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Keywords:

  • Acetylcholinesterase;
  • Fish;
  • Invertebrates;
  • Organophosphorus insecticides

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. LABORATORY STUDIES
  5. FIELD STUDIES
  6. Invertebrates
  7. SUMMARY
  8. NEW AREAS OF RESEARCH
  9. Acknowledgements
  10. REFERENCES

The majority of insecticidescurrently 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.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. LABORATORY STUDIES
  5. FIELD STUDIES
  6. Invertebrates
  7. SUMMARY
  8. NEW AREAS OF RESEARCH
  9. Acknowledgements
  10. REFERENCES

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 variety of other organisms, including many commercially and recreationally important marine fish and crustacean species that use estuaries as nursery grounds [1]. Transport of pesticides 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 Germany during the 1930s as a substitute for nicotine and as potential chemical warfare agents [2]. Because of their relatively nonpersistent characteristics in the environment, OPs have become one of the most widely used classes of insecticides worldwide. Although these compounds offer the advantage 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 intoxication caused by exposure to these compounds in the environment.

Organophosphorus insecticides are esters, amides, or thiol derivatives of either phosphoric acid or thiophosphoric acid. The majority now in use, such as azinphosmethyl, chlorpyrifos, and malathion, contain the thiono moiety (=S). The substitution of the =S for =O on the phosphorus atom increases the toxicity of the insecticide, such as is the case with malathion 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]. Household 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 industrial, commercial, and government use, the top insecticides are again OPs (chlorpyrifos and malathion [5]).

Organophosphorus insecticides produce toxicity by inhibiting cholinesterase enzymes in both vertebrate and invertebrate organisms. These enzymes are responsible for the removal 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 preganglionic and a postganglionic transmitter in the parasympathetic nervous system and as a preganglionic transmitter in the sympathetic nervous system. In critical regions of the central nervous system, ACh serves as an excitatory transmitter. When cholinesterases are inactivated by the binding of OPs, an accumulation of ACh occurs at the nerve synapse, interfering with the normal nervous system function. This produces rapid twitching of voluntary muscles followed by paralysis [6,7]. Once bound, organophosphorus compounds are considered irreversible inhibitors, as recovery usually depends on new enzyme synthesis [6].

Cholinesterases are usually divided into two broad classes: the acetylcholinesterases (AChEs) and the butyrylcholinesterases (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 inhibitory 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] reviewed 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 recently, AChE activity has been measured in a variety of crustaceans, 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 serratus [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 exposed animals [18–20]. Although the presence of many OP insecticides in the environment can be discerned using analytical chemistry techniques, AChE monitoring in the environment may offer distinct advantages over the use of analytical 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 environment, 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 invertebrate species. It discusses the relationship between OP-induced AChE inhibition and lethality as well as sublethal responses 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 research.

LABORATORY STUDIES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. LABORATORY STUDIES
  5. FIELD STUDIES
  6. Invertebrates
  7. SUMMARY
  8. NEW AREAS OF RESEARCH
  9. Acknowledgements
  10. REFERENCES

