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Environmental Toxicology and Pharmacology 25 (2008) 334–341

Carbofuran in water: Subchronic toxicity to rats
Dragica V. Brki´ a , Slavoljub Lj. Vitorovi´ b , Slavica M. Gaˇi´ a , Neˇko K. Neˇkovi´ a,∗ c c sc s s c a Institute of Pesticides and Environmental Protection, Banatska 31-b,
P.O. Box 163, 11080 Belgrade, Serbia b Faculty of Agriculture, University of Belgrade, Belgrade, Serbia

Received 31 July 2007; received in revised form 26 October 2007; accepted 4 November 2007
Available online 17 November 2007

Abstract
Carbofuran toxicity on rats was studied during subchronic exposure. Female and male rats were administered carbofuran in drinking water in concentrations of 25, 100 and 400 ppm for a period of 90 days. Clinical symptoms, water consumption, body weight gain, organ weight, pathological and histopathological changes in the liver and kidneys were observed and biochemical and haematological examinations were carried out. The results obtained show that carbofuran administered to rats caused a significant decrease in water consumption as well as in brain, serum and erythrocyte cholinesterase activities. Statistically significant increases in relation to the control were found in the serum enzyme activities.
The haematological data showed that carbofuran had no significant effect on Hb concentration and total RBC, but total WBC showed a significant statistical decrease. The histopathological changes in liver and kidneys were observed. However, cell regeneration in the liver and kidneys was found in all test groups.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Carbofuran; Water; Rats; Toxicity

1. Introduction
Carbofuran is a commonly used carbamate insecticide, nematicide and acaricide effective by contact, stomach and systemic action (Kuhr and Dorough, 1979; Machemer and Pickel,
1994).
It is highly toxic to mammals and reportedly embryotoxic and teratogenic (Gupta, 1994). It has been proven to be mutagenic after metabolic activation (Moriya et al., 1983). Carbofuran also induces micronucleus formation in mouse erythroblast and weak mutagenic response in Chinese hamster ovary cells (GeorgesGridelet et al., 1982). It has been reported to induce chromosome aberrations, micronucleus formation and sperm abnormalities in mouse (Chauhan et al., 2000). It has also been demonstrated that it is a potential endocrine disrupter (Cranmer et al.,
1978; Rawlings et al., 1998; Chatterjee et al., 2001). A most recent investigation showed that carbofuran induced oxidative stress with increased generation of free radicals such as reactive
∗

Corresponding author. Tel.: +381 11 31 61 773/30 76 133/30 76 136; fax: +381 11 31 61 773.
E-mail address: nneskovic@beotel.yu (N.K. Neˇkovi´ ). s c
1382-6689/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2007.11.002 oxygen species and reactive nitrogen species (Milatovic et al.,
2005).
Owing to its widespread use in agriculture and relatively good solubility in water carbofuran can contaminate surface and ground waters and therefore carries a risk to various consumers, as well as the environment. The available data show that carbofuran has been frequently detected as a ground water contaminant in America as well as in Europe and Asia over the last two decades (Aharonson, 1987; Kladivko et al., 1991; Chiron et al.,
1995; Matthiessen et al., 1995; Williams et al., 1995; Angelidis et al., 1996; Gallagher et al., 1996; Loewy et al., 1999; Garcia de Llasera and Bernal-Gonzales, 2001; Campbell et al., 2004;
Tariq et al., 2004).
The US Environmental Protection Agency (EPA) prescribes maximum contaminant level (MCL) for carbofuran in drinking water at 40 ␮g/L. The European Union has set a maximum admissible concentration of 0.5 ␮g/L for the sum of all pesticides and 0.1 ␮g/L for an individual compound in drinking water
(Council Directive 98/83/EC).
The occurrence of carbofuran in ground and drinking water shows the importance of determining a safety margin regarding potential harmful effects.

