Rapid assessment of organophosphorous/carbamate exposure in the bivalve mollusc Mytilus edulis using combined esterase activities as biomarkers

Article abstract of DOI:10.1016/S0166-445X(02)00051-6

Carboxylesterases in bivalve molluscs exhibit greater sensitivity to organophosphorous and carbamate pesticides than acetylcholinesterase and are present at higher levels. The aim of the present study was to combine measurement of both acetylcholinesterase and carboxylesterase activities in the marine bivalve Mytilus edulis in order to detect the effects of pesticide exposure. Spectrophotometric assays in microtitreplate format were optimised for use with M. edulis haemolymph and tissue homogenate samples. This permitted the nature and distribution of the enzymes to be determined. One predominant pharmacological form of activity consistent in its patterns of activation and inhibition with acetylcholinesterase was identified in the haemolymph with an apparent K_m for acetylthiocholineiodide of 1.33 mM. Carboxylesterase activity in the tissues was characterised by its preferential hydrolysis of the substrate analogue phenylthioacetate. Concentration-dependent inhibition of both activities was demonstrated following in vitro incubation with diisopropylfluorophosphate (DFP), paraoxon and eserine in the range 0.1-3.0 mM. When M. edulis (n=10) were exposed for 24 h to concentrations of eserine or paraoxon of 0.05-1.0 mM, the percentage inhibition of acetylcholinesterase was in each case greater than for carboxylesterase and reached statistical significance at lower concentrations. In all exposures, a proportion of carboxylesterase activity was present which remained resistant to inhibition by either organophosphorous or carbamate compounds. The ecotoxicological significance of these findings for the environmental monitoring of pesticide exposure is discussed.

Full text of DOI:10.1016/S0166-445X(02)00051-6

Aquatic Toxicology 61 (2002) 169–180
www.elsevier.com/locate/aquatox
Rapid assessment of organophosphorous/carbamate
exposure in the bivalve mollusc Mytilus edulis using
combined esterase activities as biomarkers
Tamara S. Galloway *, Nicholas Millward, Mark A. Browne,
Michael H. Depledge
Plymouth En6ironmental Research Centre, Uni6ersity of Plymouth, 6, Queen Anne Terrace, Drake Circus,
Plymouth PL4 8AA, UK
Received 19 October 2001; received in revised form 29 March 2002; accepted 1 April 2002
Abstract
Carboxylesterases in bivalve molluscs exhibit greater sensitivity to organophosphorous and carbamate pesticides
than acetylcholinesterase and are present at higher levels. The aim of the present study was to combine measurement
of both acetylcholinesterase and carboxylesterase activities in the marine bivalve Mytilus edulis in order to detect the
effects of pesticide exposure. Spectrophotometric assays in microtitreplate format were optimised for use with M.
edulis haemolymph and tissue homogenate samples. This permitted the nature and distribution of the enzymes to be
determined. One predominant pharmacological form of activity consistent in its patterns of activation and inhibition
with acetylcholinesterase was identified in the haemolymph with an apparent Km for acetylthiocholineiodide of 1.33
mM. Carboxylesterase activity in the tissues was characterised by its preferential hydrolysis of the substrate analogue
phenylthioacetate. Concentration-dependent inhibition of both activities was demonstrated following in vitro incuba-
tion with diisopropylfluorophosphate (DFP), paraoxon and eserine in the range 0.1–3.0 mM. When M. edulis (n=10)
were exposed for 24 h to concentrations of eserine or paraoxon of 0.05–1.0 mM, the percentage inhibition of
acetylcholinesterase was in each case greater than for carboxylesterase and reached statistical significance at lower
concentrations. In all exposures, a proportion of carboxylesterase activity was present which remained resistant to
inhibition by either organophosphorous or carbamate compounds. The ecotoxicological significance of these findings
for the environmental monitoring of pesticide exposure is discussed. © 2002 Elsevier Science B.V. All rights reserved.
Keywords: Acetylcholinesterase; Carboxylesterase; Mytilus edulis; Pesticide; Biomarker; Ecotoxicology
1. Introduction
Organophosphorous and carbamate com-
pounds are used extensively as pesticides in urban,
* Corresponding author. Tel.: +44-1752-232-904; fax: +
agricultural and aquacultural situations. Despite
44-1752-232-970
their rapid hydrolysis in air, water and soil, bioac-
E-mail address: tgalloway@plmouth.ac.uk (T.S. Galloway).
