An evaluation of hemolymph cholinesterase activities in thetropical scallop, Euvola (Pecten) ziczac, for the rapid assessment

of pesticide exposure

Richard Owen a,*, Lucy Buxton a, Samia Sarkis a, Megan Toaspern a,Anthony Knap a, Michael Depledge b

a Bermuda Biological Station for Research, Ferry Reach, St Georges GE01, Bermudab Plymouth Environmental Center, University of Plymouth, Plymouth PL4 8AA, UK

Abstract

The use of sequential measurements of hemolymph cholinesterase activities as a non-invasive biomarker of seasonal organo-

phosphate/carbamate exposure was investigated for the tropical scallop, Euvola (Pecten) ziczac. Overall activities of both acetyl-

cholinesterase and butyrylcholinesterase were relatively high compared to studies with bivalve tissues. Acute in vivo experiments

showed inhibition of hemolymph acetylcholinesterase activity at concentrations of the organophosphate insecticide chlorpyrifos

of 0.1, 1 and 10 ng l�1. Monthly sampling of hemolymph from scallops at two sites in Bermuda over a 15 month period showedseasonal acetylcholinesterase and butyrylcholinesterase inhibition. Direct and indirect evidence suggests that this inhibition did not

relate to biochemical or physiological changes associated with gonad maturation and spawning, but rather reflected diffuse con-

tamination of the marine environment by cholinesterase inhibitors or increased bioavailability of such inhibitors at these times.

Repetitive sampling of scallop hemolymph for cholinesterase activities represents a rapid, sensitive and non-invasive method for

assessing seasonal, sublethal pesticide exposure in these commercially important bivalves and suggests a wider use in marine pol-

lution monitoring.

� 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Biomarkers; Pesticides; Scallops; Hemolymph; Cholinesterases; Exposure assessment

1. Introduction

A wide range of chemical analysis techniques and bi-

ological effects assays are available for the detection of

exposure to, and adverse effects of anthropogenic chemi-

cal pollutants in marine ecosystems (Depledge and

Hopkin, 1995; Wells et al., 1998). However, resourcelimitations can constrain their application in day to day

environmental management. It has therefore been iden-

tified that there is a pressing need to develop and validate

a suite of rapid, sensitive, easy to use and inexpensive

assessment methods (Wells et al., 2001, amongst others).

In tropical regions, where economies are often based

on agriculture, intensive pesticide use (notably organo-

phosphate and carbamate insecticides) is widespread(Castillo et al., 1997; Mansingh et al., 1997). There is

growing concern over the biological impacts of or-

ganophosphorus and carbamate pesticides known to be

contaminants of estuarine and coastal waters and sedi-

ments (Readman et al., 1992; Mansingh et al., 1997;

Kirby et al., 2000). This is of particular concern in areas

supporting extensive fisheries or mariculture operations.

Despite this, there have been few studies investigatingpotential exposure and impact of non-organochlorine

agrochemicals in tropical marine organisms (Castillo

et al., 1997).

Since the initial work of Ellman et al. (1961), the use

of acetylcholinesterase (EC 3.1.1.7.) activity (AChE)

measurements in both freshwater and marine organisms

as biomarkers of exposure to organophosphate and

carbamate pesticides has been well documented (Vermaet al., 1979; Habig et al., 1988; Day and Scott, 1990;

Galgani and Bocquene, 1990; Bocquene et al., 1990,

1993; Morgan et al., 1990; Escartin and Porte, 1996;

Payne et al., 1996; Lundebye et al., 1997; Radenac et al.,

* Corresponding author. Fax: +1-441-297-8143.

E-mail address: rowen@bbsr.edu (R. Owen).

