Arch Environ Contam Toxicol DOI 10.1007/s00244-015-0157-4
Chronic Arsenic Toxicity in Sheep of Kurdistan Province, Western Iran Behnam Keshavarzi1,2 • Afsaneh Seradj1 • Zahra Akbari3 • Farid Moore1,2 Alireza Rahmani Shahraki4 • Mehrdad Pourjafar4
•
Received: 12 September 2014 / Accepted: 15 April 2015 Ó Springer Science+Business Media New York 2015
Abstract After the detection of arsenic (As) toxicity in sheep from Ebrahim-abad and Babanazar villages in Kurdistan province, the concentration of this element in drinking water, cultivated soil, alfalfa hay, wool, and blood samples was evaluated. Total As concentrations ranged from 119 to 310 lg/L in drinking water, 46.70–819.20 mg/ kg in soil 1.90–6.90 mg/kg in vegetation 1.56–10.79 mg/ kg in sheep’s wool, and 86.30–656 lg/L in blood samples. These very high As contents, in all parts of the biogeochemical cycle, exceed the recommended normal range for this element compared with a control area. Results indicate that As has moved through all compartments of the biogeochemical cycle by way of direct or indirect pathways. The present investigation illustrated decreased packed cell volume and hemoglobin in sheep from the As-contaminated zone. It was concluded that sheep from the contaminated areas suffer from anemia. Chronic As exposure of the liver was determined by liver function tests. For this purpose, blood aspartate transaminase (AST) and alanine transaminase (ALT) were measured. The results show that serum ALT and AST activities are increased significantly (p \ 0.01) in the sheep population exposed to
& Behnam Keshavarzi [email protected] 1
Department of Earth Sciences, College of Sciences, Shiraz University, 71454 Shiraz, Iran
2
Medical Geology Center, Shiraz University, 71454 Shiraz, Iran
3
Nuclear Science and Technology Research Institute, Atomic Energy Organization of Iran, Tehran, Iran
4
Department of Clinical Science, School of Veterinary Medicine, Shiraz University, 71454 Shiraz, Iran
As in the contaminated zone. Moreover, chronic As exposure causes injury to hepatocytes and damages the liver.
Arsenic (As) pollution in the environment has gained importance owing to its widespread toxic effects on humans, animals, birds, aquatic life, and plants through polluted groundwater and food chains (Biswas et al. 2000). At present, people in [35 countries across the globe are affected by drinking As-contaminated groundwater (Das et al. 2012). The health effects of toxic levels of As are multidimensional in both the human and animal populations (Nandi et al. 2006). Epidemiological evidence has shown that long-term chronic As exposure is associated with increased risks of skin, bladder, lung, and liver cancers (Guha Mazumder 2008; Rana et al. 2012), cardiovascular disease (States et al. 2009), diabetes mellitus (Tseng 2004), neuropathies (Ghosh et al. 2007), and ocular disease (Kundu et al. 2011). Livestock are also the likely victims of such catastrophes arising from As pollution (Biswas et al. 2000). Prolonged As ingestion leads to its accumulation in liver, kidneys, heart, and lungs and in smaller amounts in muscle, nervous system, gastrointestinal tract, and spleen because these organs are rich in oxidative enzyme systems (Benramdane et al. 1999). As exerts its toxic effects through an impairment of cellular respiration by inhibiting various mitochondrial enzymes and uncoupling oxidative phosphorylation. As toxicity mostly results from its ability to interact with sulfhydryl groups of proteins and enzymes as well as substitute phosphorus in a variety of biochemical reactions (Vutukuru et al. 2007). Some studies (Ventura-Lima et al. 2011; Das et al. 2012) have pointed out that oxidative damage to hemoglobin has been shown to cause changes in its structure and function resulting in denaturation,
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precipitation, and methemoglobin formation inside erythrocytes. Furthermore, oxidative stress in red blood cells (RBCs) is an indicator of overall oxidative stress and RBCrelated disorders (Rana et al. 2010). Among the various internal organs affected by chronic exposure to As, liver is an important target because in many animals, including mammals, inorganic As is metabolized in the liver (Das et al. 2012). Epidemiological studies have shown that chronic As exposure causes pathological alterations and liver disease including hepatomegaly, hepatoportal sclerosis, liver fibrosis, and cirrhosis (Lu et al. 2001). Once As accumulates in the liver to toxic levels, abnormal liver functions will be manifested by severe gastrointestinal problems and clinical increases of liver enzymes in serum including ALT. AST also is associated with chronic As exposure and is an indicator of hepatotoxicity (Guha Mazumder 2001). Hepatic damage releases ALT and AST into the bloodstream, and the levels of these enzymes have the potential to indicate hepatotoxicity (Escher et al. 1999). Although histopathological studies in liver tissue require more time and expertise, simple and reliable biochemical analysis of ALT and AST may be used for a rapid assessment of tissue damage (Vutukuru et al. 2007). Serum ALT and AST are usually increased in serious hepatic damage, and the extent of organ damage is dependent on the type of toxicant, its mechanism of action, and the duration of exposure (Jacobson-Kram and Keller 2001). Environmental health problems resulting from excess As in drinking water have been found in many countries such as Bangladesh (Ravenscroft et al. 2009), India (Patel et al. 2005), China (Lu and Zhang 2005), Brazil (Figueiredo et al. 2007), Argentina (Smedley et al. 2005), Chile (Ferreccio and Sancha 2006), United States (Welch et al. 2000), and Iran (Mosaferi et al. 2008). Contamination of As in Iran has been observed in several areas such as the Takab area, Zanjan province, where it is associated with gold-mining activity (Zashuran stream) (Modabberi and Moore 2004), Kurdistan province (Barati et al. 2010), the Muteh area (Keshavarzi et al. 2012), and the Kouhsorkh area (Ghassemzadeh et al. 2006). In Iran, the first case of chronic As poisoning was noted in Kurdistan province in 1986 (Mosaferi et al. 2008). It has been shown that some parts of Kurdistan province, western Iran, are at risk of geogenic As pollution because travertine springs are the main source of As in groundwater (Keshavarzi et al. 2011). A large number of livestock in As-affected areas drink/consume As-contaminated water, forage, and agricultural plants. The ingested high levels of As may be retained in blood, urine, feces, hair, and tissues of livestock and so can threaten the health of the animals. Although previous studies have shown high levels of As in the drinking water and soils of Kurdistan province, no studies have previously been performed on As toxic effects in
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animals. In the present study, clinical, hematological, biochemical, and toxicological approaches were used to investigate As toxicity in sheep.
