Evaluation of pasture allowance of manganese for ruminants

The aim of this study was to access the Mn contamination in soil, forages, and animals. Heavy metal pollution is a matter of prime significance in natural environment. Through food chain, toxicity of heavy metals and their bioaccumulation potential are transferred into humans. Higher concentrations of metallic compounds are toxic to living organisms but these are essential to maintain body metabolism. Intake of food crops polluted with heavy metals is chief food chain channel for human exposure. Animals are exposed to heavy metal stress by the intake of richly contaminated food crops; those are chief part of food chain. Samples of soil, plant, animal blood, hair, and feces were collected to find contamination through wet digestion process in lab and metal analysis. Different forages were collected to study Mn content that was our major concern in this study. The present findings also emphasized on the assessment of bio-concentration factor (BCF). Other significant indices of mobility and pollution of metal were also calculated, i.e. pollution load index (PLI), daily intake of metal (DIM), health risk index (HRI), and enrichment factor (EF). The experimentation result showed different concentrations of metal in different seasons. The Mn concentration in forages was 20.01–28.29 mg/kg and in soil was 5.27–8.90 mg/kg. Soil samples showed higher level of (PLI) Pollution load index. Bio-concentration of MN was 2.59–4.21 mg/kg. It can be concluded that regular monitoring of the metal is essential to evaluate the contamination status. Mn contents were in the safe limits in soil and plants; however, its toxic level was observed in animals.


Introduction
Manipulation and the accessibility of some crucial metals in the body of living being are produced by level of bioconcentrations of some heavy metals. In food chain, heavy metal load is measured by degree of contamination at specific area. Toxicity of heavy metals is measured by assessment of bio-magnifications in trophic levels from soil-forages-animal continuum and its bioaccumulation beside this by evaluation of contamination in fodder crops and water (Has-Schön et al. 2006;Saleemi et al. 2019). Water life is also good bioindicator of heavy metal level in water ecosystem; chemical analysis of heavy metal contents in water bodies is useful method for their bio-monitoring (Liu et al. 2007).
Group of elements that possess density greater than 5g/cm 3 are categorized as heavy metals (Stobrawa and Lorenc-Plucińska 2008). Heavy metal pollution is dangerous and risky for environment of biosphere; it includes contamination of soil, air, and water. When heavy metals interact with each other, they show different toxic effects than individual contaminant (MacFarlane and Burchett 2002). Plant anatomy and metabolic compounds are affected by heavy metals, and extinctions of species can occur in this world. Various elements that are absorbed by plants are termed as toxic elements even at low concentration. Heavy metal toxicity cause cellular death due to oxidative stress. With the passage of time, plants have evolved the detoxification mechanism to reduce the harmful effects of heavy metal accumulation and exposure (Yadav 2010). Worldwide industrial revolution has polluted the water and soil with many detrimental chemical compounds including trace metals. When these metals leach into trophic chain, these metals are harmful to plants and also cause severe damages to human health (Kumar et al. 2013). Heavy metals are persistent and non-degradable by chemical degradation or microbial degradation unlike other metalloids and organic compounds (Bolan et al. 2014). In addition to this antagonistic, synergistic and additive effects on plant growth are produced by heavy metals when they collectively show their effects to plants .
In the upper layer of the earth crust, there are some nondegradable, non-damageable, and natural metals which are found to be trace metals (Ugulu 2015). These metals are potentially toxic having long-term effects on food chain, and they should be remediate actively ). For synthesize of hormone, regulation of metabolism and to control enzyme mode of action, minerals perform a vital role. Mineral deficiencies and toxicosis are related with the metabolical functioning, effects of nutrients, and treatment of mineral-related disorders.
Livestock is major and principal part of Pakistani agricultural practices. Its contribution to net domesticated production of country is 12%; it makes possible the availability of daily used dairy products. Communities living in countryside are seriously trusting on this sector for earnings (Chandio et al. 2016). Foodbased requirements of grazing ruminants are accomplished by forage and mineral nutrients present in pasture land. On the other hand, tissue of animals, for example the kidney and liver, and animal blood samples are significant indicators of potential toxicity than forage because grazing ruminants select different nutritional and diet forages ).
Microelements are elements that required in lesser amount for animals and plants and these are present in the environment and are essential for organisms for sustainability of life, i.e. minerals and vitamins. Macronutrients and micronutrients of all types fulfill the particular nutritious needs of animals and forages (Pais and Jones Jr 1997). In soil-forage-animal continuum, heavy metals and other mineral nutrients perform a significant role in metabolic, catabolic, biochemical, biological, chemical, and enzymatic activities of living cell in organisms. This study is planned to evaluate the levels of Mn in soil, plant, and animal system and to find out the various indices of metals to determine the degree of contamination.

