Heavy metals in equine biological components

The objective of this research was to determine the concentration of heavy metals in the blood (Pb, Ni and Cd), serum (Cu and Zn) and hair (Pb, Ni, Cd, Cu and Zn) of horses raised in non-industrial and industrial areas (with steel mill), and to verify the possibility to use these data as indicators of environmental pollution. The samples were collected during summer and winter, aiming to verify animal contamination related to environment and season of the year. Copper and Zn contents determined in the serum and Cd and Ni contents obtained in the blood indicated no contamination effects of industries. For some animals, contents of Pb in the blood were higher than those considered acceptable for the species, but without relationship with industrialization and without clinical signs of Pb intoxication. The heavy metals evaluated on the hair of horses in this study were not increased with the presence of industries, but Cu and Cd contents were influenced by the season. The contents of some heavy metals in biological components analyzed were influenced by season sampling; however, serum, blood and hair may not be suitable to indicate differences in environmental contamination between the two contrasting areas. Most part of the heavy metal contents was lower or close to the reference values for horses. Serum, blood and hair components from horses may not be effective as indicators of environmental pollution with heavy metals. Industrialization and seasons have no effects on most part of heavy metals contents from those components.


Introduction
Mineral elements are essential for animal health, survival and production (Reis et al., 2010).However, if they are ingested in excessive doses or if they are toxic like heavy metals, they may be harmful and lethal.Specifically for horses, Casteel (2001) review the current concern about poisoning with metals, showing problems such as gastrointestinal upset, hepatopathies, peripheral neuropathy, intermittent colic, mild anemia, as well as diseases related to musculoskeletal system, and kidneys.
Heavy metals are present in everyday activities of men more intensely than many would ever imagine.They originate from several sources that can contaminate soil, water and plants, and consequently, animals (Hammond and Aronson, 1964;Kuno et al., 1999;Patra et al., 2007;Reis et al., 2010;Rajaganapathy et al., 2011) and humans (Duarte and Pasqual, 2000).These sources include atmospheric deposition, farming and cattle-raising residues, fertilizers and soil correctives, agrochemicals, sewage sludge, irrigation water, urban garbage composts, and urban, industrial and mining wastes.
Pastures are in general contaminated by atmospheric deposition, foundries, and various industry and urban wastes (Hammond and Aronson, 1964).Contaminated effluents from different industrial processes or small urban activities can also be responsible for contamination of water for animal use.Proximity to humans, coinciding habitats, and greater organism similarity makes it possible for animals like horses to have the potential to be used as indicators of environmental contamination.
Usually, contamination of animals by heavy metals is not a frequent scientific research topic because priority has been given to studies of the effects of these elements on human health or on some specific environmental compartment, such as soil, sediments, vegetation and water.The use of different soft tissues and blood of animals can be a way to evaluate the concentration of heavy metals in the animal body.In addition, the hair analysis has been utilized to investigate or monitor human and animal exposure to a number of toxic heavy metals (Ward and Savage, 1994;Patra et al., 2007).The objective of this research was to evaluate the heavy metal contamination in biological components of horses from two environmentally different regions and in two seasons.In addition, it was aimed to verify the possibility to use the results as indicators of environmental pollution.Horses originated from two distinct areas were used in this study, displaying the following characteristics: Non-industrial area (control area): horses selected in the rural area of the municipality of Viçosa (MG, Brazil), on farms over 3 km distant from the urban area.Only animals which were kept semi-stabled for at least one year were selected.Industrial area: animals raised close to smelters in Sete Lagoas (MG, Brazil) and Prudente de Morais (MG, Brazil).The horses had been utilized for field work and were pasture-raised for at least one year.

