosate (roundups)

During the sub-lethal concentrations exposure, no fish mortalitywas observed in three experimental groups and the control.Common carps during Roundups exposure exhibited a franticbehavior with sluggish swim patterns, irregularly aquatic surfacerespiration and elevation of opercular beat rate.

The glyphosate amounts present in water of experimentaltanks are shown in Table 1. The LC50 value of Roundups to thefish for 96 h was 22.19 ppm. Also, the values of LC10 and LC90 were18.80 and 26.89, respectively (Table 2).

3.2. AChE activity assay

Fig. 1 represents the dose-response correlation between Round-ups treatment and AChE activity of the different tissues in C. carpio.Under control conditions, the mean value of enzyme activity of thedifferent tissues including gill, muscle, liver and brain wereobtained as 17.1, 119.8, 27.4 and 53.6 nmol/min/mg protein, respec-tively. The AChE activity of gill was irregularly changed during thesampling times as a significant decrease in activity of this enzymewas observed only on day 9 (Fig. 1a). A various behavior wasobserved for the other tissues as the AChE activity was significantlyreduced from the beginning of day 5 to the end (Fig. 1b–d).

3.3. Hematological indices

Table 3 shows the erythrocyte profile of the control group andthe fishes exposed to Roundups stress during experimentalperiod. In general, a considerable change in the red blood para-meters for the fishes exposed to Roundups herbicide wasobserved compared to the control group (po0.05). The experi-mental period and Roundups dose had a significant decreaseeffect on the amounts of Hb, HCT and RBC (Table 3). Note that the

herbicide dose had a stronger effect compared with the experi-mental time (po 0.05). However, the MCV and MCH levelsshowed a significant increase trend by the increasing experimentaltime and herbicide concentration (Table 3). Moreover, the increaseof experiment time led to a slight decrease in the MCHC level of C.carpio under different doses of Roundups (po0.05).

Table 4 shows the results of WBC and its differentials duringthe experimental period. A significant decrease in the WBCnumber by increasing Roundups dose during the experimentaltime was found. However, the experimental time had no signifi-cant effect on the WBC number (p40.05). In both control andexposure groups, the percent of lymphocytes and neutrophilsrespectively decreased and increased by increasing the herbicidedose. Also, the percentage of monocytes and eosinophils were notaffected by the experimental period and Roundups dose (Table 4).

3.4. Serum enzyme assay

Fig. 2(a–d) shows the results of serum ALT, AST, LDH and ALPlevels in three experimental groups under Roundups exposureand the control. Roundups stress led to a significant increase(po0.05) in the ALT and AST activities during the sampling days(Fig. 2a and b). As considered in Fig. 2, the highest level of ALT andAST enzymes was found at 14 ppm, while both Roundups con-centration of 3.5 and 7 ppm exhibited lower amounts of theseenzymes. The plasma LDH activity was significantly increased atall experimental times (Fig. 2c) (po0.05). In addition, the ALPlevels in plasma showed a significant decrease trend by theincreasing experimental time (Fig. 2d).

4. Discussion

In the present study, the value of LC50-96h for the fish exposedto Roundups herbicide was obtained 22.19 ppm. However,Neskovic et al. (1996) reported LC50-96 h value of 620 ppm for C.carpio exposed to glyphosate. The LC50-96 h value reported in thisinvestigation also was higher than that of Oreochromis niloticus(16.8 ppm), Gambusia yucatana (17.8 ppm), Leporinus macrocepha-lus (15.2 ppm), Prochilodus lineatus (13.7 ppm) exposed to thedifferent Roundups formulations (Jiraungkoorskul et al., 2002;Osten et al., 2005; Albinati et al., 2007; Langiano and Martinez,2008). Shiogiria et al. (2012) estimated LC50-48 h about 3.74 ppmfor Piaractus mesopotamicus subjected to Roundups Ready toxi-city. This fact can probably be due to the difference in overall levelof commercial formulations of glyphosate especially used surfac-tant compounds like POEA (Peixoto, 2005). Giesy et al. (2000)previously pointed out that the surfactant POEA is more toxic forfish than the pure glyphosate. Moreover, these discrepanciesmight be attributed to the fish species and water quality para-meters such as hardness, pH and salinity (Cattaneo et al., 2011).