Study area
District Mianwali is located in south-western region of province Punjab. This district is consist of the plains of western area of salt range. It is situated near the Sakesar hill. Mianwali district has boundaries with Khushab, D.I Khan, Bhakkar, and Bannu districts. This district is part of Sargodha Division. The temperature of this area ranges from 47°C as the maximum and 19°C as the minimum per annum. In Mianwali, the maximum rain fall that occurs in July is about 6.6mm, and annual mean rain fall is about 3.3 mm. Soil condition of this area is characterized as loamy, sandy, and clay soil. Pea nut, mung, mash, mustard, Eruca, fennel, wheat, barley, and oat are important crops. Forest cover area is very low because trees are used as fuel and timber. Canal irrigation system is very less developed; only a little area is irrigated with Indus river irrigation system (Ghani et al. 2016;Qureshi et al. 2007).

Sample collection from sites
In the district of Mianwali, four sites were selected for sampling. The 3 samples of agricultural soil, forages, and animal blood, hair, and feces were taken to examine the metal profile of soil-forage-animal continuum. The samples were taken from Wan Bhachran, Mianwali, Esakhel, and Piplan. Three seasons, namely S1 (summer), S2 (autumn), and S3 (winter), were selected for sampling. The samples were taken randomly from sites.
In the district of Mianwali, four sites were selected to collect the samples. Three samples of soil were collected with equal distances in the field. Stainless steel auger was used to dig up the upper layer of soil about 12-15 cm (Siddique et al. 2019). These samples were packed into plastic bags to avoid the mixing of other chemical compounds into it. Samples were stored in laboratory and labeled and then metal analysis was performed. For each sample, three composite samples were made. The collected samples were firstly air dried and then oven dried at 72°C for 2 days. The samples were placed in incubators at 70°C temperature for 5 days Fig.1.
Sterilized apparatus were used to collect the forage samples. Forage and soil were collected from same field and place. Only those forages were selected for taking samples that are used as common feed of livestock. Three samples of each forage plant were taken from the sampling area. The samples were washed with distilled water to clear impurities and dirt. These samples were dried to eliminate moisture in the freshly collected samples. The collected samples were dried for further process. These are following species that were selected for sampling (Table 1).
Blood samples of cow, buffalo, and sheep of Mianwali was taken in 2020. Young animals within the age of 2 years were selected for sampling. Blood was collected from four sites of district Mianwali. Animal blood was calculated from 10 animals (cow, buffalo, and sheep) each from each sampling site and heavy metal evaluation was done. Sterilized syringe was used to obtain the blood samples. The grazing ruminant's blood was taken from the vein. The vacuum was created in evacuated tubes while collecting blood to minimize the extent of clotting. The blood was collected in heparinized Na-citrate voiles quickly. For 15 min, blood was centrifuged at 3000 rpm and blood plasma was separated. Polyethylene tubes were used to store the blood plasma and frozen at −20°C. The hair and feces samples were also collected and stored for further digestion process.

Digestion of samples
Soil, forages, and animal samples (blood, hair, and feces) were air dried and followed by oven dried process at 72°C for 5 days until the moisture content is removed. When plants are completely dried, standard procedure of digestion was applied to digest the samples (Siddique et al. 2019). Then 1gm sample was weighed by electrical balance and placed in a beaker of 50ml. Ten milliliters of nitric acid was added to beaker and was kept overnight. Hot plate was used for digestion of particular sample by pouring H2O2 drop wise until solution becomes transparent. Cooling at room temperature was done. For dilution purpose, 50 ml distilled water was added to the solution. To filter the solution, Whatman filter paper of 42 μm was used. In next step, atomic absorption spectrophotometer (AAS) is an apparatus through which metal analysis is done.
Blood samples collected from the Mianwali district stored and freezed at −20°C were digested with same standard procedure as applied to soil and forages (Siddique et al. 2019). Hair sample was sun dried and was cut into pieces of 1.0-2.9 cm. Deionized water was used to wash the samples and ethanol was also applied to wash. Oven-drying process was carried out for 4 h and then desiccator cooling was performed (Hashem et al. 2017). Feces samples were collected from cow, buffalo, and sheep; after air drying and oven drying, the samples were submitted for digestion (Nicholson et al. 1999).

Metal profile evaluation analysis
The prepared samples were then analyzed for Mn content by atomic absorption spectrophotometer (Perkin-Elmer Corp., 1980). Standard Mn solution was prepared to get the standardized curve. The metal analysis was done by running the samples through atomic absorption spectrophotometer. This apparatus is equipped with a graphite furnace. The amount of Mn occurring in the sample is obtained in absolute farm. While sample is being run through the atomic absorption spectrophotometer, a little quantity of sample is sprayed at the flame. Atomic resonance absorption line by element is calculated and measured. The apparatus is convenient for analysis. Any radiation that is emitted by flame had no effect on the working of apparatus. The absorption method is independent of the excitation potential of the spectral line used.