Material and Methods
Animals from non-industrialized area were only females, and those from industrial area were of both sexes.In both groups, the animals were from different breeds and ages (2-19 years, average of 9.7).Each group was formed by 20 randomly selected clinically healthy animals, from which blood, serum, and hair samples were collected twice a year, with two samplings performed: one during late winter and the other during late summer.
The two areas evaluated are located in the state of Minas Gerais, in southeastern Brazil.The climate in both regions is characterized by hot rainy summers and dry winters.Mean annual temperature and rainfall in the nonindustrial and industrial areas are 18.5 ºC and 1.220 mm and 22.9 ºC and 1.400 mm, respectively.
At the sampling places, detailed individual information data was obtained on equine (sex, age, breed, weight, main use) and farm management.After that, the animals were subjected to physical exam including nutritional status, degree of hydration, mucous membrane color, capillary refill time, and heart and respiratory rates.
The 40 animals included in the study were well hydrated with normal mucous.The heart and respiratory rates, as well as the capillary refill time were within the reference standards for the species (Loving, 2003;Southwood, 2006;Baxter, 2009).Mean body score, based on classification of Speirs (1999) was 3, on a scale from 0 to 5, meaning a good body condition.One of the horses (industrial area) was certified dead due to a snake bite.Thus, in the summer collection, total equine number accounted to 39 animals.
For the biological sampling procedure, 10 mL of blood were collected from each animal by puncturing the jugular vein to vacuum glass tubes containing sodium heparin to analyze Pb, Cd and Ni concentrations.To analyze Cu and Zn, another 10 mL of blood were collected to glass tubes without anticoagulant and immediately after collection, these samples were subjected to sedimentation at room temperature, followed by centrifugation (1720 x g, over 10 minutes) to obtain the serum, which was collected, identified and stored under refrigeration until analytical procedure.
To determine the Pb and Ni in each blood sample, 0.2 mL was taken and diluted with 1.8 mL aqueous solution containing 0.1% Triton X-100 and 0.2% nitric acid (Kuno et al., 1999).To estimate Cd contents, the blood samples were subjected to nitroperchloric digestion (3:1, v/v) at 150 °C.For content quantification of Zn and Cu, the serum samples were diluted 5 times in Milli-Q water (Jian-Xin, 1990).The elements Pb and Ni in the extracts were determined by graphite furnace atomic absorption spectrometry; Cu and Zn by flame atomic absorption spectrometry and Cd by inductively coupled plasma optical emission spectroscopy (ICP-OES).The results were expressed as µg mL -1 .
Samples of 10 g of the mane and tail hair were collected using stainless steel surgical scissors.Only samples from the nape area nearest to the skin were collected, identified, and stored in plastic bags.Then, they were cut into fragments of approximately 0.3 cm and washed four times with 1:200 (v/v) dilution of Triton X-100 solution to remove exogenous elements, rinsed twice with isopropylalcohol and allowed to drain.This was followed by rinses with Milli-Q water and two more rinses with acetone, and then they were allowed to drain.Finally, the samples were dried on a hot plate (Asano et al., 2002).For determination and digestion of the heavy metal contents, the hair samples were processed by following the technique described by Pimenta and Vital (1994).Thus, after drying, 0.4000 g of the samples was solubilized in 10 mL of concentrated nitric acid on a hot plate.After gentle boiling for 30 min, 2 mL of hydrogen peroxide (30%) were added.Once the volumes were reduced until close to drying, the solutions were cooled, transferred to 10 mL volumetric balloons and the volumes were completed with Milli-Q water.The extracts obtained were analyzed by ICP-OES and the results were expressed as µg g -1 .
In the serum, the limits of detection were 0.002 mg mL -1 for Zn and 0.004 mg mL -1 for Cu.In the blood, they were 0.002 mg mL -1 for Cd and Ni, and 0.010 mg mL -1 for Pb.In the hair, 0.001, 0.015, 0.016, 0.021 and 0.033 mg g -1 for Cd, Pb, Ni, Cu and Zn, respectively.
The experimental design consisted of a completely randomized 2 × 2 factorial arrangement (two areas and two seasons).Data obtained were subjected to statistical analysis using the SAEG software (version 9.1, 2007).A descriptive statistical analysis of the data was conducted, followed by analysis of variance (ANOVA).The effects of the areas (industrial or non-industrial) and seasons of the year (winter or summer) on the heavy metal content in the blood (Pb, Cd and Ni), serum (Cu and Zn) and hair (Cu, Zn, Pb, Cd and Ni) were assessed by the Tukey test (P<0.05).When detected, the interactions between areas and seasons evaluated were deployed.

Results
The areas and seasons evaluated affected Cu contents on serum samples.Animals from industrial area had higher (P<0.05)metal contents than those from control area and in the summer, higher contents of Cu were observed in the serum of the horses (Table 1).With respect to Zn, analysis of the deployment of the interaction indicated an important effect of season on the contents obtained (Table 2), with higher contents of this heavy metal in the winter and summer (P<0.05), for non-industrial and industrialized areas, respectively.On the other hand, in the summer sampling, there were no differences in Zn contents between the two areas evaluated, and in the winter sampling, horses from non-industrialized area had higher serum levels of the metal.
Concentrations of Pb, Cd and Ni were observed in the blood.Areas and seasons did not influence the contents of Cd (P<0.05), and 65% of the analyzed samples indicated levels below the detection limit.There was no effect of areas on the Pb and Ni contents.However, as occurred for Cu contents in the serum, higher concentrations (P<0.05) of both metals were obtained in the summer.In the winter, the concentrations obtained were lower if compared with the summer, and considering the Ni, the mean value was below the detection limit.
All heavy metals evaluated were present in the hair of the horses.The different areas and seasons studied did not influence the contents of Pb and Ni (P<0.05), with 82% and 92% of samples analyzed presenting levels below the detection limits, respectively.The contents of Cu and Cd in the hair were not affected by areas, although higher concentrations were observed in summer and winter (P<0.05),respectively.Similarly to serum, the Zn contents in the hair were influenced by the interaction area and The results presented are the mean (minimum-maximum); <dl: below the detection limit.In calculating the means and statistical analysis, the values <dl were considered as zero.According to the Tukey test (P<0.05),means of heavy metals followed by the same capital letter, and means followed by the same lowercase letter, both in the row, do not differ for the area and season, respectively.A×S -interaction evaluated between area and season of sampling (ns -not significant, *: P<0.05).For significant interactions, means were not compared, and deployments are presented in Table 2. #: death of animal.
season.Again, season influenced this heavy metal content, with higher values verified in the summer (P<0.05) for both areas evaluated.Also similarly to the serum, differences in the Zn contents in hair samples were observed only for the winter, when the horses of non-industrial area showed higher levels of the metal.