0025-326X/02/$ - see front matter � 2002 Elsevier Science Ltd. All rights reserved.PII: S0025-326X(02 )00139 -X

www.elsevier.com/locate/marpolbul

Marine Pollution Bulletin 44 (2002) 1010–1017

1998; Kirby et al., 2000). Nearly all studies of acetyl-cholinesterase inhibition in marine molluscs have

focussed on whole organism or muscle extracts of sac-

rificed bivalves (Bocquene, 1997; Galgani and Bocqu-

ene, 1990; Bocquene et al., 1990, 1993; Radenac et al.,

1998). In addition to the destructive nature of these

techniques a further disadvantage in using molluscan

tissue or muscle extracts has been the finding that

associated acetylcholinesterase activities are generallylow, suggesting limited usefulness of the tissue or

muscle extract assay in biomonitoring studies (Bocqu-

ene et al., 1990). Several studies have confirmed the

presence of cholinesterase enzymes in molluscan ad-

ductor muscle hemolymph (Winners et al., 1978; Von

Wachtendonk and Neef, 1979). Non-destructive tech-

niques involving plasma and hemolymph acetylcholin-

esterase measurements in marine organisms have beenfew in number, but have for example highlighted the

potential for their use as biomarkers of organophos-

phate exposure in crustacea (Fossi et al., 1996; Lun-

debye et al., 1997).

The aims of the work described here were to develop

a sensitive hemolymph cholinesterase inhibition assay

for the commercially important tropical scallop, Euvola

(Pecten) ziczac, that permitted repetitive, non-destruc-tive sampling of individual bivalve molluscs as part of a

long term mariculture or marine pollution monitoring

program, without the need for sacrifice. This then al-

lowed seasonal variability in enzyme activities asso-

ciated with variations in organism physiology and

anthropogenic stressors to be assessed.

2. Methods

2.1. Laboratory organophosphate exposures

Adult scallops, E. ziczac, (47–57 mm shell height)

were collected by SCUBA from culture enclosures in

Harrington Sound, Bermuda, and maintained at 22 �Cin the laboratory in filtered, flowing seawater for 10 daysprior to the experiment. For the experiment, scallops

were transferred to clean 2 l glass beakers containing 1

lm filtered seawater, one scallop transferred to eachbeaker. Beakers were placed in water baths at 22 �C andgently aerated for the experimental duration. After a 3 h

stabilization period hemolymph samples were taken

from scallops in both control and treatment groups for

analysis. Individuals (4 per treatment/control) were thenexposed to 10, 1, 0.1 and 0 ng l�1 of Dursbane (chlor-pyrifos). Chlorpyrifos was selected as a representative

organophosphate since it is known to be particularly

toxic to aquatic invertebrates, and is used both in Ber-

muda and in many other tropical regions (Castillo et al.,

1997). A survey of import and stock inventory records

showed chlorpyrifos to be used both domestically and in

insect control on the numerous golf courses present inBermuda. It is also environmentally persistent and has

been reported as a contaminant in tropical marine en-

vironments (Readman et al., 1992; Mansingh et al.,

1997). Chlorpyrifos standards were made up in acetone

with equivalent volumes of acetone (1 ll) being added toeach control beaker.

Hemolymph samples were taken over 8 h after initial

exposure, at 2 h interval. Approximately 250 ll of he-molymph was extracted from the adductor muscle of

each scallop using ac 5 cc syringe fitted with a 21 gauge

needle and stored within a 2.5 ml siliconized flip top

microcentrifuge tube at 5 �C. To minimize any potentialimpact of sampling on enzyme activity, a standardized

sample collection procedure was employed whereby

scallops were gently removed from the water, the syringe

needle carefully inserted into the adductor muscle viathe opening at the base of the shell valves, and the he-

molymph gently withdrawn, a process that was rapid

(�1 min). Measurements of acetylcholinesterase activ-ity were made within 12 h of sample collection (Von

Wachtendonk and Neef, 1979; Bocquene et al., 1990) as

outlined below. It was confirmed that cholinesterase

measurements made immediately after hemolymph col-

lection did not differ from those made on the same re-frigerated sample 12 h after collection.

2.2. Seasonal variation in hemolymph cholinesterase

activity

Two sites located in Bermuda’s inshore waters were

selected as study sites; Rabbit Island in Harrington

Sound, and Agar’s Island in the Great Sound (Fig. 1).Harrington Sound is an almost completely enclosed

body of water with high water residence time (in excess

of 140 days) that is a habitat for many of the recrea-

tionally fished bivalves in Bermuda, as well as to

other shellfish. It receives 60% of its input from saline

groundwater recharge from adjacent caves and the re-

mainder from exchange with the Northern Lagoon via a

narrow tidal inlet. Harrington Sound is bordered byresidential housing and a golf course along the north-

eastern shore. Agar’s Island is located in a much larger

body of water (The Great Sound) with much shorter

water residence time (22 days) and higher rates of water

exchange with the Northern Lagoon. There are areas of

commercial pesticide application in areas adjacent to the

Great Sound. Additionally, Mills’Creek, which receives

some limited industrial discharge from Pembroke Canaland contaminants from ship yards and marinas, is 0.5

km to the north of Agar’s Island, flowing into the Great

Sound. The stock inventory and import record survey

showed commonly used organophosphates and car-

bamates in Bermuda to include acephate, bendiocarb,

carbaryl, chlorpyrifos, diazinon, dimethoate and mala-

thion.