Materials and Methods As-affected villages, namely Ebrahim-abad (35° 460 N, 47° 190 E) and Babanazar (35° 470 N, 47° 200 E) in Bijar town of Kurdistan province (Fig. 1), western Iran, were studied as a consequence of previous reports (Keshavarzi et al. 2011) of high As content in drinking water and symptoms such as skin lesions in the local human population. A control village, namely, Shahrak Bala near Bijar city (an As safe district) was also selected for this purpose. The lithology of the study area includes sedimentary, metamorphic, and igneous rocks (Fig. 1). The oldest strata are Triassic– Jurassic volcano-sedimentary metamorphosed rocks composed of meta-andesites, metagabbros, phyllites, and quaternary sediments. Forty sheep from both the control (n = 10) and the As-affected villages (n = 30) were selected randomly from 4 herds. Environmental samples, including drinking water, agricultural soil, and alfalfa hay used for sheep, were collected from the As-contaminated areas. Blood and wool samples were also collected for determination of As content. All selected animals were examined clinically, and sex, body weight, heart rate (HR), respiratory rate (RR), and mucosal color and presence of abnormalities were recorded. A total of 96 samples were collected comprising 4 water samples, 6 soil samples, 6 plant samples, 40 wool samples, and 40 blood samples. Sampling was performed in June and July 2012. Water samples from springs, tube well water, and irrigation water were collected in acid-washed (0.50 M HCl) 1000-mL polyethylene bottles. EC, pH, Eh, and temperature field parameters of sampled waters were determined on site using portable devices. Water samples were filtered through a 0.45-lm filter paper and then acidified by concentrated HNO3 to pH of 2 in preparation for As analysis by way of inductively coupled plasma-mass spectrometry (ICP-MS) (LabWest Laboratory, Australia). Six composite soil samples were collected from villages in such a way that best represented the land on which the locally consumed alfalfa hay was grown. Soil samples were placed in sealed plastic bags. In the laboratory, the samples were airdried for a week and then were sieved through a \2-mm mesh size polyethylene sieve to remove stones, coarse materials, and other extraneous debris. The sieved samples were ground to a fine powder using an agate Tema grinder. Total As concentration was then determined using ICP-MS in a LabWest Minerals Analysis laboratory (Australia). Plant samples, 500 mg, were collected from alfalfa stems and leaves. The collected samples were washed
Arch Environ Contam Toxicol Fig. 1 Map of the study area showing Babanazar and Ebrahim-abad villages in Bijar town
with tap water, rinsed with deionized water, and air-dried at room temperature. Then 0.50 g of each dry sample was mixed with concentrated nitric acid (HNO3) and hydrogen peroxide (H2O2) and sealed in a digestion vessel. Total As concentrations were then measured using ICP-MS in a LabWest Minerals Analysis Laboratory). Approximately 30 g of wool samples were collected from grazing sheep in the study area. Wool samples were washed according to the method proposed by
Zhuang et al. (1990) which involves sequential washing with acetone, water, and acetone. The samples were dried in an oven at 35 °C and then 0.25 g of each sample was weighed and digested in extra pure nitric acid (HNO3) in a digestion bomb at approximately 150 °C. The resulting solution was diluted to volume with deionized water, and As content was measured using instrumental neutron activation analysis at the Atomic Energy Organization of Iran.
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Blood, 10 ml, was taken from the jugular vein of each animal and split into two fractions. The first fraction was mixed with ethylene diamine tetraacetic acid (EDTA) in a tube for measuring packed cell volume (PCV),and the second fraction was kept in a tube without ETDA and centrifuged for 10 min at 30009g for serum collection. Serum ALT and AST activities were estimated according to the modified international Federation for Clinical Chemistry method in AST/ALT Zist Shimi kits (Zist Shimi Company, Iran) using a UV–Vis spectrophotometer (504 nm). Bioconcentration Factor Bioconcentration is the accumulation of a metal in livestock through dietary and nondietary routes. The bioconcentration factor (BCF) is generally adopted to estimate the propensity of an organism accumulating metals by way of the oral route. It represents the capacity of a species to accumulate a compound to an extent that is greater than the background level. For long-term As exposure, the body burden of As can be expressed as (Rana et al. 2012): BCF =
Concentration of arsenic in the biological sample : Arsenic concentration of samples through oral route
Biotransfer Factor Contamination of As in plants has been shown to be an important pathway of exposure to chemicals in the agricultural food chain model. The biotransfer factor (BTF) model requires an initial quantification of chemical levels of toxic compounds in plants and sheep’s blood. BTF is used to relate either estimated daily exposure dosage or feed levels of chemicals to concentrations occurring in the blood samples. BTF can be calculated according to the method described by Liao et al. (2008). It is assumed that average intake of drinking water and forage by sheep is 2 L/days and 2 kg/days, respectively (Pathak and Bhowmik 1998).
BCF =
Fig. 2 PQF for drinking water samples
significance was set at p \ 0.05, which was used for correlation as shown in Fig. 2.