Evaluation indices
Bio concentration factor (BCF) For assessment of Mn metal (mg/kg) transport from agricultural soil and forages that are growing on this soil, a BCF is applied (Cui et al. 2004). BCF for soil to forage: Þ¼Level of metal in forage=Level of metal in soil Pollution load index (PLI) Liu et al. (2005aLiu et al. ( , 2005b) described a formula which was used to find this index.
where (M)IS (mg/kg) is the concentration of metal that occurs in soil to investigate and (M)RS is the soil reference value of metal.
Reference value for soil in Mn was taken as 25.5 suggested by Hassan et al. (2013).

Enrichment factor (EF)
Formula for enrichment factor is described by Buat-Menard and Chesselet (1979 According to Hassan et al. (2013), standard reference value for Zn was used 25.5 mg/kg.

Daily intake of metals (DIM)
Daily intake of metal (DIM) can be calculated by following equation: DIM = Cfactor × C metal × D food intake B average weight Sajjad et al. (2009).
where C metal is the concentration of metals in forages, D food intake is the daily intake of forages, and B average weight is the average body weight. For calculating this daily intake of metal, the conversion factor was taken 0.085 (Jan et al. 2010). Daily intake metal for cow was calculated by using animal body weight 600 kg and daily forage intake 12 kg, while for sheep body weight was taken 75 kg and daily forage intake 1.3 kg (Johnsen and Aaneby 2019). To calculate the DIM for buffalo, body weight was taken 550 kg and daily forage intake (TDI) was taken 12.5 kg (Yang et al. 2020).

Health risk index (HRI)
Health risk index is the ratio of daily intake of metals in the forages to oral reference dose (RfD) and was calculated by the help of the following formula (USEPA (US Environmental Protection Agency) 2002).

Health risk index HRI
where DIM is the daily intake of heavy metal and R f D is the oral reference dose.
An HRI > 1.0 for any single metal indicates that the health of consumer population is at risk or it is carcinogenic (USEPA 2013). According to USEPA (2010), oral reference dose for Mn was taken as 0.041 (mg/kg/day).

Results
The level of Mn in soil samples had variation in amounts. The Mn level varied in the range of 5.27 mg/kg to 8.90 mg/kg minimum and maximum, respectively ( Table 2). The minimum concentration of Mn was evaluated in the Soil samples of S. vulgare forage. The minimum Mn concentration was calculated during time period of S1 sampling (Fig 2). The maximum values were observed by soil of forage Z. mays during S2. Our finding for Mn concentration in soil was lower than permissible value of 437 mg/kg described by National Research Council (NRC) (2001).
The Mn concentration in forages was different in each sample (Fig. 3). The minimum amount of Mn was present in 20.1 mg/kg in B. compestris forage during S1.The maximum concentration of Mn was found in 28.29 mg/kg during the months of S3. Higher concentration of Mn was present in P. glauccum. The Mn amount in soil was below than critical limits of 200mg/kg recommended by FAO/ WHO (2001).
The Mn concentration in blood was noticed to vary from 1.00-2.16 mg/l; the minimum amount was observed in sheep of S1 and the maximum concentration was observed in cow of S3 (Table 3). The Mn level in blood of current study was higher than 0.51 mg/l (Kalita et al. 2006). The hair had Mn values ranged from minimum to maximum (1.04-2.66 mg/kg) in buffalo during S1 and cow during S3, respectively (Fig. 4). The Mn showed different concentration in feces samples varied from lower to higher (0.55-2.35 mg/kg). The minimum value was found in cow during S1. The maximum value was found in sheep during S2.

Bio-concentration factor (BCF) of Mn
Mn bio-concentration was in the range from 2.59-4.21 mg/kg. The minimum amount of bio-concentration was observed in B. compestris during S1 while the maximum concentration was found in T. alexandrium during time intervals of S2.

Pollution load index (PLI) of Mn
The level of PLI for Mn ranged from 0.207-0.349 mg/kg. The maximum range was observed in Z. mays during S2 while the minimum level was noticed in S. vulgare during S1.

Enrichment factor (EF) of Mn
The enrichment factor ranged from 0.242-0.393 mg/kg. The maximum concentration was depicted by B. compestris during intervals of S1 while the minimum amount was found by the T. alexandrium in S2 (Table 4).

Daily intake metal (DIM) of Mn
The DIM (daily intake metal) trend for Mn was found between 0.029 and 0.055 mg/kg. The lowest amount of was depicted by sheep of S1 while the highest amount was noticed in buffalo during S3 (Table 5).

