esterase activities in the bivalve mollusc adamussium colbecki as a biomarker for pollution...

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Marine Pollution Bulletin 49 (2004) 445–455

Esterase activities in the bivalve mollusc Adamussium colbeckias a biomarker for pollution monitoring in the

Antarctic marine environment

Stefano Bonacci a,*, Mark A. Browne b, Awantha Dissanayake b, Josephine A. Hagger b,Ilaria Corsi a, Silvano Focardi a, Tamara S. Galloway b

a Department of Environmental Sciences ‘‘G. Sarfatti’’, University of Siena, Via P.A. Mattioli 4, Siena I-53100, Italyb Plymouth Environmental Research Centre, University of Plymouth, Drake Circus, Plymouth PL4 8AA, UK

Abstract

Marine environments are continuously being threatened by a large number of xenobiotics from anthropogenic sources. Even in

sparsely populated and relatively pristine areas, such as Antarctica, hazardous chemicals can pose a serious environmental problem.

The main aims of the present study were to (1) validate and optimize an analytical technique utilizing a microtitre-plate photometer

to ascertain background levels of esterase activities in the Antarctic bivalve Adamussium colbecki, (2) carry out in situ monitoring of

esterase activities to assess any potential environmental impacts of the Italian Scientific Antarctic Base ‘‘Terra Nova Bay’’ on the

surrounding marine area. Results showed the presence of organophosphorous-sensitive cholinesterase (ChE) and carboxylesterase

(CbE) activities in the gills of A. colbecki and optimal assay conditions were comparable with those found for bivalve species from

temperate areas. A higher sensitivity of ChE versus acetylthiocholine activity in A. colbecki to chlorpyrifos compared to species from

temperate areas may also be inferred. The in situ study indicated no differences in the environmental quality of the three study sites

located around the Italian Base.

� 2004 Elsevier Ltd. All rights reserved.

Keywords: Biomarkers; Antarctica; Adamussium colbecki; Cholinesterases (ChEs); Carboxylesterases (CbEs); Terra Nova Bay

1. Introduction

The pollution of marine environments by the vast

number of xenobiotics has increased during the last

decade as a direct consequence of a wide variety of an-thropic activities (Dawe, 1990; Goksøyr and F€orlin,1992). Such contamination represents a serious threat to

the overall health of aquatic ecosystems (Murchelano,

1990).

Antarctica is considered to be one of the least pol-

luted areas of the world, but recent studies have detected

the presence of organic xenobiotics such as polycyclic

aromatic hydrocarbons (PAHs), polychlorinated bi-phenyls (PCBs), hexachlorobenzene (HCB) and p; p0-dichlorodiphenyl dichloroethylene (p; p0-DDE) in themarine environment (Kennicutt II, 1990; Focardi et al.,

*Corresponding author. Tel.: +39-577-232877; fax: +39-577-

232806.

E-mail address: [email protected] (S. Bonacci).

0025-326X/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.doi:10.1016/j.marpolbul.2004.02.033

1994; Kennicutt II et al., 1995; Bargagli et al., 1998a).

Additionally, pesticides are normally found in Antarctic

sea water and are often applied to biomonitoring studies

as pollution markers (Desideri et al., 1989). The sus-

ceptibility of the Antarctic marine environment to con-tamination is suggested by the slow recovery rates and

short food chains which characterize such ecosystems

(Chapman and Riddle, 2003). In addition, several

studies have indicated a difference in sensitivity of

aquatic polar organisms to pollutants in comparison

with species from temperate areas (Ling et al., 1998;

King and Riddle, 2001). In light of these studies it is

imperative that biomonitoring programs can be devel-oped to assess the anthropic impact over such an area.

In the past two decades, a wide range of biological

indicators (biomarkers) has been developed to detect

and assess the exposure to, and effects of contaminants

(Livingstone and Goldfarb, 1998) and nowadays they

are routinely used for marine pollution assessment

(Galloway et al., 2002a).

mail to: [email protected]

Fig. 1. Map of Terra Nova Bay (Ross Sea, Antarctica) showing the

sampling sites (1¼Thethys Bay; 2¼Road Bay; 3¼Ad�elie Cove)around the Italian Scientific Base.