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 in estuarine fish species. Sheepshead minnows (Cyprinodon variegatus) were exposed to selected OP insecticides (Guthion, phorate, parathion, phosphamidon, Cygon, malathion, EPN, Dursban, dichlorvos, diazinon, Dibrom, and methyl parathion) at sublethal and median lethal (40–60% mortality) concentrations (as listed in Coppage [23]). Inhibition of brain AChE was always greater at concentrations that caused mortality. 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 minnows; 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 suggested 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 exposed to lethal and sublethal concentrations of the OP insecticide 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 μg/L ranged from 59% at 24 h to 70% after 96 h. No mortality was observed at this concentration at any time point. At higher naled concentrations (25–75 μg/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 concentrations 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] investigated 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 μg/L, while the 24-h median effective concentration (EC50) for brain AChE inhibition was 0.81 μg/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 μg/L) was more than four times higher than the 24-h EC50 for inhibition of AChE in brain tissue. The highest concentration used in the 24-h sublethal tests (3.9 μg/L) caused ∼94% inhibition of brain tissue AChE activity. This concentration was much lower than the 96-h no-observed-effect concentration (19.80 μg/L) determined from the 96-h acute toxicity tests. These findings suggest that this species is able to tolerate extremely 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 different for the two species tested. In the red drum, the 96-h LC50 (6.2 μg/L) and the 24-h EC50 for brain AChE inhibition (5.2 μg/L) were quite similar. In the mummichog, however, the 96-h LC50 was 60 times greater than the 24-h EC50 (1.0 μg/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 = 6.8 μg/L) was quite similar to that in brain tissue, while mummichog muscle AChE (24-h EC50 >5μg/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 suggest that given the response observed in mummichogs, inhibition 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 × 10−8 M (∼5.1 μg/L) in dragonet muscle and 3.1 × 10−7 M (∼68.5 μg/L) in sole tissues. The IC50s for dragonet were 2.6 × 10−5 M (∼7,209 μg/L) and 6.2 × 10−7 M (∼228 μg/L) for fenitrothion and phosalone, respectively. In sole tissues, the IC50s for these two OPs were 1.2 × 10−4 M (∼33,270 μg/L) and 2.5 × 10−6 M (-919 μg/L), respectively. The authors suggested 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 synergistic in terms of AChE inhibition, while the OP-OP combinations were less so. Increasing incubation time increased the synergistic effect. The authors concluded that the mechanisms by which inhibitory effects could be enhanced was not well understood.

Enzyme inhibition and recovery. Other studies have investigated the persistence of AChE inhibition in brain tissue following 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-response fashion. Inhibition ranged from 12% at 0.005 mg/L to 58% at 0.213 mg/L. In the 2 × 24-h exposure regime, inhibition 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, activity in both treatment groups had recovered to control levels. Results of this study indicated that the time for enzyme recovery 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 chlorpyrifos 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 inhibition 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 reported 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 concentrations sufficient to cause the level of inhibition observed in this study may also affect bone strength.

Relationship between AChE inhibition and sublethal effects. A number of researchers have investigated the relationship 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 obtained during the course of the life-cycle tests. Swimming stamina was affected in fish exposed to EPN at 4.1 and 2.2 μg/L. At 2.2 μg/L, stamina was reduced by 43%, while at 4.1 μg/L, stamina was decreased by 54%. Swimming performance was not affected by Guthion concentrations up to 0.5 μg/L, a concentration that affected reproduction. Acetylcholinesterase activity was significantly depressed at all concentrations of EPN (0.25–7.9 μg/L) and Guthion (0.06–0.5 μg/L). Effects on swimming stamina were observed only when AChE inhibition was greater than 80%.

Van Dolah et al. [28] reported that red drum exposed to azinphosmethyl for 6 h at 12 μg/L had reduced swimming stamina. This concentration is approximately two times the 24-h EC50 (5.2 μg/L) for brain AChE determined in the same study. No effects on swimming stamina were observed at lower insecticide concentrations (3–6 μg/L). Swimming stamina in mummichogs was not affected at 19.4 μg/L (the highest concentration tested), which is ∼20 times higher than the 24-h EC50 (1.0 μg/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 levels of AChE inhibition. They reported that for the three species tested (brook trout, Salvelinus fontinalis; rainbow trout, Salmo gairdneri; and coho salmon, Oncorhynchus kitusch), 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 concentrations 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 activity in claw musculature. Exposed crabs showed a 90% reduction in ventral ganglion AChE activity and a greater than 80% reduction in brain AChE activity. Other crabs were exposed 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 exposure 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 incubated in the same fashion. The IC50s for crab AChE were 4.5 × 10−5 M (∼ 14,866 μg/L) for malathion and 6.9 × 10−5 M (∼20,098 ∼g/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 nondestructively 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 lower 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 neuronal control because of their observation of reduced heart rates in the crabs following dimethoate exposure.