0166-445X/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S0166-445X(02)00051-6

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T.S. Galloway et al. / Aquatic Toxicology 61 (2002) 169–180
cumulation of organophosphorous compounds in
sediments has been documented at appreciable
levels in estuaries and coastal locations, represent-
ing a potential threat to biota (Readman et al.,
1992). Organophosphorous and carbamate pesti-
cides exert acute toxicity by inhibiting acetyl-
cholinesterase (E.C 3.1.1.7), a serine hydrolase
found in neuromuscular junctions. This leads to
susceptible species in accumulation of the neuro-
transmitter acetylcholine and subsequent hyper-
polarisation of the post synaptic membrane.
Measurement of peripheral acetylcholinesterase
activity levels is a useful, dose dependent means of
monitoring exposure to organophosphorous and
carbamate compounds in humans and other verte-
brate species (Carlock et al., 1999), yet extrapola-
tion of this approach to encompass aquatic
invertebrate species that are commonly used in
the environmental monitoring of coastal loca-
tions, has been problematic (Fulton and Key,
2001). Acetylcholinesterases exhibit genetic and
molecular polymorphism and their distributions
and physiological roles differ among species
(Massoullie et al., 1993; Scaps et al., 1996; Forget
and Bocquene, 1999). As a consequence, the de-
gree of inhibition associated with toxicity is highly
variable (Lundebye et al., 1997). Baseline data is
often lacking, or has revealed large variations
both within and between populations (Rattner
and Fairbrother, 1991). Activity may be modu-
lated by seasonal and nutritional variables (Ed-
wards and Fisher, 1991; Escartin and Porte, 1997)
or by chronic toxicant exposures which can lead,
in turn, to elevated levels of activity (Thompson et
al., 1991).
years ago (Von Scramlik, 1941), there have been
relatively few published studies of the use of
acetylcholinesterase activity as a biomarker of
organophosphorous pesticide exposure in bivalves
(Najimi et al., 1997; Escartin and Porte, 1997;
Radenac et al., 1998; Basack et al., 1998; Nar-
bonne et al., 2001). This reflects low endogenous
activity (Najimi et al., 1997; Narbonne et al.,
2001) and the relative insensitivity of inhibition by
organophosphorous compounds compared with
enzymes from other species (Von Wachtendonk
and Neef, 1979; Mora et al., 1999).
In addition to their toxic action on acetyl-
cholinesterase, organophosphorous and carba-
mate compounds may also inhibit other classes of
serine hydrolase including carboxylesterases [EC
3.1.1.1] which are typically present in all tissues.
Carboxylesterases hydrolyse a wide range of en-
dogenous and exogenous esters (Parkinson, 1996).
Their tissue distribution and exact physiological
role is often unknown and varies among species
but they are believed to be important in the
hydrolytic detoxication of some organophospho-
rous compounds and play an additional protective
role as alternative sites of organophosphate bind-
ing and phosphorylation (Jokanovic et al., 1996;
Walker, 2001). Carboxylesterase activity in
bivalve molluscs exhibits higher sensitivity to
organophosphorous compounds than acetyl-
cholinesterases (Escartin and Porte, 1997) and its
measurable activity is greater (Basack et al.,
1998). It is therefore, possible that the combined
monitoring of carboxylesterase and acetyl-
cholinesterase activities may provide a more use-
ful indication of organophosphorous pesticide
exposure in bivalve species than the measurement
of acetylcholinesterase activity alone (Escartin
and Porte, 1997; Basack et al., 1998).
In coastal marine environments, the choice of
bivalve molluscs such as the blue mussel, Mytilus
edulis as sentinel species for the ecotoxicological
assessment of pollution has many advantages.
