Environmental Toxicology and Chemistry, Vol. 20, No. 1, pp.

37–45, 2001
Printed in the USA
0730-7268/01 $9.00 1 .00

Annual Review
ACETYLCHOLINESTERASE INHIBITION IN ESTUARINE FISH AND INVERTEBRATES
AS AN INDICATOR OF ORGANOPHOSPHORUS INSECTICIDE EXPOSURE AND
EFFECTS

MICHAEL H. FULTON* and PETER B. KEY

National Ocean Service, Center for Coastal Environmental Health and Biomolecular Research,
219 Fort Johnson Road, Charleston, South Carolina 29412-9110, USA

( Received 12 June 2000; Accepted 23 July 2000)

Abstract—The majority of insecticides currently in use are organophosphorus, carbamate, and synthetic pyrethroid compounds.
Organophosphorus insecticides (OPs) produce toxicity by inhibiting the cholinesterase enzymes in the nervous system. Monitoring
of acetylcholinesterase (AChE) inhibition has been widely used in terrestrial and freshwater aquatic systems as an indicator of OP
exposure and effects. This review describes the use of AChE inhibition as a biomarker in the estuarine environment, discusses the
relationship between AChE inhibition and other manifestations of OP toxicity, and highlights areas where additional research is
needed. A variety of studies with estuarine fish have suggested that brain AChE inhibition levels of .70% are associated with
mortality in most species. Selected species, however, appear capable of tolerating much higher levels (.90%) of brain inhibition.
Sublethal effects on stamina have been reported for some estuarine fish in association with brain AChE inhibition levels as low as
50%. Most studies suggest, however, that these effects are observed only when brain AChE inhibition is at near-lethal levels. A
number of field studies have successfully used AChE inhibition in fish as a biomarker in the estuarine environment. The use of
AChE inhibition as a biomarker in estuarine invertebrates has been less well studied. Although AChE inhibition has been measured
in the tissues of a variety of invertebrate species following OP exposure, the relationship between AChE inhibition and lethality
is less distinct. Additional work is needed in both fish and invertebrates to better explain species-specific differences in the relationship
between AChE inhibition and mortality and to investigate other physiological perturbations associated with AChE inhibition.

Keywords—Acetylcholinesterase Fish Invertebrates Organophosphorus insecticides

INTRODUCTION Organophosphorus insecticides are esters, amides, or thiol

Pesticide usage is a critical concern in coastal areas, where
inputs from agriculture and urbanization (resort development
and golf courses) may impact the surrounding estuaries and
marshes. Estuaries rank as one of the most productive natural
habitats, where large phytoplankton populations support a va-
riety of other organisms, including many commercially and
recreationally important marine fish and crustacean species
that use estuaries as nursery grounds [1]. Transport of pesti-
cides to these delicate ecosystems therefore creates the need
to fully understand effects in resident biota. In many areas of
the world, these sensitive ecosystems are at risk because of
the nonpoint-source runoff of pesticides from agricultural and
urban sources.
The majority of insecticides currently in use are organo-
phosphorus, carbamate, and synthetic pyrethroid compounds.
Organophosphorus insecticides (OPs) were developed in Ger-
many during the 1930s as a substitute for nicotine and as
potential chemical warfare agents [2]. Because of their rela-
tively nonpersistent characteristics in the environment, OPs
have become one of the most widely used classes of insecti-
cides worldwide. Although these compounds offer the advan-
tage of rapid degradation in the environment, they generally
lack target specificity and have high acute toxicity toward
many nontarget vertebrate and invertebrate species. Thus,
many terrestrial and aquatic organisms may be at risk for in-
toxication caused by exposure to these compounds in the en-
vironment.
* To whom correspondence may be addressed (mike.fulton@noaa.gov).
37
derivatives of either phosphoric acid or thiophosphoric acid.
The majority now in use, such as azinphosmethyl, chlorpyrifos,
and malathion, contain the thiono moiety (5S). The substi-
tution of the 5S for 5O on the phosphorus atom increases
the toxicity of the insecticide, such as is the case with mala-
thion and its oxygen analogue, malaoxon [3].
The uses of OPs are highly varied. In agriculture, OPs are
used to control insects on fruits, vegetables, grain crops, and
stored seeds [4]. The OPs chlorpyrifos and terbufos are the
two most widely used insecticides in agriculture [5]. House-
hold uses include the control of cockroaches, houseflies, and
termites along with protection of plants of horticultural interest
[4]. The most common insecticides used in home and garden
applications are the OPs diazinon and chlorpyrifos. For in-
dustrial, commercial, and government use, the top insecticides
are again OPs (chlorpyrifos and malathion [5]).
Organophosphorus insecticides produce toxicity by inhib-
iting cholinesterase enzymes in both vertebrate and inverte-
brate organisms. These enzymes are responsible for the re-
moval of the neurotransmitter acetylcholine (ACh) from the
synaptic cleft through hydrolysis [6]. In vertebrates, ACh acts
as an excitatory transmitter for voluntary muscle in the somatic
nervous system. Acetylcholine also serves as both a pregan-
glionic and a postganglionic transmitter in the parasympathetic
nervous system and as a preganglionic transmitter in the sym-
pathetic nervous system. In critical regions of the central ner-
vous system, ACh serves as an excitatory transmitter. When
cholinesterases are inactivated by the binding of OPs, an ac-
cumulation of ACh occurs at the nerve synapse, interfering
38 Environ. Toxicol. Chem. 20, 2001
with the normal nervous system function. This produces rapid
twitching of voluntary muscles followed by paralysis [6,7].
Once bound, organophosphorus compounds are considered ir-
reversible inhibitors, as recovery usually depends on new en-
zyme synthesis [6].
Cholinesterases are usually divided into two broad classes:
the acetylcholinesterases (AChEs) and the butyrylcholinester-
ases (BChEs). In fish, brain and muscle tissue contain mostly
AChEs, while liver and plasma contain mostly BChEs [6].
