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Clinicopathological and toxicological aspects of poisoning by the clomazone herbicide in sheep
Small Ruminant Research 124 (2015) 120–126
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Clinicopathological and toxicological aspects of poisoning by the clomazone herbicide in sheep M.Z. Fagundes a , M.A. Gonc¸alves a , M.P. Soares b , M.L. Martins c , R. Zanella c , F. Riet-Correa d , B.L. Anjos a,∗ a
Veterinary Patolology Laboratory (LPV), Universidade Federal do Pampa, Uruguaiana, 97500-970, Rio Grande do Sul State, Brazil Regional Diagnostic Laboratory, Universidade Federal de Pelotas, Pelotas, 96160-000, Rio Grande do Sul State, Brazil Pesticides Residues Analysis Laboratory (LARP), Universidade Federal de Santa Maria, Santa Maria, 97105-900, Rio Grande do Sul State, Brazil d Veterinary Hospital, CSTR, Universidade Federal de Campina Grande, Patos, 58700-310, Paraíba State, Brazil b c
a r t i c l e
i n f o
Article history: Received 26 April 2014 Received in revised form 5 January 2015 Accepted 10 January 2015 Available online 16 January 2015 Keywords: Diseases of sheep Herbicide poisoning Isoxazolidinone Toxic neuropathy
a b s t r a c t Clomazone (2-[(2-chlorophenyl)methyl]-4,4-dimethyl-3-isoxazolidinone) is a herbicide which has been widely used in southern Brazil in the rice cultivation and can be toxic for humans and animals. This study reports the first outbreak poisoning due to clomazone in a flock of 103 sheep, 20 of which showed mainly neurological and respiratory signs. Clomazone was detected in soil and vegetation samples and in the liver, kidney and muscles of poisoned animals. The poisoning was experimentally reproduced in three sheep by the administration of a 134 mg kg body weight dose of clomazone. In both the natural and experimental cases, the clinical signs included tachypnea, anorexia, somnolence, weakness and ataxia. Macroscopically, there were no significant changes. Histologically, vacuolization in the white matter, perineuronal vacuoles and congestion of the leptomeningeal and brain vessels were observed. Ultrastructurally, the vacuolar lesions in the brain corresponded to swelling of the dendrites and astrocytic processes. It is concluded that clomazone causes toxic neuropathy in sheep. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Nervous system diseases of small ruminants caused mainly by infectious and toxic agents cause significant economic losses in Brazil (Guedes et al., 2007); however, despite the large amounts of pesticides used in this country, the only reported neuropathies caused by insecticides or anthelmintics are related to closantel (Ecco et al., 2006; Furlan et al., 2009) and haloxon poisonings (Souza et al., 1996).
∗ Corresponding author. Tel.: +55 5534023003; fax: +55 5534134321. E-mail address: [email protected] (B.L. Anjos). http://dx.doi.org/10.1016/j.smallrumres.2015.01.006 0921-4488/© 2015 Elsevier B.V. All rights reserved.
Currently, herbicides represent the most widely used class of pesticides in Brazil, accounting for 56% of all pesticides sold in this country (Peters, 2011). The intensive use of herbicides and pesticides has worried regulatory agencies due to their deleterious effects on non-target organisms, such as plants, animals and humans (Peters, 2011). Because of the extensive agricultural activity in the southern region of the country, especially with respect to rice cultivation, herbicides such as glyphosate, paraquat and clomazone are used very intensively (Faria et al., 2009; Peters, 2011). In Brazil, glyphosate poisoning in fishes and humans (Glusczak et al., 2007; Faria et al., 2009; Modesto and Martinez, 2010) and paraquat poisoning in humans, dogs, sheep and cattle (Philbey and Morton, 2001; Serra et al., 2003; Gupta, 2012; Santos et al., 2012) have been reported.
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Clomazone (2-[(2-chlorophenyl)methyl]-4,4-dimethyl-3-isoxazolidinone) is a representative of the isoxazolidinone group of herbicides, formulated in the 1980s for control of annual grasses and broad-leaved weeds in a wide variety of crops, including rice (Gunasekara et al., 2009). In the plant body, clomazone has systemic action and is absorbed by the roots, inhibiting the formation of photosynthetic pigments and inducing death. Clomazone may be applied to crops using ground equipment or by air (USEPA, 2007). Until now, spontaneous clomazone poisoning has been reported only in freshwater fishes (Crestani et al., 2007; Moraes et al., 2007; Moraes et al., 2009; Menezes et al., 2011; Cattaneo et al., 2012), and this poisoning was reproduced experimentally in fish and rat (Miron et al., 2004; USEPA, 2007). It is believed that the toxicity of clomazone is based on the inhibition of acetylcholinesterase and its high capacity to drift enhances the risk of poisoning in animals and plants (Crestani et al., 2007). This paper reports the first outbreak of spontaneous clomazone intoxication in sheep and the experimental reproduction of this poisoning in the same species. 2. Material and methods 2.1. Epidemiology and clinical examination In November of 2011, on a farm in the municipality of Mac¸ambará, Rio Grande do Sul State, Brazil, pronounced dissection of the entire pasture occurred, which was caused by accidental drift of an herbicide applied by air to a neighboring rice field. Epidemiological and toxicological data were collected during a visit to a farm, where some animals of a sheep flock showed clinical signs of accidental exposure to a pesticide drift. Clinical evaluation was performed in a sheep that showed more severe clinical signs and was euthanized for pathological evaluation (Sheep #1). Approximately two weeks after exposure, the flock was transferred to a pesticide-free area. Four weeks post-exposure, a sheep that had clinical signs but was fully recovered was also euthanized and necropsied (Sheep #2). The surviving sheep were observed for 120 days after the accidental exposure to pesticide drift. 2.2. Pathological examination At necropsy, tissue samples of the skin, muscle, liver, rumen, omasum, abomasum, small and large intestines, pancreas, adrenal glands, kidneys, urinary bladder, lungs, lymph nodes, bone, spleen, eye, nerves of the brachial plexus, sciatic nerve, brain and spinal cord (cervical, thoracic and lumbar segments) were collected and fixed in 10% neutral-buffered formalin. The samples were embedded in paraffin, cut in 4 m sections, and stained with hematoxylin and eosin stain (HE). After fixation, 1-cm-thick serial sections were made from the brain, and transverse sections that were taken from the frontal and parietal cortices, basal ganglia, hippocampus, cerebellum, thalamus, rostral colliculus, pons and obex were examined histologically.