446 S. Bonacci et al. / Marine Pollution Bulletin 49 (2004) 445–455

Among biomarkers, the inhibition of cholinesterase

(ChEs) activities has been demonstrated to be a useful

marker for the assessment of organophosphorous (OPs)

and carbamate (CBs) insecticides exposure (Bocquen�eet al., 1990, 1997; Escartin and Porte, 1997; Doran et al.,

2001; Galloway et al., 2002b; Owen et al., 2002). Never-

theless, recent studies provide evidence that esterase

activities may be affected by a wide range of contami-

nants, including detergents, heavy metals and compo-

nents of complex mixtures of pollutants (Bocquen�e et al.,1990; Risebrought et al., 1990; Payne et al., 1996; Najimi

et al., 1997; Guilhermino et al., 1998; Cajaraville et al.,2000), similar to those present in particularly impacted

Antarctic regions (Kennicutt II et al., 1992a,b, 1995; Mc-

Donald et al., 1994, 1995; Deprez et al., 1999;Miller et al.,

1999; Stark, 2001). As a consequence, a more general use

of this biomarker for the assessment of environmental

quality has been suggested (Guilhermino et al., 1998).

Carboxylesterases (CbEs) are another class of serine-

dependent esterases which hydrolyze a wide range ofxenobiotic substrates (Maxwell, 1992; Parkinson, 1996)

and are believed to play a prominent role in detoxication

and tolerance for some OP pesticides, providing pro-

tection against neurotoxic compound poisoning (Gupta

et al., 1985; Jokanovic et al., 1996; Parkinson, 1996;

Escartin and Porte, 1997). In bivalve mussel species,

CbE activities have displayed a greater sensitivity than

ChEs to inhibition by OPs or CBs exposure (Ozretic andKrajnovic-Ozretic, 1992; Escartin and Porte, 1997;

Basack et al., 1998).

Consequently, CbE levels have been proposed as a

useful ecotoxicological biomarker for the assessment of

bivalve exposure to neurotoxic compounds.

To date, there is a general lack of knowledge con-

cerning measurements of esterase activities in Antarctic

species, both in terms of their physiological and molec-ular characteristics and of their responses to xenobiotic

exposure. Nevertheless, such information is essential if

these markers are to be successfully used in biomoni-

toring programs for pollutant risk assessment.

Adamussium colbecki is an Antarctic scallop that is

considered a key species of marine Antarctic ecosystems

(Mauri et al., 1990; Berkman and Nigro, 1992) and

meets the criteria of species in the ‘‘Mussel Watch’’program (Farrington et al., 1983). Furthermore, the

tissue composition of such bivalve has been inferred to

reflect variability of heavy metal levels in environmental

matrixes (Berkman and Nigro, 1992; Viarengo et al.,

1993; Bargagli et al., 1996) and the application of bio-

logical markers such as antioxidant alterations and

lysosomal membrane damage (Regoli et al., 1997, 2002)

has highlighted the effectiveness of enzymic responses tocontaminants in A. colbecki as tools for assessment of

aquatic environmental quality.

In light of the above information the main aims of the

present research were (1) to characterize esterase activ-

ities present in the gills of the Antarctic bivalve A. col-

becki, (2) to optimize and validate a microtitre-plate-

based spectrophotometric assay in order to measure

esterase activities in A. colbecki tissues, (3) to investigatethe sensitivity of esterase activity in an Antarctic bivalve

species to inhibition by OPs-pesticide exposure, (4) to

gain knowledge about the marine environmental quality

of the area surrounding the Italian Scientific Antarctic

Base of Terra Nova Bay using this assay.

2. Material and methods

2.1. Sampling campaign

During the Antarctic summer of 2001–2002, 15 spec-

imens of A. colbecki were randomly collected from three

coastal sites (Road Bay, Thethys Bay and Ad�elie Cove)near the Italian Antarctic Base ‘‘Baia Terra Nova’’

(BTN) (Lat. 74�410S, Long. 164�040E) (Terra Nova Bay,Ross Sea). Fig. 1 indicates the sampling sites. The Sci-

entific Station is located in a rocky, ice-free coastal area,

near the tip of a small peninsula which protects Thethys

Bay from the open sea. A sea-ice airstrip on the bay has

been used in the last eight years by an Italian Air Force

C-130 aircraft carrying personnel, equipment and sup-

plies. The BTN Base has been built and operated in such

a way to keep the anthropic impact to a minimum.Particular precautions, such as a strict waste manage-

ment plan and an emergency plan for accidental oil spills

have been taken since earlier years (Giuliani et al., 2001).

