Effects of organic pollutants on Eobania vermiculata measuredwith five biomarkers

A. Itziou • V. K. Dimitriadis

Accepted: 29 March 2012 / Published online: 11 April 2012

� Springer Science+Business Media, LLC 2012

Abstract In the present study, the effect of organic pol-

lution on land snails Eobania vermiculata was investigated.

Five pollution biomarkers (neutral red retention assay,

morphometry of lysosomes and neutral lipids, acetylcho-

linesterase activity and metallothioneins content, were

applied on tissues of the land snails. The results showed

intense differentiations between the snails treated with

organic pollutants and the control ones, as indicated by the

results obtained. Statistically significant correlations

among the results obtained emphasize the usefulness of

these biomarkers.

Keywords Eobania vermiculata � Terrestrial � Organic

pollutants � Biomarkers

Introduction

Most organic pollutants are lipophilic compounds (Cossarini-

Dunier et al. 1987) which accumulate in tissues of living

organisms (La Rocca et al. 1991). They appeared in the natural

environment as a result of the industrial development during

the last decades. Their short presence in nature did not allow

the development of special protective mechanisms in living

organisms. The toxicity of organics on organisms is enhanced

by their low water solubility, as well as by the low rate of

biodegrading these pollutants and removing them from

tissues. Organic persistence into the tissues of biota depends

merely to their ability to react with lipid membrane and cel-

lular layers. In snails, organic pollutants are accumulated

mainly in the digestive gland (Dindal and Wurzinger 1971),

while to a smaller extent in other tissues (Barker 2001).

The engagement as well as the accumulation of organic

pollutants depends on the physicochemical status of

organisms. Organisms characterized by a high lipid

reserve, may accumulate lipophilic substances more easily.

Certain organic substances enter the cells by diffusion from

the membrane. However, the uptake of several contami-

nants takes place together with many the particulate

material in the form of bound complexes (frays) (Smedes

1994). The transport of these complexes into the cell is

realized via an endocytotic or phagocytic mechanism,

dependent upon the size of the particles. Finally, the sub-

sequent intracellular behaviour includes vesicular traffic

via endocytotic or phagocytic transport of these frays to the

endosomal compartment and, hence, to the lysosomal

compartment for intracellular digestion (Moore 1990).

The biomarker neutral red retention assay (NRR) has

been applied for the evaluation of heavy metal or organic

effects both in field (Siboni et al. 2004) and in laboratory

studies (Lowe et al. 2006). The retention time of the dye

into the lysosomes is mainly influenced by the presence of

pollutants, while parameters other than pollution, such as

temperature, have little effect in this parameter (Ringwood

et al. 1998). The morphometric changes in the lysosomal

structure have been applied as a pollution biomarker with

the use of clams as bioindicator organisms, in several field

or laboratory studies (Cajaraville et al. 2000; Koukouzika

and Dimitriadis 2005), giving, however, contradictory

results on the lysosomal size and number (Marigomez and

Baybay-Villacorta 2003; Zorita et al. 2007). Various field

studies on earthworms (Gastaldi et al. 2007), and mussels

A. Itziou � V. K. Dimitriadis (&)

Department of Genetics, Development and Molecular Biology,

School of Biology, Aristotle University of Thessaloniki, 54124

Thessaloniki, Greece

e-mail: vdimitr@bio.auth.gr

123

Ecotoxicology (2012) 21:1484–1494

DOI 10.1007/s10646-012-0902-7

have proposed that heavy metals (Najimi et al. 1997) as

well as organic pollutants (Lowe 1988; Moore 1988) may

be responsible for the accumulation of neutral lipids.

However, other studies do not support the sensitivity of

these molecules in biomonitoring programs (Regoli 1992).