D.V. Brki´ et al. / Environmental Toxicology and Pharmacology 25 (2008) 334–341 c The present study aimed to investigate potentially adverse effects of different carbofuran concentrations in drinking water on rats.
2. Materials and methods
2.1. Animals
Male and female Wistar rats weighing 140–220 g at the beginning of the experiment were used. The animals were divided into four groups containing five rats each. During 90 days of exposure, the animals were kept in polyethylene cages (two animals in each) at a constant temperature (22 ± 2 ◦ C) with food and water available ad libitum. All experiments were conducted in accordance with the guidelines from NIH (USA), with adequate measures taken to minimize any discomfort to the rats.

2.2. Chemicals
Carbofuran (2,3-dihydro-2,2-dimetyl-7-benzofuranyl metylcarbamate, technical, purity 98.8%) was obtained from FMC Corporation, USA.
Other chemicals, purchased from different producers, were standard commercial products of highest purity.

2.3. Treatment
Carbofuran (doses: 25, 100 and 400 ppm) was administered to appropriate test groups of animals in drinking water. The doses were chosen based on our previous tests, i.e. oral LD50 dose (12.5 mg/kg), and those amounted to 2, 8 and
32 × LD50 . The control animals received tap water (carbofuran-free). The following parameters were taken into consideration in assessing toxic effects: body weight gain, water and food consumption, organ weight, selected haematological parameters, enzyme activity, and histopathological examinations.

2.4. Body weight gain, organ weight, food and water consumption
Animal body weight (g) and food consumption (g) were measured weekly, and water consumption (mL) daily. After the 90-day period, the animals were sacrificed by decapitation. The liver, kidneys, heart, spleen, brain, lungs and adrenal glands were removed and weighted. Blood and selected organs (brain, liver and kidneys) were prepared for biochemical analysis (enzyme activity and haematology), or histopathological examination.

2.5. Enzyme activity
The cholinesterase (ChE) activity (␮mol/mL erythrocytes/min, erythrocytes;
U/L, serum and brain) both in control and treated animals was determined

335

according to the method described by Ellman et al. (1961) and Boskovic et al. (1984).
The serum alkaline phosphatase (AP) activity (U/L) was determined according to a procedure described by McComb and Bowers (1972). The serum aspartate aminotransferase (AST, U/L) and alanine aminotransferase (ALT, U/L) were determined using methods described by Reitman and Frankel (1957) and
Wooton (1964), respectively. Test kits from Randox Laboratories Ltd., Crumlin,
UK, were used in all cases.

2.6. Haematology
Haemoglobin concentration (Hb; ␮mol/L), total white (WBC; no/L) and total red blood cells (RBC; no/L) were determined using the methods of Berg
(1945) and Biggs and MacMillan (1948).

2.7. Histopathological examination
For histopathological examination, portions of liver and kidneys were fixed in Bouin’s fixative and embedded in paraffin. The paraffin sections were stained with Ehlrich haematoxylin using eosin as counter stain (HE) (Romeis, 1948;
Humason, 1979).

2.8. Statistical analysis
The results were statistically processed using the analysis of variance and
Student’s t-test (Vukovic, 2000).

3. Results
3.1. Body weight gain and organ weights
The body weight gain in control and test animals during the experiment is presented in Fig. 1.
A statistically significant increase in the body weight gain was found only in males (400 ppm). The organ weights of control and treated animals are shown in Table 1.
3.2. Water consumption
Total and average daily water consumption (with and without carbofuran) during the 90-day exposure is presented in Fig. 2.
The average daily water consumption by test animals showed a statistically significant decrease, both in females (400 ppm) and males (100 and 400 ppm), compared with control animals.

Fig. 1. Body weight gain of rats to which carbofuran was administered for a period of 90 days. The data are expressed as percentage of control. ** Presents significant difference at P < 0.01 level (n = 5).

