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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 derivatives of either phosphoric acid or thiophosphoric acid.
inputs from agriculture and urbanization (resort development The majority now in use, such as azinphosmethyl, chlorpyrifos,
and golf courses) may impact the surrounding estuaries and and malathion, contain the thiono moiety (5S). The substi-
marshes. Estuaries rank as one of the most productive natural tution of the 5S for 5O on the phosphorus atom increases
habitats, where large phytoplankton populations support a va- the toxicity of the insecticide, such as is the case with mala-
riety of other organisms, including many commercially and thion and its oxygen analogue, malaoxon [3].
recreationally important marine fish and crustacean species The uses of OPs are highly varied. In agriculture, OPs are
that use estuaries as nursery grounds [1]. Transport of pesti- used to control insects on fruits, vegetables, grain crops, and
cides to these delicate ecosystems therefore creates the need stored seeds [4]. The OPs chlorpyrifos and terbufos are the
to fully understand effects in resident biota. In many areas of two most widely used insecticides in agriculture [5]. House-
the world, these sensitive ecosystems are at risk because of hold uses include the control of cockroaches, houseflies, and
the nonpoint-source runoff of pesticides from agricultural and termites along with protection of plants of horticultural interest
urban sources. [4]. The most common insecticides used in home and garden
The majority of insecticides currently in use are organo- applications are the OPs diazinon and chlorpyrifos. For in-
phosphorus, carbamate, and synthetic pyrethroid compounds. dustrial, commercial, and government use, the top insecticides
Organophosphorus insecticides (OPs) were developed in Ger- are again OPs (chlorpyrifos and malathion [5]).
many during the 1930s as a substitute for nicotine and as Organophosphorus insecticides produce toxicity by inhib-
potential chemical warfare agents [2]. Because of their rela- iting cholinesterase enzymes in both vertebrate and inverte-
tively nonpersistent characteristics in the environment, OPs brate organisms. These enzymes are responsible for the re-
have become one of the most widely used classes of insecti- moval of the neurotransmitter acetylcholine (ACh) from the
cides worldwide. Although these compounds offer the advan- synaptic cleft through hydrolysis [6]. In vertebrates, ACh acts
tage of rapid degradation in the environment, they generally as an excitatory transmitter for voluntary muscle in the somatic
lack target specificity and have high acute toxicity toward nervous system. Acetylcholine also serves as both a pregan-
many nontarget vertebrate and invertebrate species. Thus, glionic and a postganglionic transmitter in the parasympathetic
many terrestrial and aquatic organisms may be at risk for in- nervous system and as a preganglionic transmitter in the sym-
toxication caused by exposure to these compounds in the en-
pathetic nervous system. In critical regions of the central ner-
vironment.
vous system, ACh serves as an excitatory transmitter. When
* To whom correspondence may be addressed (mike.fulton@noaa.gov). cholinesterases are inactivated by the binding of OPs, an ac-
cumulation of ACh occurs at the nerve synapse, interfering
37
38 Environ. Toxicol. Chem. 20, 2001 M.H. Fulton and P.B. Key

with the normal nervous system function. This produces rapid in estuarine fish species. Sheepshead minnows (Cyprinodon
twitching of voluntary muscles followed by paralysis [6,7]. variegatus) were exposed to selected OP insecticides (Guth-
Once bound, organophosphorus compounds are considered ir- ion, phorate, parathion, phosphamidon, Cygon, malathion,
reversible inhibitors, as recovery usually depends on new en- EPN, Dursban, dichlorvos, diazinon, Dibrom, and methyl para-
zyme synthesis [6]. thion) at sublethal and median lethal (40–60% mortality) con-
Cholinesterases are usually divided into two broad classes: centrations (as listed in Coppage [23]). Inhibition of brain
the acetylcholinesterases (AChEs) and the butyrylcholinester- AChE was always greater at concentrations that caused mor-
ases (BChEs). In fish, brain and muscle tissue contain mostly tality. Brain inhibition of greater than 80% was observed in
AChEs, while liver and plasma contain mostly BChEs [6]. all fish that survived median lethal exposures. The authors also
Significant amounts of BChE activity, however, have been observed that given the variability observed in AChE levels
reported in the muscle tissues of several fish species [8,9]. in control fish, activity must be depressed by .13% to indicate
Neurotransmission in invertebrates has not been as well exposure to OP insecticides. Their findings suggest that brain
characterized as in vertebrates. Crustaceans contain both in- inhibition levels of 20 to 70% in live fish could be diagnostic
hibitory and excitatory motoneurons that usually employ other of OP insecticide exposure.
transmitters. When ACh is present, its primary function is as In a subsequent study, brain AChE activity was measured
a neurotransmitter for afferent nerve fibers. Welsh [10] re- in several estuarine fish species (spot, Leiostomus xanthurus;
viewed much of the early work (late 1930s to late 1950s) that Atlantic croaker, Micropogon undulatus; sheepshead min-
established the presence of cholinesterases in several species nows; and pinfish, Lagodon rhomboides) that survived median
of crustaceans, mainly crab, lobster, and crayfish. More re- lethal exposures to several OP insecticides (malathion, naled,
cently, AChE activity has been measured in a variety of crus- Guthion, and parathion, as written in [24]). In all cases, AChE
taceans, including the brain and ventral ganglion of the blue activity was inhibited by 70 to 96% [24]. Their findings sug-
crab, Callinectes sapidus [11]; the hepatopancreas of oysters gested that the response is similar for a variety of estuarine
(Crassostrea virginica) and brown shrimp (Penaeus sp.) [12]; species regardless of the OP compound tested.