Results Total As concentrations in drinking water samples are listed in Table 1 with concentrations ranging from 119 to 310 lg/L. A useful approach in evaluating the degree of pollution and potential hazard posed by water is measuring the permissible quality factor (PQF), which is calculated by dividing the maximum concentration of a polluting element by the concentration given by the drinking water guidelines by the United States Environmental Protection Agency (EPA 2009) for livestock (20 lg/L). It can be seen that the calculated PQF for As in all water samples from the study are much greater than recommended values (Fig. 2). Plants and food crops generally adsorb As from the soil. The concentrations of As in the alfalfa and soil samples from the study areas are listed in Table 1. Samples were collected near root plants. It can be seen that the As concentrations are very high in these soil samples ranging from 46.70 to 819.20 mg/kg with an average value of 275.40 mg/kg. In addition, Table 1 compares the concentration of As in the study area with the maximum concentration level (MCL) recommended by the European
Concentration of arsenic in biological samples ðmg=kgÞ : Daily animal intake of arsenic (mg/day) through contaminated drinking water
Statistical Analysis Statistical analysis was performed using SPSS software (version 16; Chicago, IL). One-sample t test was applied to examine the significance of the difference between the data of control and the As-affected villages. The level of
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Union. As concentrations in soil samples from the study area were found to be substantially greater than the recommended MCL of 20 mg/kg indicating that As is highly enriched in these cultivated soil samples. Alfalfa hay is the main forage in the study area. For this reason, alfalfa samples were collected and analyzed. The
Arch Environ Contam Toxicol Table 1 Total As concentration in drinking water, cultivated soil, and alfalfa hay samples (n = number of samples)
Samples
n
Maximum
Minimum
Mean
SD
Control village
Guideline
Drinking water (lg/L)
5
310
119
193
143.50
26.20
20a
Cultivated soil (mg/kg)
7
819.20
46.70
275.40
316.30
7.74
20b
Alfalfa hay (mg/kg)
7
6.90
1.90
5.02
2.20
0.04
0.50c
a
EPA standard in drinking water for livestock (2009)
b
Kabata-Pendias and Pendias (2001)
c
FAO (1998)
results (Table 1) indicate that the total As content of the alfalfa samples varies between 1.90 and 6.90 mg/kg with an average value of 5.02 mg/kg. According to the Food and Agricultural Organization (FAO) (1998), the normal As level in alfalfa is 0.50 mg/kg. As shown in Table 1, As concentration in all collected samples in the study area is considerably greater than the concentrations given by the FAO (1998). In alfalfa plants, As levels are closely correlated with those in the cultivated soil samples reflecting the bioaccumulating nature of alfalfa for As. Because alfalfa is used as the main forage in the study area, it is very likely that As is transferred up the food chain to sheep and then to humans. In addition, in this study the concentration of As is compared with that in the control village (Table 1). The average concentrations of As in drinking water, cultivated soils, and alfalfa samples in the contaminated area are 193 lg/L, 275.40, and 5.02 mg/kg, respectively, whereas in the control village they are 26.20 lg/L, 7.74, and 0.04 mg/kg. The results show that As content in all samples increases from the control village to the contaminated area. As in Sheep Clinical Signs Measurements from As-exposed and control sheep are listed in Table 2. There was a total of 26 female and 13 male sheep with a mean age of 2.8 and 2.1 years, respectively. The average value of body weight, body temperature, HR, and RR in As-exposed sheep were 30.70 kg, 39.22 °C, 77.60/min, and 37.46/min, respectively (Table 2). For the control area, they were 48.30 kg, 38.90 °C, 80.70/min, and 37.77/min, respectively.
Discussion Compared with the control area, a significant decrease (p \ 0.01) in the mean weight of sheep was observed in the contaminated area. These results are consistent with the known damage that As causes to alimentary tract mucosa,
damage to capillaries, and increased permeability and exudation of serum into tissue space; the resulting loss of large quantities of body fluids and proteins then leads to diarrhea, dehydration, and loss of body weight (Radostits et al. 2007). It was also found that the mean body temperature of the sheep in the As-exposed area was significantly increased (p \ 0.05). Reduction of the efficiency of the immune system due to decreased number of white blood cells can cause fever in sheep in infected areas (Radostits et al. 2007). The usual clinical signs of chronic arsenicosis—including severe weakness (53 %), diarrhea (23 %), local hair loss (37 %), congestive mucous membranes (18 %), emaciation and several ulcers in different parts of skin (33 %), languor, focal skin lesion, congested mucous membranes, and stomatitis—were all seen in the As-exposed sheep. In addition, the physical appearance of wool and hooves did not appear to be normal. Adult sheep, especially those C2 years old showed clinical signs of chronic arsenicosis. Severe hyperkeratosis and pustule formation were seen in the skin (Fig. 3). None of the control animals developed any of these signs of toxicity. As concentrations were measured in wool and blood samples from the sheep population, and the results are listed in Table 2 along with natural As concentrations in healthy tissues. The results indicate that As concentrations in wool samples from the contaminated area ranges from 1.56 to 10.79 mg/kg with an average of 4.68 mg/kg, whereas total As concentrations in wool samples from the control area range from 0.03 to 0.13 mg/kg with an average of 0.07 mg/kg. The normal concentration of As in wool is 0.50 mg/kg (Roy et al. 2009). Statistical comparison of the As contents of wool samples from the Asexposed sheep shows significantly greater levels than the control ones (p \ 0.01). As wool content is a marker of long-term exposure to inorganic As (Marchiset-Ferlay et al. 2012). High concentrations of As in wool samples are interpreted to be due to consumption of As-contaminated drinking water and forage. Radostits et al. (2007) showed that As levels in livestock organs vary with age. Figure 4 indicates a positive correlation between As levels in wool with the age of As-exposed sheep (both male and female). It is therefore concluded that As has bioaccumulated with time.
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Arch Environ Contam Toxicol Table 2 Different parameters of As-exposed and unexposed sheep in the study area
Parameter
Minimum
Maximum
Mean
Arsenic-exposed population Weight (kg)
15
55
30.70
–
Temperature (°C)
38.60
40
39.22
38.40–39.20
Heart rate (per min)
58
95
77.60
70–90
Respiratory rate (per min)
25
47
37.46
12–72
Wool (mg kg-1)
1.56
10.79
4.68
0.50
Blood (lg L-1)
86.30
656
416.90
\70
24.87
27–45
8.29
9–15
PCV (g/dl)
17
32
Hgb (L/l)
5.67
10.67
AST (U/L)
163
201
180.90
49–123
ALT (U/L)
100
131
116.40
15–44
Control population
a
Fig. 3 Skin lesions in Asaffected sheep
Fig. 4 Correlation between wool As concentration with age (male and female sheep) in Asexposed areas
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Normal range in healthy sheepa
Weight (kg)
30
63
48.30
Temperature (°C)
38.70
39.30
38.90