Potential sources of environmental pollution due to the

scientific settlement are represented by electric power

generators, a petrol station, a solid waste incinerator

and a waste water treatment plant. The Road Bay area

is theoretically the most subject to anthropic impact,receiving the waste water outfall of the Station (Bargagli

S. Bonacci et al. / Marine Pollution Bulletin 49 (2004) 445–455 447

et al., 1998a; Jim�enez et al., 1999). The Ad�elie Covesampling site is considered the most protected from

pollution, being the most distantly located with respect

to the BTN Scientific Base.After collection, organisms were quickly shipped to

the Base, the gills dissected out, immediately frozen in

liquid nitrogen to prevent enzyme deterioration and

stored at )80 �C until analysis.

2.2. Sample preparation

Gills were chosen for the measurement of esteraseactivities because this tissue exhibited a high level of

esterase activity and a higher sensitivity to contaminants

with respect to other tissues (Mora et al., 1999).

Gills were homogenized with a 1:5 ratio (w/v) of

homogenization buffer (0.1 M Tris–HCl, pH 7.2, 0.25 M

sucrose) and centrifuged at 10,000 rpm for 10 min. The

resulting pellet containing cellular debris was discarded,

while the supernatant fraction was retained, stored onice and used for immediate subsequent biochemical

determinations. All procedures were carried out at 4 �C.

2.3. Assay of enzymatic activities and total protein

quantification

Esterase activities were measured essentially by the

method of Ellman et al. (1961), modified for micro-titration plate format. In general thiocholine esters or

thioacetate derivates were hydrolysed by serine-depen-

dent esterases (cholinesterases) or carboxylesterase,

respectively, to yield thiocholine or thioacetate. Sub-

sequent combination with 5,50-dithiobis-2-nitrobenzoicacid (DTNB) forms a yellow anion 5-thio-2nitrobenzoic

acid which absorbs strongly at k ¼ 405 nm. All analyseswere performed on a OPTIMAX tunable microplatereader (Molecular Devices).

Acetylthiocholine iodide (ASCh), butyrylthiocholine

iodide (BSCh), propionylthiocholine iodide (PrSCh) and

phenylthioacetate were used as substrate analogues to

determine acetyl-, butyryl- or propionyl-cholinesterase

or CbE activities, respectively.

Optimal assay condition ranges were carried out

using a pooled sample containing gills from five animals.Each step for determination of optimum assay condi-

tions was carried out on pool aliquots of about 1 g of

tissue. For each microplate well, 30 ll of the sample or

Table 1

Optimal assay conditions for the measurements of esterase activities in gills

Substrate type Substrate concentration (mM)

ASCh 3

BSCh 3

PrSCh 3

Phenylthioacetate 3

50 ll of the buffer blank were incubated at 25 �C with150 ll DTNB (270 lM in 50 mM sodium phosphate

buffer pH 7.4) for 5 min to assess the occurrence of

endogenous cross-reaction between the sample and theDTNB. Substrate (187.5 lM final concentration) was

successively added to start the enzymatic reaction and

the progressive increase in absorbance recorded for 5

min at 405 nm. The spontaneous substrate hydrolysis

was determined as described above in the absence of the

sample. Esterase activities were initially expressed as

D absorbance units min�1, converted to nmoles hydro-lysed substrate min�1 and normalised by tissue totalprotein content.

Optimum assay conditions were subsequently deter-

mined for each substrate carrying out analyses with a

range of different substrate concentrations, reaction

buffer pH and supernatant sample volume. Concentra-

tions of substrates ranged from 187.5 lM to 3 mM,

sample volume from 10 to 50 ll, reaction buffer pH from6.0 to 9.0. Once optimum assay conditions were stan-dardized (Table 1), each sample was individually

analyzed. The inhibitory study was performed by pre-

incubation of sample homogenate in solutions containing

chlorpyrifos in the range 0.1–100 lM for 5 min before theaddiction of substrate ASCh.