tissues of the nervous system in penaeid prawns, Metapenaeus Brain AChE activities were also compared in pinfish ex-
monoceros [13]; muscle tissue of the shrimp, Palaemon ser- posed to lethal and sublethal concentrations of the OP insec-
ratus [14]; whole-body tissues of an Australian freshwater ticide malathion [25]. Brain AChE activity was measured in
shrimp, Paratya australiensis [15]; and the whole-body tissues fish that survived a near-median kill at 3.5, 24, 48, and 72 h.
of adult, larval, and embryonic grass shrimp, Palaemonetes Reductions in enzyme activity (72–79%) were similar for all
pugio [16,17]. these lethal exposure experiments regardless of the exposure
Monitoring of AChE inhibition has been widely used in time or insecticide concentration. Their data indicated that
terrestrial and freshwater aquatic ecosystems as an indicator mortality is likely when brain inhibition reaches 70 to 90%.
of OP insecticide exposure and physiological effects in ex- In a similar study, pinfish were exposed to naled at selected
posed animals [18–20]. Although the presence of many OP concentrations for varying periods of time, and then brain
insecticides in the environment can be discerned using ana- AChE activity was measured and compared to levels in control
lytical chemistry techniques, AChE monitoring in the envi- fish [26]. Inhibition at 15 mg/L ranged from 59% at 24 h to
ronment may offer distinct advantages over the use of ana- 70% after 96 h. No mortality was observed at this concentra-
lytical chemistry alone. First, AChE inhibition is the primary tion at any time point. At higher naled concentrations (25–75
mechanism by which OP insecticides produce toxicity. Thus, mg/L) that caused median kills at various time points, brain
the biomarker effect is directly linked to the compounds toxic AChE inhibition was always greater than 80%. As in the earlier
mode of action. When significant AChE inhibition is measured, study, mortality was always observed in this species when
we know not only that the organism has been exposed but also brain inhibition was in excess of 70%. They also reported that
that a sufficient dose of the compound has reached the target significant brain AChE inhibition was observed at concentra-
site to produce a physiological effect. In addition, as previously tions well below those causing mortality.
stated, most OP insecticides degrade rapidly in the environ- In all the previously described studies, the authors found
ment, and their concentrations in environmental samples may that when brain AChE inhibition reached 70 to 80%, mortality
fall below detectable levels in hours to days. Acetylcholin- was likely. Other investigators have found that this relationship
esterase inhibition in many species, however, persists for much may be less distinct in other estuarine fish. Fulton [27] inves-
longer—days to weeks [21,22]. tigated the lethal and sublethal effects of the OP insecticide
The goal of this review is to summarize studies that have azinphosmethyl in the mummichog Fundulus heteroclitus, a
investigated the use of AChE inhibition as a monitor of OP common fish in eastern U.S. estuarine tidal creek habitats. The
insecticide exposure and effects in estuarine fish and inver- 96-h median lethal concentration (LC50) for this compound
tebrate species. It discusses the relationship between OP-in- was 32.16 mg/L, while the 24-h median effective concentration
duced AChE inhibition and lethality as well as sublethal re- (EC50) for brain AChE inhibition was 0.81 mg/L. Thus, the
sponses under both laboratory and field conditions. In addition, 96-h LC50 was .39 times higher than the 24-h EC50 for brain
it describes some of the limitations associated with AChE AChE inhibition. The lowest concentration used in the acute
inhibition as a biomarker and discusses areas of needed re- toxicity tests that caused no mortality after 96 h (3.5 mg/L)
search. was more than four times higher than the 24-h EC50 for in-
LABORATORY STUDIES
hibition of AChE in brain tissue. The highest concentration
used in the 24-h sublethal tests (3.9 mg/L) caused ;94% in-
Fish hibition of brain tissue AChE activity. This concentration was
Relationship between AChE inhibition and mortality. A much lower than the 96-h no-observed-effect concentration
number of studies have examined the relationship between (19.80 mg/L) determined from the 96-h acute toxicity tests.
specific levels of OP-induced AChE inhibition and lethality These findings suggest that this species is able to tolerate ex-
Acetylcholinesterase inhibition in estuarine organisms Environ. Toxicol. Chem. 20, 2001 39

tremely high levels of brain AChE inhibition and that the sponse fashion. Inhibition ranged from 12% at 0.005 mg/L to
relationship between specific levels of brain inhibition and 58% at 0.213 mg/L. In the 2 3 24-h exposure regime, inhi-
mortality may be different than that observed in many other bition immediately following the final 24-h exposure was 16%
estuarine species. at 0.005 mg/L and 50% at 0.213 mg/L. After one week of
In a comparative study, Van Dolah et al. [28] evaluated the depuration in clean water, activity in the 0.005-mg/L group
effects of azinphosmethyl in juvenile red drum (Sciaenops had recovered to control levels, while activity in the 0.213-
ocellatus) and adult mummichogs of comparable size. Fish mg/L group was still depressed by 34%. After six weeks, ac-
were exposed to a series of azinphosmethyl concentrations, tivity in both treatment groups had recovered to control levels.
and then brain AChE activity was measured. The EC50s for Results of this study indicated that the time for enzyme re-
brain inhibition were calculated and compared to the 96-h covery was a function of the degree of initial inhibition. This
LC50s for each species. The relationship between azinphos- is likely because the recovery of the enzyme activity is largely
methyl-induced AChE inhibition and mortality was quite dif- a result of de novo synthesis of enzyme protein, and the greater
ferent for the two species tested. In the red drum, the 96-h the degree of inhibition, the more protein synthesis is required.