Health risk index (HRI) of Mn
The health risk index (HRI) of Mn varied within a range from 0.722-1.33 mg/kg. The smallest values were found sheep in S1 while the greatest value was noticed in buffalo of S3 (Table 5).  (1997). The current investigation for Mn soil concentration was found below the results by Xu et al. (2017). Our present Mn level in soil samples was greater as compared to the recorded amounts by Yaylalı-Abanuz (2011). The Mn concentration is found similar with the evaluations given by Uren (2013). The Mn usually occurred in the form of oxides with other metals; its most abundant oxide form was observed with iron metal in soil fraction. These two metals have high affinity with each other so their mobility from soil is reduced even negligible according to Yaylalı-Abanuz (2011). The Mn amounts in forage were observed similar with the investigations given by Underwood and Suttle (2010). In this study, the investigation for Mn was greater as compared to recorded values by Lindström et al. (2013). This study of Mn found lower concentration of Mn in forage as compared to the observation by Udiba et al. (2014). The current findings were found   Fig. 4 The Mn concentration in animals' blood, hair, and feces in different seasons (mg/kg) higher than those given by Spann et al. (2010). Manganese is essential for the forage growth and it is a component of chlorophyll and enzyme oxidases. Mn amount in soil varies from species to species and individual to individual; it is also affected by soil properties and drainage system (Udiba et al. 2014). Cow blood Mn concentration in this study was found lower than the suggested values of Luna et al. (2019). These findings were greater for Mn cow blood than reported values of Popovic et al. (2010). Sheep blood Mn level was found similar with those values given by Popovic et al. (2010). In this work, the Mn level in buffalo blood was found lower as compare with Abd El-Hady et al. (2006). This work for Mn in buffalo hair was lower as compare to recorded values by Huo and Shen (2020). Mn concentration in buffalo hair in this present study was lower as compared to the values given by Abd El-Hady et al. (2006). El Ashry et al. (2012 reported the similar Mn concentration of cow manure as compared to this work. Our work for cow feces Mn concentration was found in accordance with the reported work by Raghu (2013). Numerous clinical effects of supplement diet enriched with Mn cause increase in bulk and reduction in growth, nervous alterations, anemia, intestinal lesions, triglyceride and cholesterol, and increase plasma Mn concentration (McDowell 1992).

Discussion
Our present investigation for Mn bio-concentration was found greater as compared to the Sakizadeh et al. (2016). Yang et al. (2014) gave the lower values for the Mn bioconcentration than present work. This current study was found in accordance with the recorded values of Yang et al. (2013).
The amount of Mn pollution load index was observed below as compared to the Harikumar and Jisha (2010). The PLI was found similarly lower than in this present work for Mn as compared to the investigations of Sukri et al. (2018). This value of PLI Mn was lower than those values given by Izah et al. (2017).
The enrichment factor of Mn was below than the narrated values given by the Ghazzal et al. (2020). The Mn enrichment factor was observed in accordance with values Mn (PLI) with Sukri et al. (2018). In present findings, the Mn pollution load index was found lower than the values given by Enuneku et al. (2017).
The present concentration of daily intake of metal was in accordance with narrated values by Ghazzal et al. (2020). The present findings for DIM for Mn were observed greater as compared to El Ashry et al. (2012). Our results for HRI manganese were lower as compared to Ghazzal et al. (2020). These present findings for Mn health risk index were greater than those given by Khan et al. (2018).

Conclusion
It was concluded that seasonal changes gave different fluctuating concentrations of metals and sites also gave fluctuating metal readings in soil-forage-animal continuum. In soil and forage samples collected from semi-arid environment, Mn concentration was found safe according to FAO/WHO. In animal samples, Mn was found toxic according to NRC standards. Bio-concentration factor was noticed greater than 2 while pollution load index, enrichment factor, daily intake metal, and health risk index for Mn was less than 1.
Author contribution XGE, FC, JM LS, and MHS were responsible for writing the manuscript. ZIK and KA and MS supervised the study. AA, ISM, RS, MM, and MHS were responsible for conducting the experiments and the data analysis. MN and MUFA, XGE, FC, and JM were responsible for analyzing and interpreting the data. All authors read and approved the final manuscript.
Funding This work was supported by the National Natural Science Foundation of China (Nos. 51974313 and 41907405) and the Natural Science Foundation of Jiangsu Province (BK20180641).
Data availability All data generated or analyzed during this study are included in this published article.

Declarations
Ethical approval The authors declare that the manuscript has not been published previously.
Ethical statement All the study protocols were approved by the institutional animal ethics committee, University of Sargodha (Approval No. 25-A18 IEC UOS). All the experiments performed complied with the rules of the National Research Council and all methods were performed keeping in view the ethical principles regarding animals' accordance with relevant guidelines and regulations.
Consent to participate All authors voluntarily to participate in this research study.
Consent to publish All authors consent to the publication of the manuscript.

Conflict of interest
The authors declare no competing interests.