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D.V. Brki´ et al. / Environmental Toxicology and Pharmacology 25 (2008) 334–341 c Table 1
Absolute organ weight in rats to which carbofuran was administered for a period of 90 days
Sex

Treatment

Dose (ppm)

Organ weight (g)a
Liver

Kidneys

Heart

Lungs

Brain

Spleen

Adrenals

Female

Control
Carbofuran

0.0
25
100
400

7.61
7.77
8.15
7.41

±
±
±
±

0.54
0.31
0.39
1.24

1.62
1.67
1.78
1.66

±
±
±
±

0.16
0.03
0.15
0.10

0.89
0.89
0.99
0.91

±
±
±
±

0.07
0.06
0.05
0.13

1.65
1.85
1.68
1.65

±
±
±
±

0.29
0.40
0.18
0.39

1.83
1.78
1.85
1.80

±
±
±
±

0.15
0.15
0.10
0.10

0.49
0.47
0.52
0.43

±
±
±
±

0.06
0.05
0.08
0.04

0.06
0.07
0.07
0.05

±
±
±
±

0.02
0.01
0.01
0.01

Male

Control
Carbofuran

0.0
25
100
400

11.81
10.85
11.72
10.34

±
±
±
±

0.60
1.52
1.76
1.80

2.43
2.27
2.33
2.50

±
±
±
±

0.09
0.15
0.22
0.17

1.33
1.20
1.26
1.26

±
±
±
±

0.13
0.15
0.12
0.11

2.07
2.08
2.17
1.99

±
±
±
±

0.14
0.45
0.68
0.16

2.04
2.02
1.79
1.89

±
±
±
±

0.17
0.17
0.29
0.12

0.53
0.54
0.60
0.50

±
±
±
±

0.05
0.08
0.15
0.11

0.05
0.06
0.06
0.05

±
±
±
±

0.00
0.01
0.01
0.01

No statistically significant differences were found in the organ weight of control and treated animals. a Presents mean ± S.D.

Fig. 2. The water consumption of rats to which carbofuran was administered for a period of 90 days. The data are expressed as percentage of control. ** Presents significant difference at P < 0.01 level (n = 5).

3.3. Enzyme activity
The effects of carbofuran on ChE activity in the serum, brain and erythrocytes are shown in Fig. 3.

The erythrocytes ChE activity in all treated groups (all tested doses) significantly decreased compared with control animals.
The serum ChE activity in experimental animals also decreased.
Statistically significant differences, compared with the control

Fig. 3. The ChE activity of rats to which carbofuran was administered for a period of 90 days. The data are expressed as percentage of control. * Presents significant difference at P < 0.05 level (n = 5); ** presents significant difference at P < 0.01 level (n = 5).

D.V. Brki´ et al. / Environmental Toxicology and Pharmacology 25 (2008) 334–341 c 337

Fig. 4. The serum enzyme activity of rats to which carbofuran was administered for a period of 90 days. The data are expressed as percentage of control. * Presents significant difference at P < 0.05 level (n = 5); ** presents significant difference at P < 0.01 level (n = 5).

animals, were found in females (400 ppm) and males (100 ppm).
A statistically significant decrease was found in the brain ChE activity in males (400 ppm) and females (all tested doses).
The activities of AP, AST and ALT at the end of the 90-day period are shown in Fig. 4.
The AP activity increased by 25.6–48.2 (females) and
21.7–27.5% (males): the differences between the control and experimental animals were statistically significant both in females (400 ppm) and males (all doses).
The AST activity, both in the treated males and females
(doses: 100 and 400 ppm) was statistically significantly

increased. At the same time, the ALT activity increased significantly by 7.8–21.6 (females) and 16.0–52.5% (males). The differences for the 400 ppm (females), 100 and 400 ppm (males) were statistically significant.
3.4. Haematology
The haematological effects of carbofuran (Hb, WBC and
RBC) are shown in Fig. 5.
No statistically significant difference was found regarding Hb concentration and RBC count of the control and test animals. The

Fig. 5. The haematological data for the rats to which carbofuran was administered for a period of 90 days. The data are expressed as percentage of control. * Presents significant difference at P < 0.05 level (n = 5); ** presents significant difference at P < 0.01 level (n = 5).

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D.V. Brki´ et al. / Environmental Toxicology and Pharmacology 25 (2008) 334–341 c Fig. 6. Liver of control rats.

Fig. 8. Fatty degeneration in the liver of treated animals (dose: 400 ppm, males).

decrease in the WBC count in treated females (100 and 400 ppm) was statistically significant.
3.5. Histopathological changes
3.5.1. Liver
Liver had a normal structure in the control rats (Fig. 6), while hydropic degeneration (Fig. 7), fatty changes (Fig. 8) (fatty degeneration), hyperplasia Kupffer’s cells, inflammatory infiltrate, proliferation of gall tubules and necrosis were found in the liver of dosed animals (100 and 400 ppm). Pycnotic nuclei and regeneration were also found in all cases. These changes were insignificant and were not in correlation with sex and carbofuran concentration. Fig. 9. Kidney of control rats.