The sample protein concentration was determined

following the method of Bradford (1976), as modified

for microtitration plate reading (kabs ¼ 595 nm, val-ues expressed as mg protein ·ml homogenate�1). Pre-liminary experiments aiming to determine optimal

sample dilution range were carried out analyzing pools

containing gills from at least five animals and confirmed

1:4 as the optimum rate of sample dilution for the

determination of the protein concentration in gills of

A. colbecki.

2.4. Statistical analyses

All determinations were performed in quadruplicate

for each single sample or pool aliquot and results were

expressed as mean value ± 1 standard deviation. Statis-

tical significance between means was determined using

one-way analysis of variance (ANOVA for parametric

data and Kruskal–Wallis test for non-parametric data).Differences with p < 0:05 were considered as significant.Statistical analyses were carried out using Statsgraphics

5.1 software (StatSoft, USA).

of the Antarctic bivalve A. colbecki

pH range Homogenate sample volume (ll)

7.0–8.0 20

>8.5 30

7.0–8.0 20

6.5–7.5 30


3. Results and discussion

3.1. Initial screening with different substrates

Results of a preliminary screening of esterase activi-

ties in gills of A. colbecki are displayed in Fig. 2. Results

show that the highest ChE activity was obtained with

PrSCh as a substrate, whilst the hydrolysis of ASCh and

BSCh was noticeably lower. The CbE activity detected

by the hydrolyses of phenylthioacetate was higher than

each of the ChE activities.

These results are in accordance with those obtainedby Mora et al. (1999), who found PrSCh and ASCh to

be the most effective substrate for ChE activity mea-

surements in whole body homogenates of the bivalve

Corbicula fluminea. Similar results have also been re-

ported by Galloway et al. (2002a), who found the fol-

lowing rank of esterase activities substrate affinity

in whole body homogenates of Mytilus edulis: phenyl-

thioacetate >PrSCh>BSCh>ASCh. In comparisonwith other marine mollusc species, the presence of ChEs

020406080

100120140160180

ASCh BSCh PrSCh phenylthioacetate

Substrate (187.5 µM)

Spec

ific

act

ivit

y (d

elta

A m

in-1

)

Fig. 2. Substrate specificity of esterase activities in gills homogenate

preparation of A. colbecki. Mean± standard deviation of duplicate

determinations in D absorbance units·min�1.

0

20

40

60

80

100

120

140

160

180

0.1875 0.375 0.

Substrate conce

este

rase

act

ivit

y (d

elta

A m

in-1

)

BSCh

Fig. 3. Effects of substrate concentration on esterase activities in gills homoge

determinations in D absorbance units·min�1.

with substrate preference for PrSCh was assessed in the

cephalopod Sepia officinalis (Talesa et al., 1993). Other

studies, showing a different ranking of substrate pref-

erence for esterase activities in bivalve such as Mytilusgalloprovincialis (Mora et al., 1999; Talesa et al., 2001)

and C. fluminea (Basack et al., 1998) and in other mol-

lusc species (Bocquen�e et al., 1997), are in accordancewith the well-accepted point of view that a high vari-

ability with regard to esterase specificity to substrate

often occurs both in different invertebrate species as well

as in different tissues of the same organism (Bocquen�eet al., 1990; Le Bris et al., 1995; Najimi et al., 1997; Moraet al., 1999). As regard previous studies inA. colbecki, the

ChE versus ASCh activity levels we detected are of the

same order of magnitude of those measured by Corsi

et al. (in press) in gills of the Antarctic scallop.

3.2. Optimization of substrate concentration

Results showing the effects of substrate concentrationranging from 0.1875 to 3 mM on esterase activities in

gills of A. colbecki are reported in Fig. 3. For each of the

tested substrates, the highest rates of hydrolyses were

detected at the highest substrate concentration used (3

mM). CbE and ChE versus PrSCh and ASCh activities

showed a clear dose-dependence to substrate concen-

tration, while the BSCh-cleaving rate was observed to be

not very sensitive to variation in such parameter, sug-gesting that the enzyme is fully saturated already. It

should be pointed out that the order of substrate pref-

erence of esterase activities changed within the tested

range of substrates concentrations, indicating that the

initial screen was performed at suboptimal concentra-

tions. A substrate concentration equal to 3 mM was

therefore selected for all the subsequent analyses.