The determination of acetylcholinesterase (AChE) has

been applied in field and laboratory studies with the use of

vertebrates or invertebrates for the evaluation of organo-

phosphate or carbamide insecticides’ effects (Panda and

Sahu 2004; Gambi et al. 2007; Sarkar et al. 2006; Gagnaire

et al. 2008; Tryfonos et al. 2009). However, several studies

have supported the inhibition of AChE by heavy metals as

well (Viarengo 1989; Schmidt and Ibrahim 1994;

Guilhermino et al. 1998). Metallothioneins (MT) are used

as biomarkers of pollution caused by heavy metals and

have shown in many cases important correlations with

heavy metal levels in the digestive gland of land snails

(Chabicovsky et al. 2004). In certain cases, the composition

of these proteins has been reported to be induced by

organic pollutants as well (Raftopoulou et al. 2006).

The main aim of the present study was to investigate the

effect of different organic contaminants on Eobania

vermiculata using a battery of biomarkers. Furthermore,

the existence of statistical correlations has been examined

in order to support the use of this battery of biomarker in

biomonitoring of terrestrial pollution, on the land snail

Eobania vermiculata.

Materials and methods

Snails were collected from a hill near Aristotle University

of Thessaloniki (40�37057B 22�57042E) on May 2006 and

seven groups of 30–35 adult snails (28.14 ± 1.44 mm

length and 15.08 ± 1.17 mm width and 2.71 ± 0.32 gm in

weight) were placed in aerated transparent polystyrene

containers (40 9 30 9 30 cm with 30–35 individuals per

container), and maintained without food supply for 4 days

in order to be acclimatized to laboratory conditions. The

experimental animals were kept under a light: dark pho-

toperiod of 14:10 h, at a temperature of 20–22 �C, and at a

relative humidity of 80–90 %. After the acclimatization,

105 snails were divided into seven experimental groups of

15 snails each. Snails were treated with chlorpyrifos or

parathion-methyl (0.1 or 1 ppm), or with PAHs (9 ppm,

containing stock solution 1:1:1 of anthracene, phenan-

threne and naphthalin dissolved in acetone). The whole

treatment lasted for 25 days. An artificial food was pre-

pared by mixing 4 g Cerelac-with vegetables-baby food

(Nestle, Belgium) and 1 g Agar (Sigma) with solutions

containing the required concentrations of pollutants to give

100 ml of agar medium (Swaileh and Ezzughayyar, 2000).

A fungicide (methylparaben) was added to the solutions as

0.3 ml/100 ml food. Each 100 ml medium was divided

equally among four petri dishes (25 ml/dish). After cool-

ing, petri dishes were kept in the refrigerator. Control

groups consisted of animals exposed to acetone or unex-

posed animals, using distilled water in their food instead of

the above-described contaminant solutions. The bottom of

the containers was covered with a layer of damp, absorbent

paper and their feed was placed in a petri dish. The con-

tainers were cleaned and the food mixed and renewed three

times a week. The absorbent paper was changed once a

week. A set of preliminary experiments was performed in

order to test the effect of various concentrations of the

organics used in the present study. Each pollutant was

tested alone. The concentrations used caused the death of

much less than 50 % of the animals.

Stress indices

Evaluation of NRR

The NRR assay was performed according to Lowe and

Pipe (1994), with small modifications. The shell of 10

individuals was pierced and haemolymph was withdrawn

from a small hole in physiological snail buffer (50/50 of

haemolymph/buffer), as it is described by Snyman et al.

(2000). The NRR time was measured individually for ten

snails and the mean NRR time of the ten snails was the

NRR time for the whole experimental group.

Evaluation of lysosomal morphometrical parameters

Digestive glands from five snails from each sampling sta-

tion, as well as from each container, were dissected out.