These organisms are sessile filter feeders, able to
accumulate a wide range of contaminants and
reflect changes in the contaminant status of the
environment. M. edulis is found extensively in
littoral and shallow temperate waters as large
beds in both open waters and brackish estuaries
where agricultural runoff is at its highest. Al-
though the occurrence of cholinesterase activity in
molluscan haemolymph was first reported many
To use enzyme activities such as acetyl-
cholinesterase and carboxylesterase as accurate
and sensitive biomarkers of water contamination,
it is important not only that the enzymes are
adequately characterised in the species in ques-
tion, but also that the assays themselves are suffi-
ciently robust and accessible to permit their use to
make measurements of activity of adequate accu-
racy and precision to detect subtle changes in
activity. With this aim, parallel modifications to

T.S. Galloway et al. / Aquatic Toxicology 61 (2002) 169–180
171
the method of Ellman et al. (1961) using substrate
analogues specific for either cholinesterase or car-
boxylesterase activities have been adapted for use
in M. edulis tissues and body fluids. The distribu-
tion and characteristics of acetylcholinesterase
and carboxylesterase activities were then deter-
mined and the sensitivity of the enzymes to inhibi-
tion both in vitro and in vivo to
organophosphorous and carbamate compounds
was evaluated. The utility of combining these
assays as rapid and reliable means of monitoring
exposure to pesticides is discussed.
carded.
The
supernatant
fraction
was
re-centrifuged at 10 000×g and the supernatant
fraction retained and used for subsequent
analyses.
2.3. Enzyme acti6ity
Enzyme activities were assayed at 25 °C in
microtitreplate format using modifications to the
method of Ellman et al. (1961) in which thio-
choline or thioacetate derivatives are hydrolysed
by acetylcholineterase or carboxylesterase respec-
tively to yield thiocholine or thioacetate. Subse-
quent combination with DTNB forms the yellow
anion 5-thio-2-nitrobenzoic acid which absorbs
strongly at 412 nm. Initial experiments revealed
the optimum rate of hydrolysis to occur between
pH 7 and 8 and assays were subsequently con-
ducted at pH 7.4. The sample or buffer blank (50
ml) was incubated for 5 min at 25 °C with 150 ml
DTNB, 270 mM in 50 mM sodium phosphate, pH
7.4. Measurement of acetylcholinesterase activity
was initiated by the addition of acetylthiocholine,
3 mM, and the absorbance recorded at 412 nm.
Carboxylesterase activity was assayed in a similar
manner with a reduced sample size of 25 ml and
with the substitution of phenylthioacetate, 800mM
as substrate analogue. Spontaneous substrate hy-
drolysis was determined in the absence of
haemolymph. The results were calculated as nmol
substrate hydrolysed min−1 mg−1 relative to the
total protein content of the sample. Total protein
was determined using a commercial kit (BioRad)
with bovine serum albumin as standard.
2. Materials and methods
2.1. Chemicals
Acetylthiocholine iodide, butyrylthiocholine io-
dide, propoinylthiocholine iodide, phenylthioac-
etate,
5,5%-dithiobis-2-dinitrobenzoic
acid
(DTNB), tetra(monoisopropyl)pyrophosphor-te-
tramide (iso-OMPA), physostigmine (eserine), di-
isopropylfluorophosphate
(DFP),
diethyl
p-nitrophenyl phosphate (paraoxon) and phenyl-
methylsulphonylfluoride (PMSF) were from
Sigma Chemical Co., Poole, Dorset. All other
reagents and solvents were analytical grade.
2.2. Collection of organisms
Specimens of M. edulis of shell length 5–7 cm
were collected monthly between May and Septem-
ber from Whitsand Bay, Cornwall and housed in
filtered sea water in the aquarium in 10 l glass
tanks at 14=/−2C with a 12-h light:12-h dark
cycle. Samples of haemolymph were withdrawn
by syringe from the posterior adductor muscle
and transferred to small plastic centrifuge tubes
on ice. Haemolymph was centrifuged at 5000×g
for 3 min and the supernatant used for subse-
quent analysis. Tissue homogenate was prepared
by excising the whole body from the shell, placing
it on filter paper to drain excess fluid, and record-
ing the weight. The tissue was homogenised with a
1:5 ratio (w/v) of 0.1 M Tris–HCl buffer pH 7.2
containing 0.25 M sucrose, centrifuged at 1000×
g and the pellet containing cellular debris dis-
2.4. Exposure to pesticides in 6itro
Samples were incubated in the presence of the
pesticides or specific inhibitors (indicated in the
text) for 15 min at 25 °C prior to the addition of
substrate. Stock solutions were prepared at 100
mM in acetone and diluted to the desired concen-
trations with dH2O. A vehicle control was in-
cluded for each experimental run and the rate of
spontaneous hydrolysis calculated as before. The
50% inhibition concentration (IC50) for both
acetylcholinesterase and carboxylesterase activity
was determined by the addition of at least five

172
T.S. Galloway et al. / Aquatic Toxicology 61 (2002) 169–180
different concentrations of inhibitors selected to
yield between 10 and 90% inhibition. The enzyme
activity was determined and the percent inhibition
calculated and compared with the control.