LC50 (6.2 mg/L) and the 24-h EC50 for brain AChE inhibition In another study, mummichogs were exposed to chlorpyr-
(5.2 mg/L) were quite similar. In the mummichog, however, ifos in 6-h pulsed exposures [32]. In one experiment, fish were
the 96-h LC50 was 60 times greater than the 24-h EC50 (1.0 exposed to 6-h chlorpyrifos pulses daily for 4 d. In a second
mg/L), and this relationship was quite similar to that previously experiment, fish were exposed to 6-h pulses weekly for four
reported by Fulton [27]. Subsequent experiments with these weeks. Brain AChE activity was monitored as well as caudal
two species indicated that the sensitivity of red drum muscle vertebrae strength. Maximum AChE inhibition was higher for
tissue AChE (24-h EC50 5 6.8 mg/L) was quite similar to that the daily exposure regime than for the weekly exposures. In
in brain tissue, while mummichog muscle AChE (24-h EC50 general, the daily exposure regime exhibited cumulative in-
.5mg/L) was much less sensitive than brain tissue [29]. These hibition with inhibition at 96 h being greater than that at 48
findings suggest that there are clear species- and/or age-specific h. For the weekly exposure scenario, AChE inhibition at four
differences in the relationship between AChE inhibition in weeks was similar to that observed after two weeks. This
brain and muscle tissue and mortality. These studies also sug- suggests that the weekly recovery intervals allowed almost
gest that given the response observed in mummichogs, inhi- complete recovery of enzyme activity. The authors also re-
bition of AChE in the peripheral nervous system may be a ported that strength analysis of the caudal vertebrae indicated
better predictor of OP-induced mortality than brain inhibition. that vertebrae were weaker at selected time points for each of
In an in vitro study, homogenates from the dorsal muscle the exposure regimes. This suggests that chlorpyrifos concen-
of the dragonet (Callinymus lyra) and the sole (Solea solea) trations sufficient to cause the level of inhibition observed in
were exposed to dichlorvos, fenitrothion, and phosalone [30]. this study may also affect bone strength.
Dichlorvos was the most potent inhibitor in the fish tissues Relationship between AChE inhibition and sublethal ef-
with a 50% inhibitory concentration (IC50) of 2.3 3 1028 M fects. A number of researchers have investigated the relation-
(;5.1 mg/L) in dragonet muscle and 3.1 3 1027 M (;68.5 ship between AChE inhibition and other sublethal effects. In
mg/L) in sole tissues. The IC50s for dragonet were 2.6 3 1025 one study, the swimming performance of sheepshead minnows
M (;7,209 mg/L) and 6.2 3 1027 M (;228 mg/L) for feni- was measured in a stamina tunnel at the end of life-cycle
trothion and phosalone, respectively. In sole tissues, the IC50s toxicity tests with the OP insecticides EPN and Guthion [33].
for these two OPs were 1.2 3 1024 M (;33,270 mg/L) and Brain AChE inhibition was also measured. These end points
2.5 3 1026 M (;919 mg/L), respectively. The authors sug- were compared to survival, growth, and reproduction data ob-
gested that the greater sensitivity to dichlorvos for both fish tained during the course of the life-cycle tests. Swimming
tissues was because this OP is a direct cholinesterase inhibitor, stamina was affected in fish exposed to EPN at 4.1 and 2.2
which does not require activation to an oxygen analogue, such mg/L. At 2.2 mg/L, stamina was reduced by 43%, while at 4.1
as phosalone and fenitrothion. These three OPs were also tested mg/L, stamina was decreased by 54%. Swimming performance
for synergistic effects after each had been mixed with the was not affected by Guthion concentrations up to 0.5 mg/L, a
carbamates (C) carbaryl or carbofuran or with each other. The concentration that affected reproduction. Acetylcholinesterase
fish showed greater sensitivity to the combined effects of these activity was significantly depressed at all concentrations of
insecticides than a shrimp and oyster species that were also EPN (0.25–7.9 mg/L) and Guthion (0.06–0.5 mg/L). Effects
tested. In general, the OP-C combinations were highly syn- on swimming stamina were observed only when AChE inhi-
ergistic in terms of AChE inhibition, while the OP-OP com- bition was greater than 80%.
binations were less so. Increasing incubation time increased Van Dolah et al. [28] reported that red drum exposed to
the synergistic effect. The authors concluded that the mech- azinphosmethyl for 6 h at 12 mg/L had reduced swimming
anisms by which inhibitory effects could be enhanced was not stamina. This concentration is approximately two times the
well understood. 24-h EC50 (5.2 mg/L) for brain AChE determined in the same
Enzyme inhibition and recovery. Other studies have inves- study. No effects on swimming stamina were observed at lower
tigated the persistence of AChE inhibition in brain tissue fol- insecticide concentrations (3–6 mg/L). Swimming stamina in
lowing OP exposure. In one study, Atlantic salmon (Salmo mummichogs was not affected at 19.4 mg/L (the highest con-
salar) were exposed to sublethal concentrations of fenitrothion centration tested), which is ;20 times higher than the 24-h
[31]. The authors utilized two different exposure regimes. In EC50 (1.0 mg/L).