The alteration in AChE activity as a biomarker can commonlyapply for pesticide contamination in fish (Dembele et al., 2000).Recently, some studies demonstrated that herbicides could inhibitthe AChE activity (Miron et al., 2005; Modesto and Martinez,

Table 1Concentrations (mg/L) of glyphosate (nominal and measured) in water of experi-mental tank during 8 day exposure to Roundups quantified by HPLCa.

Nominal concentrations (mg/L) Day Measured concentration (mg/L)

Glyphosate (3.5) 1 3.4970.052 3.4170.074 3.2670.118 3.2170.02

Glyphosate (7) 1 7.0370.042 6.7970.0014 6.3570.158 6.2170.002

Glyphosate (14) 1 1470.032 13.8670.114 13.4170.028 13.3770.06

a Values are expressed as mean7SD (n¼3).

Table 2Lethal concentrations of Roundups to common carp, C. carpio.

24 h 48 h 72 h 96 h

Lc10 20.9570.13 (20.42–21.48) 20.57570.12 (20.13–21.02) 20.0970.13 (19.55–20.63) 18.80570.14 (18.26–19.35)Lc50 25.1270.13 (24.69–25.56) 24.27570.18 (23.92–24.63) 23.3870.12 (22.93–23.83) 22.19570.12 (21.78–22.61)Lc90 30.9570.14 (29.85–32.05) 29.19570.12 (28.41–29.98) 27.970.16 (26.89–28.91) 26.89570.15 (25.98–27.81)

Acute Roundups toxicity was determined in Common carp, after 24, 48, 72 and 96 h of toxicity.LC10, LC50 and LC90 were estimated by the Probit Analysis test.Results are expressed as mean7SD with the maximum and minimum value (n¼10 in three replicates).

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2010a,b; Moraes et al., 2011). In the current work, a dose-responseassociation between Roundups treatment and AChE activity wasgenerally found in the different tissues in C. carpio. A similar trendfor the reduction of AChE activity was observed in brain of L.obtusidens (Glusczak et al., 2006) and Rhamdia quelen (Glusczaket al., 2007), and in brain and muscle of P. lineatus (Modesto andMartinez, 2010b) after 96 h of exposure to Roundups. For the firsttime, the authors reported AChE inhibition in gill and liver tissues

of C. carpio exposed to Roundups herbicide. Xing et al. (2010)reported that juvenile C. carpio showed the inhibition of AChEactivity in brain and muscle tissues after 40 day exposure tovarious doses of atrazine herbicide. A possible explanation for theinhibition of AChE activity by glyphosate is the acetylcholineaccumulation in the synaptic space because of the receptorsstimulation. This inhibition can influence the process of choliner-gic neurotransmission (Cattaneo et al., 2011). Another hypothesis

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Fig. 1. AChE activities of Gill (a), muscle (b), liver (c) and brain (d) tissues of C. carpio after exposure to the different Roundups concentrations (0, 3.5, 7 and 14 mg/L) for 1, 3,5, 9 and 16 days. Data are reported as meanþSD (n¼6). The lowercase letters denotes there are significant differences among diverse concentrations (po0.05).

Table 3Erythrocyte profile of C. carpio subjected to different concentrations of Roundups (0 (control), 3.5, 7 and14 ppm) during experimental time.

Day Roundups concentration (mg/L) Erythrocyte profilen

RBC (�106 mm3) Hct (%) Hb (g/100 ml) MCH (pg) MCV (&m3) MCHC (%)