3. Results
3.1. Distribution and substrate preference of
enzyme acti6ity
Initial experiments sought to determine the dis-
tribution of acetylcholinesterase and car-
boxylesterase activities in preparations of
haemolymph or whole body tissue homogenate.
Using an initial adaptation of the method of
Lundebye et al. (1997), the baseline level of acetyl-
cholinesterase in haemolymph was confirmed to
be higher than in the tissues (P50.001) whilst the
reverse was true for carboxylesterase (P50.001),
(Fig. 1). Subsequently, acetylcholinesterase activ-
ity was measured only in haemolymph whilst
carboxylesterase activity was measured only in
tissue homogenate.
2.5. Exposure to pesticides in 6i6o
The extent of inhibition of enzyme activity was
assessed following in vivo exposure of test organ-
isms for 24 h groups of mussels (n=10) were
exposed to test solutions of paraoxon, DFP, eser-
ine or solvent control (acetone) in sealed, aerated
glass aquaria. The final concentration of solvent
did not exceed 0.05% (v/v). Samples were pre-
pared and assayed as described.
2.6. Statistical analysis
The limits of detection of the enzyme activities
were calculated by measuring the reaction rate for
ten replicates of an activity equivalent to zero
(buffer blank). The limits of detection were deter-
mined to be three S.D. from the mean of this rate
which gave a sensitivity of detection of 1.02 nmol
min−1 mg−1 for acetylthiocholine hydrolysis and
0.39 nmol min−1 mg−1 for phenylthioacetate hy-
drolysis. The reproducibility of the method was
assessed using ten separate measurements of a
control sample. For the acetylthiocholine hydroly-
All determinations were carried out in triplicate
and the results presented as means9standard
deviation (S.D.). Statistical analyses were per-
formed using EXCEL software to determine the
one way analysis of variance and assess statistical
significance (ANOVA, Student’s t-test, Kruskal–
Wallis). Differences between means were consid-
ered significant for PB0.05. The Michaelis
constant was calculated using the graphic method
of Eadie–Hoftsee.
Fig. 1. Acetylcholinesterase and carboxylesterase specific activities in the haemolymph and tissues of M. edulis. All values are the
mean9standard error of the mean (SEM) of ten individual mussels each assayed in triplicate and presented as nmol min−1 mg−1
.

T.S. Galloway et al. / Aquatic Toxicology 61 (2002) 169–180
173
Fig. 2. Substrate preference of haemolymph and tissue homogenate preparations. Activity was determined in the presence of
concentrations of individual substrates in the range 0.01–3.0 mM and the maximum rate recorded. Values are the mean9SEM of
triplicate determinations and are expressed as nmol min−1 mg−1
.
with 3 mM PMSF, a general inhibitor of enzyme
activities containing active site serine residues,
reduced the level of activity to negligible levels as
did denaturing the enzymes by boiling the samples
for 2 min. The hydrolysis of the substrate ana-
logues was therefore, entirely due to the action of
serine dependent enzymes and not spontaneous
hydrolysis.
sis, the mean control value was 183915.2 nmol
min−1 mg−1 representing a coefficient of varia-
tion of 8.3%. For phenylthioacetate hydrolysis,
the mean value was 95.8914.5 nmol min−1
mg−1 representing a coefficient of variation of
15.1%.