Five small pieces of five snails’ digestive glands were

placed on aluminum cryostat chucks, in a straight row

across the center of the chuck. The chuck was then placed

for 40 s in a small bath of n-hexane, which has been pre-

cooled for 2–3 min in liquid nitrogen, in order to quench

the tissue. All chucks were doubled wrapped in parafilm

and stored at -80 �C until required for sectioning, or

alternatively, were kept at -30 �C and sectioned within

1 week. N-acetyl-b-hexosaminidase was histochemically

performed according to Moore (1976) in unfixed cryostat

sections, and indicated lysosomes as purple precipitates in

the digestive cells of snails. The non pre-treated slides were

used for morphometrical analysis of lysosomes (volume

density of lysosomes-VDL, numerical density of lyso-

somes-NDL). The volume density of lysosomes/neutral

lipids refers to the volume covered by lysosomes/neutral

lipids (lm3) divided by the volume covered by the cyto-

plasm (lm3). This measurement takes into consideration

the size of each digestive tubule and that is how it is dif-

ferent from measuring only the volume of lysosomes/

Effects of organic pollutants on Eobania vermiculata 1485

123

neutral lipids. The numerical density refers to the number

of lysosomes/neutral lipids per lm3 divided by the volume

covered by the cytoplasm (lm3). That is why this param-

eter is different from measuring the number of lysosomes/

neutral lipids.

Morphometry of neutral lipids

The neutral lipids’ histochemistry was applied according to

Moore (1988). Image analysis was used in order to measure

the volume density (VDLP) and the numerical density of

neutral lipids (NDLP).

Image analysis

The quantification of the volume and numerical density of

neutral lipids and lysosomes was performed by the use of

image analysis. Slides were examined using a JVC video

camera mounted on an Olympus CX41 light microscope

with an objective lens of 1009 magnification. The image

was displayed on the computer screen and captured with

Adobe Premiere 5. Binary images segregating lipids and

lysosomes from the cytoplasm were obtained by the seg-

mentation procedure, which was manually adjusted in the

first measurement of a given section and automatic in the

others (EIKONA program). Whenever present, the diges-

tive tubule lumen and the connective tissue surrounding the

digestive tubule were eliminated from the analysis. 50

measurements per experimental group of snails were

conducted.

AChE activity

For the AChE measurements, digestive gland and haemo-

lymph were collected from five snails and the procedure

described by Dailianis et al. (2003) was followed. Four

measurements were performed for the determination of

AChE activity on samples from pooled tissues derived

from five snails.

MT content

In order to quantify MT in the digestive gland of five

snails, the spectrophotometric method according to Viar-

engo et al. (1997) was used. MT content in the samples was

measured by evaluating the sulphydryl (SH) residues

content according to Ellman (1961) with DTNB (5,5-

dithiobis-2-nitrobenzoic acid) and reduced glutathione

(GSH) as a standard. 4 measurements were performed for

the determination of MT content on samples from pooled

tissues from five snails.

Data analysis

Data on the AChE activity and MT content was tested

using non-parametric statistics (Mann–Whitney U-test,

p \ 0.05), while data on NRR assay, neutral lipids and

lysosomes were tested using Duncan’s test (p \ 0.05),

breakdown and one way ANOVA). Statistical relationships

among parameters were compared using non-parametric

Pearson test p \ 0.05). The analyses were carried out using

the STATISTICA statistical package (STATISTICA,

Microsoft Co.).

Results

Neutral red retention assay (NRR)

The statistical analysis indicated significantly lower values

of the previously mentioned biomarker in snails exposed to

both concentrations of the organophosphate insecticides

chlorpyrifos and parathion-methyl, as well as to 9 ppm of

the polycyclic aromatic hydrocarbon (PAHs) mixture,

compared to snails exposed to acetone (which was the

dilutor of the organic pollutants), and to the unexposed

ones (control snails) (Fig. 1).