R. Owen et al. / Marine Pollution Bulletin 44 (2002) 1010–1017 1011

Adult scallops, E. ziczac (69–82 mm shell height) were

deployed at each site on a sandy bottom, at 5 m water

depth. Culture enclosures were meshed with 100 poly-ethylene on sides and top, protecting animals from

predators, but allowing scallops to recess in the sand as

in their natural environment. Densities were low

(10 scallops m�2) and cages were scrubbed clean on amonthly basis allowing for optimum water flow, thus

minimizing any stress due to cage effect. Hemolymph

samples were collected for acetylcholinesterase activity

measurements from each scallop on a near monthlybasis over the period February 2000–April 2001. Bu-

tyrylcholinesterase activity measurements were initiated

in August 2000 and continued until April 2001.

2.3. Measurement of hemolymph cholinesterase activities

and protein concentrations

The methods of Ellman et al. (1961) and Lundebyeet al. (1997) were optimized to allow measurements of

acetylcholinesterase and butyrylcholinesterase activity

in scallop adductor muscle hemolymph. Hemolymph

samples were centrifuged at 10,000� g to remove he-mocytes. 50 ll of the resulting supernatant was added to

1.45 ml of 0.27 mM 5050dithiobis (2-nitrobenzoic acid)(DTNB) made up in phosphate buffer, pH 7.4. After

addition of 50 ll of the substrate analogue acetylthio-choline iodide (0.075 mM), the rate of production of

the yellow anion 5-thio-2-nitrobenzoic acid was mea-

sured spectrophotometrically at 412 nm over 1 min and

expressed as Umin�1, where U ¼ 0:001 optical densityafter blank corrections. Although measurements of the

initial endogenous reaction between hemolymph and

DTNB reported for crab hemolymph (Lundebye et al.,

1997) were made for each sample, this reaction wasfound to be negligible in scallops. Identical measure-

ments of butyrylcholinesterase activity were made using

the substrate analogue butyrylthiocholine iodide. As

cholinesterase activity measurements in marine organ-

isms are sensitive to assay temperature (Bocquene

et al., 1990), assay temperatures were carefully moni-

tored (ranging between 18 and 24 �C) to ensure that theyremained within the optimal temperature range estab-lished by these authors.

Measurements of cholinesterase activity have con-

ventionally been normalized against tissue protein con-

tent to allow for variations in enzyme concentrations

with tissue weight. Scallop hemolymph protein concen-

Fig. 1. Scallop deployment sites (r) in Bermuda.

1012 R. Owen et al. / Marine Pollution Bulletin 44 (2002) 1010–1017

trations were determined spectrophotometrically at 595nm using hemolymph (diluted 1:1 with deionized water)

and protein assay reagent (Biorad), standardised over

the range 0.1–1 mgml�1 with bovine serum albumin.

2.4. Results

Hemolymph acetylcholinesterase activities for E.

ziczac exposed to chlorpyrifos at concentrations of 10, 1and 0.1 ng l�1 under experimental conditions are shownin Fig. 2a–c. Acetylcholinesterase activities in the con-

trol group averaged 857 Umin�1 (1 sd ¼ 140 Umin�1),over the duration of the experiment. Enzyme inhibition

was exhibited at all three concentrations of chlorpyrifos.

Between 2 and 8 h at concentrations of 10 ng l�1,activities averaged 454 Umin�1 (1 sd ¼ 123 Umin�1),(Fig. 2a). Acetylcholinesterase inhibition was also evi-dent after 4 and 6 h, respectively at concentrations of 1

and 0.1 ng l�1 (Fig. 2b and c). At 1 ng l�1 mean activitywas 530 Umin�1 (1 sd ¼ 105 Umin�1), between 4 and 8h; at 0.1 ng l�1 mean activity was 479 Umin�1 (1 sd ¼91 Umin�1), between 6 and 8 h. Pairwise comparisonsshowed significant differences (p < 0:01) between con-trol and treatment data at all three concentrations dur-

ing these time periods.

