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Bulletin of EnvironmentalContamination and Toxicology ISSN 0007-4861Volume 92Number 5 Bull Environ Contam Toxicol (2014)92:574-578DOI 10.1007/s00128-014-1225-6

Effects of Permethrin on Biomarkersin Mediterranean Clams (Ruditapesdecussatus)

B. Sellami, A. Khazri, H. Louati,F. Boufahja, M. Dellali, D. Sheehan,P. Aissa, M. Ridha Driss, E. Mahmoudi& H. Beyrem

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Effects of Permethrin on Biomarkers in Mediterranean Clams(Ruditapes decussatus)

B. Sellami • A. Khazri • H. Louati • F. Boufahja •

M. Dellali • D. Sheehan • P. Aissa • M. Ridha Driss •

E. Mahmoudi • H. Beyrem

Received: 30 September 2013 / Accepted: 4 February 2014 / Published online: 12 February 2014

� Springer Science+Business Media New York 2014

Abstract The present study was focused on the assess-

ment of Catalase (CAT) and Acetylcholinesterase (AChE)

activities in Mediterranean clams (Ruditapes decussatus)

exposed to 50, 100 and 150 lg/L of Permethrin for 5, 10,

15, 20 and 25 days. In water, the measured concentrations

of Permethrin in the treated aquariums were respectively

16.66, 38.24 and 55.61 lg/L. Results showed that CAT

activity was increased after 5 days of exposure to high

concentration reaching maximum value of 10.14 lmol/

min/mg proteins after 25 days. However, no significant

changes in AChE activity after 5 days of exposure were

detected in all treated groups. AChE activity was signifi-

cantly inhibited after 10 days with 100 and 150 lg/L and

still depending on concentration and time. Maximum

inhibition of AChE activity was reached after 25 days with

the highest concentration of Permethrin. Our data indicated

that exposure to Permethrin modifies biomarker profiles

inducing oxidative stress and reducing AChE activity in

Mediterranean clams.

Keywords Permethrin � Oxidative stress � Neurotoxicity �Mediterranean clam � Ruditapes decussatus

Pyrethroids are natural insecticides causing serious envi-

ronmental problems mainly linked to their persistence and

high toxicity. These products are extremely hydrophobic

and highly immobile which increase their absorption by

sediment (Laskowski 2002). Permethrin is a synthetic

insecticide, third generation, type I, and is considered the

most used pyrethroid. Permethrin is a non polar chemical

(water solubility approximately 0.2 mg/L, log Kow value

2.88) with high affinity for soil and sediments and is

moderately persistent, with degradation occurring primar-

ily via microbial activity and photolysis (Cox 1998). Per-

methrin degrades up to 41 % within 3 days (Lee

et al. 2002). Adsorption is responsible for bioaccumulation

of Permethrin inducing many toxic effects on a wide range

of aquatic organisms (Allan et al. 2005), particularly

molluscs. Ruditapes decussatus had been widely consid-

ered for use in biomonitoring of aquatic environment

(Dellali et al. 2004). This benthic species is very abundant

and well commercialized around the Mediterranean Sea

(Mohamed et al. 2003). Bivalves are bioindicators for

extrapolating to many aquatic species that have similar

biochemical pathways. Catalase (CAT) and Acetylcholin-

esterase (AChE) are widely used in mussels and clams as

biomarkers in biomonitoring programs of aquatic envi-

ronments (Bocquene and Galgani 1991). CAT has been

considered as a non specific biomarker involving response

to metals and insecticides. The enzyme is sensitive to the

contaminants inducing cellular oxidative stress such as

B. Sellami (&) � A. Khazri � H. Louati � F. Boufahja �M. Dellali � P. Aissa � E. Mahmoudi � H. Beyrem

Laboratory of Environment Biomonitoring, Coastal Ecology

Unit, Faculty of Sciences of Bizerta, University of Carthage,

Zarzouna, 7021 Bizerta, Tunisia

e-mail: sellamibadreddine@gmail.com

H. Beyrem

e-mail: hamouda.beyrem@gmail.com

D. Sheehan

Environmental Research Institute and Department of

Biochemistry, University College Cork, Western Gateway

Building, Western Rd, Cork, Ireland

e-mail: d.sheehan@ucc.ie

M. R. Driss

Laboratoire de Chimie Analytique et Environnement, 05/UR/12-

03, Faculte des Sciences de Bizerte, Zarzouna, 7021 Bizerte,

Tunisia

e-mail: riddriss@yahoo.com

123

Bull Environ Contam Toxicol (2014) 92:574–578

DOI 10.1007/s00128-014-1225-6

Author's personal copy

pesticides, metals and environmental variables (Khessiba

et al. 2005). AChE is not implicated in the detoxification

phenomenon, but it stimulates the ionic flow of the mem-

brane and plays an essential role in the neural process. The

aim of the present study was to investigate the uptake of

Permethrin and its effects on CAT and AChE activities

following five time intervals in the clam R. decussatus

sampled from Bizerte lagoon (northern Tunisia).