Significant amounts of BChE activity, however, have been
reported in the muscle tissues of several fish species [8,9].
Neurotransmission in invertebrates has not been as well
characterized as in vertebrates. Crustaceans contain both in-
hibitory and excitatory motoneurons that usually employ other
transmitters. When ACh is present, its primary function is as
a neurotransmitter for afferent nerve fibers. Welsh [10] re-
viewed much of the early work (late 1930s to late 1950s) that
established the presence of cholinesterases in several species
of crustaceans, mainly crab, lobster, and crayfish. More re-
cently, AChE activity has been measured in a variety of crus-
taceans, including the brain and ventral ganglion of the blue
crab, Callinectes sapidus [11]; the hepatopancreas of oysters
(Crassostrea virginica) and brown shrimp (Penaeus sp.) [12];
tissues of the nervous system in penaeid prawns, Metapenaeus
monoceros [13]; muscle tissue of the shrimp, Palaemon ser-
ratus [14]; whole-body tissues of an Australian freshwater
shrimp, Paratya australiensis [15]; and the whole-body tissues
of adult, larval, and embryonic grass shrimp, Palaemonetes
pugio [16,17].
Monitoring of AChE inhibition has been widely used in
terrestrial and freshwater aquatic ecosystems as an indicator
of OP insecticide exposure and physiological effects in ex-
posed animals [18–20]. Although the presence of many OP
insecticides in the environment can be discerned using ana-
lytical chemistry techniques, AChE monitoring in the envi-
ronment may offer distinct advantages over the use of ana-
lytical chemistry alone. First, AChE inhibition is the primary
mechanism by which OP insecticides produce toxicity. Thus,
the biomarker effect is directly linked to the compounds toxic
mode of action. When significant AChE inhibition is measured,
we know not only that the organism has been exposed but also
that a sufficient dose of the compound has reached the target
site to produce a physiological effect. In addition, as previously
stated, most OP insecticides degrade rapidly in the environ-
ment, and their concentrations in environmental samples may
fall below detectable levels in hours to days. Acetylcholin-
esterase inhibition in many species, however, persists for much
longer—days to weeks [21,22].
The goal of this review is to summarize studies that have
investigated the use of AChE inhibition as a monitor of OP
insecticide exposure and effects in estuarine fish and inver-
tebrate species. It discusses the relationship between OP-in-
duced AChE inhibition and lethality as well as sublethal re-
sponses under both laboratory and field conditions. In addition,
it describes some of the limitations associated with AChE
inhibition as a biomarker and discusses areas of needed re-
search.
LABORATORY STUDIES
Fish
Relationship between AChE inhibition and mortality. A
number of studies have examined the relationship between
specific levels of OP-induced AChE inhibition and lethality
M.H. Fulton and P.B. Key
in estuarine fish species. Sheepshead minnows (Cyprinodon
variegatus) were exposed to selected OP insecticides (Guth-
ion, phorate, parathion, phosphamidon, Cygon, malathion,
EPN, Dursban, dichlorvos, diazinon, Dibrom, and methyl para-
thion) at sublethal and median lethal (40–60% mortality) con-
centrations (as listed in Coppage [23]). Inhibition of brain
AChE was always greater at concentrations that caused mor-
tality. Brain inhibition of greater than 80% was observed in
all fish that survived median lethal exposures. The authors also
observed that given the variability observed in AChE levels
in control fish, activity must be depressed by .13% to indicate
exposure to OP insecticides. Their findings suggest that brain
inhibition levels of 20 to 70% in live fish could be diagnostic
of OP insecticide exposure.
In a subsequent study, brain AChE activity was measured
in several estuarine fish species (spot, Leiostomus xanthurus;
Atlantic croaker, Micropogon undulatus; sheepshead min-
nows; and pinfish, Lagodon rhomboides) that survived median
lethal exposures to several OP insecticides (malathion, naled,
Guthion, and parathion, as written in [24]). In all cases, AChE
activity was inhibited by 70 to 96% [24]. Their findings sug-
gested that the response is similar for a variety of estuarine
species regardless of the OP compound tested.
Brain AChE activities were also compared in pinfish ex-
posed to lethal and sublethal concentrations of the OP insec-
ticide malathion [25]. Brain AChE activity was measured in
fish that survived a near-median kill at 3.5, 24, 48, and 72 h.
Reductions in enzyme activity (72–79%) were similar for all
these lethal exposure experiments regardless of the exposure
time or insecticide concentration. Their data indicated that
mortality is likely when brain inhibition reaches 70 to 90%.
In a similar study, pinfish were exposed to naled at selected
concentrations for varying periods of time, and then brain
AChE activity was measured and compared to levels in control
fish [26]. Inhibition at 15 mg/L ranged from 59% at 24 h to
70% after 96 h. No mortality was observed at this concentra-
tion at any time point. At higher naled concentrations (25–75
mg/L) that caused median kills at various time points, brain
AChE inhibition was always greater than 80%. As in the earlier
study, mortality was always observed in this species when
brain inhibition was in excess of 70%. They also reported that
significant brain AChE inhibition was observed at concentra-
tions well below those causing mortality.
In all the previously described studies, the authors found
that when brain AChE inhibition reached 70 to 80%, mortality
was likely. Other investigators have found that this relationship
may be less distinct in other estuarine fish. Fulton [27] inves-
tigated the lethal and sublethal effects of the OP insecticide
azinphosmethyl in the mummichog Fundulus heteroclitus, a
common fish in eastern U.S. estuarine tidal creek habitats. The
96-h median lethal concentration (LC50) for this compound
was 32.16 mg/L, while the 24-h median effective concentration
(EC50) for brain AChE inhibition was 0.81 mg/L. Thus, the
96-h LC50 was .39 times higher than the 24-h EC50 for brain
AChE inhibition. The lowest concentration used in the acute
toxicity tests that caused no mortality after 96 h (3.5 mg/L)
was more than four times higher than the 24-h EC50 for in-
hibition of AChE in brain tissue. The highest concentration
used in the 24-h sublethal tests (3.9 mg/L) caused ;94% in-
hibition of brain tissue AChE activity. This concentration was
much lower than the 96-h no-observed-effect concentration
(19.80 mg/L) determined from the 96-h acute toxicity tests.