3.5.2. Kidney
The kidneys of the control rats had a normal histological structure (Fig. 9). Hydropic degeneration (Fig. 10) of some tubular epithelial cells, edema in the interrenal tissue and occurrence of hyaline cylinders (Fig. 11) were found in animals exposed to
25 ppm of carbofuran. The same changes (more significant), as well as dilatation of proximal tubules, and several tubules with

hyaline and pigmentary cylinders were found in the two other groups (100 and 400 ppm). Tubular regeneration was found in all test groups.
As in the case of liver, these changes were insignificant, but correlated with sex and carbofuran concentration.

Fig. 7. Hydropic degeneration in the liver of treated animals (dose: 400 ppm, males). Fig. 10. Hydropic degeneration in the kidneys of treated animals (dose: 400 ppm, females). D.V. Brki´ et al. / Environmental Toxicology and Pharmacology 25 (2008) 334–341 c Fig. 11. Hyaline cylinders in the kidneys of treated animals (dose: 400 ppm, males). 4. Discussion
Carbofuran, like other carbamate insecticides, elicits toxicity by acetylcholinesterase inhibition (AChE) at the synapses and neuromuscular junctions. Reduced activity of AChE, as an enzymatic target, can serve as an indicator of excessive exposure to the effects of these agents (Main, 1980; Rodwell, 1989).
The inhibitory effect of AChE has therefore been thoroughly investigated and many data on this subject are available. What is common for most of these investigations is that even after the disappearance of overt signs of intoxication, enzyme activity kept decreasing in subacute, subchronic, and chronic experiments.
The decrease ranged from 50 to 90% and correlated with the substance and applied dose (Costa and Murphy, 1983; Chaudhuri et al., 1993; Jett et al., 1994; Boskovic et al., 1995, 1997; Schulz et al., 1995; Chanda and Pope, 1996).
According to literature data on subacute effects of carbofuran on rats, the decrease in AChE activity reaches as much as 52% in treated animals, compared with control. In chronic experiments (2 years) testing the dose level of 100 ppm, a moderate decrease in the activity of cholinesterase of plasma and erythrocytes was observed, while no statistically significant differences were found at a 20 ppm dose in relation to control.
Our results have shown that carbofuran administered in drinking water over the course of 90 days (doses: 25, 100 and
400 ppm) reduced the activity of ChE erythrocytes (all investigated doses), which coincides with the findings of other authors
(Costa and Murphy, 1983; Chaudhuri et al., 1993; Jett et al.,
1994; Rao et al., 1994; Schulz et al., 1995; Chanda and Pope,
1996). Similar results were obtained regarding serum and brain
ChE.
Concerning body weight gain, our results differ from those obtained by Pant et al. (1995). After carbofuran intake by food in a subchronic experiment (0.1, 0.2, 0.4 and 0.8 mg/kg) a dosedependent decrease in body weight (for doses of 0.2–0.8 mg/kg) was found. However, the method of carbofuran application and other experimental conditions should also be taken into consideration. Pant et al. (1995) reported that carbofuran administered in diet reduced food consumption probably due to its charac-