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2.3. Experimental design The experimental reproduction of the disease was performed using three 8-month-old sheep of the Ideal breed (#3, 4 and 5), weighing 26–27 kg. Each sheep received an oral dose of clomazone of 134 mg per kg body weight (mg/kg bw) diluted in 100 mL of distilled water. The dose used was adapted from the LD50 of different organophosphates for the rat (Kahn and Line, 2005). The sheep were maintained in individual pens during the experiment and were fed daily with pelleted commercial concentrate feed for sheep at an amount equivalent to 1% of their body weight. Alfalfa (Medicago sativa) and water were provided ad libitum. The sheep were submitted to daily clinical, hematological and biochemical evaluations. Serum activities of aspartate aminotransferase (AST) and ␥-glutamyltransferase (GGT) and total protein serum concentrations were determined. For all sheep, blood samples were obtained before the administration of clomazone (T0 ), 18 h (Sheep #4) and 24 h (Sheep #3 and 5) after administration (T1 ), and two days after the resolution of neurological signs in Sheep #3 (T2 ). The animals were monitored until euthanasia at 30 days after administration (Sheep #3) or death (Sheep #4 and 5). Sheep #3–5 were necropsied, and fragments of various organs were collected for histologic examination (similar to those previously described). Immediately after death, samples of the thalamus, midbrain and liver of Sheep #4 and 5 were collected and fixed in 2% glutaraldehyde and sodium cacodylate-buffered solution. The samples were dehydrated in ethanol and embedded in Epon 812. Semithin sections were stained with methylene blue, and ultrathin sections from the selected areas were stained with uranyl acetate and lead citrate prior to observation using a Zeiss EM 109 transmission electron microscope operated at 80 kilovolts. This work was approved by the Animal Research Ethics Committee of Universidade Federal do Pampa under the protocol number 009/2012. 2.4. Herbicide analysis Liver and kidney samples of Sheep #1–5 and skeletal muscle samples of Sheep #2 and #3–5 were collected and maintained at −20 ◦ C for subsequent multiresidue analysis (100 compounds). After extraction by the modified Quick Easy Cheap Effective Rugged and Safe (QuEChERS) method, the samples were analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Prestes, 2011). Soil and vegetation samples were analyzed by gas chromatography-tandem mass spectrometry (GC-MS/MS) (Anastassiades et al., 2003). 3. Results 3.1. Spontaneous poisoning The disease affected a flock of 103 Texel adult sheep grazing a pasture of 40 hectares composed mainly by native grasses, Lolium multiflorum (ryegrass) and Paspalum notatum on a farm in the municipality of Mac¸ambará, Rio
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Fig. 1. Sheep #1 showing flexion of the right forelimb due to motor weakness.
Grande do Sul State, Brazil. The pasture was neighboring to a rice field, in which an herbicide was applied preemergence by air in November 2011. On the day of herbicide application the conditions for aerial application of the product were inappropriate and the wind caused the dispersion of the product that hit the sheep pasture area. The farmer did not disclose the product or the dose that was applied. A pronounced dissection of the entire pasture occurred as a consequence of the accidental drift of the herbicide. Native grasses and leguminous species, as well as large trees showed large amounts of dry leaves by the herbicide action. During the evaluation in loco of the pasture, all members of the team reported a strong odor of herbicide and burning eyes, lips and nose. Approximately 10 days after exposure to the herbicide, 20 out of the 103 sheep showed tachypnea, anorexia, lethargy, limited mobility and weakness characterized by dragging of the hooves and flexing the hock and fetlock (Fig. 1). Some sheep showed hypotrichosis of the facial skin and crusts in the ears. Upon inspection of the area where the animals grazed, a strong smell of herbicide was noted, even at 10 days after exposure. Four weeks after exposure to the herbicide, all sheep, except one that was euthanized with severe clinical signs, recovered from clinical signs. At necropsy, the lungs of Sheep #1 showed moderate multifocal areas of consolidation and slight fibroelastic consistency, and the liver was slightly yellowish with an evident lobular pattern. Sheep #2 showed no significant gross lesions. Histologically, the lungs of Sheep #1 showed moderate thickening of the interalveolar septum with moderate proliferation of fibroblasts and collagen, mild proliferation of type II pneumocytes, emphysema, edema and moderate bronchial smooth muscle cell hypertrophy (Fig. 2A). Moderate diffuse vacuolation of hepatocytes was observed in the liver, especially in the periportal and midzonal zones. Rare hepatocytes were necrotic, and numerous neutrophils were observed within the blood vessels. The spleen showed moderate hemosiderosis and neutrophilic infiltrate surrounding the white pulp. The kidneys showed moderate diffuse congestion and rare hyaline casts within renal tubules.
Vacuolation of the white matter and severe congestion of blood vessels of the leptomeninges and brain were observed in the cerebrum, cerebellum and brainstem. Furthermore, mild perivascular edema, mild mixed inflammatory cell infiltrate around blood vessels and perineuronal vacuolization of Purkinje cells were observed in the cerebellum (Fig. 2B). Sheep #2 had similar changes in the brain, but to a lesser degree. Soil and vegetation samples contained 0.4 mg kg−1 and 0.9 mg kg−1 clomazone, respectively. In multiresidue analysis, the liver and kidney samples (Sheep #1 and 2) were positive for clomazone herbicide at values less than the minimum limit of quantification of the test (LOQm = 0.01 mg kg−1 ) but greater than the minimum limit of detection (LODm = 0.005 mg kg−1 ) (Table 1). In the skeletal muscle sample (Sheep #2), for example, a chromatographic peak corresponding to 1141.79 area units was observed for clomazone herbicide; this signal unequivocally demonstrated the presence of clomazone in the sample under the LC-MS/MS conditions employed. 3.2. Experimental poisoning Approximately 24 h after clomazone administration, Sheep #3 showed weakness, instability with frequent falls, postural changes, and ataxia with abduction and crossing of the legs when walking. Frequent falls, muscle tremors and moderate dyspnea were also observed. After falling, the animals rested in sternal recumbency for approximately 20 min before returning to a standing position. If the sheep were stimulated to walk, the signs became more accentuated. These signs gradually decreased, and on the fourth day after administration of the herbicide, the sheep were fully recovered. Sheep #4 showed sternal recumbency 12 h after administration and died 6 h later. Approximately 10 h after administration of clomazone, Sheep #5 showed weakness, falling, permanent mydriasis, lateral recumbency and muscle tremors and spasms of the hindlimbs and forelimbs, dying 20 h later. Hematological evaluations (Table 2) showed anemia in Sheep #3 and 5, a slight increase in AST in Sheep #4 and #5, a mild decrease in total plasma proteins in Sheep # 3 and 5, and leukocytosis with neutrophilia in all sheep. At necropsy of Sheep #3-5, no gross findings were observed. Histologic lesions were observed mainly in the central nervous system. In all sheep, the blood vessels of the meninges and neuropil were moderately dilated and congested, mainly in Sheep #4, which died 18 h after clomazone administration. In Sheep #3 and 5, mild hemorrhages in the Virchow-Robin space with slight swelling of endothelial cells and perivascular edema were observed. Vacuolation of the white matter was observed mainly in the brainstem. This lesion was more pronounced in Sheep #5, which died 30 h after the administration of the herbicide. Multifocal areas of congestion and moderate alveolar edema were observed in Sheep #3–5. Diffuse congestion and mild neutrophilic infiltrate were observed in the spleen in the center of the red pulp in Sheep #5. In Sheep #3 and 5, the kidney had moderate congestion in the medullary region and some granular and hyaline casts in the renal tubules. Mild interstitial infiltration by
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Fig. 2. A and B. Sheep #1. A. Lung showing thickening of the septum (arrow), moderate diffuse congestion, and areas of emphysema (*). Hematoxylin and eosin. Bar = 100 m. B. Cerebellum. Multiple perineuronal vacuoles are observed near the Purkinje cells. Hematoxylin and eosin. Bar = 20 m. C. Sheep #5. Kidney showing an area of necrosis with pyknotic nuclei and increased eosinophilia of the cytoplasm of the epithelial cells of the proximal convoluted tubules. A close-up view of the kidney showing necrotic tubular cells within a renal tubule (arrowhead) and some neutrophils in the interstitium (arrow) and in the glomerulus (G). Hematoxylin and eosin. Bar = 20 m.
neutrophils of the cortical region and acute tubular necrosis were also observed in the kidney in Sheep #5 (Fig. 2C). An ultrastructural examination of Sheep #4 and 5 showed marked swelling of astrocyte processes (Fig. 3A) and neuronal dendrites of the thalamus and midbrain (Fig. 3B). In these areas, the axons were also swollen (Fig. 3B and C), with dispersion of axoplasmic microtubules occasionally in the presence of membranes (Fig. 3B). The neuronal perikarya showed dilated cisternae of the smooth endoplasmic reticulum and mitochondria with
lysis of the cristae (Fig. 3A). The axonal mitochondria were swollen, with disruption of mitochondrial membranes and lysis of the cristae (Fig. 3C). In some mitochondria, small, round, electron-dense bodies and membrane debris were found. Hepatocytes contained swollen mitochondria with lysed cristae, lipid droplets of varying sizes, and membrane-associated vacuoles with granular content (Fig. 3D). The results of toxicological evaluation via chemical analysis of Sheep #3–5 are presented in Table 1.
