Cobalt availability in the soil plant and animal food chain: a study under a peri-urban environment

Abstract Cobalt metal is considered as an essential trace element for the animals. Present investigation was undertaken in the peri-urban area to analyze the cobalt availability in animal food chain by using different indices. Cow, buffalo and sheep samples along with forage and soil samples were collected from the three different sites of District Jhang and analyzed through atomic absorption spectrophotometer. Cobalt values differed in soil samples as 0.315-0.535 mg/kg, forages as 0.127-0.333 mg/kg and animal samples as 0.364-0.504 mg/kg. Analyzed cobalt concentration in soil, forage and animal samples was found to be deficient in concentration with respect to standard limits. Soil showed the minimum cobalt level in Z. mays while maximum concentration was examined in the forage C. decidua samples. All indices examined in this study has values lesser than 1, representing the safer limits of the cobalt concentration in these samples. Enrichment factor (0.071-0.161 mg/kg) showed the highly deficient amount of cobalt enrichment in this area. Bio-concentration factor (0.392-0.883) and pollution load index (0.035-0.059 mg/kg) values were also lesser than 1 explains that plant and soil samples are not contaminated with cobalt metal. The daily intake and health risk index ranged from 0.00019-0.00064 mg/kg/day and 0.0044-0.0150 mg/kg/day respectively. Among the animals, cobalt availability was maximum (0.0150 mg/kg/day) in the buffaloes that grazed on the C. decidua fodder. Results of this study concluded that cobalt containing fertilizers must be applied on the soil and forages. Animal feed derived from the cobalt containing supplements are supplied to the animals, to fulfill the nutritional requirements of livestock.


Introduction
Heavy metal contamination is a major ecological threat to both terrestrial and aquatic life owing to their persistency and non-degradable nature.The physical and chemical interaction of metal with the natural components of environment either precipitates them or changes their oxidation state which affects the metal mobility and bioaccumulation pattern in environment (Yasotha et al., 2021).Metal availability in soil, water and plants has significantly influence the ruminants and human health.Therefore, heavy metal monitoring in the soil, plant and animal food chain was important to ascertain the human and ecological ramifications (Miclean et al., 2019;Saleh et al., 2019) Polluted irrigation water from industrial or municipal discharges have undesirable residues of zinc, vanadium, chromium, cadmium, nickel, arsenic, copper, uranium, lead, thorium and antimony to be deposited on soil which affect the vegetation production or contaminate the groundwater which threaten the human health (Ge et al., 2021).In ecological network, earth is an eventual dealer of all existing metals because it proceeds as a sink of heavy metals as well as a supplier to plants that impart considerable effect on the living biota.Soil properties can be determined by the nature of rock that undergoes weathering process.Rocks such as mafic and ultra-mafic can degrade the adjacent soil and crops because they have prominent extent of heavy metal quantity.Metals combined with particles of soil that are negatively charged, once disengage of metal take place, it enter into the soil solution and easily accessible to plants.Transmission of metal from soil to plants rely upon the various characteristics of soil such as presence of primal matter, clay content, the acidity or alkalinity of soil as well as category of metal (Aboubakar et al., 2021).
Heavy metals may be alienated into essential and non-essential elements in accordance with the demand of plant-animal-human system.For example, Iron (Fe), Molybdenum (Mo), Copper (Cu), Manganese (Mn) and Zinc (Zn) are important for plants-animals-human being.Selenium (Se) and Cobalt (Co) are important to animalhuman being.Nickel (Ni) and Chromium (Cr) are important to plants-human being.Lead (Pb), Mercury (Hg), Cadmium (Cd) and Arsenic (As) are not needed by any one organism and serve as a toxicant metals for all the living organisms (Alysson and Fabio, 2014).
Cobalt assessment in soil plant and animal system practices prefer alternative water resources such as pond water, rain water, municipal and industrial waste water to cope up with these alarming situations.Recently researchers cited that Zn, Cr, Mn, Fe, Cd and Pb entry from wastewater irrigated soil to plants and animal products which opens the heavy metal entry into human beings (Chen et al., 2022a, b;Ge et al., 2021Ge et al., , 2022;;Ejaz et al., 2022a, b).Therefore, aim of study is to evaluate the cobalt transfer from soil and forages to animal food chain.This study focused on the cobalt availability in buffalo, cow and sheep, and possible health risks in animals through intake of these forages.This study also assessed the deficiency or toxicity status of cobalt to grazing animals by applying various indices.