Heart rate (per min) Respiratory rate (per min)
68 31
92 45
80.70 37.77
Wool (mg kg-1)
0.03
0.13
0.07
Blood (lg L-1)
42.60
58.40
50.70
PCV (g/dl)
25
38
33.44
Hgb (L/l)
8.33
12.67
11.15
AST (U/L)
54
68
60.78
ALT (U/L)
27
43
32.56
Roy et al. 2009, Dobbs 2009
Arch Environ Contam Toxicol
There is a consistent association between the mean As concentrations in wool samples in males and females (p \ 0.01) indicating a greater mean wool As concentration in the male population. This may be due to excess excretion of As from the milk in females (Biswas et al. 2000). Analysis of As in blood samples is best suited for highdose exposures (Marchiset-Ferlay et al. 2012). Quantification of As in the blood can be useful, but it depends on the kind of epidemiological study performed (Mandal et al. 2004). It could also be used to show chronic exposure to As (Hall et al. 2006). Table 2 lists the blood As concentrations in As-exposed and control animals. The total concentrations of As in the blood samples from the contaminated area vary between 86.30 and 656 ppb with a mean value of 416.90 ppb. These compare with values for the control area of 42.60, 58.40, and 50.70 ppb, respectively. Statistical comparisons shows significantly greater As content in blood samples from the contaminated area (p \ 0.01) compared with the control area. Moreover, there is a significant relationship between As levels in drinking water and blood. The results show that in all cases, the calculated BCF values in wool samples in all cases is greater compared with measured values in blood when As is consumed through drinking water (Fig. 5). The results also show that BTF in sheep wool samples is greater compared with blood when As is ingested through drinking water and forage (Fig. 5). It is therefore concluded that increased levels of As in drinking water leads to increased deposition of As in both wool and blood samples. The mean hematological indices, i.e., Hb and PCV, are significantly decreased (p \ 0.01) in sheep from the Asaffected zone (8.29 L/l and 24.87 g/dl, respectively) compared with the control area (Table 2; Fig. 6). Obviously, the animals from the As-contaminated zone suffer from hematological abnormality, as manifested by anemia, similar to the findings of Rana et al. (2008). As can interfere with metabolism and suppression of the granulopoietic activity of bone marrow by residual
toxicants. As may also cause change in structural integrity of cell membrane, decrease osmotic resistance and fragility of erythrocyte, and cause severe hematological disorders (Rana et al. 2010). The liver is the major site of As metabolism and hence exposure to As causes liver damage (Guha Mazumder et al. 2005). Research results shown that ALT and AST may be used as an indicator of liver damage (Vutukuru et al. 2007). In the present investigation, serum ALT in As-exposed animals varies between 100 to 131 U/L with a mean value of 116.40 U/L (Table 2), whereas the equivalent values for the control group are 27, 43, and 32.56 U/L, respectively. In addition, serum AST levels in the contaminated group range from 163 to 201 U/L with a mean of 180.90 U/L, which compare with the respective control group values of 54, 68, and 60.78 U/L. These results therefore show that serum AST and ALT were increased in the As-exposed animals compared with the unexposed group (Fig. 6). This increase clearly indicates that chronic As exposure causes injury to hepatocytes and damages the liver’s capacity for transporting organic anions, metabolizing drugs, cholestasis, and liver biosynthetic capacity. Our results are consistent with many findings of chronic exposure to As associated with hepatomelagy, hepatoportal sclerosis, liver fibrosis, and cirrhosis with concomitant increase in ALT and AST (Liu et al. 2002; Zhou et al. 2002). It is evident that nutritional status plays an important role in the regulation of As methylation and As-related health hazards (Milton et al. 2004). As contamination in sheep comes from different sources, such as drinking water, crops, and vegetables grown in As-affected areas (Dash et al. 2013). In the Datong basin, northern China, the use of As-contaminated groundwater for drinking and irrigation purposes has resulted in endemic As poisoning among tens of thousands of livestock and people (Guo et al. 2003). Similar cases have been reported in Argentina, Bangladesh, Canada, Hungary, India, Mexico, Poland, and the United States (Nickson et al. 1998, 2000, Jain and Ali 2000; Smith et al. 2001; Berg et al. 2001; Smedley et al. 2001; Smedley
Fig. 5 BCF and BTF in blood and wool samples of sheep that consumed As-contaminated water and alfalfa plants
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Fig. 6 As concentration in blood samples, serum PCV, serum AST, and serum ALT of sheep in the contaminated area compared with the control area
and Kinniburgh 2002; Polya et al. 2005). In this study, As concentrations in wool and blood samples also positively correlated with As levels in drinking water, cultivated soils, and alfalfa hay. A high concentration of total As in water (Table 1), which is much greater than the permissible limit, and the level of As in alfalfa hay (Table 1), which is also much greater than the allowable concentration, is interpreted to be the source of As contamination in the study area. Keshavarzi et al. (2011) indicated that the main source of As in the groundwater of Kurdistan province is travertine-forming springs.
Conclusion From this study it can be concluded that As content in drinking water, cultivated soils, alfalfa hay, and biological samples, including wool and blood samples of the sheep in
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the two endemic villages of Kurdistan province, is alarming. The most probable sources of high As concentrations in biological samples are drinking water and forage given to the animals. Approximately 78 % of the studied animals show typical symptoms of arsenicosis including diffuse or spotted melanosis or hyperkeatosis on the skin, feet, and chin. The results suggest that long-term exposure of sheep to inorganic As at the observed high levels produces severe clinical signs of toxicity and toxicopathological changes that will eventually lead to mortality. The cytotoxic effects of As are proportional to the quantitative accumulation of its residues in the organs. Our results indicate that serum levels of ALT and AST were increased in the animals exposed to As, whereas hematological indices (Hb and PCV) are significantly decreased in sheep from the contaminated area. Therefore, this study reveals that sheep in As-contaminated areas suffer from liver damage and anemia. Furthermore, high concentrations of As in wool may
Arch Environ Contam Toxicol
be considered as a biomarker of arsenicosis in livestock. Sheep may also ingest a large amount of As through daily consumption of alfalfa plants as well as the water they drink. In conclusion, the data suggest that human beings are being exposed to hazardous levels of As if they consume contaminated meat and foodstuffs grown on agricultural land. Acknowledgments The authors express their gratitude to the Medical Geology Research Center of Shiraz University for financial and logistic support. Thanks are also extended to Atomic Energy Organization of Iran for providing analytical measurements. The authors also express their gratitude to D. J. Moore for constructive comments and English correction.