1 0 1.8070.02a 42.1770.54a 11.2470.41a 62.4571.59a 234.5472.00a 26.6470.77a

3.5 1.2570.06b 38.0070.86b 8.1070.26b 64.9071.33a 305.25710.30c 21.3270.46b

3 7 1.1170.02c 32.6770.71c 6.8370.11c 64.9071.33a 295.5172.81bc 20.9370.40b

14 0.9970.01c 27.5070.34d 6.2870.16c 63.4171.44a 277.9071.75b 22.8170.40b

0 1.7170.03a 40.1770.70a 10.3070.31a 60.1871.28a 234.8171.79a 25.6270.43a

5 3.5 1.1870.02b 34.0070.86b 7.8170.14b 66.2770.56b 288.4074.44b 23.0270.51b

7 1.0570.03c 32.0070.37b 7.0770.15bc 66.2770.56b 305.1176.23b 22.0770.31b

14 0.9470.03d 27.6770.61c 6.4170.11c 68.7072.20b 296.0474.98b 23.1970.43b

0 1.7470.03a 40.3370.56a 10.3770.16a 59.8371.31a 232.5073.02a 25.7270.28a

9 3.5 1.1570.02b 36.1771.49b 8.0070.19b 69.8170.80b 315.2879.07b 22.2470.71b

7 1.0070.01c 33.5070.34b 7.4970.15b 69.8170.80bc 333.7873.60b 22.3770.51b

14 0.7570.01d 24.8370.48c 5.6370.09c 75.2670.84c 331.8673.59b 22.6970.34b

0 1.7470.03a 40.0070.37a 11.2170.25a 64.4570.99a 230.3073.90a 28.0370.70a

3.5 1.2570.02b 34.3370.21b 8.5170.14b 68.2371.52b 275.2274.16b 24.7970.46b

16 7 1.1670.02c 33.0070.68b 7.6070.11c 68.2371.52a 284.8179.64b 23.0970.70b

14 0.9970.01d 26.8370.40c 6.4870.14d 65.5971.36a 271.4972.75b 24.1570.35b

The lowercase letters show that there are significant differences among different concentrations (po0.05).n Values are means7SD (n¼6 in three replicates).

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for this fact is that AChE inhibitor-induced cholinergic hyperactiv-ity initiates the accumulation of free radicals leading to lipidperoxidation, which may be the initiator of AChE inhibition

(Yang and Dettbarn, 1996). Thus, inhibition of AChE in the muscleby Roundups might have led to an increase in its activity withreduction/depletion of intracellular ATP resulting in the generation

Table 4Changes in leukocyte profile of C. carpio exposed to different concentrations of Roundups (0 (control), 3.5, 7 and 14 ppm) during experimental time.

Day Roundups concentration (mg/L) Leukocyte profile*

WBC (�103 mm3) Neutrophil (%) Lymphocyte (%) Monocyte (%) Eosinophil (%)

1 0 22.0070.07a 22.3371.52a 74.3371.48a 2.3370.21a 1.0070.00a

3.5 21.1770.31ab 26.0070.97a 70.6770.92ab 2.0070.37a 1.3370.21a

7 20.5870.40b 33.1772.44b 63.8372.93b 1.8370.48a 1.1770.31a

14 18.8570.20c 42.3371.56c 54.0071.37c 2.6770.21a 1.0070.00a

3 0 21.7770.32a 21.0071.67a 76.0071.59a 2.3370.34a 0.6770.21a

3.5 20.3770.20ab 21.6770.42b 75.0070.37ab 2.3370.21a 1.0070.00a

7 19.9870.22b 28.6772.12c 69.3372.44b 1.3370.56a 0.6770.21a

14 19.1070.64b 39.3371.41c 58.0071.34c 1.6770.21a 1.0070.00a

5 0 21.6370.27a 23.3371.52a 73.6771.17a 1.6770.41a 1.3370.21a

3.5 19.7770.19b 31.3371.28b 66.6771.73b 2.0070.37a 1.0070.00a

7 19.2770.17c 41.1771.05c 56.3371.20c 2.3370.21a 0.6770.21a

14 18.1770.28c 46.3370.84d 51.0070.73d 2.0070.37a 0.6770.21a

9 0 21.6370.27a 23.3371.52a 73.6771.17a 1.6770.41a 1.3370.21a

3.5 19.7770.19b 31.1771.38b 66.3371.54b 2.0070.37a 1.0070.00a

7 19.3870.19b 41.3371.12c 56.0071.32c 2.3370.21a 0.6770.21a

14 18.1170.25c 46.6770.80d 50.6770.95d 2.0070.37a 0.6770.21a

16 0 21.6370.27a 19.8372.83a 77.1772.91a 1.6770.13a 1.3370.11a

3.5 19.9070.19b 31.5071.59b 66.5072.00b 2.0070.07a 1.0070.09a

7 19.2870.25b 41.6770.95c 55.6771.15c 2.3370.25a 0.6770.01a

14 18.3070.15c 45.8370.40c 51.5070.22c 2.0070.17a 0.6770.25a

The lowercase letters show that there are significant differences among different concentrations (po0.05).n Values are means7SD (n¼6 in three replicates).