75 1.5 3

ntration (mM)

ASCh

phenylthioacetate

PrSCh

nate preparation of A. colbecki. Mean± standard deviation of duplicate


Similar results have been already reported by Mora

et al. (1999), who found the increases of ChE activities

versus ASCh and versus PrSCh in whole body of M.

galloprovincialis to be proportional to substrates con-centration. In the same study, 2 and 5 mM were chosen

as optimal substrate concentrations, for measurement of

ChE versus ASCh in M. galloprovincialis and ChE ver-

sus PrSCh in C. fluminea, respectively. In the present

study, effects of higher substrate concentrations on levels

of esterase activities have not been tested. Another study

performed on specimens of the bivalveM. edulis showed

that maximum rates of hydrolysis were obtained atsubstrate concentrations equal to 3 mM ASCh and 800

lM phenylthioacetate, when haemolymph and whole

body of the bivalve were used as sources of enzymes,

respectively (Galloway et al., 2002b).

3.3. Optimization of pH

Results showing the effects of pH values ranging from6.0 to 9.0 on esterase activities in gills of A. colbecki are

reported in Fig. 4. Assay conditions were as follows:

3 mM substrate concentration, 30 ll of sample homo-genate, 25 �C temperature and pH values ranging from6 to 9 at 0.5 intervals.

The maximum activity was measured for pH values

between 7.0 and 8.0 for both ASCh- and PrSCh-

dependent ChE activities and between 6.5 and 7.5 forthe CbE activity (phenylthioacetate). When BSCh was

used as a substrate, the optimal activity occurred at

pH>8.5. A pH value of 7.4 was selected for all the

subsequent analyses. A study performed by Mora et al.

(1999) provides the best comparison with our results. A

marked effect of pH variation on ChE measurement in

bivalve tissue was shown, with optimal esterase activities

measured at pH ranging between 7.2 and 9.2 and be-

0

10

20

30

40

50

60

6.0 6.5 7.0

p

Est

eras

e ac

tivi

ty

(nm

ol m

in-1

mg

prot

-1)

Fig. 4. Effects of pH on esterase activities in gills homogenate preparation of

nmol·mg prot�1 ·min�1.

tween 8.0 and 9.2 for the detection of ChE versus ASCh

in whole body ofM. galloprovincialis and of ChE versus

PrSCh in whole body of C. fluminea, respectively. Our

results are also comparable with those reported byNajimi et al. (1997), who measured the optimal ChE

activity in M. galloprovincialis and Perna perna whole

body extract at pH ranging between 6.0 and 8.5. In

conclusion a difference in sensitivity of the various

esterase activities to pH variations has been reported

and enzymes belonging to different bivalve species have

showed different patterns of variability within the same

pH range (Najimi et al., 1997; Mora et al., 1999).

3.4. Optimization of homogenate sample volume

Results showing the effects of homogenate sample

volume ranging from 10 to 50 ll on esterase activities ingills of A. colbecki are reported in Fig. 5. Assay condi-

tions were as follows: 3 mM substrate concentration,

pH 7.4, temperature 25 �C.Each esterase activity showed reaction rates linearly

and directly proportional to the sample volume in the

range tested, in accordance with results reported by

Galloway et al. (2002b) in M. edulis. The highest values

of ChE versus ASCh and PrSCh were found at 20 ll,while the same was true at 30 ll for the BSCh-cleavingChE activity and CbE activities. Consequently, the

above sample volumes were selected for each esteraseactivity determination in the following analyses.

3.5. In vitro effects of chlorpyrifos exposure

Results obtained for A. colbecki in vitro chlorpyrifos

exposure are shown in Fig. 6.

The ChE versus ASCh activity contained in gills ex-

tract decreased in a concentration-dependent manner

7.5 8.0 8.5 9.0

H

ASCh

BSCh

PrSCh

Phenylthioacetate

A. colbecki. Mean± standard deviation of duplicate determinations in

R=0.9982; p<0.001

0

20

40

60

80

100

120

R=0.9982; p<0.001

sample volume (µl)

0

20

40

60

80

100

120

R=0.9942; p<0.001

0 10 20 30 40 50

este

rase

ver

sus

phen

ylth

ioac

etat

e ac

tivi

ty

0 10 20 30 40 50 0 10 20 30 40 50

0 10 20 30 40 50 600

20

40

60

80

100

120

140

160R=0.9973; p<0.001

sample volume (µl)

0

20

40

60

80

100

120

140

160

180

sample volume (µl)

sample volume (µl)

ChE

ver

sus

ASC

h ac

tivi

ty(n

mol

min

- 1)

(nm

ol m

in- 1

)