Fig. 2. Adductor muscle hemolymph acetylcholinesterase activities for

E. ziczac after exposure to 10, 1 and 0.1 ng l�1 of Dursban (chlor-pyrifos). Error bars ¼ �1 standard deviation, n ¼ 4 for each treat-ment/control group.

Fig. 3. Adductor muscle hemolymph acetylcholinesterase and butyr-

ylcholinesterase activities activities for E. ziczac deployed at Agar’s

Island February 2000–April 2001. Error bars ¼ �1 standard deviation,n ¼ 10.

Fig. 4. Adductor muscle hemolymph acetylcholinesterase and butyr-

ylcholinesterase activities activities for E. ziczac deployed at Harring-

ton Sound February 2000–April 2001. Error bars ¼ �1 standarddeviation, n ¼ 10.

R. Owen et al. / Marine Pollution Bulletin 44 (2002) 1010–1017 1013

Figs. 3a and 4a show adductor hemolymph acetyl-cholinesterase activities for scallops sampled at Agar’s

Island and Harrington Sound over the 15 month period

February 2000–April 2001. In general, acetylcholinest-

erase activities at both sites were comparable to those of

the control scallops in the dose response experiments,

averaging 899 Umin�1 (1 sd ¼ 132 Umin�1), and 847Umin�1 (1 sd ¼ 128 Umin�1), for Agar’s Island andHarrington Sound, respectively, over the year. How-ever, three notable periods of enzyme inhibition oc-

curred. Acetylcholinesterase activities were much lower

in Harrington Sound in February 2000 and February

2001 (averaging 458 and 525 Umin�1, respectively).Whilst there were no data for Agar’s Island in February

2000, depreciations in AChE were also seen at this site in

February–March 2001 (271 and 320 Umin�1, respec-tively). Depreciation in AChE was also evident at bothsites in August 2000 (averaging 319 and 340 Umin�1,respectively). Butrylcholinesterase activities for scallops

sampled at Agar’s Island and Harrington Sound areshown in Figs. 3b and 4b. Although the data only

spanned the period August 2000–April 2001, inhibitions

of enzyme activity were also evident at both sites in

August 2000 and February–March 2001.

3. Discussion

Acetylcholinesterase activity has conventionally

been normalized against tissue/hemolymph protein con-

tent and expressed in terms of Umin�1 mg protein�1,(Galgani and Bocquene, 1990; Lundebye et al., 1997;

Radenac et al., 1998). The rationale for this normal-

ization has been to correct for variations in enzyme

concentration as a function of weight of homogenized

tissue analysed/hemolymph protein content.Hemolymph protein concentrations were not con-

stant in either group of scallops over the duration of the

field study (Fig. 5). Protein concentrations approxi-

mated 0.5 mgml�1 for most of the year, comparableto those reported for a number of bivalve species by

Winners et al. (1978). However, concentrations rose to

approximately 2 mgml�1 in April 2000 for scallops atboth sites. Although it might reasonably be assumedthat a positive relationship between Umin�1 andhemolymph protein concentration might exist, no re-

lationship was found for data from either site (R2 ¼0:01–0:03 for linear and power fits to data). In contrast,significant inverse relationships (power functions, R2 ¼0:88 and 0.97) were found between hemolymph proteinand normalized acetylcholinesterase activities for Har-

rington Sound and Agar’s Island respectively (Fig. 6aand b).

Radenac et al. (1998) have suggested that while pro-

tein content of mussels (Mytilus edulis) may vary with

the organism’s physiology, it is mainly structural pro-

teins that are used as an energetic pool rather than active

proteins such as enzymes. If hemolymph cholinesterases

represent an approximately constant fraction of a vari-

able total hemolymph protein pool, then Umin�1 wouldbe independent of variations in total hemolymph protein

concentration. This is reflected in the lack of relation-

ship between Umin�1 and hemolymph protein con-centration above. In contrast, additions of structural

proteins to the total hemolymph protein pool would be

expected to decrease normalized enzyme activity and

subtractions increase normalized enzyme activity, as

observed in Fig. 6a and b. It should be noted that, ingeneral, hemolymph proteins are low in concentration,

contrasting with higher soluble protein values for M.

edulis adductor muscle of 10 mgml�1 (Galgani andBocquene, 1990). Thus, small changes in protein con-

centration in the scallop hemolymph might have a large

influence on protein-normalized cholinesterase activi-

ties. Consequently, when using the AChE activity bio-

Fig. 5. Hemolymph protein concentrations for scallops deployed at

Harrington Sound and Agar’s Island, February 2000–April 2001.

