Toxic effects of acephate on Paramecium caudatum with special emphasis on morphology, behaviour, and generation time

PESTICIDE Biochemistry & Physiology

Pesticide Biochemistry and Physiology 86 (2006) 131–137 www.elsevier.com/locate/ypest

Toxic effects of acephate on Paramecium caudatum with special emphasis on morphology, behaviour, and generation time J. Venkateswara Rao *, K. Srikanth, S.K. Arepalli, V.G. Gunda Toxicology Unit, Biology Division, Indian Institute of Chemical Technology, Hyderabad 500 007, India Received 4 January 2006; accepted 9 February 2006 Available online 23 March 2006

Abstract The continuous increase in the number of new chemicals as well as the discharges of solid and liquid wastes triggered the need for simple and inexpensive bioassays for routine testing. In recent years, there has been increasing development of methods (particularly rapid tests) for testing environmental samples. This paper describes the quick toxic evaluation of an organophosphorus insecticide, acephate (O,S-dimethyl acetylphosphoramidothioate) on Paramecium caudatum for acute and sub-acute toxicity studies with reference to morphology, behaviour, and its generation time. The lethal concentrations for 10 min and 2 h were determined by probit method, as 500 mg L1 and 300 mg L1, respectively. Higher concentrations of 10 min exposure caused cell lysis with disintegration of cell membrane and precipitation of protoplasm. Combination of conventional light microscopy and computerized video tracking systems were used to study the locomotor behaviour of paramecia. The test organism was under stress and exhibited an initial increase and subsequent decrease in the swimming speed when exposed to 1/4, 1/2, 3/4, and LC50 concentrations for 10 min (125, 250, 375, and 500 mg L1, respectively). Similar changes were also noticed when paramecia were exposed to LC50 for 2 h. In a separate set of experiments, the number of generations and generation time in 24 h was evaluated with respect to the different sub-lethal concentrations (30, 60, 120, and 240 mg L1). The number of generations decreased and generation time extended significantly in a concentration dependent manner. The results indicate that the Paramecium toxicity assay could be used as a complimentary system to rapidly elucidate the cytotoxic potential of insecticides. The major advantages associated with these tests are: they are inexpensive, simple, user-friendly, space saving, and seem to be attractive alternatives to conventional bioassays.  2006 Elsevier Inc. All rights reserved. Keywords: Paramecium caudatum; Acephate; Cell lysis; Cytotoxic; Morphology; Behaviour; Generation number and time

1. Introduction The extensive use of organophosphorus insecticides, during the past decades has led to a number of negative effects on terrestrial and aquatic organisms. Insecticides are being used in agriculture and they are found to be more hazardous than herbicides and fungicides. Acephate (O,S-dimethyl acetylphosphoramidothioate) a water soluble organophosphorus insecticide registered to control certain insect pests on a variety of field, fruit, and vegetable crops, in food handling

*

Corresponding author. Fax: +91 40 2719 3191/2717 3757. E-mail addresses: [email protected], [email protected] (J. Venkateswara Rao).

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establishments, on ornamental plants both in greenhouses and outdoors with residual systemic activity. Acephate and its primary metabolite, methamidophos, are toxic to various species. A number of studies were conducted on the toxicity of acephate on different organisms and indicated as a potent neurotoxicant [1]. It is also found to be mutagenic [2], carcinogenic [3], and cytotoxic [4]. Monitoring of aquatic ecosystem pollution represents one of the major activities involved in measures aimed at environmental protection. Usage of non-targeted organisms in environmental toxicology is needed to understand the wide range of toxic effects caused by the pesticides on different organisms [5]. Fish and other aquatic biota that were commonly used as bioindicators of persistent organic pollutants [6] have been