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b

b

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b bb

b b

b b

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T in

pla

sma

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/L)

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Nc 3.5 ppm 7 ppm 14 ppm

a a a a ab

aa

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a a

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ab

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b

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LDH

in p

lasm

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U/L

)

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Nc 3.5 ppm 7ppm 14 ppm

a aa a aa

a

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a

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1 3 5 9 161 3 5 9 16

1 3 5 9 16 1 3 5 9 16

ALP

in p

lasm

a (I

U/L

)

Time (day)

Nc 3.5 ppm 7ppm 14 ppm

Fig. 2. Alterations in ALT (a), AST (b), LDH (c) and ALP (d) enzyme activities in serum of C. carpio exposed to the different concentrations of Roundups (0, 3.5, 7 and 14 mg/L)during 1, 3, 5, 9 and 16 days. Data are reported as meanþSD (n¼6). The small lowercase letters illustrates there are significant differences among diverse concentrations(po0.05).

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Please cite this article as: Gholami-Seyedkolaei, S.J., et al., Effect of a glyphosate-based herbicide in Cyprinus carpio: Assessment ofacetylcholinesterase.... Ecotoxicol. Environ. Saf. (2013), http://dx.doi.org/10.1016/j.ecoenv.2013.09.011i

of reactive oxygen free radicals. This fact may have played animportant role as mediators of skeletal muscle damage andinflammation (Yang et al., 1996).

Since blood is an ideal indicator for toxic stress, analysis ofhematological profiles in fish can commonly apply to monitor thetoxic stress and functional status of the animal health. Thus,different blood characteristics of fish have been utilized in orderto determine the responses of sublethal effects (Adhikari et al.,2004). Glusczak et al. (2006) found that glyphosate can signifi-cantly affect the hematological parameters of fish.

This study showed that exposure of C. carpio to sublethal levelsof Roundups can cause considerable time- and concentration-dependent decreases in the level of Hb, HCT and RBC during 16days in relation to the control group. Moreover, an increase in MCVand MCH values and a decrease in MCHC value were observed.Decrease of Hb and Hct content in C. carpio exposed to Roundups

can be attributed to the disorders in haemopoietic processes andaccelerated disintegration of RBC cell membranes (Svobodovaet al., 1997). Moreover, this decrease can be due to enhancementof erythrocytes lysing and also hemoglobinisation or shrinkage ofRBC induced by Roundups on the erythropoietic tissue (Saravananet al., 2011b). The erythropoiesis inhibition and the destruction ofred cells are two reasons for the reduction of RBC count in C. carpioexposed with Roundups (Saravanan et al., 2011a). Since theanemia could be because of the erythropoiesis inhibition, hemo-synthesis and the increase of erythrocyte destruction in hemo-poietic organs, it could be that the decrease in levels of HCT andHb, and also RBC count may be indicators for anemia diagnosis(Svobodova et al., 1997). These findings are generally in agreementwith those reported by Glusczak et al. (2006), who obtained areduction trend in HCT percentage, number of erythrocytes andHb of L. obtusidens exposed to the various concentrations ofRoundups. However, Modesto and Martinez (2010a) obtainedthat P. lineatus treated with the higher concentrations of RoundupTransorbs for 24 and 96 h revealed an enhancement in HCTpercentage and erythrocytes number due to the release of ery-throcytes from blood deposits and/or from hemopoetic tissues intothe blood stream (Svobodova et al., 1994). This variation betweenthe results obtained for P. lineatus and C. carpio may be producedby the differences in exposure periods, the size and the fish typeand also surfactant compounds applied in herbicide formulation.The increments in MCV and MCH along with the slightly decreasedMCHC indicated that the anemia was the macrocytic nesmochro-mic type. The increase in MCV and MCH values possibly resultfrom the increase of immature RBC (Saravanan et al., 2011a).

