- Email: [email protected]
Intentional poisoning cases of animals with anticholinesterase pesticide-carbofuran in Sri Lanka
Legal Medicine 11 (2009) S500–S502
Contents lists available at ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Intentional poisoning cases of animals with anticholinesterase pesticide-carbofuran in Sri Lanka Sakunthala Tennakoon *, Bandumala Perera, Lathika Haturusinghe Government Analyst’s Department, Independence Square, Colombo-07, Sri Lanka
a r t i c l e
i n f o
Article history: Received 18 December 2008 Accepted 8 January 2009 Available online 6 March 2009 Keywords: Carbofuran Intentional poisoning HPLC Fatal Animals Birds
a b s t r a c t Carbofuran is a broad spectrum insecticide and nematicide which inhibits acetyl cholinesterase. Several intentional poisoning cases of animals and birds including crows, dogs, cow, and elephant, using carbofuran were reported in Sri Lanka. Qualitative analysis of carbofuran in biological specimens was carried out using T.L.C and GC–MS. The quantitative analysis was carried out by HPLC using Zorbax Eclips XDBC18 (150 4.6 mm I.D 5 lm particle size) column with acetonitrile: water 25:75 v/v mobile phase and UV detection at 210 nm [1]. The liquid–liquid extraction with chloroform was reproducible and sensitive. The procedure was validated in terms of linearity (0.996 < r < 0.999) for concentrations ranging from 1 to 200 lg/ml, repeatability (RSD 4.4% at 100 lg/ml, n = 10) extraction recovery (91.7% at 100 lg/g, 77.3% at 20 lg/g), limit of detection (S/N ratio = 3, 0.7 lg/ml) and limit of quantification (S/N ratio = 10, 2.4 lg/ml). The method was found satisfactory and successfully applied in fatal cases of animals and birds due to carbofuran poisoning. The fatal carbofuran levels detected were in the range of 42–910 lg/g in crow’s gizzard and contents, 50–800 lg/g in dog’s stomach contents, 13–20 lg/g in dog’s liver, 0.9 lg/g in cow’s stomach and contents, 35 lg/g in elephant’s stomach and contents. Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl methyl carbamate) is a broad spectrum carbamate insecticide, acaricide and nematicide [2]. It has been assigned a toxic class of WHO 1b, highly hazardous [2,3] and is among the most highly toxic pesticide known to birds. Cholinesterase inhibition due to carbamate pesticides is generally more rapidly reversible and of short duration [4,5]. It was first used commercially in 1967 and its use has grown rapidly over subsequent years [5]. The main metabolites of carbofuran include 3-keto carbofuran and 3-hydroxy carbofuran-7-phenol and these compounds become conjugated & excreted in the urine and bile [2,4]. The formulation of 3% carbofuran granules (curaterr, furadan etc.) is widely used in Sri Lanka for the control of insects and nematodes in paddy, cucurbits, tomato, brinjal, beet root, onion, cabbage, banana, tea, and coconut. Several cases of fatal poisoning among the animals by carbofuran following ingestion of deliberately poisoned food [6,7] and accidental ingestion of carbofuran [2] have been reported. Numerous methods for the identification and quantification of carbamates have been reported in the literature including thin layer chromatography, [3,8] high pressure liquid chromatography, [6,8] gas chromatography, [3,4,8–10] and GC–MS [3,6].
* Corresponding author. Tel.: +94 11 2693562/2784671; fax: +94 11 2692309. E-mail address: [email protected] (S. Tennakoon). 1344-6223/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.legalmed.2009.01.033
In the recent past several intentional poisoning cases of animals including crows, dogs, cow, and elephant using carbofuran were reported in Sri Lanka. Four such cases are described in this paper.