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replaced in recent years successfully by ciliates [7]. Protozoan cells are often used as bioindicators of chemical pollution, especially in aqueous environment. Among protozoans, Paramecium caudatum is one of the ciliate models, most commonly used for laboratory research [8]. This unicellular ciliate facilitates the study of physiological processes and effects of pollutants on locomotory behaviour. It has been widely used to evaluate the toxic effects of several food dyes, carcinogens, synthetic chemicals, carbamate pesticides, and pollutants [7,9–11]. Mortality is obviously not the only endpoint to consider and there is a growing interest in the development of behavioural markers to assess the sub-lethal affects of toxicant. Behaviour is considered as a promising tool in ecotoxicology [12,13] and these studies are becoming prominent in toxicity assessments in unicellular organisms [14]. Locomotion has been found to be a consistently sensitive measure of toxic stress for a wide range of environmental contamination [15]. Diverse methods have already been developed and used to measure the locomotor activity of exposed organisms. Time-lapse video techniques have been successfully used to facilitate the documentation of behaviour of normal and stressed organisms [16]. With the recent development of computer-assisted electronics, video-camera tracking systems have been greatly improved (Ethovision, Noldus, The Netherlands) and used extensively in quantification of locomotor behaviour with a high degree of precision [17,18]. Further, multidisciplinary progress in research is very much needed to increase the significance and usefulness of behavioural markers for aquatic toxicology, and aim to highlight the specific areas for consideration. The application of unicellular organisms to study the toxic effects of pesticides from contaminated wastewater is relatively new throughout the world. The toxicity of acephate to primary consumers in aquatic food chain is generally uncharacterized [19]. P. caudatum test is more sensitive to investigate the direct toxicity of compounds [20,21]. Hence, in the present paper, we have studied the toxic impacts of acephate on P. caudatum with special emphasis on locomotor behaviour, proliferation rate, and morphological abnormalities. 2. Materials and methods Stock cultures of P. caudatum were maintained in rice straw medium. Fresh cultures were initiated by seeding 100 ml of the rice straw medium with 1 ml of a stationary phase paramecium culture containing 1500–2000 organisms per ml. The cultures were maintained at room temperature (25 ± 2 C); with a photo period of 14 h light and 10 h dark, pH 7.5–8.0 with loose fitting covers.

chosen based on the initial experiments to determine the lethal concentration (LC50) for 10 min and 2 h. The required concentrations of 460, 480, 500, 520, and 540 mg L1 for 10 min, and 200, 250, 300, and 350 mg L1 for 2 h exposure were maintained in 1 ml of rice straw water in 12-well microplates for definitive tests. Twenty numbers of active paramecia were released into each well and were exposed to selected concentrations with five replicates each. Simultaneously, control experiments were also performed without toxicant. The mortality record of the paramecia was maintained (10 min and 2 h of exposure) in each concentration of the toxicant and the mortality data was observed using inverted microscope (Nikon TMS) to estimate the median-lethal concentration (LC50) using probit analysis [22]. Morphological abnormalities caused by different concentrations of acephate were observed on a glass slide and recorded as digital photographs using ‘Easy Grab’ software. The magnifications of snaps were calibrated with the aid of ocular and stage micrometers (ERMA, Tokyo, Japan). 2.2. Measurement of locomotor behavioural response of paramecium In a separate set of experiments, locomotor behavioural response of paramecium was monitored by using a singlewell test apparatus. The apparatus is a combination of a microscopic slide and a silicon sheet (0.5 mm thickness) that possess centrally located well (5 mm diameter), where the glass slide served as the bottom of the apparatus [23]. Ten microliters of different stock concentrations were added individually to the test well containing 15 ll of rice straw water along with a healthy paramecium. The final concentrations of the toxicant were maintained individually to obtain 1/4, 1/2, 3/4, and LC50 concentrations for 10 min (125, 250, 375, and 500 mg L1, respectively) in the well. The test apparatus was placed under a compound microscope (Polyvar, Reichert-Jung light microscope) attached to a CCD camera (Sony CCD IRIS, Model No: SSC-M370CE) for continuous monitoring the locomotor behaviour of test organism for 6 min with five replicates each. Speed and distance travelled parameters of individual test organism were observed using the analysis module of Ethovision-2.3 package software (Noldus Information Technology, The Netherlands). In addition, paramecia were exposed to LC50 concentration for 2 h (300 mg L1) in 100 ml of rice straw water. A paramecium with 25 ll of test solution was placed in the well to determine the altered locomotor behaviour (distance travelled in mm and velocity in mm s1) at regular intervals of 15 min. A minimum of five paramecia at each interval were used to evaluate their locomotor behaviour individually.