The substrate preference of the haemolymph
preparation was determined by comparing rates
of reaction with concentrations in the range 0.1–3
mM of the substrate analogues acetylthiocholine
iodide, butyrylthiocholine iodide, propionylthio-
choline iodide and phenylthioacetate, each of
which may be preferentially hydrolysed by differ-
ent classes of esterase (Fig. 2). Acetylthiocholine
iodide was the preferred substrate for the
haemolymph samples (P50.001). The hydrolysis
of the alternative substrate analogues was much
lower, indicating that acetylcholinesterase activity
was the predominant activity in the haemolymph.
Tissue homogenate showed a preferential ability
to hydrolyse phenylthioacetate (P50.001) with
propionyl, butyryl and acetyl thiocholine ana-
logues hydrolysed in decreasing order of
preference.
3.2. Characterisation of acetylcholinesterase
acti6ity
The concentration of acetylthiocholine iodide
was varied to give concentrations in the range
0.05–12.0 mM and the basic protocol followed as
described (Fig. 3). The Km value calculated using
the Eadie–Hofstee equation was 1.3 mM. The
maximum rate of hydrolysis, Vmax, was achieved
at 3 mM, and this concentration was used for
subsequent analysis in the knowledge that it was
not rate limiting over the 15 min time period
chosen. A distinguishing characteristic of acetyl-
cholinesterase enzymes is the occurrence of sub-
strate inhibition at high concentrations and this
phenomenon can be seen clearly in Fig. 3a. A
series of dilutions of both haemolymph and tissue
homogenate were made in buffer to confirm linear
kinetics. Reaction rates were directly proportional
to enzyme concentration across the range of dilu-
Incubation of tissue homogenate with 3 mM
iso-OMPA,
a
potent inhibitor of butyryl-
cholinesterase but not of acetylcholinesterase
(Barahona and Sanchez-Forun, 1999) inhibited
the enzyme activity by 7791.61%. Incubation

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T.S. Galloway et al. / Aquatic Toxicology 61 (2002) 169–180
tions tested confirming that Michaelis Menten
kinetics were supported in this range (Fig. 3b).
The sample volume subsequently chosen typically
yielded reaction rates for individual mussels in the
upper third of this range, allowing for the accu-
rate detection of inhibition. The presence of the
detergent Triton X 100 did not influence the
estimated for the measured activity. The maxi-
mum rate of hydrolysis, Vmax, was achieved at 800
mM, and this concentration was used for subse-
quent analysis. At higher concentrations, a precip-
itate formed on addition of the sample, making
confirmation of substrate inhibition at higher
doses impractical. A series of dilutions of tissue
homogenate were made in assay buffer to confirm
linear kinetics (Fig. 3d). Reaction rates were di-
rectly proportional to enzyme concentration
across the range of dilutions tested confirming
that Michaelis Menten kinetics were supported in
this range. A sample volume of 25 ml was chosen
for subsequent analysis which typically gave activ-
ity values for individual mussels in the upper
ranges of the assay yet was low enough to prevent
precipitation with phenylthioacetate.
measurable
activity
indicating
that
in
haemolymph the enzyme was globular rather than
membrane bound (data not shown).
3.3. Characterisation of carboxylesterase acti6ity
The concentration of phenylthioacetate was
varied to give concentrations in the range 40–800
mM and the basic protocol followed as described
(Fig. 3c). An apparent Km value of 200 mM was
Fig. 3. Characterisation of acetylcholinesterase activity in M. edulis haemolymph: (a) the relationship between substrate (acetylthio-
choline iodide) concentration and reaction rate; (b) the relationship between sample volume and reaction rate. Characterisation of
carboxylesterase activity in M. edulis tissue homogenate; (c) the relationship between substrate (phenylthioacetate) concentration and
reaction rate; (d) the relationship between sample volume and reaction rate.

T.S. Galloway et al. / Aquatic Toxicology 61 (2002) 169–180
175
Fig. 4. The effect of organophosphorous and carbamate compounds on esterase activities in M. edulis. Acetylcholinesterase activity
was determined in haemolymph and carboxylesterase in whole body tissue homogenate. Values are the mean9SEM of triplicate
determinations. Results are expressed as a percent of the vehicle control (unexposed) (a) activity; (b) eserine; (c) paraoxon; (d) DFP;
(e) Chlorpyrifos.