These findings suggest that this species is able to tolerate ex-
Acetylcholinesterase inhibition in estuarine organisms
tremely high levels of brain AChE inhibition and that the
relationship between specific levels of brain inhibition and
mortality may be different than that observed in many other
estuarine species.
In a comparative study, Van Dolah et al. [28] evaluated the
effects of azinphosmethyl in juvenile red drum (Sciaenops
ocellatus) and adult mummichogs of comparable size. Fish
were exposed to a series of azinphosmethyl concentrations,
and then brain AChE activity was measured. The EC50s for
brain inhibition were calculated and compared to the 96-h
LC50s for each species. The relationship between azinphos-
methyl-induced AChE inhibition and mortality was quite dif-
ferent for the two species tested. In the red drum, the 96-h
LC50 (6.2 mg/L) and the 24-h EC50 for brain AChE inhibition
(5.2 mg/L) were quite similar. In the mummichog, however,
the 96-h LC50 was 60 times greater than the 24-h EC50 (1.0
mg/L), and this relationship was quite similar to that previously
reported by Fulton [27]. Subsequent experiments with these
two species indicated that the sensitivity of red drum muscle
tissue AChE (24-h EC50 5 6.8 mg/L) was quite similar to that
in brain tissue, while mummichog muscle AChE (24-h EC50
.5mg/L) was much less sensitive than brain tissue [29]. These
findings suggest that there are clear species- and/or age-specific
differences in the relationship between AChE inhibition in
brain and muscle tissue and mortality. These studies also sug-
gest that given the response observed in mummichogs, inhi-
bition of AChE in the peripheral nervous system may be a
better predictor of OP-induced mortality than brain inhibition.
In an in vitro study, homogenates from the dorsal muscle
of the dragonet (Callinymus lyra) and the sole (Solea solea)
were exposed to dichlorvos, fenitrothion, and phosalone [30].
Dichlorvos was the most potent inhibitor in the fish tissues
with a 50% inhibitory concentration (IC50) of 2.3 3 1028 M
(;5.1 mg/L) in dragonet muscle and 3.1 3 1027 M (;68.5
mg/L) in sole tissues. The IC50s for dragonet were 2.6 3 1025
M (;7,209 mg/L) and 6.2 3 1027 M (;228 mg/L) for feni-
trothion and phosalone, respectively. In sole tissues, the IC50s
for these two OPs were 1.2 3 1024 M (;33,270 mg/L) and
2.5 3 1026 M (;919 mg/L), respectively. The authors sug-
gested that the greater sensitivity to dichlorvos for both fish
tissues was because this OP is a direct cholinesterase inhibitor,
which does not require activation to an oxygen analogue, such
as phosalone and fenitrothion. These three OPs were also tested
for synergistic effects after each had been mixed with the
carbamates (C) carbaryl or carbofuran or with each other. The
fish showed greater sensitivity to the combined effects of these
insecticides than a shrimp and oyster species that were also
tested. In general, the OP-C combinations were highly syn-
ergistic in terms of AChE inhibition, while the OP-OP com-
binations were less so. Increasing incubation time increased
the synergistic effect. The authors concluded that the mech-
anisms by which inhibitory effects could be enhanced was not
well understood.
Enzyme inhibition and recovery. Other studies have inves-
tigated the persistence of AChE inhibition in brain tissue fol-
lowing OP exposure. In one study, Atlantic salmon (Salmo
salar) were exposed to sublethal concentrations of fenitrothion
[31]. The authors utilized two different exposure regimes. In
the first, fish were exposed continuously for 7 d, while in the
second fish were exposed for 24 h, allowed to recover for 7
d in clean water, and then re-exposed to fenitrothion for an
additional 24 h. In the 7-d exposures, inhibition increased with
increasing insecticide concentrations in a typical dose–re-
Environ. Toxicol. Chem. 20, 2001 39
sponse fashion. Inhibition ranged from 12% at 0.005 mg/L to
58% at 0.213 mg/L. In the 2 3 24-h exposure regime, inhi-
bition immediately following the final 24-h exposure was 16%
at 0.005 mg/L and 50% at 0.213 mg/L. After one week of
depuration in clean water, activity in the 0.005-mg/L group
had recovered to control levels, while activity in the 0.213-
mg/L group was still depressed by 34%. After six weeks, ac-
tivity in both treatment groups had recovered to control levels.
Results of this study indicated that the time for enzyme re-
covery was a function of the degree of initial inhibition. This
is likely because the recovery of the enzyme activity is largely
a result of de novo synthesis of enzyme protein, and the greater
the degree of inhibition, the more protein synthesis is required.
In another study, mummichogs were exposed to chlorpyr-
ifos in 6-h pulsed exposures [32]. In one experiment, fish were
exposed to 6-h chlorpyrifos pulses daily for 4 d. In a second
experiment, fish were exposed to 6-h pulses weekly for four
weeks. Brain AChE activity was monitored as well as caudal
vertebrae strength. Maximum AChE inhibition was higher for
the daily exposure regime than for the weekly exposures. In
general, the daily exposure regime exhibited cumulative in-
hibition with inhibition at 96 h being greater than that at 48
h. For the weekly exposure scenario, AChE inhibition at four
weeks was similar to that observed after two weeks. This
suggests that the weekly recovery intervals allowed almost
complete recovery of enzyme activity. The authors also re-
ported that strength analysis of the caudal vertebrae indicated
that vertebrae were weaker at selected time points for each of
the exposure regimes. This suggests that chlorpyrifos concen-
trations sufficient to cause the level of inhibition observed in
this study may also affect bone strength.