Volume and numerical density of lysosomes (VDL,

NDL)

The volume density of lysosomes in the snails exposed to

the two concentrations (the 0.1 and 1 ppm) of the orga-

nophosphate insecticides chlorpyrifos and parathion-

methyl was statistically higher compared to the snails

exposed to acetone and to the control snails. On the

020406080

100120140

Treatment

Neutral red retention assay (NRR)

Neu

tral

red

ret

enti

on

ti

me

(min

s)

Fig. 1 ‘‘Neutral red retention’’ (NRR) assay applied on the haemo-

cytes of snails E. vermiculata exposed to 0.1 or 1 ppm of chlorpyrifos

or parathion-methyl or 9 ppm of PAHs for 25 days. Error barsindicate standard deviation. Filled star indicate significant differences

between control value and that observed after organic treatment

(Duncan’s test, p \ 0.05). Filled circle indicate statistically signifi-

cant difference between cells treated with acetone and that observed

after organic treatment (Duncan’s test, p \ 0.05)

1486 A. Itziou, V. K. Dimitriadis

123

contrary, the snails treated with 9 ppm PAHs presented

decreased lysosomal volume density in comparison with

the snails exposed to acetone and with the control snails

(Fig. 2a).

As far as the lysosomal numerical density is concerned,

the results of study revealed a statistically significant

reduction of this parameter in the case of animals exposed

to both concentrations of chlorpyrifos and parathion-

methyl as well as to 9 ppm PAHs, compared to the animals

exposed to acetone and to unexposed ones. Nevertheless,

the increase of the exposure concentration of chlorpyrifos

from 0.1–1 ppm showed a parallel increase in the number

of lysosomes (Fig. 2b).

The lysosomes of snails exposed to both concentrations

of chlorpyrifos seemed to have undergone an enlargement,

since the microscopic analysis showed less and bigger in

size lysosomes, compared to the lysosomes of control

snails (Fig. 3). Analogous enlargement was not observed in

lysosomes of snails treated with parathion-methyl and

PAHs. However, an increase of lysosomal volume density

was detected in this case as well (Fig. 3).

Volume and numerical density of neutral lipids (VDLP,

NDLP)

The volume density of lipid droplets was statistically sig-

nificantly higher in snails exposed to organic pollutants, as

measured by the statistical analysis of the results. Con-

cretely, exposure to 0.1 and 1 ppm chlorpyrifos, 0.1 and

1 ppm parathion-methyl, as well as to 9 ppm PAHs led to

higher volume density of lysosomes, compared to acetone-

exposed and non-exposed snails (Fig. 4a).

Contrary to the volume density, the numerical density of

neutral lipids presented a statistically significant reduction

in most organisms exposed to organics, compared to the

snails exposed to acetone and to non-exposed ones

(Fig. 4b). The examination of the cryotomes with the light

microscope showed an increase in the total quantity of

neutral lipids in the snails treated with organic, compared

to the controls (Fig. 5).

Acetylcholinesterase activity (AChE)

Snail exposure to organic pollutants lead to a significant

reduction of AChE activity in the digestive gland of the

treated animals, compared to the untreated ones, as well as

to those treated with acetone. Moreover, the increase in the

exposure dose of the organophosphate insecticides (chlor-

pyrifos and parathion-methyl) resulted in further suspen-

sion of the enzyme activity (Fig. 6a).

Similar differences appeared when the haemolymph was

examined. More specifically, AChE activity was signifi-

cantly reduced in snails exposed to organics for 25 days, in

comparison with the activity measured in the haemolymph

of acetone-exposed snails or of non-exposed ones. Never-

theless, the previously mentioned changes detected in the

enzyme activity did not follow the exposure dose of the

organics, since reduced values of AChE activity were

measured even at the lowest concentration of both orga-

nophosphate insecticides (Fig. 6b).

Metallothionein contents (MT)

The exposure of snails to organics led to statistically sig-

nificant increase of MT levels in the digestive gland of the

exposed animals. Moreover, in the snails treated with all

types of organic pollutants the MT levels were significantly

higher compared to MT levels in snails treated with ace-

tone, as well as in non-exposed snails. However, clear

statistical differences were not observed between the var-

ious concentrations of exposure, showing thus the

a

00,020,040,060,080,1

0,120,140,160,18

Vo

lum

e d

ensi

ty o

f ly

soso

mes

(µm

3/µm

3)

Treatment

Volume density of lysosomes (VDL)

b

00,005

0,010,015

0,020,025

0,03

Nu

mer

ical

den

sity

of

lyso

som

es(n

um

ber

/µm

3)

Treatment

Numerical density of lysosomes (NDL)

Fig. 2 Results on the morphometry of lysosomal parameters of the

digestive lysosomes of snails E.vermiculata exposed to 0.1 or 1 ppm

of chlorpyrifos or parathion-methyl or 9 ppm of PAHs for 25 days.