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Smedley PL, Zhang M, Zhang G, Luo Z (2001) Arsenic and other redox-sensitive elements in groundwater from the Huhhot Basin, Inner Mongolia. In: Cidu R (ed) Water-rock interaction, vol 1. Swets & Zeitlinger, Lisse, Holland, pp 581–584 Smedley PL, Kinniburgh DG, Macdonald DM, Nicolli HB, Barros AJ, Tullio JO et al (2005) Arsenic associations in sediments from the loess aquifer of La Pampa, Argentina. Appl Geochem 20:989–1016 Smith AH, Lingas EO, Rahman M (2001) Contamination of drinking water by arsenic in Bangladesh: a public health emergency. Bull World Health Organ 78:1093–1103 States JC, Srivastava S, Chen Y, Barchowsky A (2009) Arsenic and cardiovascular disease. Toxicol Sci 107(Suppl 2):312–323 Tseng CH (2004) The potential biological mechanisms of arsenic induced diabetes mellitus. Toxicol Appl Pharmacol 197:67–83 Ventura-Lima J, Bogo MM, Monserrat J (2011) Arsenic toxicity in mammals and aquatic animals: a comparative biochemical approach. Ecotoxicol Environ Saf 74:211–218 Vutukuru SS, Prabhath NA, Raghavender M, Yerramilli A (2007) Effects of arsenic and Chromium on the serum amino-transferases activity in Indian major carp, Labeo rohita. Int J Environ Res Public Health 4(3):224–227 Welch AH, Westjohn DB, Helsel DR, Wanty RB (2000) Arsenic in groundwater of the United States: occurrence and geochemistry. Ground Water 38:589–604 Zhou YS, Du H, Cheng ML, Liu J, Zhang XJ, Xu L (2002) The investigation of death from diseases caused by coal-burning type of arsenic poisoning. Chin J Endemiol 21:484–486 Zhuang GS, Wang YS, Tan MG, Zhi M, Pan WQ, Cheng YD (1990) Preliminary study of the distribution of toxic elements As, Cd and Hg in human hair and tissues by RNAA. Biol Trace Elem Res 26:729–736
Chronic Arsenic Toxicity in Sheep of Kurdistan Province, Western Iran Behnam Keshavarzi1,2 • Afsaneh Seradj1 • Zahra Akbari3 • Farid Moore1,2 Alireza Rahmani Shahraki4 • Mehrdad Pourjafar4
•
Received: 12 September 2014 / Accepted: 15 April 2015 Ó Springer Science+Business Media New York 2015
Abstract After the detection of arsenic (As) toxicity in sheep from Ebrahim-abad and Babanazar villages in Kurdistan province, the concentration of this element in drinking water, cultivated soil, alfalfa hay, wool, and blood samples was evaluated. Total As concentrations ranged from 119 to 310 lg/L in drinking water, 46.70–819.20 mg/ kg in soil 1.90–6.90 mg/kg in vegetation 1.56–10.79 mg/ kg in sheep’s wool, and 86.30–656 lg/L in blood samples. These very high As contents, in all parts of the biogeochemical cycle, exceed the recommended normal range for this element compared with a control area. Results indicate that As has moved through all compartments of the biogeochemical cycle by way of direct or indirect pathways. The present investigation illustrated decreased packed cell volume and hemoglobin in sheep from the As-contaminated zone. It was concluded that sheep from the contaminated areas suffer from anemia. Chronic As exposure of the liver was determined by liver function tests. For this purpose, blood aspartate transaminase (AST) and alanine transaminase (ALT) were measured. The results show that serum ALT and AST activities are increased significantly (p \ 0.01) in the sheep population exposed to
& Behnam Keshavarzi [email protected] 1
Department of Earth Sciences, College of Sciences, Shiraz University, 71454 Shiraz, Iran
2
Medical Geology Center, Shiraz University, 71454 Shiraz, Iran
3
Nuclear Science and Technology Research Institute, Atomic Energy Organization of Iran, Tehran, Iran
4
Department of Clinical Science, School of Veterinary Medicine, Shiraz University, 71454 Shiraz, Iran
As in the contaminated zone. Moreover, chronic As exposure causes injury to hepatocytes and damages the liver.
Arsenic (As) pollution in the environment has gained importance owing to its widespread toxic effects on humans, animals, birds, aquatic life, and plants through polluted groundwater and food chains (Biswas et al. 2000). At present, people in [35 countries across the globe are affected by drinking As-contaminated groundwater (Das et al. 2012). The health effects of toxic levels of As are multidimensional in both the human and animal populations (Nandi et al. 2006). Epidemiological evidence has shown that long-term chronic As exposure is associated with increased risks of skin, bladder, lung, and liver cancers (Guha Mazumder 2008; Rana et al. 2012), cardiovascular disease (States et al. 2009), diabetes mellitus (Tseng 2004), neuropathies (Ghosh et al. 2007), and ocular disease (Kundu et al. 2011). Livestock are also the likely victims of such catastrophes arising from As pollution (Biswas et al. 2000). Prolonged As ingestion leads to its accumulation in liver, kidneys, heart, and lungs and in smaller amounts in muscle, nervous system, gastrointestinal tract, and spleen because these organs are rich in oxidative enzyme systems (Benramdane et al. 1999). As exerts its toxic effects through an impairment of cellular respiration by inhibiting various mitochondrial enzymes and uncoupling oxidative phosphorylation. As toxicity mostly results from its ability to interact with sulfhydryl groups of proteins and enzymes as well as substitute phosphorus in a variety of biochemical reactions (Vutukuru et al. 2007). Some studies (Ventura-Lima et al. 2011; Das et al. 2012) have pointed out that oxidative damage to hemoglobin has been shown to cause changes in its structure and function resulting in denaturation,
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precipitation, and methemoglobin formation inside erythrocytes. Furthermore, oxidative stress in red blood cells (RBCs) is an indicator of overall oxidative stress and RBCrelated disorders (Rana et al. 2010). Among the various internal organs affected by chronic exposure to As, liver is an important target because in many animals, including mammals, inorganic As is metabolized in the liver (Das et al. 2012). Epidemiological studies have shown that chronic As exposure causes pathological alterations and liver disease including hepatomegaly, hepatoportal sclerosis, liver fibrosis, and cirrhosis (Lu et al. 2001). Once As accumulates in the liver to toxic levels, abnormal liver functions will be manifested by severe gastrointestinal problems and clinical increases of liver enzymes in serum including ALT. AST also is associated with chronic As exposure and is an indicator of hepatotoxicity (Guha Mazumder 2001). Hepatic damage releases ALT and AST into the bloodstream, and the levels of these enzymes have the potential to indicate hepatotoxicity (Escher et al. 1999). Although histopathological studies in liver tissue require more time and expertise, simple and reliable biochemical analysis of ALT and AST may be used for a rapid assessment of tissue damage (Vutukuru et al. 2007). Serum ALT and AST are usually increased in serious hepatic damage, and the extent of organ damage is dependent on the type of toxicant, its mechanism of action, and the duration of exposure (Jacobson-Kram and Keller 2001). Environmental health problems resulting from excess As in drinking water have been found in many countries such as Bangladesh (Ravenscroft et al. 2009), India (Patel et al. 2005), China (Lu and Zhang 2005), Brazil (Figueiredo et al. 2007), Argentina (Smedley et al. 2005), Chile (Ferreccio and Sancha 2006), United States (Welch et al. 2000), and Iran (Mosaferi et al. 2008). Contamination of As in Iran has been observed in several areas such as the Takab area, Zanjan province, where it is associated with gold-mining activity (Zashuran stream) (Modabberi and Moore 2004), Kurdistan province (Barati et al. 2010), the Muteh area (Keshavarzi et al. 2012), and the Kouhsorkh area (Ghassemzadeh et al. 2006). In Iran, the first case of chronic As poisoning was noted in Kurdistan province in 1986 (Mosaferi et al. 2008). It has been shown that some parts of Kurdistan province, western Iran, are at risk of geogenic As pollution because travertine springs are the main source of As in groundwater (Keshavarzi et al. 2011). A large number of livestock in As-affected areas drink/consume As-contaminated water, forage, and agricultural plants. The ingested high levels of As may be retained in blood, urine, feces, hair, and tissues of livestock and so can threaten the health of the animals. Although previous studies have shown high levels of As in the drinking water and soils of Kurdistan province, no studies have previously been performed on As toxic effects in
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animals. In the present study, clinical, hematological, biochemical, and toxicological approaches were used to investigate As toxicity in sheep.