2.1. Determination of median lethal concentration (LC50) The acute LC50 value of acephate was determined by static method. The technical grade of acephate was initially dissolved in 1 ml rice straw water and then diluted according to the test concentrations. The test concentrations were

2.3. Population growth impairment and generation time determination In a separate set of experiments, the culture was maintained in 12-well microplates for 24 h with sub-lethal

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concentrations of acephate (30, 60, 120, and 240 mg L1) in 1 ml of the rice–straw medium to evaluate the effect on population growth (number of generations) and their generation time. Paramecia were divided into 5 replicates of 20 each and were exposed to each test concentration. Cells were sampled after post exposure (24 h) and the exact number of living cells was counted under a microscope using a hemocytometer by trypan blue exclusion. For precise counting the cell mobility was minimized by adding 1–2 drops of Bouin’s solution to the cell suspension [8]. Changes in growth (proliferation) rates were examined in the presence or absence of test chemical with an inverted optical microscope (Nikon TMS) at 100· magnification [24]. Based on the data, the number of generations and time required per each generation was calculated by the following formulae (1) and (2). log N 1  log N 0 ; log 2 Time of growth Generation time ðgÞ ¼ ; Number of generations

Number of generations ðnÞ ¼

ð1Þ ð2Þ

where N1 is the number of cells at 24 h. N0 is the number of cells at T0. Time of growth = 24 h. The total number (mean) of paramecia in each concentration after 24 h was used to determine IC50 value by a linear regression, defined as the concentration of acephate required for 50% inhibition of proliferation. 2.4. Statistical analysis The LC50 was calculated using probit analysis [22] that has been recommended by OECD guideline as appropriate statistical method for toxicity data analysis [25]. After linearization of the concentration response curve by logarithmic transformation of concentrations, 95% confidence limits and slope function were calculated to provide a consistent presentation of the toxicity data. Data on locomotor behaviour [distance moved (mm) in 6 min and velocity (mm s1)] in 2 h was measured at regular intervals of 15 min each and expressed as means ± SE of 10 paramecia. Statistical significance was determined by Student’s t test, and p < 0.05 was considered significant when compared to control. 3. Results and discussion Microorganisms are highly sensitive to chemicals in aqueous environment. However, it is known that parame-

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cia are relatively resistant to high concentration of pollutants [10]. Hence, for evaluating individual xenobiotics at laboratory conditions, paramecia would be ideal and reveal the stress and toxicity on aquatic biota. The 10 min and 2 h acute toxicity tests on P. caudatum (624 h old) with technical grade >97% purity of acephate were performed and the LC50 values were found as 500 ± 6.97 and 300 ± 36.65 mg L1, respectively (Table 1). In the present experiments, we observed that acephate affected the test organism, P. caudatum in a concentration dependent manner. Paramecia exposed to >500 mg L1 for 10 min were significantly affected, as the membranes surrounding food and contractile vacuoles ruptured and their contents were thoroughly mixed up with protoplasm, appears as coagulation of proteins. Cellular volume initially increased followed by disintegration of whole protoplasm and internal membranes. Subsequently, the outer membrane was also affected and the cell contents oozed out due to internal pressure. It appears that the blebbing is common phenomena, when paramecium exposed to carcinogenic and other xenobiotics compounds [9,26]. Even our earlier studies with two organophosphorous compounds, monocrotophos, and fenthion exhibited several blebs on the surface of cell (unpublished data). Affected cells with acephate showed alteration in their shape, by developing irregular blisters of the cell membrane, which leads to cell lysis. The light microscope associated with ‘Easy Grab’ software, revealed that there was no prominent blebbing, but cell lysis occurred due to internal damage of cell organelles and rupture of outer membrane. Schematic digital photographs of blisters and cell lysis of a paramecium are presented in Fig. 1. The effect of acephate on the locomotor behaviour of paramecium was investigated for 6 min using 1/4, 1/2, 3/4, and LC50 (125, 250, 375, and 500 mg L1, respectively) concentrations for 10 min by Video tracking (computer algorithm) method for automated mobility monitoring, and compared with controls. Track paths of both controls and affected paramecia were analysed by a built in software (Ethovision-version 2.3, Noldus Information Technology, The Netherlands) to calculate the distance moved in 6 min and their velocities. The mobility tracking records (distance moved) of P. caudatum for above said concentrations of acephate along with control are presented in Fig. 2. Gradual increase of 1.52; 2.35; 2.52; 2.85-fold in the mobility was noticed with increasing concentration of 125, 250, 375, and 500 mg L1, respectively.