2. Materials and methods 2.1. Case reports (case histories) 2.1.1. Case-1 Over 300 crows were found dead in a high security area in Colombo, Sri Lanka. The cause of death was suspected to be an infectious disease or poisoning. Specimens of Gizzard & contents, Intestine, liver, and kidney of dead crows were sent for toxicological examination. 2.1.2. Case-2 In November 2007, three dogs on the same day and another dog few days later were found dead in the surrounding area of Sri Lanka Air Force Base, just outside Colombo, which is again a high security zone. This incident was highly suspicious and seems to indicate intentional poisoning. At autopsy stomach contents and liver samples of four dogs were collected and sent for toxicological analysis. On examination, sand-like substance was observed in the stomach contents and the chloroform extracts were purple in colour.
S. Tennakoon et al. / Legal Medicine 11 (2009) S500–S502
2.1.3. Case-3 An Elephant was found dead in a national park close to a pond. Remnant pieces of Pumpkins supposed to have been eaten by the elephant and two uneaten pumpkins suspected to be poisoned were found along its foot path. An empty curaterr sachet was recovered from the suspected farmer who laid the poisoned pumpkins to kill this elephant, which destroyed his cultivation. Specimens of stomach and contents of the elephant, the pumpkins suspected of containing poison and the empty sachet of the suspected poison were sent for toxicological examination.
S501
I.D, 0.25 lm film thickness). Helium was used as the carrier gas with flow rate of 24.5 ml/min. The temperature programme in the range of 80–260 °C was applied. HPLC was preformed with a GBC LC-1150 Pump, and a GBC1210 UV/visible detector. Reversed phase HPLC of carbofuran was performed using Zorbax Eclips XDB-C18 (150 4.6 mm, 5 lm particle size) column and acetonitrile: water 25:75 mobile phase at a flow rate of 1.5 ml/min. The detection wave length was 210 nm. 2.3. HPLC method validation
2.1.4. Case-4 A cow was found dead under suspicious circumstance in a garden. The owner of the cow claimed that it was given to eat poisoned pieces of jack fruit by the neighbour. The specimen of stomach and contents was sent to the toxicology laboratory for investigation. In all of these cases police investigation and findings of the toxicological analysis indicated that these deaths were caused by carbofuran mixed food being given to the innocent animals deliberately. 2.2. Experimental Biological specimens (5 g) were boiled with ethyl alcohol (50 ml) and 10% tartaric acid (2.5 ml). The filtrate was extracted three times with (10 ml 3) chloroform after adding saturated brine solution (10 ml). The dry extracts were redissolved in Acetonitrile and analysed by T.L.C, GC–MS, and HPLC. Stock solution of carbofuran (0.5 mg/ml) was prepared in acetonitrile. This solution was appropriately diluted with acetonitrile to obtain working standard solutions. The stock solution was stored at 4 °C. T.L.C was performed on glass plates precoated with silica gel 60 (E. Merck) using two independent solvent systems; (a) n-hexane: acetone: toluene 10:2:2 v/v/v, (b) n-hexane: acetone: toluene: ethyl acetate 4:4:2:2 v/v/v/v, respectively. The plates were exposed to bromine vapour for 1 min and sprayed with bromo phenol blue spray. (0.05 g bromo phenol blue was dissolved in 10 ml acetone and diluted to 100 ml with 1% AgNO3 solution in acetone: water 1:3 mixture). GC–MS analysis was carried out using Agilent 5975B instrument equipped with a capillary HP5-MS column (30 m 0.32 mm
2.3.1. Selectivity The selectivity of the method was verified against endogenous compounds due to metrics. The post-mortem specimen of stomach and contents found to be negative for any poisons was extracted with chloroform as discussed above and injected onto the HPLC column to test for the absence of interfering compounds. 2.3.2. Recovery study The extraction recovery was estimated using spiked poison free stomach and contents samples at concentration of 100 lg/g and 20 lg/g, respectively. 2.3.3. Repeatability Ten injections of 100 lg/g solution of carbofuran in acetonitrile were injected on to the column and relative standard deviation was calculated. 2.3.4. Data analysis Calibration curves for carbofuran were generated by least square linear regression of the peak area versus carbofuran concentration. The resulting slopes and intercepts were used to obtain carbofuran concentrations in biological samples. The linearity of the method was confirmed by r2 value. 2.3.5. LOD and LOQ Limit of detection (S/N ratio = 3) and limit of quantification (S/N ratio = 10) were calculated using the calibration curve of peak height versus concentration for 1–25 lg/ml standard series.