Materials and Methods

Clams R. decussatus, with weight of 1.5 ± 0.8 g and length

of 3–3.5 cm, were acquired from the site Menzel Jemil

(37�13 16.05N, 9�56 04.58E) (Tunisia). Clams were accli-

mated to laboratory conditions for 12 days prior to the

experiment. At the beginning of experiment, ten animals per

replicate (three replicates per treatment) were assigned to

5 L aquaria and maintained at 19�C ± 2 in an acclimated

room under a photoperiod 12/12. The natural seawater

(collected in Menzel Jemil) from all treatment groups was

continuously aerated and renewed three times a week. Clams

were divided into four groups including one control. Treated

groups were exposed to different concentrations of Per-

methrin (50, 100 and 150 lg/L) during 25 days these three

concentrations were selected based on earlier studies (Lee

et al. 2002; Bringolf et al. 2007; Mahmoud et al. 2012). The

same experimental conditions were considered to assess the

effective concentration of Permethrin after 2 days in the

water and clams using GC–ECD. Exposed and control ani-

mals were removed at 5, 10, 15, 20 and 25 days for bio-

chemical analyses. Chemical analysis of Permethrin in the

water was analyzed following the methods described by

Phyu et al. (2011). At the start of each test, three 50 mL

samples of test solution were taken from replicates of

experimental treatments. The 50 mL sample was combined

with 50 mL of dichloromethane (methylene chloride) in a

200 mL separatory funnel and shaken for 2 min with peri-

odic venting. This process was repeated three times for each

sample and all three extracts combined. The combined

extract was evaporated to near dryness under nitrogen gas

using a Turbo Vap Evaporator and then made up to 1 mL

with hexane prior to gas chromatography analysis.

In clams Permethrin was analyzed following the method

described by Marei et al. (1982). Briefly, one gram of clams

was mixed with 0.5 g of anhydrous sodium sulphate and

extracted for 2 min with 15 ml of hexane using a Polytron

homogenizer. After filtration through a Buchner funnel

using 0.5 g of anhydrous sodium sulfate, the residue was re-

extracted with 15 ml of hexane for 1 min with the

homogenizer prior to filtration. The combined filtrates were

concentrated to 5 ml by using a rotary evaporator. This

hexane extract was transferred to a 25 ml separatory funnel

followed by 10 ml of acetonitrile. Vigorous shaking for

2 min and then 3–5 min of standing gave the hexane phase

which was discarded and the acetonitrile phase which was

concentrated to 15 ml. The extract was purified in a chro-

matographic mini-column 40 cm 9 0.5 cm inner diameter.

The column was packed with 2 g of activated florisil and

topped with 1 g of anhydrous sodium sulfate. The extract

was eluted with 30 ml of dichloromethane and n-hexane

(1:9; v/v). The eluate was evaporated in a Kuderna-Danish

apparatus to 0.5 ml for gas chromatography analysis.

GC analysis of Permethrin was performed on an Agilent

model 6,890 gas chromatograph equipped with a 63 Ni

electron capture detector (GC–ECD). In total, 2 ll of

extract was injected in split-less mode into a PTE-5

30 m 9 0.32 mm inner diameter, 0.32 mm film thickness

capillary column, using nitrogen carrier gas with a 1 ml/

min flow rate and the following oven temperature program:

50�C initial (2 min) to 160�C at 20�C/min and to 260�C at

5�C/min and held for 10 min. The temperature of the

detector and injector were 250 and 220�C, respectively.

The PTE-5 column was used as the primary analytical

column. Quantitative and qualitative analyses were per-

formed by comparison with five external standards. The

minimum determination limit expressed on fat basis for

Permethrin was estimated to 1 ng/g. To determine the

quality of the method, the recovery study was performed on

three replicates of blank clam samples, which revealed

contamination levels below the detection limit. The percent

recovery for each analyte was corrected for background

concentration measured in unfortified sample. The recov-

ery study, done at 50, 100 and 150 lg/L levels, showed

mean values ranged from 78 % to 83 % of recovery. The

relative standard deviations were below 10 %, indicating

acceptable repeatability of the method.