Table 1 Multiresidue analysis of liver, kidney and skeletal muscle samples of the sheep naturally and experimentally poisoned by clomazone. Sheep #
1 2 3 4 5 a
Poisoning form (unit)
a
N (unit of the area)b N (unit of the area) Ec (mg/kg−1 )d E (mg/kg−1 ) E (mg/kg−1 )
Samples of the sheep Liver
Kidney
232.19
143.20
3.74
33.98
Skeletal muscle – 1141.79
400.00
63.7
1.9
255.8
10.3
15.1
88.3
51.5
96.1
Natural poisoning. Value less than the minimum limit of quantification of the test (LOQm = 0.01 mg/kg−1 ) and greater than the minimum limit of detection (LODm = 0.005 mg/kg−1 ). c Experimental poisoning. d Value above the minimum limit of quantification of the test; not evaluated. b
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Fig. 3. Transmission electron microscopy. A. Sheep #5. Brainstem. A neuron (N) with dilated cisternae in the endoplasmic reticulum (*) and tumescent mitochondria (m) with rupture of the cristae is observed. Vacuoles (V) representing very swollen astrocytic processes are observed around the neuron. Astrocyte (A). Bar = 2 m. B. Sheep #5. Brainstem. Swollen axons (*) with disappearance of microtubules and the presence of membranes (arrow) are observed. A swollen dendrite is also observed (D). Neuron (N). Bar = 2 m. C. Sheep #4. Brainstem. A swollen dilated axon (*) with a thin myelin sheath due to dilatation of the axoplasm is observed. Another axon shows a severely swollen mitochondria with an electrodense matrix and rupture of the mitochondrial membrane (white arrow). D. Sheep #4. Liver. Hepatocyte showing a membrane-limited vacuole (V) with granular content. Lipid vacuoles of different sizes (*) and tumescent mitochondria (M) with lysis of the cristae are also observed. Note the remnants of cristae within the mitochondria. Bar = 2 m.
4. Discussion The diagnosis of poisoning by clomazone among sheep in southern Brazil was based on the toxicologic analysis showing the presence of clomazone in soil, vegetation and tissues samples and on the occurrence of the disease in a pasture where the vegetation was desiccated by the accidentally application of the herbicide 10 days before. Otherwise, the clinical signs and lesions observed in the spontaneous affected sheep were similar than those observed in the experimental sheep. According to Gupta (2012), all herbicides require specific conditions for use, and application errors are the main cause of poisoning in humans and animals. In the poisoning reported herein, the exposure of sheep to clomazone at the time of application and for over 10 days thereafter occurred by accidental contact with the drift of large amounts of the product after aerial application. Clomazone residues can persist for up to 130 days in water and have been detected in 90% of water samples collected from rivers in rice growing regions (Zanella et al., 2002). Except for Sheep #4, which died after a clinical manifestation period of 6 h, experimentally and spontaneously
poisoned sheep showed nervous and respiratory signs similar to those observed in the field cases. A study examining fish exposed to water containing clomazone reported a significant reduction in acetylcholinesterase activity 12 h after exposure, with recovery to normal activity after 192 h in clean water (Crestani et al., 2007). These results suggest that clomazone inhibits acetylcholinesterase in brain and muscle tissue (Crestani et al., 2007; Cattaneo et al., 2012). Similar clinical signs have been reported in ruminants poisoned by organophosphates and carbamates, and involvement of the central nervous system was noted (Barros and Driemeier, 2007; Dalto et al., 2011). Clinical signs induced by organophosphates and carbamates are muscarinic, including cardiorespiratory disturbances, and nicotinic, characterized by flaccid paralysis, opisthotonus, tremors and muscular weakness (Osweiler, 1998; Radostits et al., 2007). However, the axonal and dendritic injuries characterized by vacuolation of the white matter and perineuronal vacuolation observed in the sheep poisoned by clomazone have not been reported in cases of organophosphate and carbamate poisoning, suggesting that these effects may be due to the direct action of the toxin on the nervous tissue.
– – 44 96 – 265 – 0–1.000
k
i
j
h
f
g
e
c
d
Erythrocyte (x106 /mm3 ). Hemoglobin(g/dl). Hematocrit (%). Mean corpuscular volume (femtoliters). Mean corpuscular hemoglobin concentration (%). Plasm protein total (g/dl). Leukocytes (/mm3 ). Neutrophils (/mm3 ). Lymphocytes (/mm3 ). Monocytes. Eosinophils (/mm3 ). a
Before administration 24 h after administration 48 h after of disappearance of the clinical signs Before administration 18 h after administration Before administration 24 h after administration 3 3 3 4 4 5 5 Referencevalues
b
Monj
48 231 220 96 414 159 64 0–750 2.016 2.618 2.904 2.688 1.794 3.604 1.792 2.000–9.000
Lymi Neuh
2.736 4.851 1.452 1.920 4.485 1.219 4.160 700–6.000 4.800 7.700 4.400 4.800 6.900 5.300 6.400 4.000–10.000
Leug
6.8 6.0 5.6 6.2 6.8 5.6 5.4 6.0–7.5 33.3 31.1 30.4 33.3 32.1 32.0 33.3 33.3 30.3 31.4 36.5 27.2 26.6 41.6 50.0 23–48 30 22 23 27 28 25 21 27–46
MCHCe MCVd Hctc Hgbb
10.0 7.3 7.0 9.0 9.0 8.0 7.0 8.0–13.0
Leucogram
Eryta
TPPf Erythrogram
Hemogram Moment of the analysis Sheep #
Table 2 Haematological values in sheep experimentally poisoned by clomazone in different periods of intoxication.