Since WBCs have a key role in the regulation of immunologicalfunction in different organisms, the changes in WBC count topollutants show the decrease in the non-specific immunity of thefish (Svobodova et al., 1994). Lower percent lymphocytes andhigher percent neutrophils are two normal responses of fishexposed to a variety of toxicants (Witeska, 2005). In this study,a significant decrease (po0.05) in WBC count and percent oflymphocytes of C. carpio was observed during exposure to sub-lethal concentration of Roundups. Similar results were obtainedby Kreutz et al. (2011) when R. quelen was subjected to glyphosate-contaminated water. However, Glusczak et al. (2006) found thatthe leukocytes count of L. obtusidens exposed to different concen-trations of Roundups insignificantly decreased compared with thecontrol group. The WBC decrease in the glyphosate-exposed R.quelen might account for a decrease in the number of cells in thecoelomic cavity (Kreutz et al., 2010). Another possible reason maybe due to extended toxic effect in kidney tissue, which is theprimary site of haematopoiesis provoking immunosuppression(Kotsanis et al., 2000). Moreover, it can also be probably becauseof the inhibition of WBC maturation and the release from tissuereservoirs by the Roundups action (Kavitha et al., 2010).

The changes in enzyme activities of aquatic animal are usuallyapplied to show the tissue damage due to disease conditionsand/or toxicant stress Nemcsok and Benedeczky (1990). In general,some enzymes such as ALT, AST, LDH and ALP have been used tomeasure pollution exposure in animals and also to scrutinize thewater pollution. Thus, it can be concluded that these enzymes areideal and sensitive biomarkers for the determining stress in thefish subjected to various pollutants present in water (Adhikariet al., 2004). ALT and AST enzymes were found in the differenttissues of liver, heart, skeletal muscle, kidney, pancreas, spleen,erythrocyte, brain and gills. They play a crucial role in protein andcarbohydrate metabolism and act as an indicator for tissue damageand cell rupture. These enzymes are released into plasma as thecells of aforementioned tissue are destroyed (Svobodova et al.,1994; Svobodova et al., 1997). In our study, the activities of ALT andAST were significantly increased for C. carpio exposed to thevarious concentrations of Roundups during the sampling days.Neskovic et al. (1996) and Jiraungkoorskul et al. (2002) previouslyreported an increase in ALT and AST levels for the fishes C. carpioand O. niloticus exposed to glyphosate-based herbicides. Thisincrease could be possibly attributed to the liver, gill and kidneydamage due to glyphosate accumulation which in turn releasesthese enzymes into blood stream. However, the damage level andseverity of the organ are in relation to the toxicant type andexposure duration (Jiraungkoorskul et al., 2002).

Level of LDH enzyme by varying chemical stress and anaerobiccapacity of tissue during the exposure period was generallychanged. Thus, LDH enzyme can be applied as a bio-indicator fordemonstrating tissue damage in fish (Saravanan et al., 2011a). Also,stress-induced alteration in ALP activity in tissues and serum hasbeen reported in fish (Das et al., 2004). In the present study, theactivity of plasma LDH was considerably increased for all theperiods of exposure. Data about the herbicide effect on LDHactivity in fish species are very scarce. However, Nunes et al.(2004) and Saravanan et al. (2011a) respectively observed anincrease in LDH activity for Gambusia holbrooki and of C. carpioafter acute exposure to stress of clofibric acid. It is proposed thatthe change of LDH activity can be probably due to protein andcarbohydrate metabolism, and structural damage of the cellmembranes or hepatic or heart tissues (Das et al., 2004;Szegletes et al., 1995). We obtained that the ALP activity in plasmahas a significant decrease trend during study time. Inhibition ofALP activity was also reported earlier in C. carpio exposed todiazinon insecticide (Banaee et al., 2008).

5. Conclusion

The results showed that high concentrations of Roundups hada considerable effect on the hematological, biochemical and serumenzymological parameters of common carps. Inhibition of AChEshowed that Roundups acts as a contaminant with anti-AChEaction. Roundups concentration had more effect than experimen-tal time on the changes of leukocyte and erythrocyte profile. Theactivities of ALT, AST, LDH and ALP enzymes in blood serum ofC. carpio were significantly altered by Roundups exposure. Thus,the alterations of studied parameters can be successfully appliedas a fast and valid technique to determine health of C. carpio in theaquatic environment contaminated by Roundups.

Acknowledgment

The authors would like to extend their appreciation for thefinancial support provided by the University of Tehran.

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Please cite this article as: Gholami-Seyedkolaei, S.J., et al., Effect of a glyphosate-based herbicide in Cyprinus carpio: Assessment ofacetylcholinesterase.... Ecotoxicol. Environ. Saf. (2013), http://dx.doi.org/10.1016/j.ecoenv.2013.09.011i

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