3.4. Inhibition of enzyme acti6ity
3.5. In 6i6o exposure to cholinesterase inhibitors
The sensitivity of the enzyme activity in
haemolymph and tissue homogenate to inhibition
by organophosphorous and carbamate com-
pounds was initially investigated in vitro. The
selected compounds were the carbamate eserine, a
potent inhibitor of cholinesterases, and the
organophosphorous compounds DFP and
paraoxon. These compounds were chosen as they
do not require metabolic activation prior to use.
Chlorpyrifos, a phosphorothioate, was also in-
cluded. In Fig. 4 the inhibition curves obtained
for both acetylcholinesterase and carboxylesterase
activities are shown. The IC50 values calculated
from these graphs are presented in Table 1. For
each compound tested, a proportion of the appar-
ent carboxylesterase activity remained insensitive
to inhibition by each of the inhibitors applied.
The inhibition concentration has been calculated
after subtraction of this pesticide resistant
activity.
The effects of eserine, paraoxon and DFP were
then determined in vivo. Initially, M. edulis (n=
10) were exposed to a single sublethal dose of 1
mM of each compound over 24 h and the acetyl-
cholinesterase and carboxylesterase activities mea-
sured as described (Fig. 5). The degree of
Table 1
Inhibition of enzyme activities by DFP, eserine, paraoxon and
chlorpyrifos following in vitro exposure
Compound
Acetylcholinesterase
(mM)
Carboxylesterase
(mM)
Eserine
Paraoxon
DFP
B0.01
1.83
0.82
Not determined*
0.4
1.1
Chlorpyrifos
15.0
10.1
Results are expressed as IC50 values and represent the mean
of three separate triplicate determinations. *, ]2.5 mM.

176
T.S. Galloway et al. / Aquatic Toxicology 61 (2002) 169–180
Fig. 6. The effect of 24 h in vivo exposure of M. edulis (n=10)
to eserine. Results are expressed as a percent of the vehicle
control (unexposed) activity.
Fig. 5. The effect of exposure of M. edulis (n=10) to a single
dose of 3 mM paraoxon, DFP or eserine for 24 h expressed as
a percentage of the vehicle control (unexposed) activity.
(Fig. 7) there was a significant difference in acetyl-
cholinesterase activity between control and ex-
posed animals for each of the treatment groups
(P50.005). Exposure to 1.0 mM resulted in a
reduction of activity to 17.991.06% relative to
the control. The inhibition of carboxylesterase
activity reached statistical significance at concen-
trations of 0.1 and 1.0 mM (P50.001). At the
highest exposure concentration (1.0 mM) 27.69
2.95% of the control activity remained.
inhibition caused by these exposure concentra-
tions was greater for acetylcholinesterase than for
carboxylesterase (P50.001). When expressed as
percent inhibition compared with the control, es-
erine, paraoxon and DFP at 1 mM inhibited
acetylcholinesterase activity in the haemolymph
by 9690.12, 8594.92 and 9090.75%, respec-
tively. Carboxylesterase activity was inhibited by
8.990.98, 81912.0 and 46 16911.4% by these
same exposure concentrations. There was a statis-
tically significant difference between each treat-
ment group and the control for both activities
(P50.001).
The concentration dependent inhibition of
acetylcholinesterase and carboxylesterase activi-
ties was then determined for the carbamate eser-
ine and the organophosphorous paraoxon in the
range 0.05–1.0 mM and the enzyme activities
determined as before (Figs. 6 and 7). For eserine
(Fig. 6) there was a significant difference in en-
zyme activity between control and exposed ani-
mals for each of the treatment groups (P50.001).
For acetylcholinesterase activity, exposure to 0.05
mM eserine caused inhibition of activity of 859
1.5% and subsequently, no significant difference
was detectable among the treatment groups them-
selves (P=0.07). Carboxylesterase activity varied
significantly between each of the treatment con-
centrations (P50.002) and reached a level of
3990.73% inhibition relative to the control at 0.5
mM eserine. Following exposure to paraoxon
4. Discussion
This study has sought to characterise the mea-
surable esterase activity in selected tissues and
body fluids of M. edulis in order that their utility
Fig. 7. The effect of 24 h in vivo exposure of M. edulis (n=5)
to paraoxon. Results are expressed as a percent of the vehicle
control (unexposed) activity.