Study area
Present research was conducted in District Jhang which is located at a distance of nearly 40 km from Gojra and Toba Tek Singh, 76 km to Faisalabad city and 86 km from Chiniot (Figure 1).The temperature in this study area varies between 52-4.5 ο C. Approximately, 150-350mm is the rainfall annually and moisture (humidity) is about 32-80% (Khan et al., 2022).

Sampling locations
Three selected sites of this study were: Jhang (Jh-I) irrigated with ground-water, Shorkot (Sh-II) irrigated with canal-water and Ahmad Pur Sial (Aps-III) irrigated with sewage water, were selected in this research work.Soil, fodder plants and animal samples (blood, hair and feces) were collected randomly from these sampling locations.Sampling was done in the District Jhang during 2019-2020.

Soil sampling
Five replicates of each soil sample were collected from each site.Soil sample (100g) used to grow collected forages were sampled from 0-20cm depth.Firstly, these samples were dried in an open air and placed them at 75 o C in an oven for almost 3-4 days.Properly labeled them and stored in sealed bags (Chen et al., 2022c).

Forage sampling
Aerial parts of these forages were collected that were mostly consumed by the grazing animals.Five replicates of forage randomly collected from each site and make a composite sample (Table 1).Forage sample were rinsed with distilled water to eliminate all the dust contamination deposit on the surface of these plants.These sample were dried in open air and then in oven (70-75 o C) to remove all the moisture content.After oven drying process, forages were grinded into a fine powder and stored in sealedlabeled bags.

Animal sampling
Cow, Buffalo and Sheep were the selected animal categories in this study.10 animals from each category of selected animal were used to collect blood, hair and fecal samples.All the animals were about 3-5 years in age and mainly feed on these collected forages.
Blood (5ml) was sampled from the animal jugular vein into the test tubes.Blood plasma was separated by centrifuging the blood samples at 3500rpm (15 minutes) then stored at -20 o C for digestion process.Collected hair  samples from the animal body were washed with acetone to remove externally deposited contaminants and cut into small pieces.Both hair and fecal samples were air-dried and kept in oven for at least 3-4 days (Chen et al., 2022b).

Sample digestion
Two gram weighted each samples with a mixture of H 2 SO 4 and H 2 O 2 (2:1) was taken in a digestion flask.Heat this mixture in a digestion chamber for 30 minutes until yellow color of the solution appeared.Further, add H 2 O 2 in the digestion flask to completely digest the samples and finally a colorless solution was obtained in the flask.Allow this solution to cool down at a room temperature to filter it.Add distilled water in this filtered solution to make its final volume 50ml and stored it for next step (Ge et al., 2021).

Cobalt analysis
After digestion, all the sample solutions were examined in the atomic absorption spectrophotometer to evaluate the cobalt concentration in the respective samples.Cobalt concentration measured for solid samples was in mg/kg but the liquid samples were measured in mg/l.