Fig. 1. Chromatograms of (A) Poison free stomach and content. (B) Poison-free stomach and content spiked with carbofuran 100 lg/g. (C) Stomach contents of a dog.
S502
S. Tennakoon et al. / Legal Medicine 11 (2009) S500–S502
Table 1 HPLC method validation parameters. Selectivity Percentage recovery R.S.D Linearity
Verified against endogenous compounds due to the matrices 91.7% at 100 lg/g, 77.3% at 20 lg/g 4.4% at 100 lg/ml 1–25 lg/ml r2 = 0.999 10–200 lg/ml r2 = 0.996 0.7 lg/ml (14 ng on column) 2.4 lg/ml (48 ng on column)
LOD LOQ
Conflict of interest All authors hereby confirm that there are no any financial and personal relationships with other people or organisations that could inappropriately influence our work. The above work was not carried out on a grant or other funding. Therefore there is no conflict of interest.
Table 2 Carbofuran concentrations detected in biological specimens. Carbofuran concentration (lg/g) Gizzard and contents Case 1
Crow (1) Crow (2)
Case 2
Dog Dog Dog Dog
Case 3
Elephant
Case 4
Cow
Stomach and contents
Stomach contents
Liver
800 200 50 56
15 20 13 14
910 42
(1) (2) (3) (4)
animals were not submitted for analysis. Hence it was not possible to detect the fatal blood carbofuran levels of the above animals. The low cost, easy access, free availability and high toxicity of carbofuran to birds and other animals seem to be preferred by those who poisoned animals and birds by mixing their food with carbofuran.
34
Acknowledgement We thank Asst. Govt. Analysts, Mrs. W.D.V. Karunarathna, & Mr. U.S.S. Udugampala of the Govt. Analyst’s department for giving technical support in carrying out this work. We also wish to extend our thanks to Ms. Sumathy Rajasingham for proof reading & Ms. Nadeera Hansamali for the support given in computer work.
0.9
References
3. Results and discussion HPLC Chromatograms of extracts obtained from poison free stomach and contents, stomach and contents spiked with carbofuran and stomach contents of the dog are presented in Fig. 1. The method was found to be acceptable for quantitative analysis of carbofuran in biological specimens with respect to all the validation parameters (Table 1). In Case 3 carbofuran was identified in the remnant pieces of pumpkins said to be eaten by the elephant and pumpkins suspected to be poisoned (Table 2). The concentration of carbofuran in gizzard and contents, stomach, and contents, stomach contents and in liver of animals could not be compared with previous fatal reports as the literature lacks data on the concentration of carbofuran in animals’ post-mortem specimens. However Malgorzaba Klys et al. reported total concentration of carbofuran in liver specimen of a human fetus as 2.5 lg/g [4]. Kiyoshi Ameno et al. reported human fatal blood concentration range from 0.32 to 29.3 lg/ml [3]. Therefore determination of fatal levels of carbofuran in animals is most valuable research opportunity. The blood samples of above
[1] U.S. Department of Labor (2007). Carbofuran (Furadan) (Method PV2127). [Retrieved November 20, 2007. Available from:]. [2] Summer Evert (2007). Environmental fate of carbofuran. Environmental monitoring branch, Department of pesticide regulation, retrieved March 13, 2008. [Available from: ]. [3] Ameno Kiyoshi, Lee Sang-Ki, In Sang-Whan, Yang Ja-Yeol, Yoo Young-Chan, Ameno Setsuko, Kubota Takako, Kinoshita Hiroshi, Ijiri Iwao. Blood carbofuran concentrations in suicidal ingestion cases. J Forensic Sci Int 2001;116:59–61. [4] Klys Malgorzata, Kosun´ Jacek, Pach Janusz, Kamen´czak Aleksandra. Carbofuran poisoning of pregnant woman and fetus per ingestion. J Forensic Sci 1989;34(6):1413–6. [5] Satar Salim, Satar Serdar, Sebe Ahmet, Yesilagac Hasan. Carbofuran poisoning among farm workers. Mt Sinai J Med 2005;72(6):389–92. [6] Kwon YK, Wee SH, Kim JH. Pesticide poisoning events in wild birds in Korea from 1998 to 2002. J Wildl Dis 2004;40(4):737–40. [7] Taylor MJ, Sharp EA, Giela A. (2006). Pesticide poisoning of animals in 2006, A report of investigations in Scotland. Scottish agricultural science agency; 2006. [Retrieved March 13, 2008. Available from: ]. [8] Osselton Michael David, Snelling Robert Duncan. Chromatographic identification of pesticides. J Chromatogr 1986;368:265–71. [9] Zhong WZ, Lemley AT. Quantitative determination of ppb levels of carbamate pesticides in water by capillary gas chromatography. J Chromatogr 1984;299:269–74. [10] Mayer William J, Greenberg Mark S. Determination of some carbamate pesticides by high-performance liquid chromatography with electrochemical detection. J Chromatogr 1981;208:295–304.