9.9 7.0 6.3 9.0 10.5 6.0 4.2 8.0–11.0
Eosk
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All spontaneously and experimentally intoxicated sheep had moderate congestion of blood vessels with mild hemorrhages and edema in addition to swelling of the endothelial cells in most regions of the central nervous system. These changes may be related to vascular changes induced directly by the toxin. The vascular changes may also be responsible for the neutrophilic infiltration observed in the liver (Sheep #1) and spleen (Sheep #1 and 5). The histologic lesions of the nervous system observed in Sheep #1 and 2 were more marked than those observed in the experimental cases, which was most likely due to a longer period of exposure to clomazone. Additionally, in the spontaneous outbreak, sheep may have been exposed to the pesticide by both ingestion and the respiratory route. Sheep #1 and 2 had interstitial pneumonia, suggesting that the primary toxic effects of clomazone occur in the respiratory epithelium and indicating that the respiratory system is an important route of ingestion of the product. A similar situation has been reported in paraquat poisoning (Serra et al., 2003). However, the muscarinic action may also be responsible for these signs, as described in cases of poisoning by organophosphates and carbamates in herbivores (Pugh, 1975; Summers et al., 1994; Radostits et al., 2007; Barros and Driemeier, 2007). Hypotrichosis with ulcerative dermatitis was observed in the skin of some sheep during the outbreak, which may have occurred through contact with the drift of clomazone. Thus, skin absorption should be considered as an absorption pathway of the product, similar to that reported in paraquat poisoning (Serra et al., 2003). The fatty degeneration observed in hepatocytes of Sheep #4 was confirmed by ultrastructural evaluation and may have been caused by the direct action of the product in hepatocytes, similar to the lesions described by Crestani et al. (2007) in the liver of fish poisoned by clomazone. The mitochondrial damage in hepatocytes was similar to that observed in both neurons and astrocytes of Sheep #4 and #5. The routes of clomazone poisoning in mammals have not been described; however, the manufacturer mentioned the risk of ingestion by oral, respiratory and cutaneous routes (USEPA, 2007). In the present outbreak, the sheep presented nervous and respiratory signs as well as skin lesions, suggesting that the toxin was ingested by digestive, respiratory and cutaneous routes. The elimination route of clomazone has not been described (USEPA, 2007); however, the acute tubular necrosis in the kidneys observed in Sheep #1 and 5 demonstrates the nephrotoxic action of clomazone and suggests that the kidneys have an important role in the elimination of this herbicide. Reduced hematocrit and red blood cells, similar to that observed in the spontaneously and experimentally poisoned sheep (Table 2), has been reported in humans (Santi et al., 2011) and catfish (Crestani et al., 2006; Crestani et al., 2007; Santi et al., 2011; Pereira, 2012) poisoned by clomazone and are due to injury of the cell membrane caused by increased levels of oxidative stress. Lesions in the erythrocyte cell membrane also explain the hemosiderosis and the presence of neutrophils in the splenic parenchyma observed in Sheep #1 and 5. The elevation of AST activity
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was most likely due to the degenerative necrotic lesions in predominantly centrilobular hepatocytes (Sheep #1), as it was also observed in fish exposed to clomazone. There was significant reduction in lesions after these fish were removed from the contaminated water (Crestani et al., 2007). Clomazone was detected in soil and vegetation samples and also in samples of the liver, kidney and skeletal muscle of affected sheep that died of poisoning. However, Sheep #3, which fully recovered 30 days after administration, also showed a significant amount of clomazone in the liver, kidney and skeletal muscle, suggesting that there is a risk associated with ingestion of contaminated meat and offal from sheep exposed to clomazone. No data could be found on the maximum acceptable clomazone limits in meat destined for human consumption in Brazil. The results of this research indicate the necessity of better supervision of the use of clomazone and other herbicides in Brazil because these herbicides may be public health hazards. Conflict of interest The authors declare no conflict of interest. Acknowledgements To Coordination for the Improvement of Higher Education Personnel-CAPES for the scholarship of the first author and the company Topographia e Planejamento Rural S/S Ltda by collaboration in the epidemiological and toxicological data collection. References Anastassiades, M., Lehotay, S.J., Stajnbaher, D., Schenck, F.J., 2003. Fast and easy multiresidue method employing acetonitrile extraction/partitioning and dispersive solid-phase extraction for the determination of pesticide residues in produce. J. AOAC Int. 86, 412–431. Barros, C.S.L., Driemeier, D., 2007. Intoxicac¸ão por organofosforados e carbamatos. In: Riet-Correa, F., Schild, A.L., Lemos, R.A.A., Borges, J.R.J. (Eds.), Doenc¸as de Ruminantes e Equídeos. , 4th ed. Pallotti Gráfica e Editora, Santa Maria, pp. 80–85. Cattaneo, R., Moraes, B.S., Loro, V.L.P.A., Menezes, C., Sartori, G.M.S., Clasen, B., de Avila, L.A., Marchesan, E., Zanella, R., 2012. Tissue biochemical alterations of Cyprinus carpio exposed to commercial herbicide containing clomazone under rice-field conditions. Arch. Environ. Contam. Toxicol. 62, 97–106. Crestani, M., Menezes, C., Glusczak, L., Miron, D.S., Lazzari, R., Duarte, M.F., Morsch, V.M., Pippi, A.L., Vieira, V.P., 2006. Effects of clomazone herbicide on hematological and some parameters of protein and carbohydrate metabolism of silver catfish Rhamdia quelen. Ecotoxicol. Environ. Saf. 65, 48–55. Crestani, M., Menezes, C., Glusczak, L., Miron, D.S., Spanevell, R., Silveira, A., Gonc¸alves, F.F., Zanella, R., Loro, V.L., 2007. Effect of clomazone herbicide on biochemical and histological aspects of silver catfish (Rhamdia quelen) and recovery pattern. Chemosphere 67, 2305–2311. Dalto, A.G.C., Albornoz, L., Gonzalez, P.C.S., Bitencourt, A.P.G., Gomes, D.C., Pedroso, P.M.O., Bandarra, P.M., 2011. Intoxicac¸ão por organofosforados em bezerros no Uruguai. Acta Sci. Vet. 39, 1–4. Ecco, R., Barros, C.S.L., Grac¸a, D.L., Gava, A., 2006. Closantel toxicosis in kid goats. Vet. Rec. 159, 564–566. Faria, N.M.X., Rosa, J.A.R., Facchini, L.A., 2009. Intoxicac¸ões por agrotóxicos entre trabalhadores rurais de fruticultura. Rev. Saúde Pública 43, 335–344.