T.S. Galloway et al. / Aquatic Toxicology 61 (2002) 169–180
177
as accurate and sensitive biomarkers of
organophosphorous and carbamate exposure
might be determined. An additional consideration
for their use in environmental monitoring has
been the need for such assays to be accessible to
personnel who may have limited experience of
biochemical techniques or access to specialised
monitoring equipment (Depledge, 2000).
The choice of a common endpoint for the de-
tection of cholinesterase or carboxylesterase activ-
ity was made to simplify both reagent preparation
and detection, the only difference between the
methods being the nature of the substrate ana-
logue. Both methods proved reproducible and
sensitive for the detection of small changes in
activity in microliter sample volumes. The added
advantage of using haemolymph is the opportu-
nity to employ repeated, non-destructive sam-
pling. For reasons of simplicity in sample
preparation, only whole body tissue homogenate
rather than the dissection of individual tissues was
considered.
cholinesterase purified from the gill of Mytilus
species (Mora et al., 1999). The hydrolysis of
other substrate analogues, albeit at considerably
lower levels, is in agreement with previous reports
of four distinct cholinesterase activities in the
haemolymph of M. edulis, only one of which
could be classified as an acetylcholineterase based
on its substrate preference and susceptibility to
inhibitors (Von Wachtendonk and Neef, 1979).
Thus, interpretation of acetylcholinesterase activ-
ity measurements in M. edulis haemolymph may
be made on the basis that a single predominant
pharmacological form is present.
Many different substrate analogues have been
used in the measurement of carboxylesterase ac-
tivities which typically exhibit a level of promiscu-
ity in their substrate reactions. Phenylthioacetate
was chosen as a substrate for monitoring car-
boxylesterase activity in this study because of its
preferential hydrolysis (Fig. 2), ease of detection
using parallel methodology to the detection of
acetylcholinesterase activity and because hydroly-
sis of this substrate by acetylcholinesterase is neg-
4.1. Characterisation of enzyme acti6ities
ligible
(Basack
et
al.,
1998)
allowing
differentiation of the two activities. Car-
boxylesterase activity as measured by this method
was higher in whole body tissue homogenate than
in haemolymph in agreement with previous re-
ports (Basack et al., 1998). High levels of esterase
activity have been reported in the digestive gland
(Ozretic and Krajnovic-Ozretic, 1992) and gill tis-
sues (Escartin and Porte, 1997) of Mytilus species
although for the purposes of this study, the exact
location of individual carboxylesterases was not
determined. The measured activity could be inhib-
ited completely with PMSF, a general inhibitor of
serine hydrolases and was also inhibited partially
by iso-OMPA, confirming that this activity is
distinct from acetylcholinesterase. Although the
predominant activity in the tissue preparation was
carboxylesterase, some cholinesterase activity was
also present showing an order of thiocholine sub-
strate preference of butyryl\propionylꢀacetyl.
An alternative conclusion, that a single broad-
spectrum activity was present, able to hydrolyse
each derivative cannot be ruled out but is less
likely. Carboxylesterases are in general specific for
simple carboxylic esters and lack the anionic bind-
In higher vertebrates, esterases are generally
classified into three groups, cholinesterases,
arylesterases and carboxylesterases base on their
catalytic properties, inhibition specificities and tis-
sue distributions (Mounter and Whittaker, 1953).
The differentiation of these enzyme activities in
many invertebrate species remains largely unde-
scribed and the vertebrate classification system
may be inappropriate to use (Walker and Thomp-
son, 1991). Bearing this in mind, the results pre-
sented here verify the existence of one
predominant enzyme activity, classifiable as an
acetylcholinesterase on the basis of its substrate
preference, pH optimum and high dose substrate
inhibition in the haemolymph of M. edulis (Von
Wachtendonk and Neef, 1979). The level of activ-
ity was comparatively low, a characteristic re-
ported for acetylcholinesterases in mollusc
haemolymph (Winners et al., 1977; Najimi et al.,
1997; Narbonne et al., 2001). The apparent Km of
1.33 mM for acetylthiocholine iodide calculated
for this unpurified preparation is in the same
order of magnitude as values reported for acetyl-

178
T.S. Galloway et al. / Aquatic Toxicology 61 (2002) 169–180
ing site considered necessary for interaction with
cholinesterase derivatives (Maxwell, 1992).