Contents lists available at ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Intentional poisoning cases of animals with anticholinesterase pesticide-carbofuran in Sri Lanka Sakunthala Tennakoon *, Bandumala Perera, Lathika Haturusinghe Government Analyst’s Department, Independence Square, Colombo-07, Sri Lanka
a r t i c l e
i n f o
Article history: Received 18 December 2008 Accepted 8 January 2009 Available online 6 March 2009 Keywords: Carbofuran Intentional poisoning HPLC Fatal Animals Birds
a b s t r a c t Carbofuran is a broad spectrum insecticide and nematicide which inhibits acetyl cholinesterase. Several intentional poisoning cases of animals and birds including crows, dogs, cow, and elephant, using carbofuran were reported in Sri Lanka. Qualitative analysis of carbofuran in biological specimens was carried out using T.L.C and GC–MS. The quantitative analysis was carried out by HPLC using Zorbax Eclips XDBC18 (150 4.6 mm I.D 5 lm particle size) column with acetonitrile: water 25:75 v/v mobile phase and UV detection at 210 nm [1]. The liquid–liquid extraction with chloroform was reproducible and sensitive. The procedure was validated in terms of linearity (0.996 < r < 0.999) for concentrations ranging from 1 to 200 lg/ml, repeatability (RSD 4.4% at 100 lg/ml, n = 10) extraction recovery (91.7% at 100 lg/g, 77.3% at 20 lg/g), limit of detection (S/N ratio = 3, 0.7 lg/ml) and limit of quantification (S/N ratio = 10, 2.4 lg/ml). The method was found satisfactory and successfully applied in fatal cases of animals and birds due to carbofuran poisoning. The fatal carbofuran levels detected were in the range of 42–910 lg/g in crow’s gizzard and contents, 50–800 lg/g in dog’s stomach contents, 13–20 lg/g in dog’s liver, 0.9 lg/g in cow’s stomach and contents, 35 lg/g in elephant’s stomach and contents. Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl methyl carbamate) is a broad spectrum carbamate insecticide, acaricide and nematicide [2]. It has been assigned a toxic class of WHO 1b, highly hazardous [2,3] and is among the most highly toxic pesticide known to birds. Cholinesterase inhibition due to carbamate pesticides is generally more rapidly reversible and of short duration [4,5]. It was first used commercially in 1967 and its use has grown rapidly over subsequent years [5]. The main metabolites of carbofuran include 3-keto carbofuran and 3-hydroxy carbofuran-7-phenol and these compounds become conjugated & excreted in the urine and bile [2,4]. The formulation of 3% carbofuran granules (curaterr, furadan etc.) is widely used in Sri Lanka for the control of insects and nematodes in paddy, cucurbits, tomato, brinjal, beet root, onion, cabbage, banana, tea, and coconut. Several cases of fatal poisoning among the animals by carbofuran following ingestion of deliberately poisoned food [6,7] and accidental ingestion of carbofuran [2] have been reported. Numerous methods for the identification and quantification of carbamates have been reported in the literature including thin layer chromatography, [3,8] high pressure liquid chromatography, [6,8] gas chromatography, [3,4,8–10] and GC–MS [3,6].