339

teristic odour, which further reflected on the animals’ weight increase rate.
In our investigations the observed increase in AP activity in serum was correlated with the doses of carbofuran used, and can be related to hystopathological changes in the liver of experimental animals (Poole and Leslie, 1987). These results correspond to the data reported by Pach et al. (1998), indicating a statistically significant increase in AP activity in acute human poisoning with carbofuran. The increase was also found to coincide with a serious liver lesion found in over 61% of the patients.
Considering the effects of transaminases (ALT and AST), our results showed that carbofuran administered in drinking water in the course of 90 days caused an increase in AST activity for all three studied doses in males and the 400 ppm dose in females.
An increase in ALT activity was found in the groups of 100 and
400 ppm as well.
Gupta et al. (1993) found an increase in transaminases (ALT and AST) activity (as a result of an acute effect of carbofuran at
1.5 mg/kg rate), in relation to control values. The AST activity increased 3-fold compared to control, while AST activity almost doubled. The authors attributed the increase in serum transaminase activity to an enzyme leakage from tissue to serum, which most probably occurs as a result of considerably reduced levels of high-energy phosphates (ATP and ADP) and phosphocreatine, these being indispensable for maintaining permeability, integrity and other important characteristics of cell membranes
(stability, electrophysiological characteristics, etc.). After carbofuran poisoning a substantial amount of energy is required, and the level of ATP decreases by 40–60%. The lack of highenergy phosphates probably affects membrane characteristics, provoking the leakage of enzymes into tissue and concentration increase in serum (Gupta et al., 1991, 1994).
The increase in enzyme activity was similarly explained by Maraschin (1987) and Vandenberghe (1996); increase in transaminase activities in serum points to an enzyme leakage into the blood due to destroyed hepatocyte membrane (necrosis), or changed membrane permeability. Determination of specific liver enzymes is a sensitive and reliable test for detecting mildto-serious liver damages (severe necrosis). However, a mild and temporary activity increase often occurs without pathohistological signs of hepatocyte membrane damage, or they are minimal and hardly visible.
The most recent investigations have shown increased ALT and AST activities in fish exposed to carbofuran, compared to control fish. The increased activity of transaminases provides oxaloacetic acid and pyruvate, ␣-ketoglutarate and glutaric acid so as to meet the increased energy demand during carbofuran poisoning. The oxaloacetic acid, pyruvate and ␣-ketoglutarate might have been channeled into the citric acid cycle (Begum,
2004).
Our experiments indicate that the administered doses of carbofuran (25, 100 and 400 ppm) had no statistically significant effect on the observed haematological parameters. The observed changes were mostly uncorrelated with the dosages, so that no reliable conclusions can be drawn from the acquired results.
Similar results were obtained also in some investigations involving farmers (Hussain et al., 1990).

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D.V. Brki´ et al. / Environmental Toxicology and Pharmacology 25 (2008) 334–341 c Histopathological investigation of the liver and kidneys of control animals and those dosed with carbofuran in drinking water for 90 days were carried out in order to improve our understanding of biochemical and other investigated parameters. The most evident change in the liver was a hydropic degeneration of hepatocytes predominantly in the centrilobular region, while fatty changes of small droplet type were rare, and hyperplasia of Kupffer’s cells occurred sporadically (moderately). Regenerative changes (anizonucleosis, two nucleus hepatocytes, increased miotic activity) were present in all cases.
These changes corresponded with the toxic damage of liver, which was found to be low. An evident correlation with dose and sex was not found.
Regarding location, the most frequent injuries of the liver in our investigations were centrilobular. Such location of injuries is attributed to the fact that this region is particularly sensitive to damage by toxic compounds because it has an ample amount of smooth endoplasmic reticulum with mixed-function oxidases—enzymes participating in the metabolic pathways of toxic substances (Vandenberghe, 1996).
The most frequently observed morphological change in kidney was hydropic degeneration of the epithelium of the investigated tubules (mostly without visible cell necrosis), edem of intersticium and the occurrence of hyaline cylinder in collection tubules. These changes occurred in all cases, while poor to moderate dilatation of proximal tubules, tubular necrosis and tubular regeneration were observed in the groups of 100 and
400 ppm.
The pathological toxic damages of the kidneys were found to be of low degree, the most outstanding being to the proximal tubules. The degree of changes correlated with carbofuran doses and the length of exposure.
Our results showed that all three doses have some toxic effects so that the level of carbofuran safety agrees with the present limit of 0.1 ␮g/L in drinking water.
Histopathological data do not always correspond to biochemical changes, those changes may be assumed to carry little biological significance and the environmental risk of carbofuran may consequently be considered minimal.
However, these findings are significant in so far as the very presence of toxic effects suggests caution and a need for further investigation of carbofuran effects on some other parameters possibly significant in evaluating the risk to humans and the environment. References
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