For the Biochemical analysis clams were dissected and

soft body of ten specimens from each aquarium were

homogenized in a Tris buffer (Tris 50 mM, NaCl 150 mM,

pH 7.4). The homogenates were centrifuged at 9,000g for

30 min. The supernatants were frozen at -70�C until bio-

chemical analysis. Total proteins were determined following

the method of Bradford (1976) using cattle serum albumin as

a standard. CAT activity was determined by the method of

Claiborne (1985). AChE activity was determined using the

method of Ellman et al. (1961). Measurements of AChE

activity were initiated by the addition of acetylthiocholine

and then the absorbance was read at 412 nm. Results were

expressed in lmol/min/mg of hydrolyzed substrate relative

to the total protein content. This method was adapted to a

microplate reader by Bocquene and Galgani (1991). The

results were expressed as mean ± SE. Analysis of variance

(ANOVA) was performed using the software STATISTI-

CA� 8.0. After testing ANOVA assumptions, statistical

significance was evaluated through two-way ANOVA.

Bull Environ Contam Toxicol (2014) 92:574–578 575

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Whenever ANOVA detected significant differences, post

hoc comparisons were made using the Tukey HSD test. A

probability level of less than 0.05 was considered significant

(95 % confidence interval).

Results and Discussion

Permethrin levels were ten times higher in clams exposed

to 100 lg/L (7.12 ± 2.09 lg/g) than in the control

(0.87 ± 0.03 lg/g). In water, the measured concentrations

in the treated aquariums were 16.66, 38.24, and 55.61 lg/L

respectively corresponding to nominal concentrations 50,

100 and 150 lg/L. Nominal values decreased after 48 h

respectively to 33.32 %, 38.24 % and 37.07 %. Therefore,

effect concentrations have been calculated on the basis of

measured values. CAT activity measured in control clams

remained unchanged during the entire experiment (Fig. 1)

and no significant difference (p [ 0.05) was detected.

Clams treated 16.66, 38.24 and 55.61 lg/L revealed that

Permethrin induced concentration and time dependent

response (Fig. 1). CAT activity in clams treated with 16.66

and 38.24 lg/L was similar to the control group after the

first period of exposure (5 days). However, with 55.61 lg/

L the activity of this biomarker was two-fold higher than

control group (p \ 0.001). After 10, 15, 20 and 25 days of

exposure, CAT activity increased uniformly with signifi-

cant differences (p \ 0.05) detected in all treated groups.

After 25 days of exposure, CAT activity was increased to

7.06 with 16.66 lg/L, to7.21 lmol/min/mg of proteins

with 38.24 lg/L and still maximum with 55.61 lg/L

reaching 10.14 lmol/min/mg of proteins. Factorial two-

way ANOVA showed significant differences in CAT

activity between concentrations (F = 572.53, p \ 0.0001)

and within days of exposure (F = 372.41, p \ 0.0001).

Similarly, the interaction of the concentration and time of

exposure showed a statistically significant difference

(F = 41.53, p \ 0.0001) which suggest that the response

of clams to different concentrations of Permethrin is

depending on the time of exposure. Acethylcholinesterase

activity in clams was determined after 5, 10, 15, 20 and

25 days in order to assess neurotoxicity effect of Per-

methrin (Fig. 2). During 25 days: of the exposure no sig-

nificant (p [ 0.05) variation of the AChE activity was

observed between control clams and AChE activity varied

between 3.01 ± 0.40 and 3.07 ± 0.82 lmol min/mg of

proteins. After 5 days of exposure, treated groups did not

show any modification in AChE activity. However, expo-

sure to 16.66 lg/L decreased AChE activity to 8 % after

after 10 days and to 61 % after 25 days. After 10 days of

exposure to 38.24 lg/L, inhibition was decreased from

3.05 to 1.67 lmol/min/mg of proteins and reached maxi-

mum of inhibition after 25 days of exposure with 55.24 lg/

L (p \ 0.001).This study investigated firstly the uptake of

Permethrin in R. decussatus after a short term exposure.

Our results confirmed the high uptake and accessibility of

Permethrin into clams. These results are similar with other

studies reporting the bioaccumulation of Permethrin in

marine invertebrates (Werner and Hilgert 1992).

Additionally, the analytical check of dissolved Per-

methrin concentrations in treated aquaria showed that the

actual concentrations in the water were lower than the

nominal levels. Nominal concentrations of Permethrin

were decreased to 33.32 %, 38.24 % and 37.07 %. These

values are approximately similar to those found by

Lee et al. (2002). Our study confirmed concentration and

time-dependent responses of CAT activity after exposure

to Permethrin. After 5 days of exposure to 16.66 and

Fig. 1 Evolution of CAT activity of clams R. decussatus exposed to

different concentrations of Permethrin for 5, 10, 15, 20 and 25 days.