the first, fish were exposed continuously for 7 d, while in the Both of these studies suggest that swimming stamina in fish
second fish were exposed for 24 h, allowed to recover for 7 is affected only at very high levels of brain AChE inhibition.
d in clean water, and then re-exposed to fenitrothion for an In an earlier study, however, Post and Leasure [22] reported
additional 24 h. In the 7-d exposures, inhibition increased with that significant effects on swimming stamina in three species
increasing insecticide concentrations in a typical dose–re- of salmonids were observed in conjunction with much lower
40 Environ. Toxicol. Chem. 20, 2001 M.H. Fulton and P.B. Key

levels of AChE inhibition. They reported that for the three serve an apparent hormetic effect of increased AChE activity
species tested (brook trout, Salvelinus fontinalis; rainbow in larvae exposed at the 0.01-mg/L concentration that may
trout, Salmo gairdneri; and coho salmon, Oncorhynchus ki- interfere with the ability to distinguish between animals ex-
tusch), fish that had brain AChE activity reduced by ;50% posed to low concentrations and unexposed animals.
experienced stamina reductions of 23 to 44%. Another study [37] examined the effect of dichlorvos on
AChE activity in two commercial bivalves, Manila clam (Rud-
Invertebrates itapes philippinarum) and Japanese oyster (Crassostrea gi-
The use of AChE as a biomarker of OP exposure in marine gas). Both organisms were exposed for 6 h to 0.1 and 1.0 mg/
invertebrates is not as well established as in fish. Because of L dichlorvos. Oyster gills and clam tissue were sampled from
the lack of a clearly defined organ (such as brains in fish), 0 to 48 h for AChE activity. The activity was lowest in oyster
researchers have relied on a variety of techniques to determine gill after 4 h of exposure to 1.0 mg/L with 87% inhibition and
AChE levels in these organisms. Various investigators have after 6 h to 0.1 mg/L with 83% inhibition. Recovery was not
used hemolymph, nerve ganglion, muscle tissue, and whole- observed at the end of the experiment. The AChE decrease
body tissues of invertebrates to evaluate the effects of OPs on was slower in clam tissue with only 64% inhibition after 10
AChE levels. Blue crabs, Callinectes sapidus, were exposed h from the 1.0-mg/L concentration and 42% inhibition after 8
to DEF(S,S,S,-tri-n-butyl phosphorotrithioate) at concentra- h from the 0.1-mg/L concentration. The response to dichlorvos
tions of 1.0 to 10.0 mg/L for 96 h and then assayed for AChE was more rapid in oyster gill than clam tissue. While no an-
activity [11]. In control crabs, high AChE activity was found imals died at these concentrations, the authors suggest that the
in both the ventral ganglion and the brain with negligible ac- decrease in AChE activity indicated an early deleterious effect
tivity in claw musculature. Exposed crabs showed a 90% re- that may affect other functions such as growth.
duction in ventral ganglion AChE activity and a greater than Galgani and Bocquene [14] found that AChE from whole
80% reduction in brain AChE activity. Other crabs were ex- mussel, Mytilus edulis, was less sensitive to five organophos-
posed for 48 h to 4.0 mg/L, then transferred to clean seawater phates than the enzyme from the muscle tissues of the shrimp
for 10 d. Control AChE activity levels were significantly higher Palaemon serratus and two fish species. The OPs used in this
in the ganglion and brain after this period. This study not only in vitro test were malathion, parathion, paraoxon, temephos,
showed significant anticholinesterase effects from DEF ex- and dichlorvos in concentrations of milligrams per liter. These
posure in vulnerable tissues of C. sapidus but also indicated observations may be due, in part, to species-specific differ-
that these effects may persist for a significant period of time ences in sensitivity. They may also, however, be an indication
after a single short-term exposure. of the need to perform the AChE assay on other than the whole
In a later study, Habig et al. [34] incubated C. sapidus body, as discussed in the following research, or even the need
ganglia tissue with malathion and parathion and compared their to account for other less sensitive classes of cholinesterases
sensitivity to catfish (Ictalurus punctatus) neural tissues in- present in the organism (see also the following discussion).
cubated in the same fashion. The IC50s for crab AChE were While the previous studies indicate the potential usefulness
4.5 3 1025 M (;14,866 mg/L) for malathion and 6.9 3 1025 of bivalves as biomonitors of OP exposure, Bocquene et al.
M (;20,098 mg/L) for parathion. Crab tissue was about three [38] determined the presence of two cholinesterases in the
times more sensitive to malathion and 10 times more sensitive oyster Crassostrea gigas. The A cholinesterases were mem-
to parathion than catfish tissue. brane bound, and the B cholinesterases were soluble enzymes.
Another crab species (Carcinus maenas) was exposed in The A group was highly sensitive in vitro to the OP compounds
vivo to dimethoate for 18 h at concentrations from 0.5 to 2.0
DFP (ki 5 2.0 3 103/M/min) and paraoxon (ki 5 3.0 3 105/
mg/L [35]. For this assay, the hemolymph was nondestruc-
M/min), while the B group was considered to be almost in-
tively extracted and analyzed for AChE activity. At the highest
sensitive. The presence of this insensitive cholinesterase sug-
concentration, a 30% reduction in AChE activity was detected.
gests that improved use of AChE activity in oyster as a bio-
In comparing baseline AChE levels, the authors found that C.
marker of inhibitory effects can be achieved if these two clas-
maenas hemolymph activity at 210.7 nmol/min/mg P was low-
ses are isolated from the total cholinesterase activity.