Furlan, F.H., Lucioli, J., Borelli, V., Fonteque, J.H., Stolf, L., Traverso, S.D., Gava, A., 2009. Intoxicac¸ão por closantel em ovinos e caprinos no Estado de Santa Catarina. Pesq. Vet. Bras. 29, 89–93. Glusczak, L., Miron, S.D., Silveira, M.B., Rodrigues, S.R., Schetinger, M.R.C., Morsch, V.M., Loro, V.L., 2007. Acute effects of glyphosate herbicide on metabolic and enzymatic parameters of silver catfish (Rhamdia quelen). Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 146, 519–524. Guedes, K.M.R., Riet-Correa, F., Dantas, A.F.M., Simões, S.V.D., Miranda Neto, E.G., Nobre, V.M.T., Medeiros, R.M.T., 2007. Doenc¸as do sistema nervoso central em caprinos e ovinos no semi-árido. Pesq. Vet. Bras. 27, 29–38. Gupta, P.K., 2012. Toxicity of herbicides. In: Gupta, R.C. (Ed.), Veterinary Toxicology Basic and Clinical Principles. , 2nd ed. Academic Press, San Diego, pp. 631–652. Gunasekara, A.S., Dela Cruz, I.D., Curtis, M.J., Claassen, V.P., Tjeerdema, R.S., 2009. The behavior of clomazone in the soil environment. Pest Manag. Sci. 65, 711–716. Kahn, C.M., Line, S., 2005. The Merck Veterinary Manual, 9th ed. Merck & CO., INC, Whitehouse Station. Menezes, C.C., Loro, V.L., Fonseca, M.B., Cattaneo, R.P.A., Miron, D.S., 2011. Oxidative parameters of Rhamdia quelen in response to commercial herbicide containing clomazone and recovery pattern. Pest. Biochem. Physiol. 100, 145–150. Miron, D.S., Silva, L.V.F., Golombieski, J.I., Machado, S.L.O., Marchezan, E., Baldisserotto, B., 2004. Lethal concentration of clomazone, metsulfuron-metil, and quinclorac for silver catfish. Rhamdia quelen fingerlings. Ciên. Rur. 34, 1465–1469. Modesto, K.A., Martinez, C.B.R., 2010. Roundup® causes oxidative stress in liver and inhibits acetylcholinesterase in muscle and brain of the fish Prochilodus lineatus. Chemosphere 78, 294–299. Moraes, B.S., Loro, V.L., Glusczak, L., Pretto, A., Menezes, C., 2007. Effects of four rice herbicides on some metabolic and toxicology parameters of teleost fish (Leporinus obtusidens). Chemosphere 68, 1597–1601. Moraes, B.S., Loro, V.L., Pretto, A., Fonseca, M.B., Menezes, C., Marchesan, E., Reimche, G.B., Avila, L.A., 2009. Toxicological and metabolic parameters of the teleost fish (Leporinus obtusidens) in response to commercial herbicides containing clomazone and propanil. Pestic. Biochem. Physiol. 95, 57–62. Osweiler, G.D., 1998. Inseticidas e Moluscidas. In: Osweiler, G.D. (Ed.), Toxicologia Veterinária. Artes Médicas, Porto Alegre, pp. 259–282. Pereira, L., 2012. Efeitos dos herbicidas clomazone e ametrina em parâmetros funcionais da espécie de peixe neotropical Prochilodus lineatus. Universidade Federal de São Carlos, PhD Thesis. Peters, L.P., 2011. Efeito do herbicidas ametrina e clomazone no sistema antioxidante bacteriano. Universidade Federal de São Carlos, MSc Thesis. Philbey, A.W., Morton, A.G., 2001. Paraquat poisoning in sheep from contaminated water. Aust. Vet. J. 79, 842–843. Prestes, O.D., 2011. Método rápido para determinac¸ão simultânea de resíduos de agrotóxicos e medicamentos veterinários em alimentos de origem animal por LC-MS/MS. Universidade Federal de Santa Maria, PhD Thesis. Pugh, W.S., 1975. An outbreak of organophosphate poisoning (Thimet) in cattle. Can. Vet. J. 16, 56–58. Radostits, O.M., Gay, C.C., Blood, D.C., Hinchcliff, K.W., Constable, P.D., 2007. Veterinary Medicine: A textbook of the diseases of cattle, horses, sheep, pigs and goats, 10th ed. W.B. Saunders, Philadelphia. Santi, A., Menezes, C., Duarte, M.M.F., Leitemperger, J., Lopes, T., Loro, V.L., 2011. Oxidative stress biomarkers and acetylcholinesterase activity in human erythrocytes exposed to clomazone (in vitro). Interdiscip. Toxicol. 4, 149–153. Santos, A., Maia, F., Coutinho, M., Rodrigues, A.L., 2012. Aspetos gerais da intoxicac¸ão por paraquat em animais domésticos. Rev. Lusófona Ciên. Med. Vet. 5, 43–55. Serra, A., Domingos, F., Prata, M., 2003. Intoxicac¸ão Por Paraquat. Acta Méd. Port. 16, 25–32. Souza, M.V., Grac¸a, D.L., Cervo, D., 1996. Neurotoxidade tardia experimentalmente induzida por haloxon em ovinos. Arq. Bras. Med. Vet. Zootec. 48, 275–286. Summers, B.A., Cummings, J.F., De Lahunta, A., 1994. Veterinary Neuropathology. Mosby Year Books, Saint Louis. USEPA-United States Environmental Protection Agency, 2007. Clomazone summary document, registration review environmental, Washington. Zanella, R., Primel, E.G., Machado, S.L.O., Goncalves, F.F., Marchezan, E., 2002. Monitoring of the herbicide clomazone in environmental water samples by solid-phase extraction and high performance liquid chromatography with ultraviolet detection. Chromatography 55, 573–577.
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Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Clinicopathological and toxicological aspects of poisoning by the clomazone herbicide in sheep M.Z. Fagundes a , M.A. Gonc¸alves a , M.P. Soares b , M.L. Martins c , R. Zanella c , F. Riet-Correa d , B.L. Anjos a,∗ a
Veterinary Patolology Laboratory (LPV), Universidade Federal do Pampa, Uruguaiana, 97500-970, Rio Grande do Sul State, Brazil Regional Diagnostic Laboratory, Universidade Federal de Pelotas, Pelotas, 96160-000, Rio Grande do Sul State, Brazil Pesticides Residues Analysis Laboratory (LARP), Universidade Federal de Santa Maria, Santa Maria, 97105-900, Rio Grande do Sul State, Brazil d Veterinary Hospital, CSTR, Universidade Federal de Campina Grande, Patos, 58700-310, Paraíba State, Brazil b c
a r t i c l e
i n f o
Article history: Received 26 April 2014 Received in revised form 5 January 2015 Accepted 10 January 2015 Available online 16 January 2015 Keywords: Diseases of sheep Herbicide poisoning Isoxazolidinone Toxic neuropathy
a b s t r a c t Clomazone (2-[(2-chlorophenyl)methyl]-4,4-dimethyl-3-isoxazolidinone) is a herbicide which has been widely used in southern Brazil in the rice cultivation and can be toxic for humans and animals. This study reports the first outbreak poisoning due to clomazone in a flock of 103 sheep, 20 of which showed mainly neurological and respiratory signs. Clomazone was detected in soil and vegetation samples and in the liver, kidney and muscles of poisoned animals. The poisoning was experimentally reproduced in three sheep by the administration of a 134 mg kg body weight dose of clomazone. In both the natural and experimental cases, the clinical signs included tachypnea, anorexia, somnolence, weakness and ataxia. Macroscopically, there were no significant changes. Histologically, vacuolization in the white matter, perineuronal vacuoles and congestion of the leptomeningeal and brain vessels were observed. Ultrastructurally, the vacuolar lesions in the brain corresponded to swelling of the dendrites and astrocytic processes. It is concluded that clomazone causes toxic neuropathy in sheep. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Nervous system diseases of small ruminants caused mainly by infectious and toxic agents cause significant economic losses in Brazil (Guedes et al., 2007); however, despite the large amounts of pesticides used in this country, the only reported neuropathies caused by insecticides or anthelmintics are related to closantel (Ecco et al., 2006; Furlan et al., 2009) and haloxon poisonings (Souza et al., 1996).
∗ Corresponding author. Tel.: +55 5534023003; fax: +55 5534134321. E-mail address: [email protected] (B.L. Anjos). http://dx.doi.org/10.1016/j.smallrumres.2015.01.006 0921-4488/© 2015 Elsevier B.V. All rights reserved.
Currently, herbicides represent the most widely used class of pesticides in Brazil, accounting for 56% of all pesticides sold in this country (Peters, 2011). The intensive use of herbicides and pesticides has worried regulatory agencies due to their deleterious effects on non-target organisms, such as plants, animals and humans (Peters, 2011). Because of the extensive agricultural activity in the southern region of the country, especially with respect to rice cultivation, herbicides such as glyphosate, paraquat and clomazone are used very intensively (Faria et al., 2009; Peters, 2011). In Brazil, glyphosate poisoning in fishes and humans (Glusczak et al., 2007; Faria et al., 2009; Modesto and Martinez, 2010) and paraquat poisoning in humans, dogs, sheep and cattle (Philbey and Morton, 2001; Serra et al., 2003; Gupta, 2012; Santos et al., 2012) have been reported.