Relationship between AChE inhibition and sublethal ef-
fects. A number of researchers have investigated the relation-
ship between AChE inhibition and other sublethal effects. In
one study, the swimming performance of sheepshead minnows
was measured in a stamina tunnel at the end of life-cycle
toxicity tests with the OP insecticides EPN and Guthion [33].
Brain AChE inhibition was also measured. These end points
were compared to survival, growth, and reproduction data ob-
tained during the course of the life-cycle tests. Swimming
stamina was affected in fish exposed to EPN at 4.1 and 2.2
mg/L. At 2.2 mg/L, stamina was reduced by 43%, while at 4.1
mg/L, stamina was decreased by 54%. Swimming performance
was not affected by Guthion concentrations up to 0.5 mg/L, a
concentration that affected reproduction. Acetylcholinesterase
activity was significantly depressed at all concentrations of
EPN (0.25–7.9 mg/L) and Guthion (0.06–0.5 mg/L). Effects
on swimming stamina were observed only when AChE inhi-
bition was greater than 80%.
Van Dolah et al. [28] reported that red drum exposed to
azinphosmethyl for 6 h at 12 mg/L had reduced swimming
stamina. This concentration is approximately two times the
24-h EC50 (5.2 mg/L) for brain AChE determined in the same
study. No effects on swimming stamina were observed at lower
insecticide concentrations (3–6 mg/L). Swimming stamina in
mummichogs was not affected at 19.4 mg/L (the highest con-
centration tested), which is ;20 times higher than the 24-h
EC50 (1.0 mg/L).
Both of these studies suggest that swimming stamina in fish
is affected only at very high levels of brain AChE inhibition.
In an earlier study, however, Post and Leasure [22] reported
that significant effects on swimming stamina in three species
of salmonids were observed in conjunction with much lower
40 Environ. Toxicol. Chem. 20, 2001
levels of AChE inhibition. They reported that for the three
species tested (brook trout, Salvelinus fontinalis; rainbow
trout, Salmo gairdneri; and coho salmon, Oncorhynchus ki-
tusch), fish that had brain AChE activity reduced by ;50%
experienced stamina reductions of 23 to 44%.
Invertebrates
The use of AChE as a biomarker of OP exposure in marine
invertebrates is not as well established as in fish. Because of
the lack of a clearly defined organ (such as brains in fish),
researchers have relied on a variety of techniques to determine
AChE levels in these organisms. Various investigators have
used hemolymph, nerve ganglion, muscle tissue, and whole-
body tissues of invertebrates to evaluate the effects of OPs on
AChE levels. Blue crabs, Callinectes sapidus, were exposed
to DEF(S,S,S,-tri-n-butyl phosphorotrithioate) at concentra-
tions of 1.0 to 10.0 mg/L for 96 h and then assayed for AChE
activity [11]. In control crabs, high AChE activity was found
in both the ventral ganglion and the brain with negligible ac-
tivity in claw musculature. Exposed crabs showed a 90% re-
duction in ventral ganglion AChE activity and a greater than
80% reduction in brain AChE activity. Other crabs were ex-
posed for 48 h to 4.0 mg/L, then transferred to clean seawater
for 10 d. Control AChE activity levels were significantly higher
in the ganglion and brain after this period. This study not only
showed significant anticholinesterase effects from DEF ex-
posure in vulnerable tissues of C. sapidus but also indicated
that these effects may persist for a significant period of time
after a single short-term exposure.
In a later study, Habig et al. [34] incubated C. sapidus
ganglia tissue with malathion and parathion and compared their
sensitivity to catfish (Ictalurus punctatus) neural tissues in-
cubated in the same fashion. The IC50s for crab AChE were
4.5 3 1025 M (;14,866 mg/L) for malathion and 6.9 3 1025
M (;20,098 mg/L) for parathion. Crab tissue was about three
times more sensitive to malathion and 10 times more sensitive
to parathion than catfish tissue.
Another crab species (Carcinus maenas) was exposed in
vivo to dimethoate for 18 h at concentrations from 0.5 to 2.0
mg/L [35]. For this assay, the hemolymph was nondestruc-
tively extracted and analyzed for AChE activity. At the highest
concentration, a 30% reduction in AChE activity was detected.
In comparing baseline AChE levels, the authors found that C.
maenas hemolymph activity at 210.7 nmol/min/mg P was low-
er than Callinectes sapidus homogenate activity (555 nmol/
min/mg P) and synaptosomal activity (833 nmol/min/mg P) as
reported by Habig et al. [34]. They concluded that since AChE
is primarily a membrane-bound enzyme, lower activity would
be expected in the hemolymph. They also suggested a direct
effect of reduced AChE activity in C. maenas on cardiac neu-
ronal control because of their observation of reduced heart
rates in the crabs following dimethoate exposure.
Several papers have examined the effects of the OP com-
pound dichlorvos used in marine fish farms to control ecto-
parasites. After treatment, waters containing this product may
be discharged into surrounding estuarine areas. McHenery et
al. [36] exposed larval lobster (Homarus gammarus) to di-
chlorvos for 24 h at 10-fold intervals from 0.001 to 100 mg/
L. They observed 50% AChE inhibition at 2.7 mg/L. This value
was about 10-fold lower than the 24-h LC50 of 28.3 mg/L
determined for lobster larvae in this study. The authors sug-
gested that AChE activity in the lobster may provide a sensitive
method for determining OP exposure. They did, however, ob-
M.H. Fulton and P.B. Key
serve an apparent hormetic effect of increased AChE activity
in larvae exposed at the 0.01-mg/L concentration that may
interfere with the ability to distinguish between animals ex-
posed to low concentrations and unexposed animals.
