Inter-Relationship of Soil-Forage-Plasma, and Milk Chromium: A Case Study in an Arid Region of Pakistan

Muhammad Danish1*, Nazir Ahmad2, Ihsan Sharif3, Mirza Naveed Shahzad4, Syed Sibtain Raza Rizvi5 and Muhammad Faizan Nazar1 1Department of Chemistry, University of Gujrat, Gujrat, Pakistan 2Lab of Organometallics, Catalysis & Ordered Materials, Wuhan University of Technology, China 3Department of Chemistry, University of Sargodha, Sargodha, Pakistan 4Department of Statistics, University of Gujrat, Gujrat, Pakistan 5Livestock Experiment Station, Kalurkot, Bhakkar, Pakistan


Introduction
Metals contamination has been causing great threat to the terrestrial as well as aquatic environment since long time. Polluted water contains undesirable material and deposits them on the soil when used for irrigation. Metals are transferred from such soil to plants and finally the herbivores. Transfer of metals from soil to plants depends upon metal speciation which in turn depends upon the feature of soil such as pH, organic matter and clay contents [1][2][3][4].
Heavy metals are physically and chemically interrelated with natural components of the environment which consequences in altering their flow from soil to herbivores. These metals interrelate with precise compounds that may cause their precipitation or change their oxidation state. These metals bound with natural compounds resulting in increase or decrease in their mobility [5]. The high levels of bio-available heavy metals found in water, soil and fodder have significant impact on livestock. The monitoring of metal levels is helpful in ascertaining risk to human health as well as in the evaluation of environmental quality [6][7][8].
Heavy metal such as chromium (Cr) is considered as one of the elements destructive to the environment and is potential bioaccumulative toxins of dairy production system. In nature it is present as chromites rocks that may form complexes under different environmental conditions. Major source of Cr contamination to the environment is tanning while others include electroplating, polishing, paints and pigment formation. Trivalent chromium is insoluble in water and is oxidized into hexavalent in presence of large amount of oxygen. Hexavalent chromium is potent for cancer, asthma, bronchitis and also destructive for DNA [9]. However, Cr is an essential metal and plays a very important role in glucose metabolism. It increases the activity of insulin because of its presence in glucose tolerance factor [10]. Although Cr is essential to maintain the metabolic systems of human body, it can lead to poisoning at higher level. The objective of this study was to investigate the Cr concentration speciation in soil and to check the accumulation of Cr in milk that may result in health problem of the consumers. Freidman non-parametric test shows that there seems some dependency of milk chromium on sources.

Sampling duration and species
The study was conducted during the months of October, December, February and April 2010-11 at livestock farm of Kalurkoat, District Bhakkar, South Western Punjab, Pakistan. This livestock farm was established in 1979 with total area about 3649 acres. It is located between 31°10´ and 32°22´ N. and 70°47´ and 72° E. Total number of Sahiwal cows breed in this station is 512 that graze in the open ranch.
At the time of survey dominant forages species were Triffolium alexandrium, Cichorium intybus, Chinopodium morale, Medicago sativa, Avena sativa, Brassica compastrus and Alf alfa etc. Mostly the animals of Kalurkoat livestock grazed on the above mentioned forages. collected from each acre. In this way 20 samples were collected each time. Soil samples were taken from depth of 20 cm. From the same site 20 samples of different forages were collected which were mostly grazed by animals. The forage samples were plucked from the height of 3-6 cm from ground to stimulate the grazing behavior of animals [11]. These samples were dried under shade, heated at 70°C for 72 hrs in an electric oven and stored in plastic bags.
Blood samples were collected by puncturing the jugular vein with a syringe and needle and transferred into evacuated tubes containing heparin as an anticoagulant. The plasma was separated by centrifugation and transferred into polyethylene tubes and frozen at -20oC for Cr detection. For milk sampling, the animals' teat were thoroughly washed and then dried. Milk was collected in 125 ml nalgene bottles using the first drawn milk [12].