er than Callinectes sapidus homogenate activity (555 nmol/
Bocquene et al. [30] determined 1-h IC50 values for the
min/mg P) and synaptosomal activity (833 nmol/min/mg P) as
shrimp Palaemon serratus and oyster C. gigas after exposure
reported by Habig et al. [34]. They concluded that since AChE
is primarily a membrane-bound enzyme, lower activity would to dichlorvos, fenitrothion, and phosalone. Dichlorvos was the
be expected in the hemolymph. They also suggested a direct most potent inhibitor with an IC50 of 1.1 3 1026 M (;243.08
effect of reduced AChE activity in C. maenas on cardiac neu- mg/L) for shrimp muscle and 7.3 3 1028 M (;16.13 mg/L)
ronal control because of their observation of reduced heart for oyster gill. The IC50s for shrimp were both greater than
rates in the crabs following dimethoate exposure. 1024 M for fenitrothion (;27,725 mg/L) and phosalone
Several papers have examined the effects of the OP com- (;36,780 mg/L). For oysters, the IC50s for these two OPs
pound dichlorvos used in marine fish farms to control ecto- were 1.4 3 1024 M (;38,815 mg/L) and 5 3 1024 M (;183,900
parasites. After treatment, waters containing this product may mg/L), respectively. The authors stated that the greater sen-
be discharged into surrounding estuarine areas. McHenery et sitivity to dichlorvos for both invertebrates was due to this
al. [36] exposed larval lobster (Homarus gammarus) to di- OP being a direct cholinesterase inhibitor that does not require
chlorvos for 24 h at 10-fold intervals from 0.001 to 100 mg/ activation to an oxygen analogue, as is the case with phosalone
L. They observed 50% AChE inhibition at 2.7 mg/L. This value and fenitrothion. These three OPs were tested for synergistic
was about 10-fold lower than the 24-h LC50 of 28.3 mg/L effects after each had been mixed with one of two carbamates
determined for lobster larvae in this study. The authors sug- (C) carbaryl or carbofuran or with each other. As with two
gested that AChE activity in the lobster may provide a sensitive fish species discussed earlier, the OP-C combinations were
method for determining OP exposure. They did, however, ob- highly synergistic in terms of AChE inhibition, whereas the
Acetylcholinesterase inhibition in estuarine organisms Environ. Toxicol. Chem. 20, 2001 41

OP-OP combinations were less inhibitory. The mechanisms rates of AChE and that fenitrooxon may be more rapidly hy-
by which inhibitory effects were enhanced remains unclear. drolyzed in P. vannamei.
Most research utilizing AChE activity as a biomarker of Studies with other OPs have also found a lack of AChE
OP exposure in invertebrates has been with shrimp species. In inhibition in shrimp following exposure to concentrations
one of the earlier papers, Coppage and Matthews [24] exposed based on their respective LC50 values. Rompas et al. [42]
the pink shrimp, Penaeus duorarum, to 1,000 mg/L malathion increased fenithrothion concentrations to 50 times higher than
for 48 h. The shrimp were moribund with AChE levels in the the 24-h LC50 in order to achieve a 50% inhibition in AChE
ventral nerve cord reduced an average of 75% in comparison in tiger shrimp (P. japonicus) larvae. Diazinon levels 300
to controls. times higher than the 24-h LC50 were required in the same
Another pink shrimp study [39] exposed the animals to shrimp species to achieve a 50% reduction in AChE activity.
methyl parathion (MPT) and methyl paraoxon. Acetylcholin- There was no indication of the physiological condition (swim-
esterase activity in the ventral nerve cord was significantly ming or moribund) of the shrimp prior to analysis.
depressed in moribund shrimp after MPT exposure for 6 h to Key [43] was not able to obtain a 24-h EC50 for AChE
1.3mg/L and not depressed after 74 h to methyl paraoxon at inhibition in adult grass shrimp (Palaemonetes pugio) after
0.98 mg/L. The excised ventral nerve cord was exposed in exposure to malathion. For malathion-exposed grass shrimp,
vitro, resulting in 100% inhibition after 1 h to 60 mg/L MPT no more than six times the 48-h LC50 could be used for the
and 100% inhibition after 1 h to 300 mg/L methyl paraoxon. highest concentration without complete mortality. At this con-
In the in vivo MPT study, AChE activity in exposed non- centration, the shrimp were either dead or moribund. There-
moribund shrimp was not significantly different from the con- fore, a 50% reduction in AChE levels could not be approached
trol. The authors concluded that there was not a direct rela- without total mortality in the exposed shrimp. Adult shrimp
tionship between AChE inhibition and death in these shrimp surviving malathion exposure did not have more than a 30%
and that analysis of AChE in the ventral nerve cord is not a reduction in AChE. Pulse dose exposures, representing more
reliable indicator of OP exposure [39]. of a field situation of OP exposure, of malathion to larval grass
shrimp did not produce significant reductions in AChE activity.
The nervous tissue of the penaeid shrimp, Metapenaeus
One 6-h pulse and four 6-h pulse doses of 1.88, 7.50, and 30.0
monoceros, was assayed for AChE activity following in vivo
mg/L malathion caused a trend toward reduced activity at the
exposure to MPT and phosphamidon [13]. After 48 h, enzyme
highest concentration, but it was not significant [43, 44]. A
activity in the shrimp was depressed 63.6% following exposure
24-h continuous-exposure EC50 for newly hatched and 18-d-
to the 48-h LC50 of 0.12 mg/L MPT. Exposure to a sublethal
old larval grass shrimp exposed to malathion was obtained,
dose of 0.04 mg/L MPT for 48 h also significantly depressed
however, with results being 7.33 and 22.04 mg/L, respectively.