Error bars indicate standard deviation. a Volume density of

lysosomes (VDL); b numerical density of lysosomes (NDL). Filledstar indicate significant differences between pairs of mean values

(Duncan’s test, p \ 0.05). Filled circle indicate statistically signifi-

cant difference between cells treated with acetone and that observed

after organic treatment (Duncan’s test, p \ 0.05). Open circleindicate statistically significant difference between cells treated with

0.1 and 1 ppm of chlorpyrifos (Duncan’s test, p \ 0.05)

Effects of organic pollutants on Eobania vermiculata 1487

123

sensitivity of the present biomarker even from the lower

concentrations (Fig. 7).

Correlations

The results of the correlation analysis among the biological

parameters are reported in Table 1. In brief, the most sig-

nificant correlations were reported between AChE activity

in digestive gland and haemolymph, VDL and MT content,

VDLP and MT content, NRR and VDL, VDL and NDL,

VDL and VDLP, NRR and NDL, NDL and VDLP.

Discussion

The lysosomal system constitutes a particular objective for

the toxic effects of pollutants on a subcellular level and has

been used for the study of dangerous substances’ effect on

organisms (Moore 1990, Scott-Fordsmand et al. 2000,

Spungeon et al. 2004). Previous studies on toxic and

pathological repercussions of organic pollutants on lyso-

somes have shown that the lysosomal system constitutes a

sensitive objective of this type of pollutants (Moore et al.

1982; Moore 1985; Moore et al. 1986). The direct reaction

of lysosomes provides useful information, that contribute

to diagnostic methods for damage localisation in tissues as

well as useful indicators for further pathological studies

(Moore 1990).

The results of the present study showed decreased NRR

detention times in the snails that were exposed in all

organic pollutants. The reduction of lysosomal membrane

stability, in cells such as haemocytes, reflects the damaging

consequences of organic pollutants, like the ones used in

the present study. It is known that the exposure to organic

Aceton Chlorpyrifos 0.1 ppm

Chlorpyrifos 1 ppm Parathion-methyl 0.1 ppm

Parathion-methyl 1ppm PAHs 9 ppm

dcba

e f g h

i j k l

Fig. 3 Histochemistry for the lysosomal enzyme N-acetyl-b-hexos-

aminidase in cryosections of the digestive gland of snails exposed to

acetone and snails exposed to 0.1 ppm chlorpyrifos, 1 ppm chlor-

pyrifos, 0.1 ppm parathion-methyl, 1 ppm parathion-methyl and

9 ppm PAHs for 25 days. Animals treated with the organophosphate

insecticides chlorpyrifos and parathion-methyl displayed bigger and

less in number lysosomes (c–j), while animals treated with PAHs

displayed less in number lysosomes (k–l) compared to those treated

with acetone (a–b). 12009

1488 A. Itziou, V. K. Dimitriadis

123

xenobiotics can lead to increased production of reactive

oxygen species, with the endolysosomal system constitut-

ing the basic place of production of reactive oxygen spe-

cies. The production of ROS takes place through several

potential mechanisms, both direct and indirect, such as

redox cycling, redox reactions with O2 and ROS, autoxi-

dation, enzyme induction, disruption of membrane-bound

electron transport, and depletion of antioxidant defenses

(Livingstone 2001). The effect of oxidative stress on

membranes, on proteins (e.g. carbonyls) or on DNA leads

undeniably to decreased protein synthesis, to tissue damage

and to pathophysiological conditions (Cajaraville et al.