Sample preparation
For each set of processed sample, blanks (deionized water and reagents) were included throughout the entire sample preparation and analytical process.
Soil samples were dried at 70°C and grounded in an agate mortar. One gram of dry and powdered soil was transferred to digestion flask and 10 ml of conc. HNO 3 was then added. This mixture of soil and acid heated for 30 minutes. Further 10 ml of conc. HNO 3 was added and heated until mixture became clear. Then 10 ml of conc. HCl was added and again heated till solution was reduced to half of its volume. At the end solution was filtered and diluted with distilled water up to 20 ml [13].
One gram of each forage samples was digested with the help of HNO 3 and HClO 4 (3:1) at 250°C for 3-4 hours until solution become colorless and thick white fumes appeared in the flask. The contents of flask were diluted with distilled water, filtered and sample was made up to 20 ml [14].
The hyperanized blood was centrifuged and then 2 ml of blood plasma was taken and mixed with 2 ml of HNO 3 . The mixture was digested by heating at 120°C until all the organic matter was dissolved. The digestion process was enhanced by adding 2 ml of H 2 O 2 to the reaction mixture and heated again. The digested sample was cooled, filtered and diluted up to 20 ml with distilled water [15].
Milk, 10 ml of was taken in dry pyrex digestion flask, 10 ml of 65% HNO 3 and 3 ml of 30% H 2 O 2 was added and heated until the solution become transparent. The mixture was filtered with Whattman filter paper 40 and made up volume to 20 ml using distilled water [16].

Sample analysis
The concentrations of chromium in soil, forage, plasma and milk were quantified by flame atomic absorption spectrophotometry using a Shimadzu atomic absorption spectrophotometer (AA-6300) coupled with a graphite furnace atomizer (GFA-EX7i). The standard burner and air-acetylene was applied as fuel. The wavelength of Cr was 267.2 nm. The values were expressed in mg/L of blood or milk and mg/kg of soil or forage.

Statistical analysis
Statistical analysis was performed for the descriptive statistics to define the basic characteristics of the chromium concentration. To determine the significance difference among the mean concentration with respect to the time and source non-parametric Friedman test was applied. Pearson's correlation was established to describe the association between the different categories.

Chromium concentration in soil, forage, blood plasma and milk
It is evident from statistical analysis that effect of sampling time on soil Cr was significant. The concentration of Cr in soil samples ranged from 6.7-10 mg/kg (Figure 1a). The higher amount of Cr in soil samples was found during the 1st sampling (October) and lower amount of soil Cr was found during the 4th sampling (April). The concentration of Cr in soil samples was found lower than the value reported by many authors [17][18][19].
The concentration of forage Cr varied from 2.9-4.0 mg/kg ( Figure  1b). The higher amount of forage Cr was found during February and the lower amount during December. The concentration of Cr in forage samples was found higher than the values reported in literature [20,21].
The concentration of blood Cr fluctuated from 3.5-4.3 mg/L ( Figure  1c). The higher and lower amounts of blood Cr were found during February and December respectively. The quantity of blood Cr was found higher than the value suggested by authors [21,22].
Range of milk Cr diverged from 0.4-0.6 mg/L (Figure 1d). The higher amount of milk Cr was found during the 1st sampling in October and during the 3rd sampling in February. The concentration of Cr in milk samples was found lower than that reported in literature [21,[23][24][25][26].
Freidman test, a substitute of analysis of variance (ANOVA) was applied to observe the possible difference in the mean concentration of Cr due to different sources (Soil, forage, milk and blood) and in different months (October, December, February and April). One of the basic assumptions of the ANOVA is the homogeneity of the variances, but that is strongly rejected by the Levene's test. Due to this reason the analysis was performed by the non-parametric Freidman test [27]. The results of this test showed that the p-values less than the level of significance (even than 0.005). Therefore the average effect on concentration level of Cr in different months and due to different sources is significantly not same.
Since the equality of averages is rejected, so Bonferroni multiple comparison test is applied to determine which average Cr concentration is different and which averages are same in different months ( Table 1). The same treatment is used for different sources (Table 2).
Concentration level of Cr is same where lower bound and upper bound contain zero or p-value greater than 0.05 as in October and in February. Concentration level is different where lower bound and upper bound does not contain zero or p-value greater than 0.05 as in October and in December.