In other species, the combined monitoring of
measures of behaviour and neurological function
has helped to define the toxicological significance
of exposure (Beauvais et al., 2000). Adequate
means of assessing behavioural changes in
bivalves would no doubt be of benefit in interpre-
tation of this kind of data, as would further
insight into the physiological roles of the enzymes.
Although the acetylcholinesterase activity de-
scribed here has been defined as such based on
substrate preference and inhibitor sensitivity,
there is no indication other than this that it is
involved in neuromuscular activity. The toxic ef-
fects of organophosphorous pesticide exposure
can also be evaluated by measuring other forms
of toxicity which can occur in addition to their
effects on esterase targets, for example the alkylat-
ing potential of compounds such as dichlorvos
can cause damage to DNA, detectable using as-
says of genotoxicity (Sbrana and Musio, 1995).
4.2. Interpretation of combined esterase acti6ity
measurements
Characterisation of the relative sensitivities of
the two enzyme activities to carbamate and
organophosphorous compounds following in vitro
incubation confirmed the sensitivity of both en-
zymes to the exposure-dependent effects of these
compounds. The sensitivity of the enzymes was
similar, with carboxylesterase slightly more sensi-
tive to paraoxon and chlorpyrifos. The inhibition
seen at high concentrations of the phosphoroth-
ioate chlorpyrifos may be due to the presence of
chlorpyrifos oxon or other contaminants in the
preparation. The manufacturer’s quoted purity
was 99.5%. Carboxylesterase activity was insensi-
tive to the carbamate eserine except at high con-
centrations. Similar patterns of inhibition were
also evident following in vivo exposures. Both
enzyme activities showed sensitivity in vitro to
concentrations of pesticide in the same order of
magnitude although the percent inhibition of
acetylcholinesterase activity was consistently
greater. Acetylcholinesterase activity could be
used to detect in vivo exposure to 0.05 mM
eserine and 0.01 mM paraoxon with statistical
significance. Despite the similar in vitro sensitivi-
ties of the enzymes, the percent inhibition of
carboxylesterase activity was smaller at was sig-
nificantly inhibited only by higher amounts of
pesticide. The observation of a high percentage of
pesticide resistant carboxylesterase activity is
striking and has been reported by others in inver-
tebrate (Maxwell, 1992; Boone and Chambers,
1996; Basack et al., 1998) and vertebrate studies
(Lassiter et al., 1999). The inhibition of enzyme
activity seen in tissue preparations following in
vivo exposure provides a net reflection of the
sensitivity of the organism in terms of uptake,
biotransformation and detoxification patterns.
Conversely, the relative insensitivity of the en-
zyme to environmentally relevant pesticide con-
centrations may make it difficult to accurately
measure small fluctuations in activity against a
varying background of inter-individual activities.
5. Conclusions
The measurement of acetylcholinesterase activ-
ity in M. edulis haemolymph using the described
methodology provides a rapid, relatively inexpen-
sive and reliable means of non-destructively as-
sessing the exposure of mussels (and potentially
other invertebrates) to organophosphorous and
carbamate pesticides. Inhibition of activity is ex-
posure concentration dependent and the interpre-
tation of inhibition can be made on the basis that
one predominant pharmacological form of the
enzyme is present. Parallel determination of car-
boxylase activity can be achieved with the need
for just one additional reagent and with minimal
extra expenditure of time. However, the interpre-
tation of carboxylesterase activity must take ac-
count of the large percentage of pesticide resistant
activity apparent using the method described.
Whilst these assays provide a useful means of
assessing the exposure of bivalves to pesticides,
interpretation of the ecotoxicological significance
of such exposure should be made in conjunction
with other multi-level indicators of toxicity, such
as physiological and behavioural changes of evi-
dence of genotoxic damage.

T.S. Galloway et al. / Aquatic Toxicology 61 (2002) 169–180
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Acknowledgements
This work was supported financially by The
Nuffield Foundation, DEFRA and the University
of Plymouth. Many thanks to Sarah MacMahon
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