AChE levels by 34.9%. These levels were still significantly
The author hypothesized that several factors could lead to the
depressed (although to a lesser extent) 7 d after shrimp from
limited inhibition in adults exposed to concentrations at or
both exposures were placed in clean water. Acetylcholines-
above the LC50. He suggested, for example, that specific path-
terase levels were also reduced (53.61%) after a 48-h exposure
ways essential to shrimp function and survival could be ex-
to the 48-h LC50 for phosphamidon (1.2 mg/L) and reduced
periencing detrimental inhibition not apparent in the assay and
by 28.03 % at a sublethal exposure of 0.4 mg/L. A more rapid that enzymes other than AChE may be inhibited by malathion
recovery was observed in these shrimp when placed in clean [43].
water for 7 d. Thus, recovery of AChE activity in these shrimp Lund et al. [17] exposed two late stages of grass shrimp
took 7 d or longer even when the exposures were at sublethal (P. pugio) embryos to malathion and chlorpyrifos. The 24-h
concentrations [13]. malathion EC50s for stage VI and stage VII embryos were
The shrimp Palaemon serratus was exposed to phosalone 55.53 and 29.93 mg/L, respectively. The 24-h chlorpyrifos
for 29 d at concentrations ranging from 0.1 up to 1,000 mg/ EC50s for stages VI and VII embryos were much lower at
L. After 12 h, the shrimp in the highest concentration had an 0.49 and 0.36 mg/L, respectively. The differences in sensitivity
AChE inhibition in muscle homogenates of 91.8%. No shrimp were seen as differences in the persistence of the two insec-
at this exposure concentration survived beyond 12 h. Only ticides as well as the inherent toxicity of the compounds [17].
those shrimp exposed at the lowest concentration survived for Key [43] also found much lower 24-h EC50s for chlorpyrifos
29 d. Significant AChE inhibition (42.3%) was also observed than malathion in newly hatched larvae (0.42 mg/L), 18-d-old
in these shrimp [40]. larvae (0.27 mg/L), and adult grass shrimp (1.42 mg/L). Six-
The difference in the response of two shrimp species to hour pulse doses to chlorpyrifos yielded interesting results in
AChE inhibition was explored by Lignot et al. [41]. Juvenile grass shrimp larvae [16]. Acetylcholinesterase was signifi-
Penaeus stylirostris were exposed to fenitrothion at sublethal cantly reduced after 6 h of exposure to 1.6 mg/L chlorpyrifos,
concentrations of 4, 6, and 8 mg/L for 24 h. Acetylcholines- but the next highest exposure of 0.4 mg/L had significantly
terase activity decreased in muscle tissue by 18, 13, and 16%, higher AChE activity than the control group. After four 6-h
respectively. P. vannamei were exposed to higher concentra- doses, the 0.4-mg/L group was once again significantly higher
tions from 10 to 30 mg/L for 24 h, but no decrease in activity than controls. This effect was most likely the result of a phys-
was detected in the muscle tissue. However, in moribund P. iological compensatory response due to insecticide stress and
vannamei, AChE activity significantly decreased with in- was similar to that observed by McHenery et al. [36], when
creased fenitrothion concentrations (up to 30% at 30 mg/L). lobster larvae were exposed to low concentrations of dichlor-
This lack of inhibition was similar to the one reported by vos as discussed previously.
Schoor and Brausch [39] in P. duorarum exposed to lethal Palaemonetes pugio larvae were also subjected to 6-h pulse
concentrations of methyl parathion. Lignot et al. [41] theorized doses of azinphosmethyl at concentrations of 0.15, 0.60, and
that the difference in AChE activity between the two penaeids 2.40 mg/L [45]. After one dose, the AChE activity significantly
can be attributed to different affinities and phosphorylation decreased at all three concentrations. After four doses, the
42 Environ. Toxicol. Chem. 20, 2001 M.H. Fulton and P.B. Key

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 % Acetylcholinesterase
date (mg/L) at 24 h (mg/L) inhibition % Mortality

6/7–11/88 3.44 0.57 47 0


6/11–15/88 0.57 0.55 22 0
6/23–27/88 0.00 0.00 0 0
6/3–7/89 1.73 1.12 63 0
6/11–15/89 0.37 0.21 0 0
6/15–19/89 2.46 0.72 85 0
6/23–27/89 7.00 1.60 98 17

activity was significantly decreased in the two highest con- of normal, but no additional mortality was observed. The au-
centrations. As with chlorpyrifos, azinphosmethyl pulse dose thors also noted that activity in fish collected 69 d after the
exposures reduced AChE activity in larvae significantly more last treatment was still depressed by 62%.
than malathion. Key [43] also found much lower 24-h EC50s Acetylcholinesterase and BChE activity was measured in
for azinphosmethyl than malathion in newly hatched larvae the muscle tissue of dab (Limanda limanda) collected from a
(0.35 mg/L), 18-d-old larvae (0.55 mg/L), and adult grass series of stations in the North Sea [50]. Both AChE and BChE
shrimp (3.29 mg/L). Cochran and Burnett [46] also exposed activity were depressed in fish from nearshore stations in com-
adult grass shrimp to azinphosmethyl at a concentration of 2.0 parison to those from offshore sites. The authors hypothesized
mg/L for 24 h. They found that AChE activity at this concen- that the observed effects were related to river inputs and the
tration was not significantly different; however, variability in accumulation of contaminants. They suggested that the effects
this study was high. may have been due to the presence of OP or carbamate com-
Other marine invertebrates examined as potential indicator pounds.