(nm

ol m

in-1

)(n

mol

min

- 1)

ChE

ver

sus

PrS

Ch

acti

vity

ChE

ver

sus

BSC

h ac

tivi

ty

Fig. 5. Effects of variations in sample volume on esterase activities in gills homogenate preparation of A. colbecki. Mean± standard deviation of

duplicate determinations in nmol·min�1.


within the exposure range (0.1–100 lM) with significantinhibition occurring even at the lowest concentration(0.1 M chlorpyrifos). At the highest pesticide doses (60

and 100 lM), only 8.37% of the control activity re-

mained. The inhibition reached suggest the possible

formation of the chlorpyrifos oxon active form and/or

the presence of other esterase-inhibitors in the prepa-

ration. Similar results were obtained by Galloway et al.

0

10

20

30

40

50

60

70

80

90

100

0 0.1 0.5 1

Chlorpyryfos

Res

idua

l C

hE v

ersu

s A

SCh

acti

vity

(%

)

Fig. 6. In vitro effects of chlorpyrifos 5 min incubation on ChE versus ASC

expressed as the % of unexposed samples activity and represent the mean of

(2002b), who observed a concentration-dependent

inhibitory effect of 15 min in vitro exposure to chlor-pyrifos upon both ChE versus ASCh and CbE activities,

measured in haemolymph and whole body tissue homo-

genate of the bivalve species M. edulis. Comparison

between results lead us to infer a higher sensitivity of

ChE versus ASCh activity in A. colbecki to chlorpyrifos

compared to species from temperate areas. For instance,

15 30 60 100

concentration (µM)

h activity in gills homogenate preparation of A. colbecki. Values are

triplicate determinations in pool of at least five specimens.


exposure to 15 lM chlorpyrifos elicited a 83.5% inhi-

bition in the Antarctic scallop, whilst doses between 10

and 100 lM resulted in markedly lower reduction of

esterase activities in M. edulis (Galloway et al., 2002b).The tissue composition of A. colbecki has been demon-

strated to reflect environmental quality (Berkman and

Nigro, 1992). In particular, A. colbecki species has a

marked ability to concentrate high levels of certain

contaminants such as cadmium (Mauri et al., 1990) and

mercury, especially in the gills (Bargagli et al., 1998b). In

view of this, the results of the present study highlight the

potential of ChE activity in A. colbecki as a biomarkerof contamination.

Nevertheless, further research is needed to extrapo-

late the importance of ASCh cleaving-ChE activity

reduction in A. colbecki obtained in the laboratory to

both in vivo and in situ exposure to OPs and to other

widespread marine pollutants before it may be routinely

used for biomonitoring purposes.

3.6. In situ study: determination of esterases activity

Results showing values of esterase activities of A.

colbecki collected in the three Antarctic sites are re-

ported in Table 2. Esterase activity levels were highest in

specimens from the site of Ad�elie Cove for both BSCh-and PrSCh-cleaving activities and CbE activity, while

the highest ASCh-cleaving ChE activity was recorded insamples from Thethys Bay. No statistically significant

differences were found for each enzymatic activity be-

tween samples from different sampling stations (Krus-

kal–Wallis) and observed discrepancies in esterase

activities between sites are most likely due to random

interspecific variations.

It is a well-accepted point of view that all human

settlements in Antarctica are potential sources of heavymetals and xenobiotic pollution, even if such environ-

mental contamination is generally low and can be de-

tected only within a few kilometers of scientific stations

(Bargagli, 2000). Several studies have been performed to

assess the anthropic impact of the Italian Base BTN on

the surrounding area. For instance, a research program

by Focardi et al. (1993) measured significantly lower con-

centrations of dichlorodiphenyltrichloroethane (DDT)in fish species (Pagothenia bernacchii, Cryodracus

antarticus and Dissostychus mawsoni) caught around

the Italian Base than in samples collected near to the

Table 2

Esterase activities measured in gills of the Antarctic bivalve A. colbecki from

Sampling site ChE versus ASCha ChE versus BS

Thethys Bay 17.4± 7.6 29.6± 12.7

Road Bay 16.0± 1.8 22.8± 1.8

Ad�elie Cove 10.9± 3.7 29.7± 9.6

aAll values are mean± standard deviation (46 n6 6) and expressed as nm

Japanese Antarctic Station, Syowa (Subramanian et al.,

1983); on the contrary, PCBs contents were higher.