* Corresponding author. Tel.: +94 11 2693562/2784671; fax: +94 11 2692309. E-mail address: [email protected] (S. Tennakoon). 1344-6223/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.legalmed.2009.01.033
In the recent past several intentional poisoning cases of animals including crows, dogs, cow, and elephant using carbofuran were reported in Sri Lanka. Four such cases are described in this paper.
2. Materials and methods 2.1. Case reports (case histories) 2.1.1. Case-1 Over 300 crows were found dead in a high security area in Colombo, Sri Lanka. The cause of death was suspected to be an infectious disease or poisoning. Specimens of Gizzard & contents, Intestine, liver, and kidney of dead crows were sent for toxicological examination. 2.1.2. Case-2 In November 2007, three dogs on the same day and another dog few days later were found dead in the surrounding area of Sri Lanka Air Force Base, just outside Colombo, which is again a high security zone. This incident was highly suspicious and seems to indicate intentional poisoning. At autopsy stomach contents and liver samples of four dogs were collected and sent for toxicological analysis. On examination, sand-like substance was observed in the stomach contents and the chloroform extracts were purple in colour.
S. Tennakoon et al. / Legal Medicine 11 (2009) S500–S502
2.1.3. Case-3 An Elephant was found dead in a national park close to a pond. Remnant pieces of Pumpkins supposed to have been eaten by the elephant and two uneaten pumpkins suspected to be poisoned were found along its foot path. An empty curaterr sachet was recovered from the suspected farmer who laid the poisoned pumpkins to kill this elephant, which destroyed his cultivation. Specimens of stomach and contents of the elephant, the pumpkins suspected of containing poison and the empty sachet of the suspected poison were sent for toxicological examination.
S501
I.D, 0.25 lm film thickness). Helium was used as the carrier gas with flow rate of 24.5 ml/min. The temperature programme in the range of 80–260 °C was applied. HPLC was preformed with a GBC LC-1150 Pump, and a GBC1210 UV/visible detector. Reversed phase HPLC of carbofuran was performed using Zorbax Eclips XDB-C18 (150 4.6 mm, 5 lm particle size) column and acetonitrile: water 25:75 mobile phase at a flow rate of 1.5 ml/min. The detection wave length was 210 nm. 2.3. HPLC method validation
2.1.4. Case-4 A cow was found dead under suspicious circumstance in a garden. The owner of the cow claimed that it was given to eat poisoned pieces of jack fruit by the neighbour. The specimen of stomach and contents was sent to the toxicology laboratory for investigation. In all of these cases police investigation and findings of the toxicological analysis indicated that these deaths were caused by carbofuran mixed food being given to the innocent animals deliberately. 2.2. Experimental Biological specimens (5 g) were boiled with ethyl alcohol (50 ml) and 10% tartaric acid (2.5 ml). The filtrate was extracted three times with (10 ml 3) chloroform after adding saturated brine solution (10 ml). The dry extracts were redissolved in Acetonitrile and analysed by T.L.C, GC–MS, and HPLC. Stock solution of carbofuran (0.5 mg/ml) was prepared in acetonitrile. This solution was appropriately diluted with acetonitrile to obtain working standard solutions. The stock solution was stored at 4 °C. T.L.C was performed on glass plates precoated with silica gel 60 (E. Merck) using two independent solvent systems; (a) n-hexane: acetone: toluene 10:2:2 v/v/v, (b) n-hexane: acetone: toluene: ethyl acetate 4:4:2:2 v/v/v/v, respectively. The plates were exposed to bromine vapour for 1 min and sprayed with bromo phenol blue spray. (0.05 g bromo phenol blue was dissolved in 10 ml acetone and diluted to 100 ml with 1% AgNO3 solution in acetone: water 1:3 mixture). GC–MS analysis was carried out using Agilent 5975B instrument equipped with a capillary HP5-MS column (30 m 0.32 mm
2.3.1. Selectivity The selectivity of the method was verified against endogenous compounds due to metrics. The post-mortem specimen of stomach and contents found to be negative for any poisons was extracted with chloroform as discussed above and injected onto the HPLC column to test for the absence of interfering compounds. 2.3.2. Recovery study The extraction recovery was estimated using spiked poison free stomach and contents samples at concentration of 100 lg/g and 20 lg/g, respectively. 2.3.3. Repeatability Ten injections of 100 lg/g solution of carbofuran in acetonitrile were injected on to the column and relative standard deviation was calculated. 2.3.4. Data analysis Calibration curves for carbofuran were generated by least square linear regression of the peak area versus carbofuran concentration. The resulting slopes and intercepts were used to obtain carbofuran concentrations in biological samples. The linearity of the method was confirmed by r2 value. 2.3.5. LOD and LOQ Limit of detection (S/N ratio = 3) and limit of quantification (S/N ratio = 10) were calculated using the calibration curve of peak height versus concentration for 1–25 lg/ml standard series.