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Clomazone (2-[(2-chlorophenyl)methyl]-4,4-dimethyl-3-isoxazolidinone) is a representative of the isoxazolidinone group of herbicides, formulated in the 1980s for control of annual grasses and broad-leaved weeds in a wide variety of crops, including rice (Gunasekara et al., 2009). In the plant body, clomazone has systemic action and is absorbed by the roots, inhibiting the formation of photosynthetic pigments and inducing death. Clomazone may be applied to crops using ground equipment or by air (USEPA, 2007). Until now, spontaneous clomazone poisoning has been reported only in freshwater fishes (Crestani et al., 2007; Moraes et al., 2007; Moraes et al., 2009; Menezes et al., 2011; Cattaneo et al., 2012), and this poisoning was reproduced experimentally in fish and rat (Miron et al., 2004; USEPA, 2007). It is believed that the toxicity of clomazone is based on the inhibition of acetylcholinesterase and its high capacity to drift enhances the risk of poisoning in animals and plants (Crestani et al., 2007). This paper reports the first outbreak of spontaneous clomazone intoxication in sheep and the experimental reproduction of this poisoning in the same species. 2. Material and methods 2.1. Epidemiology and clinical examination In November of 2011, on a farm in the municipality of Mac¸ambará, Rio Grande do Sul State, Brazil, pronounced dissection of the entire pasture occurred, which was caused by accidental drift of an herbicide applied by air to a neighboring rice field. Epidemiological and toxicological data were collected during a visit to a farm, where some animals of a sheep flock showed clinical signs of accidental exposure to a pesticide drift. Clinical evaluation was performed in a sheep that showed more severe clinical signs and was euthanized for pathological evaluation (Sheep #1). Approximately two weeks after exposure, the flock was transferred to a pesticide-free area. Four weeks post-exposure, a sheep that had clinical signs but was fully recovered was also euthanized and necropsied (Sheep #2). The surviving sheep were observed for 120 days after the accidental exposure to pesticide drift. 2.2. Pathological examination At necropsy, tissue samples of the skin, muscle, liver, rumen, omasum, abomasum, small and large intestines, pancreas, adrenal glands, kidneys, urinary bladder, lungs, lymph nodes, bone, spleen, eye, nerves of the brachial plexus, sciatic nerve, brain and spinal cord (cervical, thoracic and lumbar segments) were collected and fixed in 10% neutral-buffered formalin. The samples were embedded in paraffin, cut in 4 m sections, and stained with hematoxylin and eosin stain (HE). After fixation, 1-cm-thick serial sections were made from the brain, and transverse sections that were taken from the frontal and parietal cortices, basal ganglia, hippocampus, cerebellum, thalamus, rostral colliculus, pons and obex were examined histologically.
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2.3. Experimental design The experimental reproduction of the disease was performed using three 8-month-old sheep of the Ideal breed (#3, 4 and 5), weighing 26–27 kg. Each sheep received an oral dose of clomazone of 134 mg per kg body weight (mg/kg bw) diluted in 100 mL of distilled water. The dose used was adapted from the LD50 of different organophosphates for the rat (Kahn and Line, 2005). The sheep were maintained in individual pens during the experiment and were fed daily with pelleted commercial concentrate feed for sheep at an amount equivalent to 1% of their body weight. Alfalfa (Medicago sativa) and water were provided ad libitum. The sheep were submitted to daily clinical, hematological and biochemical evaluations. Serum activities of aspartate aminotransferase (AST) and ␥-glutamyltransferase (GGT) and total protein serum concentrations were determined. For all sheep, blood samples were obtained before the administration of clomazone (T0 ), 18 h (Sheep #4) and 24 h (Sheep #3 and 5) after administration (T1 ), and two days after the resolution of neurological signs in Sheep #3 (T2 ). The animals were monitored until euthanasia at 30 days after administration (Sheep #3) or death (Sheep #4 and 5). Sheep #3–5 were necropsied, and fragments of various organs were collected for histologic examination (similar to those previously described). Immediately after death, samples of the thalamus, midbrain and liver of Sheep #4 and 5 were collected and fixed in 2% glutaraldehyde and sodium cacodylate-buffered solution. The samples were dehydrated in ethanol and embedded in Epon 812. Semithin sections were stained with methylene blue, and ultrathin sections from the selected areas were stained with uranyl acetate and lead citrate prior to observation using a Zeiss EM 109 transmission electron microscope operated at 80 kilovolts. This work was approved by the Animal Research Ethics Committee of Universidade Federal do Pampa under the protocol number 009/2012. 2.4. Herbicide analysis Liver and kidney samples of Sheep #1–5 and skeletal muscle samples of Sheep #2 and #3–5 were collected and maintained at −20 ◦ C for subsequent multiresidue analysis (100 compounds). After extraction by the modified Quick Easy Cheap Effective Rugged and Safe (QuEChERS) method, the samples were analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Prestes, 2011). Soil and vegetation samples were analyzed by gas chromatography-tandem mass spectrometry (GC-MS/MS) (Anastassiades et al., 2003). 3. Results 3.1. Spontaneous poisoning The disease affected a flock of 103 Texel adult sheep grazing a pasture of 40 hectares composed mainly by native grasses, Lolium multiflorum (ryegrass) and Paspalum notatum on a farm in the municipality of Mac¸ambará, Rio
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Fig. 1. Sheep #1 showing flexion of the right forelimb due to motor weakness.
Grande do Sul State, Brazil. The pasture was neighboring to a rice field, in which an herbicide was applied preemergence by air in November 2011. On the day of herbicide application the conditions for aerial application of the product were inappropriate and the wind caused the dispersion of the product that hit the sheep pasture area. The farmer did not disclose the product or the dose that was applied. A pronounced dissection of the entire pasture occurred as a consequence of the accidental drift of the herbicide. Native grasses and leguminous species, as well as large trees showed large amounts of dry leaves by the herbicide action. During the evaluation in loco of the pasture, all members of the team reported a strong odor of herbicide and burning eyes, lips and nose. Approximately 10 days after exposure to the herbicide, 20 out of the 103 sheep showed tachypnea, anorexia, lethargy, limited mobility and weakness characterized by dragging of the hooves and flexing the hock and fetlock (Fig. 1). Some sheep showed hypotrichosis of the facial skin and crusts in the ears. Upon inspection of the area where the animals grazed, a strong smell of herbicide was noted, even at 10 days after exposure. Four weeks after exposure to the herbicide, all sheep, except one that was euthanized with severe clinical signs, recovered from clinical signs. At necropsy, the lungs of Sheep #1 showed moderate multifocal areas of consolidation and slight fibroelastic consistency, and the liver was slightly yellowish with an evident lobular pattern. Sheep #2 showed no significant gross lesions. Histologically, the lungs of Sheep #1 showed moderate thickening of the interalveolar septum with moderate proliferation of fibroblasts and collagen, mild proliferation of type II pneumocytes, emphysema, edema and moderate bronchial smooth muscle cell hypertrophy (Fig. 2A). Moderate diffuse vacuolation of hepatocytes was observed in the liver, especially in the periportal and midzonal zones. Rare hepatocytes were necrotic, and numerous neutrophils were observed within the blood vessels. The spleen showed moderate hemosiderosis and neutrophilic infiltrate surrounding the white pulp. The kidneys showed moderate diffuse congestion and rare hyaline casts within renal tubules.