organisms in AChE biomarker studies include copepods. For- The effects of insecticides present in nonpoint-source ag-
get et al. [47] exposed Tigriopus brevicornis to several com- ricultural runoff on brain AChE activity in caged mummichogs
binations of OP insecticides that included dichlorvos mixed was evaluated during two growing seasons (1988–1989) at
with each of copper, arsenic, and cadmium; malathion mixed tidal creek sites adjacent to agricultural fields located south of
with each of the same three metals; and a dichlorvos–malathion Charleston, South Carolina [27,51,52]. A series of 96-h in situ
mixture. Each mixture contained the two compounds in equal bioassays were conducted in 1988 and 1989 at three sites. Two
fractions of their LC50 for the copepod. All combinations, of the sites (EXP 1 and EXP 2) were located adjacent to farms
except those containing cadmium, reduced AChE activity after with extensive tomato crops under cultivation. The third site
96 h of exposure by at least 65% when mixture levels were (REF) was located in close proximity to the other two sites
only one-fourth of the LC50. The dichlorvos–malathion mix- but was located within the drainage basin of rural, low-density
ture, at a 50th and a 100th of the LC50, was additive in its housing and upland forest. It received no direct inputs of ag-
reduction of AChE activity. The authors stated that the ad- ricultural runoff and served as a reference site. During each
ditivity potential does not decrease when lethal concentrations of the bioassays, water samples were collected and analyzed
are reduced to sublethal concentrations [47]. for insecticides being applied to the adjacent farms. These
insecticides included endosulfan (an organochlorine), fenval-
FIELD STUDIES
erate (a synthetic pyrethroid), and azinphosmethyl (an OP
Fish compound). Brain AChE activity was depressed in caged
A number of studies have evaluated the response of AChE mummichogs deployed at the EXP 2 site after five of the seven
in estuarine fish exposed to OP insecticides in the environment. field deployments in 1988 and 1989 (Table 1). In all cases
Williams and Sova [48] compared brain AChE activity levels where inhibition was observed, significant concentrations of
in menhaden (Brevoortia tyrannus) and Atlantic croakers from azinphosmethyl were measured in water samples collected at
a polluted area of the Ashley River near Charleston, South the EXP 2 site. Measured inhibition levels ranged from 0 to
Carolina, to levels in fish of the same species collected from 98% for the seven bioassays, while azinphosmethyl concen-
reference sites. Acetylcholinesterase activity in moribund men- trations ranged from 0.00 to 7.00 mg/L. Significant brain AChE
haden was depressed by ;50% in comparison to reference site inhibition was observed at all azinphosmethyl concentrations
fish. Apparently healthy menhaden from the polluted site $0.57 mg/L. Mortality was observed in only one of the field
showed inhibition of 17%. Croakers from the polluted area deployments when brain AChE activity was reduced to 2% of
had 36% brain AChE inhibition. Analysis of water samples normal. The authors also compared the effects on brain AChE
from the polluted area indicated the presence of at least two observed in the field study with those observed under labo-
AChE-inhibiting compounds. ratory conditions. They calculated a laboratory-derived 24-h
In another study, caged mummichogs were exposed to four EC50 (for azinphosmethyl-induced brain AChE inhibition) and
consecutive applications of chlorpyrifos [49]. After the first compared this to a field-derived EC50 (based on the inhibition
application, brain AChE in treated fish was inhibited by 74% measured in the field-deployed mummichogs and the 24-h av-
relative to the controls. Inhibition increased to .90% after the erage azinphosmethyl concentration quantified in water sam-
second treatment, and 19% mortality was observed. After the ples). They found excellent agreement between these two ap-
fourth application, AChE activity in treated fish was only 1% proaches, with the laboratory-derived value being 0.90 mg/L,
Acetylcholinesterase inhibition in estuarine organisms Environ. Toxicol. Chem. 20, 2001 43

while the field-derived value was 1.13 mg/L. The results of The relationship between AChE inhibition and mortality in
this study suggest that brain AChE inhibition is a sensitive invertebrates is generally less well established than that in fish.
indicator of OP insecticide exposure for this species and that Although inhibition of AChE has been measured in a variety
significant inhibition can be detected at concentrations well of invertebrates following OP exposure, mortality is often ob-
below those that cause mortality. They also indicate that a served in association with very low levels of inhibition. This
simple 24-h laboratory exposure may be an excellent predictor may be due, in part, to the fact that AChE activity is most
of OP-induced AChE inhibition in the field. often measured in invertebrates using whole-body homoge-
nates rather than specific neurological tissue preparations. Us-
Invertebrates ing this approach, significant inhibition in invertebrates typi-
Escartin and Porte [53] evaluated the potential of using the cally is only observed at near-lethal concentrations. The use
mussel Mytilus galloprovincialis as a monitor for the effects of AChE inhibition in invertebrates as a biomarker of OP
of pesticide runoff in an agricultural area of the Ebro Delta, exposure has potential; however, more research is needed to
Spain. Laboratory-derived IC50 values were determined for clarify the relationships between OP exposure, AChE inhibi-
gills and digestive glands after in vitro exposure to fenitrothion tion, and mortality.