Statistical analysis
The presented data was analyzed statistically with the help of Two-way-ANOVA and SPSS-20 software.Further, mean concentrations were also calculated in the collected samples.
2.9.Pollution Load Index (PLI) Liu et al. (2005) determined the heavy metal contamination in the soil by using PLI index (Equation 1): (M) SS = Metal concentration in soil sample (mg/kg) (M) RS= Metal reference value in soil as 9.07mg/kg (Singh et al., 2010)

Enrichment Factor (EF)
Buat-Menard and Chesselet (1979) mentioned the EF formula as (Equation 2):  4): C metal means conc. of metals in forages CF means conversion factor whose value was 0.085 (Jan et al., 2010).D food intake means daily food intake whose value for cows was 12kg, for sheep was 1.3kg (Johnsen and Aaneby, 2019) and 12.5kg was recommended for buffalo (Briggs and Briggs, 1980) BW= Average body weight of animals whose value was 550kg for buffalo (Briggs and Briggs, 1980), 600kg for cows and 75 kg in case of sheeps (Johnsen and Aaneby, 2019).

Soil analysis
According to the analysis of variance, the cobalt concentration demonstrated the non-significant effect in the all the sources of variation (p˃0.05)such as Soil, Site and Soil * Site (Table 2).The observed concentration of Co was between the ranged of 0.315-0.535mg/kg (Table 3).Highest concentration of this range was present in the soil of Cobalt assessment in soil plant and animal system Aps-III that raises P. glaucum while lowered concentration was present in the Z. mays soil of Jh-I (Figure 2).

Forage analysis
The analysis of variance pointed out the non-significant impact of cobalt on all the observed Site and Forage interactions (Table 2).The Co concentration in the forages was varied as 0.127-0.333mg/kg (Table 3).Highest concentration was accessed in the canal water irrigated Sh-II in which C. decidua was grown but the Z. mays presents lowest concentration in ground water irrigated, Jh-I site (Figure 3).

Animal analysis
The significantly affected variations for Co were noted in the Site, Animal, Site*Animal (p<0.01) and Site*Source treatments (Table 2).The observed reading for cobalt in animal blood was found between the 0.390-0.504mg/l (Table 4).At the Sh-II site, sheep blood showed the concentration of Co as maximum and minimum was located in the cow blood at Jh-I (Figure 4).Cobalt variation in the animal hair was ascertained between the limits of 0.372-0.477mg/kg (Table 4).Highest concentration was encountered in the buffalo reared on the Aps-III whereas Jh-I buffalo showed the lowered content of cobalt (Figure 4).Co content in fecal samples lied within the range of 0.364-0.504mg/kg (Table 4).The least amount of present range is revealed in the cows of Aps-III but the buffalo of Sh-II represented the highest concentration of cobalt (Figure 4).

Pollution load index
The observed PLI for Co concentration in all the sites were lowered than 1which highlighted that the sites are non-contaminated with this metal.The PLI concentrations expressed for cobalt were varied from 0.035-0.059mg/ kg.The Z. mays depicted the minimum concentration at Jh-I sampling point while higher PLI level for cobalt was existed in P. glaucum collected from Aps-III (Table 5).

Enrichment factor
The results of cobalt displayed the EF range within 0.071-0.161mg/kg.The minimum EF was noticed in A. nilotica sampled from the Sh-II but the C. decidua implied the maximum enrichment cultivated on the Sh-II (Table 5).

Bio-concentration factor
Analyzed BCF level for Co was fluctuated between the ranges of 0.392-0.883(Table5).Greatest BCF level of this range was present in the C. decidua of Sh-II while minimal value was analyzed in A. nilotica of the same site.