Vacuolation of the white matter and severe congestion of blood vessels of the leptomeninges and brain were observed in the cerebrum, cerebellum and brainstem. Furthermore, mild perivascular edema, mild mixed inflammatory cell infiltrate around blood vessels and perineuronal vacuolization of Purkinje cells were observed in the cerebellum (Fig. 2B). Sheep #2 had similar changes in the brain, but to a lesser degree. Soil and vegetation samples contained 0.4 mg kg−1 and 0.9 mg kg−1 clomazone, respectively. In multiresidue analysis, the liver and kidney samples (Sheep #1 and 2) were positive for clomazone herbicide at values less than the minimum limit of quantification of the test (LOQm = 0.01 mg kg−1 ) but greater than the minimum limit of detection (LODm = 0.005 mg kg−1 ) (Table 1). In the skeletal muscle sample (Sheep #2), for example, a chromatographic peak corresponding to 1141.79 area units was observed for clomazone herbicide; this signal unequivocally demonstrated the presence of clomazone in the sample under the LC-MS/MS conditions employed. 3.2. Experimental poisoning Approximately 24 h after clomazone administration, Sheep #3 showed weakness, instability with frequent falls, postural changes, and ataxia with abduction and crossing of the legs when walking. Frequent falls, muscle tremors and moderate dyspnea were also observed. After falling, the animals rested in sternal recumbency for approximately 20 min before returning to a standing position. If the sheep were stimulated to walk, the signs became more accentuated. These signs gradually decreased, and on the fourth day after administration of the herbicide, the sheep were fully recovered. Sheep #4 showed sternal recumbency 12 h after administration and died 6 h later. Approximately 10 h after administration of clomazone, Sheep #5 showed weakness, falling, permanent mydriasis, lateral recumbency and muscle tremors and spasms of the hindlimbs and forelimbs, dying 20 h later. Hematological evaluations (Table 2) showed anemia in Sheep #3 and 5, a slight increase in AST in Sheep #4 and #5, a mild decrease in total plasma proteins in Sheep # 3 and 5, and leukocytosis with neutrophilia in all sheep. At necropsy of Sheep #3-5, no gross findings were observed. Histologic lesions were observed mainly in the central nervous system. In all sheep, the blood vessels of the meninges and neuropil were moderately dilated and congested, mainly in Sheep #4, which died 18 h after clomazone administration. In Sheep #3 and 5, mild hemorrhages in the Virchow-Robin space with slight swelling of endothelial cells and perivascular edema were observed. Vacuolation of the white matter was observed mainly in the brainstem. This lesion was more pronounced in Sheep #5, which died 30 h after the administration of the herbicide. Multifocal areas of congestion and moderate alveolar edema were observed in Sheep #3–5. Diffuse congestion and mild neutrophilic infiltrate were observed in the spleen in the center of the red pulp in Sheep #5. In Sheep #3 and 5, the kidney had moderate congestion in the medullary region and some granular and hyaline casts in the renal tubules. Mild interstitial infiltration by
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Fig. 2. A and B. Sheep #1. A. Lung showing thickening of the septum (arrow), moderate diffuse congestion, and areas of emphysema (*). Hematoxylin and eosin. Bar = 100 m. B. Cerebellum. Multiple perineuronal vacuoles are observed near the Purkinje cells. Hematoxylin and eosin. Bar = 20 m. C. Sheep #5. Kidney showing an area of necrosis with pyknotic nuclei and increased eosinophilia of the cytoplasm of the epithelial cells of the proximal convoluted tubules. A close-up view of the kidney showing necrotic tubular cells within a renal tubule (arrowhead) and some neutrophils in the interstitium (arrow) and in the glomerulus (G). Hematoxylin and eosin. Bar = 20 m.
neutrophils of the cortical region and acute tubular necrosis were also observed in the kidney in Sheep #5 (Fig. 2C). An ultrastructural examination of Sheep #4 and 5 showed marked swelling of astrocyte processes (Fig. 3A) and neuronal dendrites of the thalamus and midbrain (Fig. 3B). In these areas, the axons were also swollen (Fig. 3B and C), with dispersion of axoplasmic microtubules occasionally in the presence of membranes (Fig. 3B). The neuronal perikarya showed dilated cisternae of the smooth endoplasmic reticulum and mitochondria with
lysis of the cristae (Fig. 3A). The axonal mitochondria were swollen, with disruption of mitochondrial membranes and lysis of the cristae (Fig. 3C). In some mitochondria, small, round, electron-dense bodies and membrane debris were found. Hepatocytes contained swollen mitochondria with lysed cristae, lipid droplets of varying sizes, and membrane-associated vacuoles with granular content (Fig. 3D). The results of toxicological evaluation via chemical analysis of Sheep #3–5 are presented in Table 1.
Table 1 Multiresidue analysis of liver, kidney and skeletal muscle samples of the sheep naturally and experimentally poisoned by clomazone. Sheep #
1 2 3 4 5 a
Poisoning form (unit)
a
N (unit of the area)b N (unit of the area) Ec (mg/kg−1 )d E (mg/kg−1 ) E (mg/kg−1 )
Samples of the sheep Liver
Kidney
232.19
143.20
3.74
33.98
Skeletal muscle – 1141.79
400.00
63.7
1.9
255.8
10.3
15.1
88.3
51.5
96.1
Natural poisoning. Value less than the minimum limit of quantification of the test (LOQm = 0.01 mg/kg−1 ) and greater than the minimum limit of detection (LODm = 0.005 mg/kg−1 ). c Experimental poisoning. d Value above the minimum limit of quantification of the test; not evaluated. b
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Fig. 3. Transmission electron microscopy. A. Sheep #5. Brainstem. A neuron (N) with dilated cisternae in the endoplasmic reticulum (*) and tumescent mitochondria (m) with rupture of the cristae is observed. Vacuoles (V) representing very swollen astrocytic processes are observed around the neuron. Astrocyte (A). Bar = 2 m. B. Sheep #5. Brainstem. Swollen axons (*) with disappearance of microtubules and the presence of membranes (arrow) are observed. A swollen dendrite is also observed (D). Neuron (N). Bar = 2 m. C. Sheep #4. Brainstem. A swollen dilated axon (*) with a thin myelin sheath due to dilatation of the axoplasm is observed. Another axon shows a severely swollen mitochondria with an electrodense matrix and rupture of the mitochondrial membrane (white arrow). D. Sheep #4. Liver. Hepatocyte showing a membrane-limited vacuole (V) with granular content. Lipid vacuoles of different sizes (*) and tumescent mitochondria (M) with lysis of the cristae are also observed. Note the remnants of cristae within the mitochondria. Bar = 2 m.
4. Discussion The diagnosis of poisoning by clomazone among sheep in southern Brazil was based on the toxicologic analysis showing the presence of clomazone in soil, vegetation and tissues samples and on the occurrence of the disease in a pasture where the vegetation was desiccated by the accidentally application of the herbicide 10 days before. Otherwise, the clinical signs and lesions observed in the spontaneous affected sheep were similar than those observed in the experimental sheep. According to Gupta (2012), all herbicides require specific conditions for use, and application errors are the main cause of poisoning in humans and animals. In the poisoning reported herein, the exposure of sheep to clomazone at the time of application and for over 10 days thereafter occurred by accidental contact with the drift of large amounts of the product after aerial application. Clomazone residues can persist for up to 130 days in water and have been detected in 90% of water samples collected from rivers in rice growing regions (Zanella et al., 2002). Except for Sheep #4, which died after a clinical manifestation period of 6 h, experimentally and spontaneously
poisoned sheep showed nervous and respiratory signs similar to those observed in the field cases. A study examining fish exposed to water containing clomazone reported a significant reduction in acetylcholinesterase activity 12 h after exposure, with recovery to normal activity after 192 h in clean water (Crestani et al., 2007). These results suggest that clomazone inhibits acetylcholinesterase in brain and muscle tissue (Crestani et al., 2007; Cattaneo et al., 2012). Similar clinical signs have been reported in ruminants poisoned by organophosphates and carbamates, and involvement of the central nervous system was noted (Barros and Driemeier, 2007; Dalto et al., 2011). Clinical signs induced by organophosphates and carbamates are muscarinic, including cardiorespiratory disturbances, and nicotinic, characterized by flaccid paralysis, opisthotonus, tremors and muscular weakness (Osweiler, 1998; Radostits et al., 2007). However, the axonal and dendritic injuries characterized by vacuolation of the white matter and perineuronal vacuolation observed in the sheep poisoned by clomazone have not been reported in cases of organophosphate and carbamate poisoning, suggesting that these effects may be due to the direct action of the toxin on the nervous tissue.