Values are given as mean ± SD (n = 10). Asterisk significantly

different from control (p \ 0.05)

Fig. 2 Evolution of AChE activity of clams R. decussatus exposed to

different concentrations of Permethrin for 5, 10, 15, 20 and 25 days.

Values are given as mean ± SD (n = 10). Asterisk significantly

different from control (p \ 0.05)

576 Bull Environ Contam Toxicol (2014) 92:574–578

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38.24 lg/L CAT activity was not induced by Permethrin

indicating that those concentrations cannot generate ROS

after short-term exposure. These results are similar to those

of Schreck et al. (2008) who reported that a low concen-

tration of mixture 11 lg/kg cythoalothrine (pyrethroid) and

168 lg/kg chlorpyrifos (organophosphorous) did not

induce CAT activity after 3 days of exposure in the worms

(Aporrectodea caliginosa nocturna). These authors sug-

gested that the metabolism of these products does not

generate ROS. Similarly, Barata et al. (2005) observed that

endosulfan did not induce this enzyme activity in Daphnia

magna at low concentration (200 lg/L). In the present

study, only high concentration of Permethrin (55.61 lg/L)

induced significant CAT activity in R. decussatus after

5 days. This may due to the incapacity of the non-enzy-

matic antioxidant mechanisms to neutralize the ROS gen-

erated by high Permethrin concentration.

Indeed, if non-enzymatic antioxidant mechanisms are

compromised, or if the levels of ROS exceed them, specific

enzymes such as CAT are induced. After 10 days of Per-

methrin exposure, R. decussatus CAT activity increased

significantly in all treated groups. During the rest of the

exposure period (10–25 days), CAT activity was linearly

induced following an increased rate of generated ROS.

These results are in agreement with those observed by

Jebali et al. (2007) who reported that the increase in R.

decussatus CAT activity was related to the ROS level and

the presence of the pollutants in the aquatic environment.

AChE hydrolyzes the neurotransmitter, acetylcholine, in

the cholinergic synapse of the central and peripheral ner-

vous system (Sturm et al. 2007). It constitutes a useful tool

for the pesticides study (Jemec et al. 2007; Reinecke and

Reinecke 2007). AChE inhibition in R. decussatus can be

seen as an early warning biomarker from contaminants

risks (Dellali et al. 2004). In the present study, no signifi-

cant modification of AChE activity was detected after

5 days in all treated groups. These results are in agreement

with those observed by Romeo et al. (2006) who reported

that the carbofuran (carbamate) does not have an effect on

AChE activity in Hexaplex trunculus after 3 days of

exposure. After 10 days of exposure, Permethrin has an

inhibiting effect on AChE activity which is dependent to

time and concentration; where the low concentration

16.66 lg/L slightly inhibited the AChE activity and the

greater inhibition was detected at higher concentrations

38.24 and 55.61 lg/L. Results of two way ANOVA for

AChE suggests significant differences between concentra-

tions (F = 327.009, p \ 0.0001), within days of exposure

(F = 210.885, p \ 0.0001) and between interaction of the

concentrations and days of exposure (F = 34.229,

p \ 0.0001). Xing et al. (2010) showed that AChE was

significantly decreased in the common carp, Cyprinus

carpio L. exposed to sub-lethal concentrations

(1.16–11.6 lg/L for 20 days). In the same way, Amanullah

et al. (2010) showed a reduction of AChE activity on the

freshwater mussel Lamellidens marginalis exposed to sub-

lethal concentrations of chlorpyrifos (5 ppm for 30 days).

Because of its uptake and persistence, Permethrin can act

following two manners: slight modification in AChE

activity with low concentration and dramatic effect with

high concentration. The effect of low concentrations was

recorded by Laguerre et al. (2009) who reported that AChE

inhibition by pesticides depends upon the concentration. In

addition, our results are comparable with those observed by

Romeo et al. (2006) who showed that gradual Lindane rates

of 40, 60 and 80 lg/L inhibited the activity of this enzyme

in Hexaplex trunculus following a slight inhibition with

40 lg/L and a strong inhibition with 60 and 80 lg/L. Our

study showed that the effect of the Permethrin on AChE

activity depends also upon the exposure time. This time-

dependent response may be explained by the persistence of

Permethrin in clams during the 25 days of exposure. It can

be also related to the uptake of this insecticide in these

molluscs. Allan et al. (2005) recorded that Permethrin has a

capacity of adsorption to the water particles and sediment

which increase then its half-life and its bioavailability in

benthic invertebrates. In conclusion, the results of the

Permethrin contamination showed that this insecticide

altered two biomarkers profiles. CAT and AChE assessed

in this study could be a quick tool for the assessment of the

ecotoxicity of Permethrin contamination on R. decussatus.

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