Another study [37] examined the effect of dichlorvos on
AChE activity in two commercial bivalves, Manila clam (Rud-
itapes philippinarum) and Japanese oyster (Crassostrea gi-
gas). Both organisms were exposed for 6 h to 0.1 and 1.0 mg/
L dichlorvos. Oyster gills and clam tissue were sampled from
0 to 48 h for AChE activity. The activity was lowest in oyster
gill after 4 h of exposure to 1.0 mg/L with 87% inhibition and
after 6 h to 0.1 mg/L with 83% inhibition. Recovery was not
observed at the end of the experiment. The AChE decrease
was slower in clam tissue with only 64% inhibition after 10
h from the 1.0-mg/L concentration and 42% inhibition after 8
h from the 0.1-mg/L concentration. The response to dichlorvos
was more rapid in oyster gill than clam tissue. While no an-
imals died at these concentrations, the authors suggest that the
decrease in AChE activity indicated an early deleterious effect
that may affect other functions such as growth.
Galgani and Bocquene [14] found that AChE from whole
mussel, Mytilus edulis, was less sensitive to five organophos-
phates than the enzyme from the muscle tissues of the shrimp
Palaemon serratus and two fish species. The OPs used in this
in vitro test were malathion, parathion, paraoxon, temephos,
and dichlorvos in concentrations of milligrams per liter. These
observations may be due, in part, to species-specific differ-
ences in sensitivity. They may also, however, be an indication
of the need to perform the AChE assay on other than the whole
body, as discussed in the following research, or even the need
to account for other less sensitive classes of cholinesterases
present in the organism (see also the following discussion).
While the previous studies indicate the potential usefulness
of bivalves as biomonitors of OP exposure, Bocquene et al.
[38] determined the presence of two cholinesterases in the
oyster Crassostrea gigas. The A cholinesterases were mem-
brane bound, and the B cholinesterases were soluble enzymes.
The A group was highly sensitive in vitro to the OP compounds
DFP (ki 5 2.0 3 103/M/min) and paraoxon (ki 5 3.0 3 105/
M/min), while the B group was considered to be almost in-
sensitive. The presence of this insensitive cholinesterase sug-
gests that improved use of AChE activity in oyster as a bio-
marker of inhibitory effects can be achieved if these two clas-
ses are isolated from the total cholinesterase activity.
Bocquene et al. [30] determined 1-h IC50 values for the
shrimp Palaemon serratus and oyster C. gigas after exposure
to dichlorvos, fenitrothion, and phosalone. Dichlorvos was the
most potent inhibitor with an IC50 of 1.1 3 1026 M (;243.08
mg/L) for shrimp muscle and 7.3 3 1028 M (;16.13 mg/L)
for oyster gill. The IC50s for shrimp were both greater than
1024 M for fenitrothion (;27,725 mg/L) and phosalone
(;36,780 mg/L). For oysters, the IC50s for these two OPs
were 1.4 3 1024 M (;38,815 mg/L) and 5 3 1024 M (;183,900
mg/L), respectively. The authors stated that the greater sen-
sitivity to dichlorvos for both invertebrates was due to this
OP being a direct cholinesterase inhibitor that does not require
activation to an oxygen analogue, as is the case with phosalone
and fenitrothion. These three OPs were tested for synergistic
effects after each had been mixed with one of two carbamates
(C) carbaryl or carbofuran or with each other. As with two
fish species discussed earlier, the OP-C combinations were
highly synergistic in terms of AChE inhibition, whereas the
Acetylcholinesterase inhibition in estuarine organisms
OP-OP combinations were less inhibitory. The mechanisms
by which inhibitory effects were enhanced remains unclear.
Most research utilizing AChE activity as a biomarker of
OP exposure in invertebrates has been with shrimp species. In
one of the earlier papers, Coppage and Matthews [24] exposed
the pink shrimp, Penaeus duorarum, to 1,000 mg/L malathion
for 48 h. The shrimp were moribund with AChE levels in the
ventral nerve cord reduced an average of 75% in comparison
to controls.
Another pink shrimp study [39] exposed the animals to
methyl parathion (MPT) and methyl paraoxon. Acetylcholin-
esterase activity in the ventral nerve cord was significantly
depressed in moribund shrimp after MPT exposure for 6 h to
1.3mg/L and not depressed after 74 h to methyl paraoxon at
0.98 mg/L. The excised ventral nerve cord was exposed in
vitro, resulting in 100% inhibition after 1 h to 60 mg/L MPT
and 100% inhibition after 1 h to 300 mg/L methyl paraoxon.
In the in vivo MPT study, AChE activity in exposed non-
moribund shrimp was not significantly different from the con-
trol. The authors concluded that there was not a direct rela-
tionship between AChE inhibition and death in these shrimp
and that analysis of AChE in the ventral nerve cord is not a
reliable indicator of OP exposure [39].
The nervous tissue of the penaeid shrimp, Metapenaeus
monoceros, was assayed for AChE activity following in vivo
exposure to MPT and phosphamidon [13]. After 48 h, enzyme
activity in the shrimp was depressed 63.6% following exposure
to the 48-h LC50 of 0.12 mg/L MPT. Exposure to a sublethal
dose of 0.04 mg/L MPT for 48 h also significantly depressed
AChE levels by 34.9%. These levels were still significantly
depressed (although to a lesser extent) 7 d after shrimp from
both exposures were placed in clean water. Acetylcholines-
terase levels were also reduced (53.61%) after a 48-h exposure
to the 48-h LC50 for phosphamidon (1.2 mg/L) and reduced
by 28.03 % at a sublethal exposure of 0.4 mg/L. A more rapid
recovery was observed in these shrimp when placed in clean
water for 7 d. Thus, recovery of AChE activity in these shrimp
took 7 d or longer even when the exposures were at sublethal
concentrations [13].