2000; Dailianis et al. 2003; Domouhtsidou and Dimitriadis

2001; Kirchin et al. 1992; Livingstone 2001). The results of

present study manifest the unfavourable effect of organics

on the lysosomal system of cells involved in defence

reactions. Organic pollutants suspend the activity of

detoxification enzymes, even if some reports support their

activation for the confrontation of organics (Baturo and

Lagadic 1996), cause histopathological damage in the

digestive gland (Zupan and Kalafatic 2003) and non

reversible cell lysis (Roses et al. 1999).

The changes observed in digestive gland lysosomes of

various organisms have been related in several cases of

exposure to organic pollutants. However, controversial

results exist with regard to the effect of organic pollutants

on the size and the number of lysosomes. Certain studies in

the past recorded reduction in the size and in the number of

these organelles after short exposure of mussels in pollu-

tants, while increase in the size with simultaneous reduc-

tion in the number was presented after long-lasting effect

of pollution (Moore 1988; Marigomez and Baybay-Villa-

corta 2003). Alternatively, various pollutants may possibly

cause the formation of smaller and more abundant lyso-

somes (Marigomez et al. 1996; Sokolova et al. 2005;

Guerlet et al. 2006) and more specifically after effect of

organic pollutants (Cajaraville et al. 1991).

In the present study increase in the size of lysosomes

and reduction in their number was reported mainly after the

exposure of snails to both concentrations of chlorpyrifos

(0.1 and 1 ppm). The lysosomal destabilisation caused by

organic xenobiotics and the consequent lysosomal inflation

probably reflects the changes in the lysosomal membrane,

and precisely the increase of membrane penetrability and

fluidity (Moore and Viarengo 1987). Consequently, the

entry of different sublayers into the lysosomal system is

enhanced, with imminent osmotic changes that lead to

increase of lysosomal size (Cajaraville et al. 1989).

According to Cajaraville and Pal (1995), the reduction or

the increase of lysosomal size can be considered as a result

of different degree of toxicity or different mechanism of

toxic action of each pollutant. The changes that the

digestive gland lysosomes undergo after the effect of pol-

lution may therefore depend on the species of pollutant, the

dose, the duration of exposure or from combination of all

previous factors.

Controversial results have been recorded with regard to

the fluctuation of neutral lipids’ quantity after organic

substances’ effect (Cajaraville et al. 1990; Gastaldi et al.

2007; Radwan et al. 2008; Koukouzika and Dimitriadis,

2008). The previously mentioned studies support the fact

that both increase or reduction of neutral lipids are possible

due to the effect of pollutants, which could be attributed to

a complex reaction of molluscs to the pollutant factors.

Thus, the reduction of neutral lipids is probably caused by

increased consumption of lipids, as a source of energy, in

stressed organisms, in order to activate cellular processes

of detoxification (Guerlet et al. 2006). Alternatively, cer-

tain pollutants can strengthen the activity of lipases, thus

helping in the split of lipids (Gil et al. 1989).

a

00,050,1

0,150,2

0,250,3

Vo

lum

e d

ensi

ty o

f n

eutr

al li

pid

s( µ

m3/

µm3)

Treatment

Volume density of neutral lipids (VDLP)

b

00,0020,0040,0060,0080,01

0,0120,0140,0160,018

Nu

mer

ical

den

sity

of

neu

tral

lip

ids

(nu

mb

er/µ

m3)

Treatment

Numerical density of neutral lipids (NDLP)

Fig. 4 Results on the morphometry of neutral lipid parameters of the

digestive lysosomes of snails E.vermiculata exposed to 0.1 or 1 ppm

of chlorpyrifos or parathion-methyl or 9 ppm of PAHs for 25 days.