Materials and Methods As-affected villages, namely Ebrahim-abad (35° 460 N, 47° 190 E) and Babanazar (35° 470 N, 47° 200 E) in Bijar town of Kurdistan province (Fig. 1), western Iran, were studied as a consequence of previous reports (Keshavarzi et al. 2011) of high As content in drinking water and symptoms such as skin lesions in the local human population. A control village, namely, Shahrak Bala near Bijar city (an As safe district) was also selected for this purpose. The lithology of the study area includes sedimentary, metamorphic, and igneous rocks (Fig. 1). The oldest strata are Triassic– Jurassic volcano-sedimentary metamorphosed rocks composed of meta-andesites, metagabbros, phyllites, and quaternary sediments. Forty sheep from both the control (n = 10) and the As-affected villages (n = 30) were selected randomly from 4 herds. Environmental samples, including drinking water, agricultural soil, and alfalfa hay used for sheep, were collected from the As-contaminated areas. Blood and wool samples were also collected for determination of As content. All selected animals were examined clinically, and sex, body weight, heart rate (HR), respiratory rate (RR), and mucosal color and presence of abnormalities were recorded. A total of 96 samples were collected comprising 4 water samples, 6 soil samples, 6 plant samples, 40 wool samples, and 40 blood samples. Sampling was performed in June and July 2012. Water samples from springs, tube well water, and irrigation water were collected in acid-washed (0.50 M HCl) 1000-mL polyethylene bottles. EC, pH, Eh, and temperature field parameters of sampled waters were determined on site using portable devices. Water samples were filtered through a 0.45-lm filter paper and then acidified by concentrated HNO3 to pH of 2 in preparation for As analysis by way of inductively coupled plasma-mass spectrometry (ICP-MS) (LabWest Laboratory, Australia). Six composite soil samples were collected from villages in such a way that best represented the land on which the locally consumed alfalfa hay was grown. Soil samples were placed in sealed plastic bags. In the laboratory, the samples were airdried for a week and then were sieved through a \2-mm mesh size polyethylene sieve to remove stones, coarse materials, and other extraneous debris. The sieved samples were ground to a fine powder using an agate Tema grinder. Total As concentration was then determined using ICP-MS in a LabWest Minerals Analysis laboratory (Australia). Plant samples, 500 mg, were collected from alfalfa stems and leaves. The collected samples were washed
Arch Environ Contam Toxicol Fig. 1 Map of the study area showing Babanazar and Ebrahim-abad villages in Bijar town
with tap water, rinsed with deionized water, and air-dried at room temperature. Then 0.50 g of each dry sample was mixed with concentrated nitric acid (HNO3) and hydrogen peroxide (H2O2) and sealed in a digestion vessel. Total As concentrations were then measured using ICP-MS in a LabWest Minerals Analysis Laboratory). Approximately 30 g of wool samples were collected from grazing sheep in the study area. Wool samples were washed according to the method proposed by
Zhuang et al. (1990) which involves sequential washing with acetone, water, and acetone. The samples were dried in an oven at 35 °C and then 0.25 g of each sample was weighed and digested in extra pure nitric acid (HNO3) in a digestion bomb at approximately 150 °C. The resulting solution was diluted to volume with deionized water, and As content was measured using instrumental neutron activation analysis at the Atomic Energy Organization of Iran.
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Blood, 10 ml, was taken from the jugular vein of each animal and split into two fractions. The first fraction was mixed with ethylene diamine tetraacetic acid (EDTA) in a tube for measuring packed cell volume (PCV),and the second fraction was kept in a tube without ETDA and centrifuged for 10 min at 30009g for serum collection. Serum ALT and AST activities were estimated according to the modified international Federation for Clinical Chemistry method in AST/ALT Zist Shimi kits (Zist Shimi Company, Iran) using a UV–Vis spectrophotometer (504 nm). Bioconcentration Factor Bioconcentration is the accumulation of a metal in livestock through dietary and nondietary routes. The bioconcentration factor (BCF) is generally adopted to estimate the propensity of an organism accumulating metals by way of the oral route. It represents the capacity of a species to accumulate a compound to an extent that is greater than the background level. For long-term As exposure, the body burden of As can be expressed as (Rana et al. 2012): BCF =
Concentration of arsenic in the biological sample : Arsenic concentration of samples through oral route
Biotransfer Factor Contamination of As in plants has been shown to be an important pathway of exposure to chemicals in the agricultural food chain model. The biotransfer factor (BTF) model requires an initial quantification of chemical levels of toxic compounds in plants and sheep’s blood. BTF is used to relate either estimated daily exposure dosage or feed levels of chemicals to concentrations occurring in the blood samples. BTF can be calculated according to the method described by Liao et al. (2008). It is assumed that average intake of drinking water and forage by sheep is 2 L/days and 2 kg/days, respectively (Pathak and Bhowmik 1998).
BCF =
Fig. 2 PQF for drinking water samples
significance was set at p \ 0.05, which was used for correlation as shown in Fig. 2.