Table 1 Median lethal concentrations of acephate at different exposure tenures against P. caudatum Duration of exposure

10 min 2h

Regression equation (Log + 2)

Acute toxicity range 98% confidence limit

Y ¼ ðY  bxÞ þ bx

Upper (mg L1)

Lower (mg L1)

Y = 118.78 + 6.30x Y = 4.37 + 2.09x

516.67 370.81

489.33 227.14

LC

50

(mg L1)

500.00 ± 6.97 300 ± 36.65

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Fig. 1. Schematic pictures showing blisters (A–E) and lysis (F) of P. caudatum exposed to higher concentrations (>350 mg L1) of acephate (1400·). Arrows indicating rupture of outer membrane.

The mean values of velocities (mm s1) for every 10 s in 6 min track are presented in Fig. 3. The velocity of control paramecia was calculated as 5.46 ± 0.10 mm s1. However, the toxicant acephate has significantly enhanced the velocities of exposed paramecia in a concentration dependent manner. The lower concentrations (<125 mg L1) of acephate did not exhibit any intra cellular damage but showed an initial enhancement in velocity and subsequently regained their normal velocity after few min. About 40% of the exposed paramecia at 375 mg L1 affected the internal membranes, which might have reduced their velocities. However, more than 80% paramecia exposed to 500 mg L1 showed initial increase in their velocities but declined with in few min, due to bulging of cells.

Fig. 2. The mobility tracking records (distance moved) of P. caudatum exposed to 1/4, 1/2, 3/4, and LC50 (125, 250, 375, and 500 mg L1, respectively) concentrations for 6 min by Video tracking system for automated mobility monitoring and compared with controls. Each value is means ± SE of five independent observations.

Such alterations in the locomotor behaviour were observed earlier in Paramecium tetraurelia exposed to pertussis toxin with enhancement in turn angels and in case of exposure to deltamethrin, backward swimming was enhanced. These toxic substances were already shown as G-protein modulator and voltage-gated channel agonists, respectively, [21]. In a separate set of experiments, paramecia were exposed to a single concentration, 300 mg L1 (LC50 for 2 h) and their altered velocities were monitored at regular intervals of 15 min (Fig. 4). It is evident from the figure that the initial velocity was 3.85-fold than control value and subsequently reduced to equalize that of control around 45 min. Further length of exposure caused more than 50% inhibition in their velocities and immobilization at the end of 2 h exposure (Fig. 4, inset). Characteristic increase in the velocity compared to that of control shows the negative response of the organism to the acephate. Gradual decrease in the swimming speed, with the increased time of exposure, may be due to the effect of the pesticide on the cellular metabolism. Our earlier studies indicated that the ‘trichocyst exocytosis’ was a common phenomena in paramecium, when treated with sub-lethal concentrations of mercury and chromium (unpublished data), where as acephate treated paramecia did not show any exocytosis. Exocytosis occurs due to variation in calcium flux in the cells [27,28]. Since, such exocytosis does not occur, it may be anticipated that acephate has a different mode of action.