Fig. 1. Chromatograms of (A) Poison free stomach and content. (B) Poison-free stomach and content spiked with carbofuran 100 lg/g. (C) Stomach contents of a dog.
S502
S. Tennakoon et al. / Legal Medicine 11 (2009) S500–S502
Table 1 HPLC method validation parameters. Selectivity Percentage recovery R.S.D Linearity
Verified against endogenous compounds due to the matrices 91.7% at 100 lg/g, 77.3% at 20 lg/g 4.4% at 100 lg/ml 1–25 lg/ml r2 = 0.999 10–200 lg/ml r2 = 0.996 0.7 lg/ml (14 ng on column) 2.4 lg/ml (48 ng on column)
LOD LOQ
Conflict of interest All authors hereby confirm that there are no any financial and personal relationships with other people or organisations that could inappropriately influence our work. The above work was not carried out on a grant or other funding. Therefore there is no conflict of interest.
Table 2 Carbofuran concentrations detected in biological specimens. Carbofuran concentration (lg/g) Gizzard and contents Case 1
Crow (1) Crow (2)
Case 2
Dog Dog Dog Dog
Case 3
Elephant
Case 4
Cow
Stomach and contents
Stomach contents
Liver
800 200 50 56
15 20 13 14
910 42
(1) (2) (3) (4)
animals were not submitted for analysis. Hence it was not possible to detect the fatal blood carbofuran levels of the above animals. The low cost, easy access, free availability and high toxicity of carbofuran to birds and other animals seem to be preferred by those who poisoned animals and birds by mixing their food with carbofuran.
34
Acknowledgement We thank Asst. Govt. Analysts, Mrs. W.D.V. Karunarathna, & Mr. U.S.S. Udugampala of the Govt. Analyst’s department for giving technical support in carrying out this work. We also wish to extend our thanks to Ms. Sumathy Rajasingham for proof reading & Ms. Nadeera Hansamali for the support given in computer work.
0.9
References
3. Results and discussion HPLC Chromatograms of extracts obtained from poison free stomach and contents, stomach and contents spiked with carbofuran and stomach contents of the dog are presented in Fig. 1. The method was found to be acceptable for quantitative analysis of carbofuran in biological specimens with respect to all the validation parameters (Table 1). In Case 3 carbofuran was identified in the remnant pieces of pumpkins said to be eaten by the elephant and pumpkins suspected to be poisoned (Table 2). The concentration of carbofuran in gizzard and contents, stomach, and contents, stomach contents and in liver of animals could not be compared with previous fatal reports as the literature lacks data on the concentration of carbofuran in animals’ post-mortem specimens. However Malgorzaba Klys et al. reported total concentration of carbofuran in liver specimen of a human fetus as 2.5 lg/g [4]. Kiyoshi Ameno et al. reported human fatal blood concentration range from 0.32 to 29.3 lg/ml [3]. Therefore determination of fatal levels of carbofuran in animals is most valuable research opportunity. The blood samples of above
[1] U.S. Department of Labor (2007). Carbofuran (Furadan) (Method PV2127). [Retrieved November 20, 2007. Available from: