Journal of Environmental Science and Health Part B, 40:813–825, 2005Copyright C© Taylor & Francis Inc.ISSN: 0360-1234 (Print); 1532-4109 (Online)DOI: 10.1080/03601230500227509

Sublethal Effects ofMonocrotophos on LocomotorBehavior and Gill Architectureof the Mosquito Fish,Gambusia affinis

J. Venkateswara Rao, Ghousia Begum, V. Sridhar,and N. Chakra ReddyToxicology Unit, Biology Division, Indian Institute of Chemical Technology, Hyderabad,India

Subacute studies of monocrotophos [Dimethyl (E)-1-methyl-2-(methyl-carbamoyl) vinylphosphate] on mosquito fish, Gambusia affinis, were carried out in vivo for 24 days toassess the locomotor behavior, structural integrity of gill, and targeted enzyme acetyl-cholinesterase (AChE, EC: 3.1.1.7) interactions. Monocrotophos (MCP) can be rated asmoderately toxic to G. affinis, with a median lethal concentration (LC50) of 20.49 ±2.45 mgL−1. The fish exposed to sublethal concentration of LC10 (7.74 mgL−1) were un-der stress and altered their locomotor behavior, such as distance traveled per unit time(m min−1) and swimming speed (cm sec−1) with respect to the length of exposure. Inhibi-tion in the activity of brain AChE and deformities in the primary and secondary lamellaeof gill may have resulted in failure of exchange of gases. The maximum inhibition of 95%of AChE activity was observed on days 20 and 24.

Morphological aberrations in the gills were also studied during exposure to thesublethal concentration of monocrotophos for a period ranging from 8 to 24 days. Theextent of damage in gill was dependent on the duration of exposure. The findings revealedthat inhibition in brain AChE activity and structural alteration in gill were responsiblefor altering the locomotor behavior of exposed fish.

Key Words: Gambusia affinis; Monocrotophos; Acetylcholinesterase; Gill; Locomotorbehavior.

Received December 23, 2004.Address correspondence to J. Venkateswara Rao, Scientist, Toxicology Unit, BiologyDivision, Indian Institute of Chemical Technology, Hyderabad 500 007, India; E-mail:jv@iict.res.in

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INTRODUCTION

The use of pesticides has increased severalfold in India and is likely to in-crease in the forthcoming years. It is a well-known fact that indiscriminateuse of pesticides in agriculture has resulted in widespread distribution in theenvironment and also has a direct impact on nontargeted organisms. Monocro-tophos, Dimethyl (E)-1-methyl-2-(methyl-carbamoyl) vinyl phosphate (MCP),commonly known as Azodrin, is one of the extensively used organophospho-rus (OP) insecticides in agricultural and animal husbandry. A number of stud-ies conducted on the toxicity of MCP on different organisms indicated thatit is a potent neurotoxicant.[1−4] Pesticides either are directly applied to soilto control soilborne pests or are deposited on soil as runoff from foliar ap-plications. As a result, fish are continuously exposed to comparatively lowconcentrations of pesticides, affecting their behavioral responses. Mortalityis obviously not the only endpoint to consider, and there is a growing in-terest in the development of behavioral markers to assess the sublethal ef-fects of toxicants. Behavior is considered a promising tool in ecotoxicology,[5−7]

and these studies are becoming prominent in toxicity assessments in unicel-lular organisms, insects, fish, and even rodents.[8−13] Locomotion has beenfound to be a consistently sensitive measure of toxic stress for a wide rangeof environmental contamination.[14] Effects of pesticides on the locomotor be-havior of fish are lacking, due to methodological difficulties involved. Di-verse methods have already been developed and used to measure the lo-comotor activity of exposed organisms.[15] Time-lapse video techniques havebeen successfully used to facilitate the documentation of behaviors of nor-mal and stressed organisms.[16] With the recent development of computer-assisted electronics, video-camera tracking systems have been greatly im-proved (Ethovision, Noldus, the Netherlands) and used extensively in quan-tification of locomotor behavior with a high degree of precision.[9,17] The toxiceffects of organophosphorus compounds result from their ability to inhibit theenzyme acetylcholinesterase (AChE), which in turn disrupts the transmis-sion of nerve impulses. The gill is an important organ for respiration andosmoregulation of fish. The effects on neurotransmitter enzyme and struc-tural integrity of the gill influence the alteration in locomotor behavior offish.