Fuoco et al. (1991) observed that sea water samples

collected in Terra Nova Bay exhibited a decrease in totalPCB concentration in stations located at increasing

distances from the Italian Scientific Base; on the whole,

the studied area showed low PCB contamination. In the

frame of a biomonitoring study where sea water samples

taken from Terra Nova Bay were analyzed for the

presence of organic compounds, Desideri et al. (1989)

observed that the area where the Italian Scientific Sta-

tion is located exhibited a substantial homogeneouspollution state, higher than in neighboring areas more

distant from the anthropic settlement. Particularly, one

of the sampling station situated closer to the Base

showed the highest levels of total PCBs, total hetero-

compounds and total aromatic hydrocarbons such as

benzenic hydrocarbons and PAHs, while even levels of

aliphatic hydrocarbons were one of the higher in com-

parison with the other sampling sites. Interestingly, thepresence of pesticides was observed in almost all studied

areas even if at very low concentration, with samples

from the site situated nearest to the Base showing the

highest levels of lindane and heptachlor. Desideri et al.

(1990) identified biogenic and anthropogenic com-

pounds in samples of matrixes such as sea water, sea

sediment and pack-ice belongings from Terra Nova Bay

and observed that samples taken near the scientific set-tlement had almost always the highest concentration of

n-paraffins, phthalates, alkenes and PAHs. Lower levelsof polychlorinated naphthalenes (PCNs) were observed

by Corsolini et al. (2002) in krill and fish species col-

lected from the Ross Sea in Terra Nova Bay than in

samples from other locations than Antarctica, while

PCDD/F congener concentrations were often less than

the limits of detection. Colombini and Fuoco (1990)determined the total content of PCBs in sea water

samples from Terra Nova Bay and results showed low

contamination in the area under observation. Bargagli

et al. (1998b) detected very low concentrations of total

mercury in the finest fraction of recent marine sediments

from 12 sampling sites located in the surroundings of the

BTN Base and the absence of local mercury deposition

and pollution by the scientific settlement was suggested.Low concentrations of metals of toxicological interest

were determined in sediment samples collected along the

coast of Terra Nova Bay (Giordano et al., 1999); levels

three sampling sites near the Italian Antarctic Base

Cha ChE versus PrSCha Carboxylesterasea

37.3± 12.9 80.0± 17.7

30.3± 0.5 79.4± 3.4

39.3± 11.9 81.8± 23.5

ol·min�1 ·mg prot�1.


of Cd and Pb were lower than in theoretically polluted

areas and values of Ni, Cr, Sn and total organic carbon

were generally characteristic of areas not impacted by

anthropogenic inputs (Larsen and Gaudette, 1995; Borgand Jonsson, 1996). Mazzucotelli et al. (1989) observed

lower levels of trace metals in samples of sediment col-

lected in Terra Nova Bay than in sediments of non-

Antarctic seas. High levels of cadmium were found in

invertebrates such as sponges and mollusks sampled in

an area on the inner continental shelf in Terra Nova

Bay, but it was suggested to be due to both the rapid

regeneration of the element and to its natural occurrenceand bioavailability in the highly productive Antarctic

coastal area (Bargagli et al., 1996). Other biomonitoring

programs carried out on both biotic and abiotic matri-

ces have showed very low concentrations of trace metals

and organochlorine pollutants in samples collected in

the surroundings of the Italian Station (Capelli et al.,

1990; Focardi et al., 1992, 1995).

With regard to studies undertaken utilizing a bio-marker approach, Jim�enez et al. (1999) found enhanceddetoxifying monooxygenase (MFO)-dependent enzy-

matic activities in fish from the site receiving the waste

water from the Base with respect to those measured in

samples from theoretically less polluted areas, and sug-

gested it was due to organic pollutant contamination. A

study relying on both biomarkers and classic chemical

analyses in fish samples demonstrated a slight contam-ination by organochlorine and PAHs in the Road Bay

compared to a pristine area (on the Gerlache Inlet

continental shelf, 5 km from the Base) and higher MFO

activities were also measured (Bargagli et al., 1998a).

However only slight differences in antioxidant defense

enzymatic activities and no significant reductions in

lysosomal membrane stability were measured between

specimens of A. colbecki translocated from GerlacheInlet to the Road Bay (Regoli et al., 1998). Conse-

quently, it was inferred that the anthropic impact caused

by the Base TNB did not have marked deleterious effects

on marine organisms.