Discussion
Copper contents in serum were affected by areas and seasons (Table 1).Even though data show higher Cu contents in the industrial area, as also observed for pastureraised animals by Maia et al. (2006), the mean contents for both areas were close to or within normality range expected for horses, considering 1.04-1.77µg mL -1 , as defined by Thompson (1992) or considering the 0.5-1.5 μg mL -1 used by Wichert et al. (2002).Thus, the results do not show clear effects of industrial activities on the serum Cu contents of horses.
Zinc contents in the serum were not affected by industrial area (Table 1).On the other hand, the control area showed higher contents than the industrialized area when sampling was carried during winter, and no differences were observed between the two areas in the summer (Table 2).In fact, the contents measured were lower or close to the reference values (0.77-1.19 μg mL -1 ) defined by Thompson (1992) for horses or those adopted (0.6-1.2 μg mL -1 ) by Wichert et al. (2002) in their study with 106 horses from Bavaria.In a first analysis, these results raise a concern, since this element is essential for enzymatic metabolism, and it is involved in protein synthesis and carbohydrate metabolism (Marçal et al., 2003a).However, Wichert et al. (2002) reported that 31% of the animals studied also presented Zn contents in serum below reference values, but as verified in the present study, no animals showed specific signs of zinc deficiency.This lower concentration of Zn in the serum may be related to absence of mineral supplementation or to small amounts of salt mixed in the feed.Other factors related to low Zn serum contents include environment and physiology, which can lead to variations in the concentration of Zn in horses (Auer et al., 1988).Birick et al. (2005) showed that seasonal and diet changes have important effects on the concentration of this element in equine serum.On the other hand, the mean contents obtained in this study were higher than those considered normal for equines (0.47±0.09 µg mL -1 ) by Auer et al. (1988), who studied 83 clinically healthy horses in Australia.
Although industrialization did not affect the levels of Cu and Zn in the serum, some animals presented high contents of these heavy metals in their serum, surpassing the highest reference values of 1.77 and 1.19 μg mL -1 defined by Thompson (1992) for Cu and Zn, respectively.For Cu contents, all highest values were obtained in the summer for five animals in industrialized area and for two animals in control area.For Zn contents, only one animal per area showed higher values than the references, but in different seasons.This result, especially for Cu, may suggest some effect of industrialization on the environmental contamination, affecting only a few individuals.
The lead toxicity values in blood for farm animals are always lower than those in humans (Marçal et al., 2003b).In horses, the concentration of 0.25 µg mL -1 is accepted as maximum limit, while in humans, a maximum of 0.60 µg mL -1 is adopted (Gilman, 1991).In a study carried out in India with 288 horses from three different areas (industrial, highway adjacent and rural zone), the mean Pb contents in serum were 0.47±0.02,0.55±0.02and 0.38±0.03µg mL -1 , respectively (Dey and Dwivedi, 2004).These authors emphasized the non-observation of clinical signs associated with intoxication by this heavy metal in the studied animals.On the other hand, a study conducted with horses living on farmland in the vicinity of non-ferrous metal smelters in China (Liu, 2003) showed signs of intoxication by Pb in 10 horses, which presented mean contents in the serum of 0.28±0.10µg mL -1 .In addition, Palacios et al. (2002) reported Pb contents of 0.20 to 0.89 mg kg -1 in blood samples taken from six dead horses with diagnosis of lead poisoning.Thompson (1992) considers normal animals with serum Pb contents lower than 0.10 µg mL -1 , but concentrations equal to or higher than 0.30 µg mL -1 , when accompanied by clinical signs, are compatible with intoxication in horses.
Some horses included in this study showed blood Pb contents higher than those considered acceptable for the species, but without relationship with industries presence.The maximum value (0.60 μg mL -1 ) indicated by Gilman (1991) was surpassed by 15 animals in industrial area, but also by 15 animals in control area.In all cases, these high According to the Tukey Test (P<0.05), in the same area, means of heavy metals followed by the same capital letter in the row do not differ among themselves, and means followed by the same lowercase letter in the row, do not differ for season.
This work was approved by the Ethics Committee of Veterinary Department of Universidade Federal de Viçosa, and the assays are in agreement with the Veterinary Professional Ethics Code, with the Ethical Principles for Animal Research established by Colégio Brasileiro de Experimentação Animal and with current Brazilian legislation.

Table 1 -
Mean heavy-metal contents in biological components of horses, as a function of the area and season of sampling

Table 2 -
Zinc contents in biological components of horses after deploying interactions of areas and seasons of sampling