Several papers have examined the effects of the OP compound dichlorvos used in marine fish farms to control ectoparasites. After treatment, waters containing this product may be discharged into surrounding estuarine areas. McHenery et al. [36] exposed larval lobster (Homarus gammarus) to dichlorvos for 24 h at 10-fold intervals from 0.001 to 100 μg/L. They observed 50% AChE inhibition at 2.7 μg/L. This value was about 10-fold lower than the 24-h LC50 of 28.3 μg/L determined for lobster larvae in this study. The authors suggested that AChE activity in the lobster may provide a sensitive method for determining OP exposure. They did, however, observe an apparent hormetic effect of increased AChE activity in larvae exposed at the 0.01-μg/L concentration that may interfere with the ability to distinguish between animals exposed to low concentrations and unexposed animals.

Another study [37] examined the effect of dichlorvos on AChE activity in two commercial bivalves, Manila clam (Ruditapes 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 animals 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 differences 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 membrane bound, and the B cholinesterases were soluble enzymes. The A group was highly sensitive in vitro to the OP compounds DFP (ki = 2.0 × 103/M/min) and paraoxon (ki = 3.0 × 105/M/min), while the B group was considered to be almost insensitive. The presence of this insensitive cholinesterase suggests that improved use of AChE activity in oyster as a biomarker of inhibitory effects can be achieved if these two classes 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 × 10−6 M (∼243.08 μg/L) for shrimp muscle and 7.3 × 10−8 M (-16.13 μg/L) for oyster gill. The IC50s for shrimp were both greater than 10−4 M for fenitrothion (∼27,725 μg/L) and phosalone (∼36,780 μg/L). For oysters, the IC50s for these two OPs were 1.4 × 10−4M(∼38,815 μg/L) and 5 × 10−4M(∼183,900 μg/L), respectively. The authors stated that the greater sensitivity 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 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 μg/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. Acetylcholinesterase activity in the ventral nerve cord was significantly depressed in moribund shrimp after MPT exposure for 6 h to 1.3μg/L and not depressed after 74 h to methyl paraoxon at 0.98 μg/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 μg/L methyl paraoxon. In the in vivo MPT study, AChE activity in exposed non-moribund shrimp was not significantly different from the control. The authors concluded that there was not a direct relationship 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. Acetylcholinesterase 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 μg/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 μg/L for 24 h. Acetylcholinesterase activity decreased in muscle tissue by 18, 13, and 16%, respectively. P. vannamei were exposed to higher concentrations from 10 to 30 μg/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 increased fenitrothion concentrations (up to 30% at 30 μg/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 rates of AChE and that fenitrooxon may be more rapidly hydrolyzed 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 (swimming 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 concentration, the shrimp were either dead or moribund. Therefore, 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 μg/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 μg/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 pathways essential to shrimp function and survival could be experiencing 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 μg/L, respectively. The 24-h chlorpyrifos EC50s for stages VI and VII embryos were much lower at 0.49 and 0.36 μg/L, respectively. The differences in sensitivity were seen as differences in the persistence of the two insecticides 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 μg/L), 18-d-old larvae (0.27 μg/L), and adult grass shrimp (1.42 μg/L). Six-hour pulse doses to chlorpyrifos yielded interesting results in grass shrimp larvae [16]. Acetylcholinesterase was significantly reduced after 6 h of exposure to 1.6 μg/L chlorpyrifos, but the next highest exposure of 0.4 μg/L had significantly higher AChE activity than the control group. After four 6-h doses, the 0.4-μg/L group was once again significantly higher than controls. This effect was most likely the result of a physiological 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 μg/L [45]. After one dose, the AChE activity significantly decreased at all three concentrations. After four doses, the activity was significantly decreased in the two highest concentrations. 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 μg/L), 18-d-old larvae (0.55 μg/L), and adult grass shrimp (3.29 μg/L). Cochran and Burnett [46] also exposed adult grass shrimp to azinphosmethyl at a concentration of 2.0 μg/L for 24 h. They found that AChE activity at this concentration was not significantly different; however, variability in this study was high.