Results of the present work suggest that anthropo-

genic activities at the Italian Scientific Base are not

causing any significant contamination of neurotoxic

pollutants in the surrounding marine environment.Nevertheless, it is a well-accepted point of view that

esterase activities may be influenced by environmental

factors not connected with contaminants exposure, such

as water temperature, pH and salinity (Baslow and

Nigrelli, 1964; Hogan, 1970; Edwards and Fisher, 1991).

The above and other water-quality factors (i.e. dissolved

oxygen content) are also reported to affect sensitivity of

aquatic biota to pollutants exposure (Zitko and Carson,1976; Escartin and Porte, 1997).

Therefore, even if samples have been obtained syn-

chronously, it cannot be ruled out that the integrated

influence of such factors may have differentially affected

enzymatic activities in specimens from the three sam-

pling sites, perhaps masking or strengthening differences

in esterase levels between samples belonging from dif-

ferent areas.Moreover, esterase activity levels may also be indi-

rectly linked to other enzymatic systems such as that of

mixed function oxidase (MFO), which have been ob-

served to mediate the first step in the metabolism

of certain OP pesticides to their much more potent

oxon analogues, eliciting a bioactivation phenomena

(Takimoto et al., 1987; Habig and Di Giulio, 1991;

Dauberschmidt et al., 1997; Neri et al., 2003) which mayfinally result in enhanced neurotoxic effect of such

chemicals. Being even such metabolizing enzymes af-

fected by physiological and environmental variations as

well as by exposure to dangerous and widespread

aquatic pollutants such as PAHs, polychlorinated di-

oxins (PCDDs) and PCBs (De Matteis, 1994; Bend,

1994; Livingstone and Goldfarb, 1998; Sheehan and

Power, 1999), their influence on esterase activities mighthave presumably added another factor of uncertainty to

the interpretation of results in the present study.

In marine bivalves the presence of MFO enzymatic

activities have been observed in several bivalve species

(Livingstone et al., 1985; Yawetz et al., 1992; Sol�e et al.,1994; Michel et al., 1995) and their existence may be

presumed in A. colbecki even if, to date, there is only one

study reporting the presence of MFO-linked enzymaticactivities in the Antarctic scallop (Focardi et al., 1994)

and further investigations are still needed.

Finally, it has also been inferred that esterase activi-

ties in A. colbecki are not significantly affected by in vivo

exposure to heavy metals such as Zn2þ (Corsi et al., inpress) and studies concerning effects of complex mixture

of pollutants on such enzymes in the Antarctic bivalve

are still lacking. Consequently a broader deductionconcerning the whole anthropic impact of the Base BTN

on the surrounding aquatic area is still too early.

4. Conclusions

Our results have clearly shown the presence of ChE

and CbE activities in gills of the Antarctic bivalve A.

colbecki. The present study emphasizes the importance

in identifying and standardizing the optimal conditions

for measuring esterase activities. The current method

used a microtitre-plate is a reliable and quick method forassessing ChE and CbE activities in gills from a bivalve

species which has been used in several biomonitoring

studies of the Antarctic (Regoli et al., 1997, 2002).

Moreover, results showed that ChE activities were

inhibited by the OP pesticide chlorpyrifos following an

in vitro exposure, in parallelism with past laboratory

studies performed on bivalve species from temperate

areas (Ozretic and Krajnovic-Ozretic, 1992; Galloway


et al., 2002b; Brown et al., 2004) highlighting the po-

tential of such ecotoxicological tools in A. Colbecki, for

the assessment of the environmental marine quality of

the Antarctic ecosystem. Further studies will be carriedout to characterize these esterase activities and to im-

prove our knowledge about the sensitivity of the en-

zymes to a wider range of pesticides, heavy metals and

widespread water contaminants, with the aim to rou-

tinely apply the measurements of such enzymatic activ-

ities as biomarkers of pollution in the Antarctic sea.

Acknowledgements

The present study was supported financially by funds

of the PNRA (Programma Nazionale di Ricerche in

Antartide) Italian Antarctic Program and is part of S.

Bonacci’s Ph.D. work of research. The work was per-

formed in part at the University of Plymouth, UK. We

thank Dr. Mauro Alberti for helpful advice.

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