Results Total As concentrations in drinking water samples are listed in Table 1 with concentrations ranging from 119 to 310 lg/L. A useful approach in evaluating the degree of pollution and potential hazard posed by water is measuring the permissible quality factor (PQF), which is calculated by dividing the maximum concentration of a polluting element by the concentration given by the drinking water guidelines by the United States Environmental Protection Agency (EPA 2009) for livestock (20 lg/L). It can be seen that the calculated PQF for As in all water samples from the study are much greater than recommended values (Fig. 2). Plants and food crops generally adsorb As from the soil. The concentrations of As in the alfalfa and soil samples from the study areas are listed in Table 1. Samples were collected near root plants. It can be seen that the As concentrations are very high in these soil samples ranging from 46.70 to 819.20 mg/kg with an average value of 275.40 mg/kg. In addition, Table 1 compares the concentration of As in the study area with the maximum concentration level (MCL) recommended by the European
Concentration of arsenic in biological samples ðmg=kgÞ : Daily animal intake of arsenic (mg/day) through contaminated drinking water
Statistical Analysis Statistical analysis was performed using SPSS software (version 16; Chicago, IL). One-sample t test was applied to examine the significance of the difference between the data of control and the As-affected villages. The level of
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Union. As concentrations in soil samples from the study area were found to be substantially greater than the recommended MCL of 20 mg/kg indicating that As is highly enriched in these cultivated soil samples. Alfalfa hay is the main forage in the study area. For this reason, alfalfa samples were collected and analyzed. The
Arch Environ Contam Toxicol Table 1 Total As concentration in drinking water, cultivated soil, and alfalfa hay samples (n = number of samples)
Samples
n
Maximum
Minimum
Mean
SD
Control village
Guideline
Drinking water (lg/L)
5
310
119
193
143.50
26.20
20a
Cultivated soil (mg/kg)
7
819.20
46.70
275.40
316.30
7.74
20b
Alfalfa hay (mg/kg)
7
6.90
1.90
5.02
2.20
0.04
0.50c
a
EPA standard in drinking water for livestock (2009)
b
Kabata-Pendias and Pendias (2001)
c
FAO (1998)
results (Table 1) indicate that the total As content of the alfalfa samples varies between 1.90 and 6.90 mg/kg with an average value of 5.02 mg/kg. According to the Food and Agricultural Organization (FAO) (1998), the normal As level in alfalfa is 0.50 mg/kg. As shown in Table 1, As concentration in all collected samples in the study area is considerably greater than the concentrations given by the FAO (1998). In alfalfa plants, As levels are closely correlated with those in the cultivated soil samples reflecting the bioaccumulating nature of alfalfa for As. Because alfalfa is used as the main forage in the study area, it is very likely that As is transferred up the food chain to sheep and then to humans. In addition, in this study the concentration of As is compared with that in the control village (Table 1). The average concentrations of As in drinking water, cultivated soils, and alfalfa samples in the contaminated area are 193 lg/L, 275.40, and 5.02 mg/kg, respectively, whereas in the control village they are 26.20 lg/L, 7.74, and 0.04 mg/kg. The results show that As content in all samples increases from the control village to the contaminated area. As in Sheep Clinical Signs Measurements from As-exposed and control sheep are listed in Table 2. There was a total of 26 female and 13 male sheep with a mean age of 2.8 and 2.1 years, respectively. The average value of body weight, body temperature, HR, and RR in As-exposed sheep were 30.70 kg, 39.22 °C, 77.60/min, and 37.46/min, respectively (Table 2). For the control area, they were 48.30 kg, 38.90 °C, 80.70/min, and 37.77/min, respectively.
Discussion Compared with the control area, a significant decrease (p \ 0.01) in the mean weight of sheep was observed in the contaminated area. These results are consistent with the known damage that As causes to alimentary tract mucosa,
damage to capillaries, and increased permeability and exudation of serum into tissue space; the resulting loss of large quantities of body fluids and proteins then leads to diarrhea, dehydration, and loss of body weight (Radostits et al. 2007). It was also found that the mean body temperature of the sheep in the As-exposed area was significantly increased (p \ 0.05). Reduction of the efficiency of the immune system due to decreased number of white blood cells can cause fever in sheep in infected areas (Radostits et al. 2007). The usual clinical signs of chronic arsenicosis—including severe weakness (53 %), diarrhea (23 %), local hair loss (37 %), congestive mucous membranes (18 %), emaciation and several ulcers in different parts of skin (33 %), languor, focal skin lesion, congested mucous membranes, and stomatitis—were all seen in the As-exposed sheep. In addition, the physical appearance of wool and hooves did not appear to be normal. Adult sheep, especially those C2 years old showed clinical signs of chronic arsenicosis. Severe hyperkeratosis and pustule formation were seen in the skin (Fig. 3). None of the control animals developed any of these signs of toxicity. As concentrations were measured in wool and blood samples from the sheep population, and the results are listed in Table 2 along with natural As concentrations in healthy tissues. The results indicate that As concentrations in wool samples from the contaminated area ranges from 1.56 to 10.79 mg/kg with an average of 4.68 mg/kg, whereas total As concentrations in wool samples from the control area range from 0.03 to 0.13 mg/kg with an average of 0.07 mg/kg. The normal concentration of As in wool is 0.50 mg/kg (Roy et al. 2009). Statistical comparison of the As contents of wool samples from the Asexposed sheep shows significantly greater levels than the control ones (p \ 0.01). As wool content is a marker of long-term exposure to inorganic As (Marchiset-Ferlay et al. 2012). High concentrations of As in wool samples are interpreted to be due to consumption of As-contaminated drinking water and forage. Radostits et al. (2007) showed that As levels in livestock organs vary with age. Figure 4 indicates a positive correlation between As levels in wool with the age of As-exposed sheep (both male and female). It is therefore concluded that As has bioaccumulated with time.