Error bars indicate standard deviation. a Volume density of neutral

lipids (VDLP); b numerical density of neutral lipids (NDLP). Filledstar indicate significant differences between control value and that

observed after organic treatment (Duncan’s test, p \ 0.05). Filledcircle indicate statistically significant difference between cells treated

with acetone and that observed after organic treatment (Duncan’s test,

p \ 0.05)

Effects of organic pollutants on Eobania vermiculata 1489

123

The results of present study showed increase of neutral

lipids after the exposure of snails E. vermiculata to organic

pollutants. Researches on mussel tissues have mainly cor-

related the accumulation of neutral lipids with the presence

of organic pollution (Lowe 1988; Moore 1988; Marigomez

and Baybay-Villacorta 2003). This reaction has been

attributed to the lipophilic character of organic substances,

which leads to the accumulation of intracellular lipids

(Marigomez and Baybay-Villacorta 2003). Alternatively,

according to previous studies, an increase in neutral lipids

may be attributed to a disturbance of their metabolism

deriving from the pollutants (Moore 1988). A characteristic

increase of lipids has been reported in hydrocarbon-

exposed clams (Wolf et al. 1981; Carles et al. 1986). This

may either have been due to the induction of lipid com-

position from the hydrocarbons (Carles et al. 1986) or to

the exclusion of their intracellular transport in the Golgi

apparatus or even of their transport between the endo-

plasmic reticulum and the Golgi apparatus (Raber and

Carter, 1986). The last conclusion was supported partly by

changes that have been observed in the morphology of

Golgi apparatus after the effect of organics (Cajaraville

et al. 1990; Carles et al. 1986).

The effect of organophosphates on land snails has been

studied by Rorke and Gardner (1974), who recorded a

powerful inhibition of AChE in haemolymph of the land

snail Helix aspersa, caused by various organophosphate

and carbamide insecticides. Moreover, an inhibition of

AChE activity was also reported in H. aspersa exposed to

the organophosporic plant-protection substance dimethoate

either in the food or in an artificial sublayer (Coeurdassier

et al. 2001). Nevertheless, Young and Wilkins (1989) did

not observe any inhibition of the enzyme’s activity in slugs

D. reticulatum when these were exposed to increasing

Aceton Chlorpyrifos 0.1 ppm

Chlorpyrifos 1 ppm Parathion-methyl 0.1 ppm

Parathion-methyl 1 ppm PAHs 9 ppm

a b c d

e f g h

i j k l

Fig. 5 ‘‘Oil Red’’ staining on cryosections of the digestive gland of

snails Eobania vermiculata. Transversely cut digestive tubules of

snails exposed to acetone and snails exposed to 0.1 ppm chlorpyrifos,

1 ppm chlorpyrifos, 0.1 ppm parathion-methyl, 1 ppm parathion-

methyl and 9 ppm PAHs for 25 days. Animals treated with

chlorpyrifos or PAHs displayed bigger in size and less in number

neutral lipids (c–f, k, l), compared to controls (a, b), while snails

treated with both concentrations of parathion-methyl displayed more

numerous neutral lipids (g–j), compared to controls (a, b). 12009

1490 A. Itziou, V. K. Dimitriadis

123

concentrations of the carbamide insecticide methiocarb,

and attributed this resistance to a difference in the sensi-

tivity of the five isoenzymes of AChE. The results of

present study showed important inhibition of AChE

activity in snails E. vermiculata exposed to organic pol-

lutants, with the most important reduction detected in the

higher exposure concentrations (1 ppm) of the organo-

phosphate insecticides chlorpyrifos and parathion-methyl

in the digestive cells. The previous observation is probably

related to the particularly fast interaction of these organic

substances with AChE, as competitive inhibitors of this

enzyme, as it has been also proposed by Essawy et al.