Correlation among soil, forage, blood plasma and milk
Pearson method was used to find the correlation between soil, forage, blood plasma and milk Cr concentrations. The correlation coefficient (r) value between soil and forage Cr was found to be -0.15, soil and blood 0.14, soil and milk 0.19, forage and blood -0.31, forage and milk -0.08, blood and milk -0.33. There was found a negative and significant relationship between soil and forage, and forage and plasma Cr levels whereas a positive and significant was established between soil and plasma, soil and milk, and blood and milk Cr levels. A negative

Chromium concentration in various blood samples during different sampling times
Sampling period also affected the blood plasma Cr concentration significantly (Figure 2). It is evident from the Friedman test that there is significant effect of sampling time and blood source on milk chromium. As the p-values of month are less than the level of significance (0.0001) it is concluded that the different months (October, December, February and April) affect the accumulation of this mineral in plants, blood and eventually in milk based on the results of Bonferroni multiple comparison test (Table 3).
*The mean difference is significant at the 0.05 level It was also assumed that different sources of blood (Young, Lacto and Dry) may affect the concentration level of Cr or there might be any interaction between months and sources of blood which play the role in the concentration level. So we can conclude that the average effect of different month on concentration level is different, when we consider only blood with three level of source (Young, Lacto and Dry).
These results show that there is continuous fluctuation in concentration of Cr uptake by plants. It decreases from October to December and again increases, becomes maximum in February. There is again decrease in its concentration in April. The same trend is found in the blood plasma and milk of the selected cows. However these ranges do not enter the dangerous limits. We were expecting that there could be more Cr accumulation in the blood of sucklers as they intake through milk as well as grazing. However, this is not the case and the  Cr concentrations in their blood followed the same trends as that of other cows.
The Cr concentration range for livestock requirement is 0.3 to 1.6 mg/kg [21]. There is no threshold limit of maximum chromium concentration but different reports suggested a concentration between 0.03 to 1.0 mg/kg [28,29]. A range of 0.156-0.285 mg/g of chromium was reported in the salt-range Pakistan [30] whereas its concentration was reported 0.0003 to 0.0006 mg/L in central Punjab Pakistan [21]. The higher level are toxic to livestock and badly affects the reproductive potential of ruminants [31,32]. The tolerance limit of Cr in milk is reported 0.3 mg/kg (GB/T 1461-94). The concentration of Cr in milk samples was found (0.4-0.6 mg/L) near the range of the certified value (0.39 mg/kg) [33] while in soil, forage and blood plasma samples was found higher than the normal values. It means that transfer of Cr to milk is independent of its concentration in soil, forage and blood.

Conclusion
In the present case the concentration of Cr ranged from 6.7-10 mg/kg, 2.9-4.0 mg/kg, 3.5-4.3 mg/L, and 0.4-0.6 mg/L in soil, forage, blood plasma and cows' milk respectively. The concentration of Cr in milk samples was found (0.4-0.6 mg/L) near the range of the certified value (0.39 mg/kg) while in soil, forage and blood plasma samples was found higher than the normal values. It means that transfer of Cr to milk is independent of its concentration in soil, forage and blood. Although there is no adverse effect of higher concentration of Cr in forage and blood plasma, yet it can be decreased using the plants which absorb less quantity of chromium. Furthermore, a dietary supplementation of area specific metals and minerals mixture is suggested in order to safeguard the environment and preserve human health.