Fig. 6. Plot of hemolymph protein against normalized acetylcholin-

esterase activity for scallops deployed at Harrington Sound and Agar’s

Island.

1014 R. Owen et al. / Marine Pollution Bulletin 44 (2002) 1010–1017

marker, it is advisable not to normalize values againsthemolymph protein concentrations, but rather to ex-

press them in terms of Umin�1.Three periods of acetylcholinesterase activity depre-

ciation were found within the field study: February

2000, August 2000 and February–March 2001. An

initial conclusion is that these periods of AChE depre-

ciation reflect environmental exposure to organophos-

phates/carbamates. However, it is necessary to becautious because the synchronous, high levels of inhi-

bition at both sites may indicate a common physiolog-

ically related mediation of enzyme activity. For

example, inhibition may relate in some way to bio-

chemical or physiological changes associated with gonad

maturation and spawning. Gonad maturation and

spawning occurs in this species between late Autumn

and May in local waters (Manuel, 2001) and the syn-chronous enzyme inhibition observed in February–

March could reflect this. However, indirect and direct

evidence suggests this is unlikely. In general, the local

scallop population does not show synchronicity in

gametogenesis and spawning, such that various stages

of the reproductive cycle are seen at any one time

throughout these months; for this reason, these animals

have been termed as ‘‘dribble spawners’’ (Manuel,2001). Acetylcholinesterase activities in E. ziczac were

neither consistently low nor highly variable during the

December–April period. Additionally, scallops in the

same size range maintained in the laboratory for a

month and spawned in April 2001 showed no inhibition

of acetylcholinesterase activity immediately prior to

spawning when gonad maturation was complete (1232

Umin�1, 1 sd ¼ 171 Umin�1). While a physiologicallyrelated influence upon AChE cannot be ruled out, we

have as yet no evidence for this, although further work

should address this possibility.

Assuming that the observed depreciations in AChE

do not reflect a physiological influence upon enzyme

activity, then impacts of one or more environmental

contaminants present at these times might be implicated.

Several authors (Day and Scott, 1990; Morgan et al.,1990; Escartin and Porte, 1996; Lundebye et al., 1997)

have suggested that inhibition of acetylcholinesterase

activity in excess of 20% from normal reflects exposure

to organosphosphates and/or carbamates. From the

field data sets obtained here, the normal range of acet-

ylcholinesterase enzyme activity for E. ziczac hemol-

ymph in unexposed conditions averages 872 Umin�1 (1sd ¼ 129 Umin�1). This is comparable to control valuesfor the unexposed experimental scallops sampled in

August (857 Umin�1 (1 sd ¼ 76 Umin�1)). In August2000, scallops in Harrington Sound and Agar’s Island

exhibited 63% and 61% inhibition, respectively, relative

to this reference value. Inhibitions of 47% and 40% were

evident in February 2000 and February 2001 at Har-

rington Sound and inhibitions of 69% and 63% were

also evident in February–March 2001 at Agar’s Island.This may indicate the presence of one or more organo-

phosphates/carbamates or other AChE inhibiting con-

taminants in these marine habitats at these times.