(2008), after the study of snails E. vermiculata, which is in

agreement with Pessah and Sokolove (1983) and Young

and Wilkins (1989). The neurotoxic action of organics is

owed to the inhibition of AChE activity and the accumu-

lation of acetylcholine in the synaptic connections that

can lead to changes of movement ability (Wedgwood and

Bailey 1988) and diet (Bailey 1989). Consequently,

changes in the structure of the nervous system connected

with the repression of AChE in snails can potentially lead

to other biological responses (behavior of feeding, sur-

vival, locomotion, production of mucus, production of

energy, and reproduction). Indeed, organophosphates are

considered as non reversible inhibition factors of AChE,

because of the fact that the time required for the reacti-

vation of the enzyme after organic effect may be bigger

than the time required for the composition of new AChE

(Hyne and Maher 2003). The suspensive effect of the

organophosphate pollutants can often be long lasting,

proposing that in such cases the reorganization is realized

mainly from the composition of the enzyme (Engenheiro

et al. 2005).

Metallothioneins constitute biomarkers of heavy metals,

while some studies on clams support the induction of MT

from organic pollutants as well (Raftopoulou et al. 2006).

The results of the present study showed a statistically

significant increase of MT in the digestive gland of the land

snails exposed to the all organic pollutants that were used.

The organic pollutants, such as the organophosphate

insecticides have the ability to cause oxidant stress via the

production of reactive oxygen species (ROS), by affecting

the effectiveness of antioxidant enzymes (Gultekin et al.

2000; Tuzmen et al. 2008). The induction of MT compo-

sition, under ROS creation circumstances, leads to the

conclusion that these proteins are probably involved in the

protection against the oxidative stress and act as detectors

of free oxygen species (Bauman et al. 1991). Therefore,

they play an important role in the cellular defence mech-

anisms of organisms against the free OH- species. More-

over, MTs can be connected with organic species, as

previous studies have shown, strengthening the opinion

that these metalloproteins have the ability to detect a pleiad

a

0100200300400500600700

Ace

tylc

ho

lines

tera

se a

ctiv

ity

(U/m

gr

pro

tein

)

Treatment

Acetylcholinesterase activity (AChE) in the digestive gland

b

0100200300400500600700800

Ace

tylc

ho

lines

tera

se a

ctiv

ity

(U/m

gr

pro

tein

)

Treatment

Acetylcholinesterase activity (AChE) in the haemolymph

Fig. 6 ‘‘Acetyl-cholinesterase activity’’ (AChE) assay applied (a) in

the digestive gland or (b) in the haemolymph of snails E. vermiculataexposed to 0.1 or 1 ppm of chlorpyrifos or parathion-methyl or 9 ppm

of PAHs for 25 days. Error bars indicate standard deviation. Filledstar indicate significant differences between control value and that

observed after organic treatment (Mann–Whitney U test, p \ 0.05).

Filled circle indicate statistically significant difference between cells

treated with acetone and that observed after organic treatment (Mann–

Whitney U test, p \ 0.05)

050

100150200250300350400450

Met

allo

thio

nei

n c

on

ten

t ( µ

gr/

gr

wet

tis

sue)

Treatment

Metallothionein content (MT) in the digestive gland

Fig. 7 ‘‘Metallothionein contents’’ (MT) assay applied in the diges-

tive gland of snails E. vermiculata exposed to 0.1 or 1 ppm of

chlorpyrifos or parathion-methyl or 9 ppm of PAHs for 25 days.

Error bars indicate standard deviation. Filled star indicate significant

differences between control value and that observed after organic

treatment (Mann–Whitney U test, p \ 0.05). Filled circle indicate

statistically significant difference between cells treated with acetone

and that observed after organic treatment (Mann–Whitney U test,

p \ 0.05)

Effects of organic pollutants on Eobania vermiculata 1491

123

of species that includes peroxides, hydroxyls and organic

radicals (Sato and Bremmer 1993).

Conclusions

The results of the present study showed that organic pol-

lutants may cause significant changes in organisms as

shown by a wide range of pollution biomarkers. This

became apparent through the intense differentiations

detected at the tissues of snails affected by pollutants,

compared to non exposed animals. The statistically sig-

nificant correlations recorded among the applied biomark-

ers clearly show the value of these biomarkers to detect

effects of organic pollutants in studies on the land snail

E. vermiculata.

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