Daily intake of metal & health risk index
Cobalt concentration varied from 0.00019-0.00064mg/kg/day for the daily intake indices.Least daily intake value was perceived by the sheep of Jh-I whosever feed on Z. mays.Maximum daily intake was observed in the buffaloes that grazed on the C. decidua located in the Sh-II site.Health risk index value for the cobalt metal was assorted between the ranges of 0.0044-0.0150mg/kg/day.Highest HRI value was commenced by the buffaloes of Sh-II area while minimal risk of health index was noticed in the sheep animal that nurtured on the Z. mays forage cultivated at Jh-I (Table 6).FAO (2001FAO ( , 2000) ) suggested the standard values of cobalt as 100 mg/kg and 10mg/kg respectively in soil which were quite higher than present concentrations.These standards suggested that cobalt concentration in the soil samples was found within safe level.Wu et al. (2020) declared that Co amount in soil of control (6.85-7.18mg/kg)and experimental pastures (6.83-7.13mg/kg) of Weining.These reported concentrations are higher against our study while lesser concentration was regarded by Patel et al. (2019) in both industrial (0.019mg/kg) and nonindustrial area (0.017mg/kg).Numerous studies reported in the India (Sharma et al., 2018), Moa city (Rizo et al., 2011), Peshawar (Jan et al., 2010) and in Siberia (Syso et al.,  Cobalt assessment in soil plant and animal system 2017) showed elevated cobalt level as compared to this study.Alejandro et al. (2020) study corroborated that acidic nature of soil promotes the binding of cobalt with the manganese oxide which reduces its transportation to plants.Alengebawy et al. (2021) mentioned that level of Co, Zn, Pb and Cd in soil is enhanced by fungicidal, herbicidal and sewage water applications.That's why, current study area also showed greater concentration at Aps-III site due to these man-induced activities but within the standard limits.

Discussion
Cobalt concentrations in forages were placed within the acceptable limit of 52mg/kg, according to the FAO (2001) criteria.Alghobar et al. (2014) ascertained the greater cobalt level (7-8mg/kg) then studied values of this study.Patel et al. (2019) and Syso et al. (2017) mentioned the Co content as 0.219 & 0.1 mg/kg respectively that was found to be lowered than current study.Conversely, various studies conducted by Marijanusic et al. (2017) and Rizwan et al. (2019) showed higher content of Co than investigated results.Co level in plant was antagonistically effected by concentration of Mn and Fe concentration.Little amount of Co was absorbed by the plants because Ca 2+ competes with the binding site of cobalt and overcome their toxicity effects.On the other hand, formation of organic complexes also reduces its movement from root to the aerial parts (Lwalaba et al., 2020).Bisen et al. (2019) observed the elevated cobalt concentration in both cyclic (0.76-1.12mg/l) and non-cyclic buffalo blood (0.76-1.27 mg/l).Omoniwa et al. (2017) determined the lowered concentration (0.135mg/l) in animal serum of Zaria.In addition to this study, an increased Co concentration between 1.34-1.61mg/lwas noted by the Tahir et al. (2017) in blood samples.The emitted concentration of both Zn and Pb decreases the availability of cobalt (Duan et al., 2022).The decreased concentration of cobalt in blood was attributed the higher Zn and Pb level in the study area.2009) study, similar Co concentration observed in sheep wool of Poland (0.98mg/kg), lowered level was found in the sheep hair of Greece (0.65mg/kg) whereas greater Co content was revealed in the hair of sheep (2.31mg/kg) at Syria.. Demirel et al. (2013) measured the Co amount in animal feces as 0.10mg/kg that was lowered against this investigation.Ukpe and Chokor (2018) declared the mean Co value (0.43mg/kg) within the analyzed range of present results.This study also highlighted that Co content in animal wastes was significantly higher in relation to intake of metal through feed.These stated outcomes showed correspondence with the existing investigation.
Present PLI values were much lowered than reference level of Co (9.1mg/kg) given by Dutch standard (2000).Similarly, El-Alfy et al. (2017) studied the pollution load for cobalt in soil irrigated with Kitchener drain of Egypt (0.30mg/kg) that was compatible to current result.The studied range in this study was lowered than the PLI observed by Kalavrouziotis et al. (2012) and Likuku et al. (2013).Hussain et al. (2015) evaluated the value 3.008 as an average PLI for cobalt level that was considered to be much greater with respect to present analysis.Rizo et al. (2011) found least enrichment of Co in urban soil of Cuba.Ezemokwe et al. (2017) and Hussain et al. (2015) suggested the higher enriched value of Co (2.61 &1.53mg/kg respectively) in their studied samples with respect to current investigation.Similarly, Likuku et al. (2013) also analyzed the higher enriched value (0.28-2.11) in Selebi Phikwe Region of Botswana.With respect to Barbieri (2016) standards, studied range represented the highly deficient amount of Co enrichment in the study area.
BCF is actually the comparison of heavy metal content absorb by plants to their amount present in soil.Elamin and Saeed (2019) evaluated the soil to plant transfer of Co in three different irrigating systems: 0.62mg/kg in  -Vila et al. (2015).Different studies also stated that Co and Ni were poorly transferred from root to the plant shoot.Therefore, all the BCF values were less than 1 and showed that plants cannot accumulate the cobalt concentration in their aerial parts.
As compared to the present range of Co daily intake, a higher concentration was calculated by Khan et al. (2019) in the buffaloes (0.001-0.03 mg/kg/day).Roggeman et al. (2013) calculated the Co intake in cows that varied from 0.1-0.9mg/kg/day and found to be higher than the investigated Co intake in different animals.Khan et al. (2019) study on buffaloes estimated the HRI values as 0.240-0.697mg/kg/day that was considered to be greater than observed HRI of animals in this research.In this study, the measured HRI level for cobalt was considered to be much lowered than HRI standards specified by USEPA (2002).