In addition to organophosphates and carbamates,

exposure of marine organisms to a number of inor-

ganic contaminants may potentially be associated with

acetylcholinesterase inhibition. Acetylcholinesterase re-

sponses to metal exposure in vivo and in vitro has beenshown to be variable in fish tissues for Cd (Olson and

Christensen, 1980; Gill et al., 1991). Cu has been shown

to be an inhibitor of AChE (Mukherjee and Bhattach-

arya, 1974; Olson and Christensen, 1980). However, the

range of concentrations at which inhibition has been

reported for these metals far exceed concentrations re-

ported for the inshore waters of Bermuda (Connelly,

1997), Table 1, which were not associated with anydistinct seasonal trends. While it has been suggested

(Bocquene et al., 1995; Payne et al., 1996) that heavy

metals at environmental concentrations are not signifi-

cant cholinesterase inhibitors, studies have shown that

fish exposed to industrial effluents or receiving waters

from paper mill activity are associated with brain AChE

inhibition (Mukherjee and Bhattacharya, 1974; Payne

et al., 1996). While industrial activity in Bermuda isnegligible, it cannot be ruled out that the observed pe-

riods of hemolymph AChE inhibition in this study

reflect influence of xenobiotics other than organophos-

phates and carbamates. Analyses of sediments, water

and biota have to date not been undertaken for or-

ganophosphate and carbamate compounds in Ber-

muda’s inshore marine environment. We advocate the

approach that the hemolymph cholinesterase assay beused as a sensitive, rapid and inexpensive tool to provide

initial, unconfirmed evidence of exposure to biota by

these compounds on spatial and/or temporal scales. This

Table 1

Inorganic and organic contaminants in the Great Sound and Har-

rington Sound

Cd Cu TbT PAH

Great Sound

seawater

(nmol l�1)

0.2a 4.0a 0.04b n/a

Great Sound

sediments

(lg g�1)

5.2a 74.1a n/a 0.1c

Harrington

Sound sea-

water

(nmol l�1)

0.2a 5.3a n/a n/a

Harrington

Sound sedi-

ments (lg g�1)

0.2a 13.8a n/a n/a

aConnelly (1997).b Connelly et al. (2001).c Burns et al. (1990).

R. Owen et al. / Marine Pollution Bulletin 44 (2002) 1010–1017 1015

then allows targeting of chemical analyses of sediments,water and biota by conventional high resolution tech-

niques, an approach envisaged for exposure assessment

at these sites.

In general, protein-normalized acetylcholinesterase

activities for months not including March and April

(periods of high variation in hemolymph protein) av-

eraged between 1200 and 5100 Umin�1 mg protein�1.These values are comparable to the range of values re-ported by Bocquene et al. (1993) for a number of fish

species in the North Atlantic and English Channel (1100–

4300 Umin�1 mg protein�1) but are considerably higherthan mussel adductor extract activities (which averaged

only 228 Umin�1 mg protein�1) and scallop adductormuscle extract activities (which were in the range 396–

590 Umin�1 mg protein�1, Bocquene, 1997). The lowrange of acetylcholinesterase activities for molluscantissues has led to the conclusion that the tissue or muscle

extract assay for molluscs is of limited value (Bocquene

et al., 1990). In contrast, the relatively high scallop he-

molymph acetylcholinesterase activities reported in this

study demonstrate that the hemolymph assay as applied

to this commercially important organism may be of far

greater use in providing early warning of a sublethal

pollution effect in coastal waters. The experimental re-sults of this study further showed that at environmentally

relevant concentrations of the organosphosphate insec-

ticide chlorpyrifos, substantial inhibition of acetylcho-

linesterase activity occurs. These findings confirm earlier

reports of similar AChE inhibition at sublethal concen-

trations of organophosphates for other aquatic inverte-

brates (Day and Scott, 1990; Escartin and Porte, 1996).

Additionally, the ability to sample the same scallop non-invasively on a seasonal basis makes this particular ad-

aptation of the classical cholinesterase inhibition assay

extremely attractive as part of a mariculture or marine

pollution monitoring program.

It should be noted that results of incubation experi-

ments of whole organism extracts of mussels and several

freshwater invertebrates have shown that the sensitivity

of the inhibitory effect is variable, dependent upon thepesticide to which the organism is exposed (Galgani and

Bocquene, 1990; Day and Scott, 1990). Clearly more

experimental work is needed to assess the sensitivity of

the scallop hemolymph acetylcholinesterase biomarker

assay to a range of organosphosphates and carbamates

(both individually and in combination) as well as to

other non-pesticide organic contaminants before fully

incorporating this tool in environmental managementpractices.

Acknowledgements

We gratefully acknowledge the helpful comments

made by Tamara Galloway (PERC) and assistance of

Giles Watson (PERC). This work was funded by theMinistry of the Environment, Bermuda Government.

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