Table 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
Field test dateMaximum azinphosmethyl concentration (μg/L)Azinphosmethyl concentration at 24 h (μg/L)% Acetylcholinesterase inhibition% Mortality
6/7–11/883.440.57470
6/11–15/880.570.55220
6/23–27/880.000.0000
6/3–7/891.731.12630
6/11–15/890.370.2100
6/15–19/892.460.72850
6/23–27/897.001.609817

Other marine invertebrates examined as potential indicator organisms in AChE biomarker studies include copepods. Forget et al. [47] exposed Tigriopus brevicornis to several combinations 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 mixture, at a 50th and a 100th of the LC50, was additive in its reduction of AChE activity. The authors stated that the additivity potential does not decrease when lethal concentrations are reduced to sublethal concentrations [47].

FIELD STUDIES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. LABORATORY STUDIES
  5. FIELD STUDIES
  6. Invertebrates
  7. SUMMARY
  8. NEW AREAS OF RESEARCH
  9. Acknowledgements
  10. REFERENCES

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 menhaden 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% of normal, but no additional mortality was observed. The authors 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 comparison 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 compounds.

The effects of insecticides present in nonpoint-source agricultural 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-hinsitu 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 agricultural 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), fenvalerate (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 concentrations ranged from 0.00 to 7.00 μg/L. Significant brain AChE inhibition was observed at all azinphosmethyl concentrations ≥0.57 μg/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 laboratory 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 average azinphosmethyl concentration quantified in water samples). They found excellent agreement between these two approaches, with the laboratory-derived value being 0.90 μg/L, while the field-derived value was 1.13 μg/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

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. LABORATORY STUDIES
  5. FIELD STUDIES
  6. Invertebrates
  7. SUMMARY
  8. NEW AREAS OF RESEARCH
  9. Acknowledgements
  10. REFERENCES

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 μM (∼83,175 μg/L) for fenitrothion and much lower at 5.8 μM (∼1,514 μg/L) for fenitrooxon, indicating that fenitrothion requires activation to its oxygen analogue 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

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. LABORATORY STUDIES
  5. FIELD STUDIES
  6. Invertebrates
  7. SUMMARY
  8. NEW AREAS OF RESEARCH
  9. Acknowledgements
  10. REFERENCES

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 indications, however, that this relationship may not hold true for all species. For example, the results from the previously described 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 understand 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. Recovery of AChE activity in fish exposed to OPs appears to be a function largely of the degree of the initial inhibition as enzyme 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; however, in most cases these effects have been observed only at near-lethal concentrations.

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 observed 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. Using this approach, significant inhibition in invertebrates typically 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 inhibition, and mortality.

NEW AREAS OF RESEARCH

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. LABORATORY STUDIES
  5. FIELD STUDIES
  6. Invertebrates
  7. SUMMARY
  8. NEW AREAS OF RESEARCH
  9. Acknowledgements
  10. REFERENCES

Although OP-induced AChE inhibition in estuarine organisms 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., Ellman method) since these approaches cannot distinguish between these esterases. Because BChE is typically more sensitive 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 invertebrates 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 invertebrates is the possibility that OPs may affect neurological development. 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 researchers 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 estuarine fish and invertebrates is unknown; however, they clearly 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.

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. LABORATORY STUDIES
  5. FIELD STUDIES
  6. Invertebrates
  7. SUMMARY
  8. NEW AREAS OF RESEARCH
  9. Acknowledgements
  10. REFERENCES

The National Ocean Service (NOS) does not approve, 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, recommends, 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.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. LABORATORY STUDIES
  5. FIELD STUDIES
  6. Invertebrates
  7. SUMMARY
  8. NEW AREAS OF RESEARCH
  9. Acknowledgements
  10. REFERENCES
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