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¼ LC

35

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300

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Fig. 3. The change in velocity (mm s1) of P. caudatum at regular intervals of 10 s on exposure to 1/4, 1/2, 3/4, and LC50 (125, 250, 375, and 500 mg L1, respectively) concentrations for 6 min by Video tracking system. Symbol —d— indicates controls and —h— represents treated. Each value is means ± SE of five independent observations.

plication of paramecia in a concentration dependent manner. The test concentrations (30–240 mg L1) have shown significant decrease (p < 0.001) in proliferation of P. caudatum. Acephate showed a significant inhibitory effect on the proliferation rates of P. caudatum and IC50 value was determined to be 100.30 mg L1 (Fig. 5, inset). Toxicant concentration at 350 mg L1 totally inhibited the growth of paramecia. The proliferation of P. caudatum was significantly affected by the action of acephate as the generation number and concentration of acephate are inversely proportional (Table 2). The generation time gradually increased with the increase of toxicant concentration. Acephate is also known to affect the growth rate of an aquatic bacterium Chromobacterium lividum [29]. Fig. 4. Change in the velocity (mm s1) of P. caudatum for every 15 min interval, when exposed to LC50 conc. (300 mg L1 for 2 h). Each value is means ± SE of five independent observations. (Inset) Percent variation in velocity in comparison to control velocity (5.46 ± 0.10 mm s1). * Indicates significant differences from control values (p < 0.05), x indicates significant differences from control values (p < 0.01), and m indicates significant differences from control values (p < 0.001).

In continuation, the effect of sub-lethal concentrations (30, 60, 120, and 240 mg L1) of acephate on population growth rate of P. caudatum was evaluated with and without toxicant for 24 h. It is evident from the Fig. 5 that the toxicant, acephate has significantly affected the multi-

4. Conclusions The prime focus of the present paper is to develop a simple and reliable evaluation method to detect the toxic effects of insecticides at laboratory conditions. The effect of toxicant on several biological properties can be studied on paramecia considered as a single cell cum organism, where as such wide range tests may not be possible to perform with human cell lines [9]. Preliminary toxicological evaluations of any compound subjected to behavioural, morphological alterations, proliferation rates, and survival of paramecia in short duration would be more appropriate

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Percent reduction

180 160

Total number of cells

140

60

Linear Fit: (Y) = A + B * xscale(X) A = -0.75; B = 25.36 -1

55

IC value = 100.30 mg L 50

50 45 40 35

120

11

0

Concentration in mg L

-1 100

(log)

100 80 60 40 20 0 Control

30

60

120

240

-1

Concentration (mg L ) Fig. 5. Effect of acephate on population growth (proliferation) of P. caudatum at sub-lethal concentrations (30, 60, 120, and 240 mg L1). x Indicates significant differences from control values (p < 0.01). (Inset) Inhibition of population growth (IC50) in P. caudatum as a function of concentration for acephate.

Table 2 Effect of acephate on P. caudatum generation number (n) and generation time (g) after 24 h of growth Acephate concentrations (mg L1)

Generation no. (n) ± SE

Generation time (g) ± SE

30 60 120 240 Control

2.46 ± 0.12 2.31 ± 0.12 2.13 ± 0.11 1.80 ± 0.09 3.14 ± 0.15

9.74 ± 0.48 10.38 ± 0.51 11.29 ± 0.56 13.37 ± 0.66 7.63 ± 0.38

Each value is mean of five independent assays ± standard error. Values are significant from control values at p < 0.01.

than in using higher organisms. These ciliates are preferred for their simplicity in operation and also cost effectiveness. We can conclude that the paramecium toxicity test could be considered as rapid and novel method for evaluating the effects of environmental pollutants, based on the derived LC50 values (10 min and 2 h), and sub-lethal effect on population growth and generation time in 24 h. Hence, further experiments are warranted to study the mode of action of acephate on paramecia. Acknowledgments The authors are thankful to the Director, IICT for providing the facilities and constant encouragement through out the study. The authors V.G. Gunda and S.K. Arepalli are also thankful to Council of Scientific and Industrial Research (CSIR) for providing Junior Research Fellowship. IICT Communication no: 060102.

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