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Arch Environ Contam Toxicol Table 2 Different parameters of As-exposed and unexposed sheep in the study area
Parameter
Minimum
Maximum
Mean
Arsenic-exposed population Weight (kg)
15
55
30.70
–
Temperature (°C)
38.60
40
39.22
38.40–39.20
Heart rate (per min)
58
95
77.60
70–90
Respiratory rate (per min)
25
47
37.46
12–72
Wool (mg kg-1)
1.56
10.79
4.68
0.50
Blood (lg L-1)
86.30
656
416.90
\70
24.87
27–45
8.29
9–15
PCV (g/dl)
17
32
Hgb (L/l)
5.67
10.67
AST (U/L)
163
201
180.90
49–123
ALT (U/L)
100
131
116.40
15–44
Control population
a
Fig. 3 Skin lesions in Asaffected sheep
Fig. 4 Correlation between wool As concentration with age (male and female sheep) in Asexposed areas
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Normal range in healthy sheepa
Weight (kg)
30
63
48.30
Temperature (°C)
38.70
39.30
38.90
Heart rate (per min) Respiratory rate (per min)
68 31
92 45
80.70 37.77
Wool (mg kg-1)
0.03
0.13
0.07
Blood (lg L-1)
42.60
58.40
50.70
PCV (g/dl)
25
38
33.44
Hgb (L/l)
8.33
12.67
11.15
AST (U/L)
54
68
60.78
ALT (U/L)
27
43
32.56
Roy et al. 2009, Dobbs 2009
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There is a consistent association between the mean As concentrations in wool samples in males and females (p \ 0.01) indicating a greater mean wool As concentration in the male population. This may be due to excess excretion of As from the milk in females (Biswas et al. 2000). Analysis of As in blood samples is best suited for highdose exposures (Marchiset-Ferlay et al. 2012). Quantification of As in the blood can be useful, but it depends on the kind of epidemiological study performed (Mandal et al. 2004). It could also be used to show chronic exposure to As (Hall et al. 2006). Table 2 lists the blood As concentrations in As-exposed and control animals. The total concentrations of As in the blood samples from the contaminated area vary between 86.30 and 656 ppb with a mean value of 416.90 ppb. These compare with values for the control area of 42.60, 58.40, and 50.70 ppb, respectively. Statistical comparisons shows significantly greater As content in blood samples from the contaminated area (p \ 0.01) compared with the control area. Moreover, there is a significant relationship between As levels in drinking water and blood. The results show that in all cases, the calculated BCF values in wool samples in all cases is greater compared with measured values in blood when As is consumed through drinking water (Fig. 5). The results also show that BTF in sheep wool samples is greater compared with blood when As is ingested through drinking water and forage (Fig. 5). It is therefore concluded that increased levels of As in drinking water leads to increased deposition of As in both wool and blood samples. The mean hematological indices, i.e., Hb and PCV, are significantly decreased (p \ 0.01) in sheep from the Asaffected zone (8.29 L/l and 24.87 g/dl, respectively) compared with the control area (Table 2; Fig. 6). Obviously, the animals from the As-contaminated zone suffer from hematological abnormality, as manifested by anemia, similar to the findings of Rana et al. (2008). As can interfere with metabolism and suppression of the granulopoietic activity of bone marrow by residual
toxicants. As may also cause change in structural integrity of cell membrane, decrease osmotic resistance and fragility of erythrocyte, and cause severe hematological disorders (Rana et al. 2010). The liver is the major site of As metabolism and hence exposure to As causes liver damage (Guha Mazumder et al. 2005). Research results shown that ALT and AST may be used as an indicator of liver damage (Vutukuru et al. 2007). In the present investigation, serum ALT in As-exposed animals varies between 100 to 131 U/L with a mean value of 116.40 U/L (Table 2), whereas the equivalent values for the control group are 27, 43, and 32.56 U/L, respectively. In addition, serum AST levels in the contaminated group range from 163 to 201 U/L with a mean of 180.90 U/L, which compare with the respective control group values of 54, 68, and 60.78 U/L. These results therefore show that serum AST and ALT were increased in the As-exposed animals compared with the unexposed group (Fig. 6). This increase clearly indicates that chronic As exposure causes injury to hepatocytes and damages the liver’s capacity for transporting organic anions, metabolizing drugs, cholestasis, and liver biosynthetic capacity. Our results are consistent with many findings of chronic exposure to As associated with hepatomelagy, hepatoportal sclerosis, liver fibrosis, and cirrhosis with concomitant increase in ALT and AST (Liu et al. 2002; Zhou et al. 2002). It is evident that nutritional status plays an important role in the regulation of As methylation and As-related health hazards (Milton et al. 2004). As contamination in sheep comes from different sources, such as drinking water, crops, and vegetables grown in As-affected areas (Dash et al. 2013). In the Datong basin, northern China, the use of As-contaminated groundwater for drinking and irrigation purposes has resulted in endemic As poisoning among tens of thousands of livestock and people (Guo et al. 2003). Similar cases have been reported in Argentina, Bangladesh, Canada, Hungary, India, Mexico, Poland, and the United States (Nickson et al. 1998, 2000, Jain and Ali 2000; Smith et al. 2001; Berg et al. 2001; Smedley et al. 2001; Smedley
Fig. 5 BCF and BTF in blood and wool samples of sheep that consumed As-contaminated water and alfalfa plants
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Fig. 6 As concentration in blood samples, serum PCV, serum AST, and serum ALT of sheep in the contaminated area compared with the control area
and Kinniburgh 2002; Polya et al. 2005). In this study, As concentrations in wool and blood samples also positively correlated with As levels in drinking water, cultivated soils, and alfalfa hay. A high concentration of total As in water (Table 1), which is much greater than the permissible limit, and the level of As in alfalfa hay (Table 1), which is also much greater than the allowable concentration, is interpreted to be the source of As contamination in the study area. Keshavarzi et al. (2011) indicated that the main source of As in the groundwater of Kurdistan province is travertine-forming springs.
Conclusion From this study it can be concluded that As content in drinking water, cultivated soils, alfalfa hay, and biological samples, including wool and blood samples of the sheep in
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the two endemic villages of Kurdistan province, is alarming. The most probable sources of high As concentrations in biological samples are drinking water and forage given to the animals. Approximately 78 % of the studied animals show typical symptoms of arsenicosis including diffuse or spotted melanosis or hyperkeatosis on the skin, feet, and chin. The results suggest that long-term exposure of sheep to inorganic As at the observed high levels produces severe clinical signs of toxicity and toxicopathological changes that will eventually lead to mortality. The cytotoxic effects of As are proportional to the quantitative accumulation of its residues in the organs. Our results indicate that serum levels of ALT and AST were increased in the animals exposed to As, whereas hematological indices (Hb and PCV) are significantly decreased in sheep from the contaminated area. Therefore, this study reveals that sheep in As-contaminated areas suffer from liver damage and anemia. Furthermore, high concentrations of As in wool may
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be considered as a biomarker of arsenicosis in livestock. Sheep may also ingest a large amount of As through daily consumption of alfalfa plants as well as the water they drink. In conclusion, the data suggest that human beings are being exposed to hazardous levels of As if they consume contaminated meat and foodstuffs grown on agricultural land. Acknowledgments The authors express their gratitude to the Medical Geology Research Center of Shiraz University for financial and logistic support. Thanks are also extended to Atomic Energy Organization of Iran for providing analytical measurements. The authors also express their gratitude to D. J. Moore for constructive comments and English correction.
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