and fenitrooxon for 15 min. Gills were more susceptible to
inhibition by the compounds than digestive glands. In gills, NEW AREAS OF RESEARCH
the IC50 was 300 mM (;83,175 mg/L) for fenitrothion and Although OP-induced AChE inhibition in estuarine organ-
much lower at 5.8 mM (;1,514 mg/L) for fenitrooxon, indi- isms has been under study since the 1960s, additional work
cating that fenitrothion requires activation to its oxygen ana- is needed in several important areas. The relationship between
logue in order to cause significant AChE inhibition. In terms AChE inhibition in various fish tissues and mortality requires
of field-collected mussels from the agricultural region, AChE further study since the relationship does not appear uniform
activity in the gills was lowest in April, corresponding to when for all species tested. Some species appear to be able to tolerate
irrigation channels from the farm fields are flushed into the very high levels of brain inhibition without the associated
mussel breeding grounds of the delta. As fenitrothion was the mortality observed in most species. The relationship between
pesticide of choice in this region, it was considered a factor the sensitivity of brain and muscle AChE also appears to be
in the lowered AChE activity. However, no water samples from species specific. In some species, AChE sensitivity in muscle
the region were analyzed for OPs, and the authors stated that tissue mirrors that in brain tissue, while in others muscle tissue
higher water temperatures can also cause reductions in AChE is much more insensitive. In addition, BChE activity has been
activity. measured in the muscle tissues of some estuarine fish. The
In an earlier study, Bocquene et al. [54] collected the mussel presence of this enzyme can influence the interpretation of
species M. edulis along the French coast for AChE activity inhibition data obtained using standard techniques (e.g., Ell-
analysis. Mean activity level for all the samples taken was 228 man method) since these approaches cannot distinguish be-
units/min/mg P with highest activity in the north of France tween these esterases. Because BChE is typically more sen-
and lowest activity in the south and Mediterranean coast. As sitive than AChE [8,9], these approaches can overestimate the
with the study mentioned previously, no water samples were effects on the neurological target enzyme. Future studies
analyzed for the presence of OP compounds in the areas in should account for these complicating factors.
which the mussels were collected, so the authors were unable The relationship between AChE inhibition and mortality in
to directly link the reductions in AChE to OP or carbamate estuarine invertebrates has not been widely studied. Existing
exposure. data suggest that this relationship is highly variable among
invertebrates. Additional research is needed to determine
SUMMARY
whether these observed differences are due to differences in
In general, most studies with estuarine fish species have analytical techniques or reflect truly species-specific responses.
indicated that brain AChE inhibition levels in excess of 70% The possibility that OP-induced toxicity in estuarine inverte-
are well correlated with imminent mortality. There are indi- brates is influenced by factors other than a compound’s potency
cations, however, that this relationship may not hold true for as an AChE inhibitor also merits further study.
all species. For example, the results from the previously de- Another area that deserves study in both fish and inverte-
scribed field and laboratory studies with mummichogs appear brates is the possibility that OPs may affect neurological de-
to suggest that this species, in particular, may be able to tolerate velopment. Recent studies in mammalian and avian systems
very high levels of brain AChE and that inhibition in the suggest that both AChE and the neurotransmitter ACh have a
peripheral nervous system (muscle) may be a better predictor role in the development of the nervous system [55,56]. These
of mortality. The discrepancies point out the need to under- studies suggested that AChE may play a direct role in neuronal
stand the specific relationship between AChE inhibition and differentiation and that AChE activity appears in tissues of the
lethality for any species that is being considered for use as a nervous system while axons are developing and prior to the
bioindicator. In all cases, it appears that significant levels of formation of synapses [55,57]. Some AChE inhibitors have
brain AChE inhibition can be measured in fish exposed to OPs been found to suppress neurite outgrowth [58,59]. Other re-
at concentrations well below those causing mortality. Recov- searchers have reported that ACh released from the developing
ery of AChE activity in fish exposed to OPs appears to be a axons regulates growth and differentiation in central nervous
function largely of the degree of the initial inhibition as en- system neurons [56]. The relevance of these findings to es-
zyme recovery requires de novo synthesis of the enzyme. Other tuarine fish and invertebrates is unknown; however, they clear-
sublethal physiological effects (e.g., reduced stamina) have ly suggest that studies are needed to evaluate these phenomena
been measured in fish concurrent with AChE inhibition; how- in estuarine species. Any compound capable of inhibiting
ever, in most cases these effects have been observed only at AChE has the potential to influence the levels of both AChE
near-lethal concentrations. and ACh.
44 Environ. Toxicol. Chem. 20, 2001 M.H. Fulton and P.B. Key

As this review has demonstrated, monitoring of AChE in ticide induced acetylcholinesterase inhibition. Aquat Toxicol 48:
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18. Grue CE, Hart ADM, Mineau P. 1991. Biological consequences
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Acknowledgement—The National Ocean Service (NOS) does not ap- Cholinesterase Inhibiting Insecticides: Their Impact on Wildlife
prove, recommend, or endorse any proprietary product or material and the Environment, Vol 2—Chemicals in Agriculture. Elsevier,
mentioned in this publication. No reference shall be made to NOS, New York, NY, USA, pp 233–254.
or to this publication furnished by NOS, in any advertising or sales 20. Edwards CA, Fisher SW. 1991. The use of cholinesterase mea-
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mentioned herein or which has as its purpose any intent to cause Insecticides: Their Impact on Wildlife and the Environment, Vol
directly or indirectly the advertised product to be used or purchased 2: Chemicals in Agriculture. Elsevier, New York, NY, USA, pp
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