– – 44 96 – 265 – 0–1.000
k
i
j
h
f
g
e
c
d
Erythrocyte (x106 /mm3 ). Hemoglobin(g/dl). Hematocrit (%). Mean corpuscular volume (femtoliters). Mean corpuscular hemoglobin concentration (%). Plasm protein total (g/dl). Leukocytes (/mm3 ). Neutrophils (/mm3 ). Lymphocytes (/mm3 ). Monocytes. Eosinophils (/mm3 ). a
Before administration 24 h after administration 48 h after of disappearance of the clinical signs Before administration 18 h after administration Before administration 24 h after administration 3 3 3 4 4 5 5 Referencevalues
b
Monj
48 231 220 96 414 159 64 0–750 2.016 2.618 2.904 2.688 1.794 3.604 1.792 2.000–9.000
Lymi Neuh
2.736 4.851 1.452 1.920 4.485 1.219 4.160 700–6.000 4.800 7.700 4.400 4.800 6.900 5.300 6.400 4.000–10.000
Leug
6.8 6.0 5.6 6.2 6.8 5.6 5.4 6.0–7.5 33.3 31.1 30.4 33.3 32.1 32.0 33.3 33.3 30.3 31.4 36.5 27.2 26.6 41.6 50.0 23–48 30 22 23 27 28 25 21 27–46
MCHCe MCVd Hctc Hgbb
10.0 7.3 7.0 9.0 9.0 8.0 7.0 8.0–13.0
Leucogram
Eryta
TPPf Erythrogram
Hemogram Moment of the analysis Sheep #
Table 2 Haematological values in sheep experimentally poisoned by clomazone in different periods of intoxication.
9.9 7.0 6.3 9.0 10.5 6.0 4.2 8.0–11.0
Eosk
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All spontaneously and experimentally intoxicated sheep had moderate congestion of blood vessels with mild hemorrhages and edema in addition to swelling of the endothelial cells in most regions of the central nervous system. These changes may be related to vascular changes induced directly by the toxin. The vascular changes may also be responsible for the neutrophilic infiltration observed in the liver (Sheep #1) and spleen (Sheep #1 and 5). The histologic lesions of the nervous system observed in Sheep #1 and 2 were more marked than those observed in the experimental cases, which was most likely due to a longer period of exposure to clomazone. Additionally, in the spontaneous outbreak, sheep may have been exposed to the pesticide by both ingestion and the respiratory route. Sheep #1 and 2 had interstitial pneumonia, suggesting that the primary toxic effects of clomazone occur in the respiratory epithelium and indicating that the respiratory system is an important route of ingestion of the product. A similar situation has been reported in paraquat poisoning (Serra et al., 2003). However, the muscarinic action may also be responsible for these signs, as described in cases of poisoning by organophosphates and carbamates in herbivores (Pugh, 1975; Summers et al., 1994; Radostits et al., 2007; Barros and Driemeier, 2007). Hypotrichosis with ulcerative dermatitis was observed in the skin of some sheep during the outbreak, which may have occurred through contact with the drift of clomazone. Thus, skin absorption should be considered as an absorption pathway of the product, similar to that reported in paraquat poisoning (Serra et al., 2003). The fatty degeneration observed in hepatocytes of Sheep #4 was confirmed by ultrastructural evaluation and may have been caused by the direct action of the product in hepatocytes, similar to the lesions described by Crestani et al. (2007) in the liver of fish poisoned by clomazone. The mitochondrial damage in hepatocytes was similar to that observed in both neurons and astrocytes of Sheep #4 and #5. The routes of clomazone poisoning in mammals have not been described; however, the manufacturer mentioned the risk of ingestion by oral, respiratory and cutaneous routes (USEPA, 2007). In the present outbreak, the sheep presented nervous and respiratory signs as well as skin lesions, suggesting that the toxin was ingested by digestive, respiratory and cutaneous routes. The elimination route of clomazone has not been described (USEPA, 2007); however, the acute tubular necrosis in the kidneys observed in Sheep #1 and 5 demonstrates the nephrotoxic action of clomazone and suggests that the kidneys have an important role in the elimination of this herbicide. Reduced hematocrit and red blood cells, similar to that observed in the spontaneously and experimentally poisoned sheep (Table 2), has been reported in humans (Santi et al., 2011) and catfish (Crestani et al., 2006; Crestani et al., 2007; Santi et al., 2011; Pereira, 2012) poisoned by clomazone and are due to injury of the cell membrane caused by increased levels of oxidative stress. Lesions in the erythrocyte cell membrane also explain the hemosiderosis and the presence of neutrophils in the splenic parenchyma observed in Sheep #1 and 5. The elevation of AST activity
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was most likely due to the degenerative necrotic lesions in predominantly centrilobular hepatocytes (Sheep #1), as it was also observed in fish exposed to clomazone. There was significant reduction in lesions after these fish were removed from the contaminated water (Crestani et al., 2007). Clomazone was detected in soil and vegetation samples and also in samples of the liver, kidney and skeletal muscle of affected sheep that died of poisoning. However, Sheep #3, which fully recovered 30 days after administration, also showed a significant amount of clomazone in the liver, kidney and skeletal muscle, suggesting that there is a risk associated with ingestion of contaminated meat and offal from sheep exposed to clomazone. No data could be found on the maximum acceptable clomazone limits in meat destined for human consumption in Brazil. The results of this research indicate the necessity of better supervision of the use of clomazone and other herbicides in Brazil because these herbicides may be public health hazards. Conflict of interest The authors declare no conflict of interest. Acknowledgements To Coordination for the Improvement of Higher Education Personnel-CAPES for the scholarship of the first author and the company Topographia e Planejamento Rural S/S Ltda by collaboration in the epidemiological and toxicological data collection. References Anastassiades, M., Lehotay, S.J., Stajnbaher, D., Schenck, F.J., 2003. Fast and easy multiresidue method employing acetonitrile extraction/partitioning and dispersive solid-phase extraction for the determination of pesticide residues in produce. J. AOAC Int. 86, 412–431. Barros, C.S.L., Driemeier, D., 2007. Intoxicac¸ão por organofosforados e carbamatos. In: Riet-Correa, F., Schild, A.L., Lemos, R.A.A., Borges, J.R.J. (Eds.), Doenc¸as de Ruminantes e Equídeos. , 4th ed. Pallotti Gráfica e Editora, Santa Maria, pp. 80–85. Cattaneo, R., Moraes, B.S., Loro, V.L.P.A., Menezes, C., Sartori, G.M.S., Clasen, B., de Avila, L.A., Marchesan, E., Zanella, R., 2012. Tissue biochemical alterations of Cyprinus carpio exposed to commercial herbicide containing clomazone under rice-field conditions. Arch. Environ. Contam. Toxicol. 62, 97–106. Crestani, M., Menezes, C., Glusczak, L., Miron, D.S., Lazzari, R., Duarte, M.F., Morsch, V.M., Pippi, A.L., Vieira, V.P., 2006. Effects of clomazone herbicide on hematological and some parameters of protein and carbohydrate metabolism of silver catfish Rhamdia quelen. Ecotoxicol. Environ. Saf. 65, 48–55. Crestani, M., Menezes, C., Glusczak, L., Miron, D.S., Spanevell, R., Silveira, A., Gonc¸alves, F.F., Zanella, R., Loro, V.L., 2007. Effect of clomazone herbicide on biochemical and histological aspects of silver catfish (Rhamdia quelen) and recovery pattern. Chemosphere 67, 2305–2311. Dalto, A.G.C., Albornoz, L., Gonzalez, P.C.S., Bitencourt, A.P.G., Gomes, D.C., Pedroso, P.M.O., Bandarra, P.M., 2011. Intoxicac¸ão por organofosforados em bezerros no Uruguai. Acta Sci. Vet. 39, 1–4. Ecco, R., Barros, C.S.L., Grac¸a, D.L., Gava, A., 2006. Closantel toxicosis in kid goats. Vet. Rec. 159, 564–566. Faria, N.M.X., Rosa, J.A.R., Facchini, L.A., 2009. Intoxicac¸ões por agrotóxicos entre trabalhadores rurais de fruticultura. Rev. Saúde Pública 43, 335–344.
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