In the present study, we have made an attempt to evaluate the sub-lethal aquatic toxicity of monocrotophos on the mosquito fish, Gambusia affi-nis, chosen as an experimental model because of its wide availability andsuitability to evaluate the toxicity of xenobiotics.[18−20] The work focusedmainly on studying the altered locomotor behavior (distance traveled perunit time in m min−1 and swimming speed in cm sec−1) and morphologi-cal differences in gill tissue with special emphasis on target enzyme AChEinteraction.

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MATERIALS AND METHODS

All the reagents used in the present study were of analytical grade and wereused without further purification. The test compound monocrotophos, synthe-sized at our institute, was of 99% purity. The fish species Gambusia affinis(order: Cyprinodontiformes, family: Poeciliidae) were collected from AndhraPradesh Fisheries Department, Medchal (Hyderabad) and were brought to thelaboratory in large aerated drums. They were acclimatized for seven days ina cement tank of 8′ × 6′ × 4′ and fed wheat bran daily. Fish weighing 125 ±5 mg were transferred to a 40-L glass aquarium (60 × 30 × 30 cm) for an addi-tional seven days and fed commercial dry feed pellets (Hello Fish Dry Pellets;CVM Products, Beijing, China) for conditioning. The water in the aquariumwas renewed daily and was aerated with a Jumbo-Jet aquarium air pump(Super-8300, made in India). The natural photoperiod of 13:11 L:D (24 h) wasmaintained. The average values for water quality data in exposure tanks weretemperature 26 ± 2◦C, pH 7.10 ± 0.05, dissolved oxygen 8.15 ± 0.064 mgL−1.[21]

The acute median lethal concentration(LC50) value of MCP was determinedin the laboratory using the semistatic method.[22] The test concentrations werechosen based on the initial experiments to determine the lethal concentration(LC50) for 96 h. The required concentrations (10, 20, 30, and 40 mgL−1) weremaintained in 40 L of water by mixing the toxicant directly into the water andwere renewed daily without aeration. Fish starved for two days were releasedinto each aquarium (10 fish in each aquarium) and were exposed to the aboveconcentrations with two replicates. The control experiments without toxicantwere also performed simultaneously. The mortality record of the fish wasmaintained (during 96 h of exposure without aeration and further seven daysof observation) in each concentration of the toxicant, and the data were usedto estimate the median lethal concentration (LC50) by using a computerizedprogram developed by the method of Finney.[23]

Further, the sublethal test concentration 7.74 mgL−1 (LC10) was main-tained in 40 L of water (five replicates) and 50 fish starved for two days were re-leased into each aquarium. The desired concentration of the toxicant was main-tained and renewed daily until the end of exposure tenure (24 days) withoutaeration, and fish were provided food pellets. The aquarium holding/possessingthe same number of fish without toxicant was maintained as a control. Duringthe exposure tenure, the altered locomotor behavior of fish was monitored atregular intervals at day 4, 8, 12, 16, 20, and 24 by computer-assisted electron-ics, video-camera tracking system (EthoVision, Noldus Information Technology,Wageningen, the Netherlands). Before recording the behavior, fish from controland treated lots were acclimatized individually for 10 min in a recording glassaquarium (15 × 15 × 15 cm), containing 2.5 L of treated/untreated water. Thethree sides of the aquarium and bottom were made opaque to avoid the mirrorimage of the test organism and visual disturbances.

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The behavior of fish was recorded for 5 min in a fixed monitoring arena(internal diameter of aquarium and height of the water, 15 × 15 × 11 cm)with a high-resolution CCD camera, Sony CCD-IRIS (Model No. SSC-M370CE)mounted 20 cm away from the leftover plain side of the recording aquar-ium. The analog video sequence was digitized using EthoVision software in-terfaced with a personal computer. A minimum of 10 fish from each testinterval were used to evaluate individually for determining their locomotorbehavior (distance traveled per unit time in m min−1 and swimming speed incm sec−1).

Immediately, after behavior recording of each lot, five numbers of fish weredissected out and their brains were homogenized (10% w v−1) in 0.1 M phos-phate buffer (pH-7.5) using Potter-Elvehjam homogenizer fitted with a Teflonpestle for estimating AChE activity. The homogenates were centrifuged at5000 × g for 10 min, and the supernatant was further centrifuged at 5000 × gfor 10 min. The resultant supernatant was used as the enzyme source for theestimation of AChE activity. All the enzyme preparations were carried out at4◦C. Protein was estimated by the method of Lowry et al.[24] The AChE activitywas estimated at different intervals of day 4, 8, 12, 16, 20, and 24 in braintissues by the method described by Ellman et al.[25]

The remaining five fish were sacrificed and their representative samples ofgill (pair-III) from either side of the fish were dissected out and placed in nor-mal saline. After rinsing (two to three times) they were placed on a glass slideand observed under a compound microscope (Polyvar, Reichert-Jung light mi-croscope) attached to EthoVision version 2.3 (Noldus Information Technology,the Netherlands) through a CCD camera (Sony CCD-IRIS, Model No. SSC-M370CE). The digital photographs were stored in the computer system. Themagnifications of the photographs were calibrated with the aid of ocular andstage micrometers (ERMA, Tokyo, Japan).

Statistical AnalysisData on locomotor behavior were expressed as mean ± SE of 10 fish from

each interval and were evaluated individually. Statistical significance was de-termined by Student’s t-test, and P < 0.05 was considered significant as com-pared to control.

RESULTS AND DISCUSSION

Acute toxicity of monocrotophos on Gambusia affinis was carried out by thesemistatic method and its LC50 value for 96 h is presented in Table 1. It isevident from the results that the monocrotophos can be rated as moderatelytoxic to the fish G. affinis, with an LC50 value of 20.49 mgL−1. It is evident fromthe earlier results that monocrotophos is comparatively more toxic to other fish

Sublethal Effects of MCP on Gambusia affinis 817

Table 1: Acute toxicity of monocrotophos on Gambusia affinis.

Acute toxicity range98% confidence limit

CompoundRegression equationY = (y − bx) + bX

Upper(mgL−1)

Lower(mgL−1)

Median LC50(mgL−1)

Monocrotophos Y = −4.83 + 2.96X 25.30 15.68 20.49 ± 2.45

(i.e., Labeo rohita, Mystus vittatus, and Channa punctata), and their calculatedLC50 values were 3.558 mgL−1, 2.274 mgL−1 and 3.285 mgL−1, respectively.[26]

The mosquito fish, G. affinis, after exposure to the higher concentrations (30and 40 mgL−1) of MCP used for determining the lethal concentration, exhibitedabnormal behaviors such as erratic swimming with jerky movements, loss ofequilibrium, and secretion of a copious amount of mucus from the whole body.Similar behavioral changes were reported previously in the fish exposed to OPpesticides.[27−30] However, no significant difference was observed in the rate ofopercular movements of treated fish when compared to control.

The video tracking (computer algorithm) method for automated behavioralmonitoring implemented in this present study has the potential for generatingresults that may be utilized for evaluating risks related to contamination by ordischarge of pesticides into stream ecosystems. Hence, a sublethal concentra-tion of 7.74 mgL−1 was used for behavioral monitoring of fish under laboratoryconditions. It is evident from Figure 1 that the locomotor behavior of fish wasgreatly influenced during the exposure tenure. It is apparent from the data thatthe mobility (distance traveled) of fish was gradually decreased significantly

Figure 1: In vivo effect of monocrotophos on locomotor behavior (distance traveled, mmin−1) of Gambusia affinis during sublethal exposure (LC10 conc. 7.74 mgL−1), tenure of 24days.

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(P < 0.05) by the action of toxicant from day 8 to day 24 in comparison to thecontrol value. Similar behavioral changes such as hypoactivity and lethargicconditions were observed in an air-breathing fish Anabas testudineus (Bloch)when exposed to sublethal concentration of monocrotophos.[31]

The average swimming speed (cm sec−1) of fish at regular intervals duringthe sublethal exposure revealed that the swimming speed was also consider-ably reduced with the effect of the toxicant (Fig. 2). The percent reduction inswimming speed at regular intervals was calculated based on the control valuesand presented in inset A. The gradual reduction in the swimming speed wasobserved with a prolonged exposure and reached up to 61.65% on day 24. Theestimated time required for 50% reduction in the swimming (moving) speed wascalculated by converting the time period into log values, thus taken on x-axisand plotted linear regression against percent reduction on y-axis (inset B). Thederived time value from the linear regression for inhibiting 50% of locomotoractivity in the exposed fish is 19.85 days (which is equivalent to 19 days, 20 h,

Figure 2: Percent decrease in locomotor activity (swimming speed in cm sec−1) ofmosquito fish, Gambusia affinis, at regular intervals of 24 days’ exposure to the sublethalconcentration (LC10 conc. 7.74 mgL−1) of monocrotophos. Inset A: Percent change in theirswimming speed. Inset B: Determination of time required to reduce 50% of their mobility.

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24 min). Earlier findings indicated that food deprivation, anoxic conditions, andinhibition of AChE enzyme were responsible for the reduction in locomotor ac-tivity of rats.[32, 33] In the present experiments the exposed fish were not facingsuch anoxic conditions or the deficiency of food. Therefore, the altered locomo-tor activity (swimming speed) of fish may be due to the toxicant-induced stress,which is an indication of neurotoxicity.

The AChE activity of brain in control fish was estimated in all the inter-vals and its mean value was 0.088 ± 0.004 µmoles acetylthiocholine hydrolysismin−1 mg−1 protein. The fish were exposed to a sublethal concentration of MCP(7.74 mgL−1) for 24 days, and its effect on percent inhibition of brain AChE ac-tivity was estimated at regular intervals (4 days, up to 24 days) (see Fig. 3).On day 4, about 50% (48.86 ± 2.44) inhibition of the brain AChE activity wasrecorded, subsequently, the inhibition was increased and reached maximum(95.46 ± 4.77%) on day 20 and was maintained on day 24. A similar patternof AChE inhibition was reported earlier in Oreochromis mosambicus exposedto profenofos and monocrotophos.[28,30] It is apparent from the earlier resultsthat the fish can survive even after 90% inhibition of true cholinesterases inbrain AChE when exposed to parathion.[34] There was no significant differencein brain AChE activity of control fish during the same intervals. Inhibition ofbrain AChE activity by monocrotophos and accumulation of acetylcholine (ACh)at synaptic junctions may be responsible for altering their locomotor behavior.

Respiratory distress is one of the early symptoms of pesticide poison-ing, and the gill takes part in metabolism and elimination of xenobiotics.[35]

The gill is made of primary lamellae arranged in double rows. White tuftsof secondary lamellae are aroused from these filaments and lined with a

Figure 3: In vivo effect of monocrotophos on brain acetylcholinesterase activity inGambusia affinis during sublethal exposure up to 24 days.

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Figure 4: Digital microphotographs of the gill lamellae (via CCD camera of EthoVision) offish exposed to LC10 concentration (7.74 mgL−1) of monocrotophos at different exposureintervals. (A) Photograph of a healthy mosquito fish gill. The tips seen in this photograph arecalled the secondary lamellae arisen from primary lamellae. These filaments transferoxygen from the water to the fish (220X). (B) Bulging formation of intraepithelial edema insecondary lamellae on day 8 (240X). (C) Erosion of secondary lamellae at distal andthickening of lamellae at proximal end of filaments on day 12 (230X). (D) The curvatureand breakages in primary lamellae at the distal end; lesions and erosions of secondarylamellae on day 16. (E) Erosion resulted in shortened lengths of primary lamellae on day 20(250X). (F) Entire distraction of primary and secondary lamellae on day 24 (260X).

squamous epithelium (Fig. 4A). Different degrees of degenerative changes ingill, lamellae, and rakers were noticed during 24 days of exposure of sub-lethal concentration (7.74 mgL−1) of MCP (Figs. 4 and 5). A bulging for-mation of large subepithelial spaces and intraepithelial edema within thesecondary lamellae was observed on day 8 (Fig. 4B). A thick coating ofmucus covering the entire gill filaments and lamellae was observed dur-ing the exposure tenure of days 8 to 12. An excess amount of mucus se-cretion might be to protect the respiratory epithelial cells and avoid the

Sublethal Effects of MCP on Gambusia affinis 821

erosion and damage of the respiratory surface, which would affect thereduction in gaseous exchange between blood and water. The excessive mu-cus secretion and lifting up of the epithelium and clavate lamella was noted asa defense response toward the toxicant.[28] Further degenerative changes werenoticed on day 12: the erosion of secondary lamellae at distal and the thick-ening of lamellae at the proximal end of filaments were due to inflammationof epithelial cells, resulting in irregular shape and disassociation of epithe-lium; this reduces the availability of the water space and obstructs the bloodcapillaries (Fig. 4C). The curvature and breakages in primary lamellae at thedistal end, as well as lesions and erosions of secondary lamellae, were evident

Figure 5: Digital microphotographs of the gill rakers of fish (via CCD camera of EthoVision)exposed to LC10 concentration (7.74 mgL−1) of monocrotophos at different exposureintervals. (A) Control—normal arrangements of rakers on the gill arch (170X). (B) Disruptionin primary epithelium on the surface of rakers on day 8 (220X). (C) The diameter of therakers has enlarged and elongated on day 12 (240X). (D & E) Developed outer growthprojections on the cartilage filament of rakers on day 16 and prominent on day 20 (240X).(F) Deep lesions and erosions destroying the basic structure of gill raker on day 24 (240X).

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on day 16 (Fig. 4D). Total degeneration of secondary lamellae and shortenedlength of primary lamellae were prominent on day 20 (Fig. 4E). Severe damagewas noticed on day 24 necrosis and rupture of epithelium and erosion of sec-ondary lamellae resulting in hypoxia may lead to respiratory failure (Fig. 4F).Related discrepancies were noticed in other species of fish (Oreochromis mosam-bicus) when exposed to profenofos concentration of 0.108 mgL−1. Exposure re-sulted in gill lesions, including hyperplasia and desquamation of the epitheliumand thrombosis in the secondary gill lamellae.[28] Similar observations such asnecrosis and exudation were noted in the gill of Lepomis macrochirous exposedto 0.05 mgL−1 malathion[36] and to different concentrations of diazinon.[37] Theabove observed alterations may have affected the locomotor behavior of fish dueto variation in respiratory surface of gill and the effect on O2 uptake.

The mildly distorted shapes of rakers were due to disruption in the primaryepithelium on day 8 in comparison to controls (Fig. 5B). The gill rakers werefurther damaged on day 12 onward—i.e., they lost their shape and appeared tobe thickened and elongated, which may hamper the function of the rakers inpreventing the entry of dust and food particles into the gills (Fig. 5C). The outergrowth projections on the cartilage filament of rakers were prominent on days16 and 20 (Figs. 5D and 5E). The deep lesions and erosions clearly appearedon day 24 with destruction of the basic structure of gill including rakers andarches (Fig. 5F).

CONCLUSION

The study showed that the effects of monocrotophos at sublethal concentrationsfor a prolonged period include not only direct effects on vital organs but alsoeffects on the locomotor behavior of the mosquito fish, Gambusia affinis. Itconcluded that the MCP caused inhibition in the target enzyme AChE andaltered the structural integrity of the respiratory organ (gill) and thus mighthave been responsible for the change in the locomotor behavior of fish. Basedon the current findings, further research is necessary to study the effect ofother pesticides and their combinations on the behavior of fish in the laboratoryas well as under field conditions before implementing such insecticides in thecurrent scenario.

ACKNOWLEDGMENTS

The authors are thankful to the director of the Indian Institute of ChemicalTechnology for providing the facilities and for constant encouragement through-out the study. We would like to express our sincere gratitude to B. Venkateshamand P. Narsireddy for their invaluable assistance during the experiments (IICTcommunication No. 041110).

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