The shrimp Palaemon serratus was exposed to phosalone
for 29 d at concentrations ranging from 0.1 up to 1,000 mg/
L. After 12 h, the shrimp in the highest concentration had an
AChE inhibition in muscle homogenates of 91.8%. No shrimp
at this exposure concentration survived beyond 12 h. Only
those shrimp exposed at the lowest concentration survived for
29 d. Significant AChE inhibition (42.3%) was also observed
in these shrimp [40].
The difference in the response of two shrimp species to
AChE inhibition was explored by Lignot et al. [41]. Juvenile
Penaeus stylirostris were exposed to fenitrothion at sublethal
concentrations of 4, 6, and 8 mg/L for 24 h. Acetylcholines-
terase activity decreased in muscle tissue by 18, 13, and 16%,
respectively. P. vannamei were exposed to higher concentra-
tions from 10 to 30 mg/L for 24 h, but no decrease in activity
was detected in the muscle tissue. However, in moribund P.
vannamei, AChE activity significantly decreased with in-
creased fenitrothion concentrations (up to 30% at 30 mg/L).
This lack of inhibition was similar to the one reported by
Schoor and Brausch [39] in P. duorarum exposed to lethal
concentrations of methyl parathion. Lignot et al. [41] theorized
that the difference in AChE activity between the two penaeids
can be attributed to different affinities and phosphorylation
Environ. Toxicol. Chem. 20, 2001 41
rates of AChE and that fenitrooxon may be more rapidly hy-
drolyzed in P. vannamei.
Studies with other OPs have also found a lack of AChE
inhibition in shrimp following exposure to concentrations
based on their respective LC50 values. Rompas et al. [42]
increased fenithrothion concentrations to 50 times higher than
the 24-h LC50 in order to achieve a 50% inhibition in AChE
in tiger shrimp (P. japonicus) larvae. Diazinon levels 300
times higher than the 24-h LC50 were required in the same
shrimp species to achieve a 50% reduction in AChE activity.
There was no indication of the physiological condition (swim-
ming or moribund) of the shrimp prior to analysis.
Key [43] was not able to obtain a 24-h EC50 for AChE
inhibition in adult grass shrimp (Palaemonetes pugio) after
exposure to malathion. For malathion-exposed grass shrimp,
no more than six times the 48-h LC50 could be used for the
highest concentration without complete mortality. At this con-
centration, the shrimp were either dead or moribund. There-
fore, a 50% reduction in AChE levels could not be approached
without total mortality in the exposed shrimp. Adult shrimp
surviving malathion exposure did not have more than a 30%
reduction in AChE. Pulse dose exposures, representing more
of a field situation of OP exposure, of malathion to larval grass
shrimp did not produce significant reductions in AChE activity.
One 6-h pulse and four 6-h pulse doses of 1.88, 7.50, and 30.0
mg/L malathion caused a trend toward reduced activity at the
highest concentration, but it was not significant [43, 44]. A
24-h continuous-exposure EC50 for newly hatched and 18-d-
old larval grass shrimp exposed to malathion was obtained,
however, with results being 7.33 and 22.04 mg/L, respectively.
The author hypothesized that several factors could lead to the
limited inhibition in adults exposed to concentrations at or
above the LC50. He suggested, for example, that specific path-
ways essential to shrimp function and survival could be ex-
periencing detrimental inhibition not apparent in the assay and
that enzymes other than AChE may be inhibited by malathion
[43].
Lund et al. [17] exposed two late stages of grass shrimp
(P. pugio) embryos to malathion and chlorpyrifos. The 24-h
malathion EC50s for stage VI and stage VII embryos were
55.53 and 29.93 mg/L, respectively. The 24-h chlorpyrifos
EC50s for stages VI and VII embryos were much lower at
0.49 and 0.36 mg/L, respectively. The differences in sensitivity
were seen as differences in the persistence of the two insec-
ticides as well as the inherent toxicity of the compounds [17].
Key [43] also found much lower 24-h EC50s for chlorpyrifos
than malathion in newly hatched larvae (0.42 mg/L), 18-d-old
larvae (0.27 mg/L), and adult grass shrimp (1.42 mg/L). Six-
hour pulse doses to chlorpyrifos yielded interesting results in
grass shrimp larvae [16]. Acetylcholinesterase was signifi-
cantly reduced after 6 h of exposure to 1.6 mg/L chlorpyrifos,
but the next highest exposure of 0.4 mg/L had significantly
higher AChE activity than the control group. After four 6-h
doses, the 0.4-mg/L group was once again significantly higher
than controls. This effect was most likely the result of a phys-
iological compensatory response due to insecticide stress and
was similar to that observed by McHenery et al. [36], when
lobster larvae were exposed to low concentrations of dichlor-
vos as discussed previously.
Palaemonetes pugio larvae were also subjected to 6-h pulse
doses of azinphosmethyl at concentrations of 0.15, 0.60, and
2.40 mg/L [45]. After one dose, the AChE activity significantly
decreased at all three concentrations. After four doses, the
42 Environ. Toxicol. Chem. 20, 2001
Table 1. Summary of insecticide-related effects on brain acetylcholinesterase activity and survival observed
in mummichogs deployed at the EXP 2 site (near Charleston, SC, USA) during 1988 and 1989
Maximum
azinphosmethyl Azinphosmethyl
Field test concentration concentration
date (mg/L) at 24 h (mg/L)
6/7–11/88 3.44 0.57
6/11–15/88 0.57 0.55
6/23–27/88 0.00 0.00
6/3–7/89 1.73 1.12
6/11–15/89 0.37 0.21
6/15–19/89 2.46 0.72
6/23–27/89 7.00 1.60
activity was significantly decreased in the two highest con-
centrations. As with chlorpyrifos, azinphosmethyl pulse dose
exposures reduced AChE activity in larvae significantly more
than malathion. Key [43] also found much lower 24-h EC50s
for azinphosmethyl than malathion in newly hatched larvae
(0.35 mg/L), 18-d-old larvae (0.55 mg/L), and adult grass
shrimp (3.29 mg/L). Cochran and Burnett [46] also exposed
adult grass shrimp to azinphosmethyl at a concentration of 2.0
mg/L for 24 h. They found that AChE activity at this concen-
tration was not significantly different; however, variability in
this study was high.