Conclusion
In this work, cobalt concentration was varied within sites and animal samples.The cobalt concentration available to animals is occupied by the forage intake as well as the presence of antagonistic metals (zinc & lead).Research work concluded that as soil and forages samples are deficient of cobalt metal.Therefore, minimum amount of this metal is transferred from soil and forage to animals and deficiency of cobalt level was prevailed in the studied animal samples.The poor absorption of cobalt in the animal blood exposed the lead and zinc toxicity in the study area.Study suggested that precautionary measures regarding the emissions of antagonistic metals must be carried out in the present area and to fulfill the nutritive value of farm animals and their products, cobalt containing fertilizers are recommended for the soil/forage system while a high quality animal feed containing cobalt supplements must be accomplished.

Figure 1 .
Figure 1.Map of study area.
cobalt in soil and plants are 9.1mg/kg (Dutch Ministry of Housing, Spatial Planning and Environment, 2000) and 50mg/kg (FAO, 2001) respectively 2.11.Bio-Concentration Factor (BCF) Cui et al. (2004) gave the BCF index to examine the metal transfer from soil to forage tissues by following formula (Equation 3): represents the analyzed metal concentration in forage samples.(M) Soil represents the analyzed metal concentration in soil samples.Daily intake of metals (DIM) To determine DIM values, equation was (Equation

Figure 2 .
Figure 2. Cobalt concentration in soil samples with respect to various sites.

Figure 4 .
Figure 4. Cobalt concentrations in different animal samples with respect to various sites.
Vorobyov et al. (2019) also stated the higher concentration of Co metal in sheep and goat hairs (4.25 & 3.16mg/kg) of Volga region.Against Patkowska-Sokoła et al. (

Table 1 .
Enlist of collected forage samples.

Table 2 .
ANOVA table for cobalt availability in sampled soil, forage and animal samples.

Table 3 .
Mean Co concentration (mg/kg) in soil and forage samples with respect to various sites (Mean±S.E).
Figure 3. Cobalt concentration in forage samples with respect to various sites.

Table 4 .
Mean Co concentration (mg/kg) in animal samples with respect to various sites (Mean±S.E).

Table 5 .
PLI, EF and BCF for Co concentration with respect to various sites.

Table 6 .
Taha et al. (2013)ha (2015)k index of Co metal in animals with respect to various sites./kg in treated waste water + River Nile water source.All observed concentration of these three treatments was appeared to be within the investigated range.Alghobar and Suresha (2015)appraised the Co transfer in plant as 0.50 in all sources of irrigation (ground water & waste water) which was lowered than observed range of existing study.Similarly, lesser concentration (0.023-0.066) was also reported by theTaha et al. (2013)in the plant Sorghum color of Sudan country.An elevated accumulation of cobalt in sampling sites (0.56-4.95) was measured by the study of Lago