Other marine invertebrates examined as potential indicator
organisms in AChE biomarker studies include copepods. For-
get et al. [47] exposed Tigriopus brevicornis to several com-
binations of OP insecticides that included dichlorvos mixed
with each of copper, arsenic, and cadmium; malathion mixed
with each of the same three metals; and a dichlorvos–malathion
mixture. Each mixture contained the two compounds in equal
fractions of their LC50 for the copepod. All combinations,
except those containing cadmium, reduced AChE activity after
96 h of exposure by at least 65% when mixture levels were
only one-fourth of the LC50. The dichlorvos–malathion mix-
ture, at a 50th and a 100th of the LC50, was additive in its
reduction of AChE activity. The authors stated that the ad-
ditivity potential does not decrease when lethal concentrations
are reduced to sublethal concentrations [47].
FIELD STUDIES
Fish
A number of studies have evaluated the response of AChE
in estuarine fish exposed to OP insecticides in the environment.
Williams and Sova [48] compared brain AChE activity levels
in menhaden (Brevoortia tyrannus) and Atlantic croakers from
a polluted area of the Ashley River near Charleston, South
Carolina, to levels in fish of the same species collected from
reference sites. Acetylcholinesterase activity in moribund men-
haden was depressed by ;50% in comparison to reference site
fish. Apparently healthy menhaden from the polluted site
showed inhibition of 17%. Croakers from the polluted area
had 36% brain AChE inhibition. Analysis of water samples
from the polluted area indicated the presence of at least two
AChE-inhibiting compounds.
In another study, caged mummichogs were exposed to four
consecutive applications of chlorpyrifos [49]. After the first
application, brain AChE in treated fish was inhibited by 74%
relative to the controls. Inhibition increased to .90% after the
second treatment, and 19% mortality was observed. After the
fourth application, AChE activity in treated fish was only 1%
M.H. Fulton and P.B. Key
% Acetylcholinesterase
inhibition % Mortality
47 0
22 0
0 0
63 0
0 0
85 0
98 17
of normal, but no additional mortality was observed. The au-
thors also noted that activity in fish collected 69 d after the
last treatment was still depressed by 62%.
Acetylcholinesterase and BChE activity was measured in
the muscle tissue of dab (Limanda limanda) collected from a
series of stations in the North Sea [50]. Both AChE and BChE
activity were depressed in fish from nearshore stations in com-
parison to those from offshore sites. The authors hypothesized
that the observed effects were related to river inputs and the
accumulation of contaminants. They suggested that the effects
may have been due to the presence of OP or carbamate com-
pounds.
The effects of insecticides present in nonpoint-source ag-
ricultural runoff on brain AChE activity in caged mummichogs
was evaluated during two growing seasons (1988–1989) at
tidal creek sites adjacent to agricultural fields located south of
Charleston, South Carolina [27,51,52]. A series of 96-h in situ
bioassays were conducted in 1988 and 1989 at three sites. Two
of the sites (EXP 1 and EXP 2) were located adjacent to farms
with extensive tomato crops under cultivation. The third site
(REF) was located in close proximity to the other two sites
but was located within the drainage basin of rural, low-density
housing and upland forest. It received no direct inputs of ag-
ricultural runoff and served as a reference site. During each
of the bioassays, water samples were collected and analyzed
for insecticides being applied to the adjacent farms. These
insecticides included endosulfan (an organochlorine), fenval-
erate (a synthetic pyrethroid), and azinphosmethyl (an OP
compound). Brain AChE activity was depressed in caged
mummichogs deployed at the EXP 2 site after five of the seven
field deployments in 1988 and 1989 (Table 1). In all cases
where inhibition was observed, significant concentrations of
azinphosmethyl were measured in water samples collected at
the EXP 2 site. Measured inhibition levels ranged from 0 to
98% for the seven bioassays, while azinphosmethyl concen-
trations ranged from 0.00 to 7.00 mg/L. Significant brain AChE
inhibition was observed at all azinphosmethyl concentrations
$0.57 mg/L. Mortality was observed in only one of the field
deployments when brain AChE activity was reduced to 2% of
normal. The authors also compared the effects on brain AChE
observed in the field study with those observed under labo-
ratory conditions. They calculated a laboratory-derived 24-h
EC50 (for azinphosmethyl-induced brain AChE inhibition) and
compared this to a field-derived EC50 (based on the inhibition
measured in the field-deployed mummichogs and the 24-h av-
erage azinphosmethyl concentration quantified in water sam-
ples). They found excellent agreement between these two ap-
proaches, with the laboratory-derived value being 0.90 mg/L,
Acetylcholinesterase inhibition in estuarine organisms
while the field-derived value was 1.13 mg/L. The results of
this study suggest that brain AChE inhibition is a sensitive
indicator of OP insecticide exposure for this species and that
significant inhibition can be detected at concentrations well
below those that cause mortality. They also indicate that a
simple 24-h laboratory exposure may be an excellent predictor
of OP-induced AChE inhibition in the field.
Invertebrates
Escartin and Porte [53] evaluated the potential of using the
mussel Mytilus galloprovincialis as a monitor for the effects
of pesticide runoff in an agricultural area of the Ebro Delta,
Spain. Laboratory-derived IC50 values were determined for
gills and digestive glands after in vitro exposure to fenitrothion
and fenitrooxon for 15 min. Gills were more susceptible to
inhibition by the compounds than digestive glands. In gills,
the IC50 was 300 mM (;83,175 mg/L) for fenitrothion and
much lower at 5.8 mM (;1,514 mg/L) for fenitrooxon, indi-
cating that fenitrothion requires activation to its oxygen ana-
logue in order to cause significant AChE inhibition. In terms
of field-collected mussels from the agricultural region, AChE
activity in the gills was lowest in April, corresponding to when
irrigation channels from the farm fields are flushed into the
mussel breeding grounds of the delta. As fenitrothion was the
pesticide of choice in this region, it was considered a factor
in the lowered AChE activity. However, no water samples from
the region were analyzed for OPs, and the authors stated that
higher water temperatures can also cause reductions in AChE
activity.
In an earlier study, Bocquene et al. [54] collected the mussel
species M. edulis along the French coast for AChE activity
analysis. Mean activity level for all the samples taken was 228
units/min/mg P with highest activity in the north of France
and lowest activity in the south and Mediterranean coast. As
with the study mentioned previously, no water samples were
analyzed for the presence of OP compounds in the areas in
which the mussels were collected, so the authors were unable
to directly link the reductions in AChE to OP or carbamate
exposure.
SUMMARY
In general, most studies with estuarine fish species have
indicated that brain AChE inhibition levels in excess of 70%
are well correlated with imminent mortality. There are indi-
cations, however, that this relationship may not hold true for
all species. For example, the results from the previously de-
scribed field and laboratory studies with mummichogs appear
to suggest that this species, in particular, may be able to tolerate
very high levels of brain AChE and that inhibition in the
peripheral nervous system (muscle) may be a better predictor
of mortality. The discrepancies point out the need to under-
stand the specific relationship between AChE inhibition and
lethality for any species that is being considered for use as a
bioindicator. In all cases, it appears that significant levels of
brain AChE inhibition can be measured in fish exposed to OPs
at concentrations well below those causing mortality. Recov-
ery of AChE activity in fish exposed to OPs appears to be a
function largely of the degree of the initial inhibition as en-
zyme recovery requires de novo synthesis of the enzyme. Other
sublethal physiological effects (e.g., reduced stamina) have
been measured in fish concurrent with AChE inhibition; how-
ever, in most cases these effects have been observed only at
near-lethal concentrations.
Environ. Toxicol. Chem. 20, 2001 43
The relationship between AChE inhibition and mortality in
invertebrates is generally less well established than that in fish.
Although inhibition of AChE has been measured in a variety
of invertebrates following OP exposure, mortality is often ob-
served in association with very low levels of inhibition. This
may be due, in part, to the fact that AChE activity is most
often measured in invertebrates using whole-body homoge-
nates rather than specific neurological tissue preparations. Us-
ing this approach, significant inhibition in invertebrates typi-
cally is only observed at near-lethal concentrations. The use
of AChE inhibition in invertebrates as a biomarker of OP
exposure has potential; however, more research is needed to
clarify the relationships between OP exposure, AChE inhibi-
tion, and mortality.
NEW AREAS OF RESEARCH
Although OP-induced AChE inhibition in estuarine organ-
isms has been under study since the 1960s, additional work
is needed in several important areas. The relationship between
AChE inhibition in various fish tissues and mortality requires
further study since the relationship does not appear uniform
for all species tested. Some species appear to be able to tolerate
very high levels of brain inhibition without the associated
mortality observed in most species. The relationship between
the sensitivity of brain and muscle AChE also appears to be
species specific. In some species, AChE sensitivity in muscle
tissue mirrors that in brain tissue, while in others muscle tissue
is much more insensitive. In addition, BChE activity has been
measured in the muscle tissues of some estuarine fish. The
presence of this enzyme can influence the interpretation of
inhibition data obtained using standard techniques (e.g., Ell-
man method) since these approaches cannot distinguish be-
tween these esterases. Because BChE is typically more sen-
sitive than AChE [8,9], these approaches can overestimate the
effects on the neurological target enzyme. Future studies
should account for these complicating factors.
The relationship between AChE inhibition and mortality in
estuarine invertebrates has not been widely studied. Existing
data suggest that this relationship is highly variable among
invertebrates. Additional research is needed to determine
whether these observed differences are due to differences in
analytical techniques or reflect truly species-specific responses.
The possibility that OP-induced toxicity in estuarine inverte-
brates is influenced by factors other than a compound’s potency
as an AChE inhibitor also merits further study.
Another area that deserves study in both fish and inverte-
brates is the possibility that OPs may affect neurological de-
velopment. Recent studies in mammalian and avian systems
suggest that both AChE and the neurotransmitter ACh have a
role in the development of the nervous system [55,56]. These
studies suggested that AChE may play a direct role in neuronal
differentiation and that AChE activity appears in tissues of the
nervous system while axons are developing and prior to the
formation of synapses [55,57]. Some AChE inhibitors have
been found to suppress neurite outgrowth [58,59]. Other re-
searchers have reported that ACh released from the developing
axons regulates growth and differentiation in central nervous
system neurons [56]. The relevance of these findings to es-
tuarine fish and invertebrates is unknown; however, they clear-
ly suggest that studies are needed to evaluate these phenomena
in estuarine species. Any compound capable of inhibiting
AChE has the potential to influence the levels of both AChE
and ACh.
44 Environ. Toxicol. Chem. 20, 2001
As this review has demonstrated, monitoring of AChE in
estuarine species can play an important role in assessing the
risk of OPs in the environment. Additional work is needed,
however, in a variety of areas to facilitate the interpretation
of depressed AChE in estuarine organisms and to validate the
use of this biomarker in estuaries.
Acknowledgement—The National Ocean Service (NOS) does not ap-
prove, recommend, or endorse any proprietary product or material
mentioned in this publication. No reference shall be made to NOS,
or to this publication furnished by NOS, in any advertising or sales
promotion which would indicate or imply that NOS approves, rec-
ommends, or endorses any proprietary product or proprietary material
mentioned herein or which has as its purpose any intent to cause
directly or indirectly the advertised product to be used or purchased
because of NOS publication.
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