405 1Department of Veterinary Pharmacology and Toxicology, Federal University of Agriculture, Makurdi, Nigeria. 2Gan-Rovet Animal Hospital, Warri, Nigeria. 3Department of Veterinary Physiology and Biochemistry, Federal University of Agriculture, Makurdi, Nigeria. 4Department of Veterinary Medicine, Federal University of Agriculture, Makurdi, Nigeria. *Corresponding author at: Gan-Rovet Animal Hospital, Warri, Nigeria. E-mail: kshimaelyx@gmail.com. Fidelis Aondover Gberindyer1, Felix Kundu Shima2*, Victor Masekaven Ahur3, Solomon Tsekohol Agu3, Thaddaeus Ternenge Apaa4 and Matthew Terzungwe Tion4* Keywords Dog, Environmental pollutants, Exposure, Health risk, Potentially toxic metals. Veterinaria Italiana 2022, 58 (4), 405‑411. doi: 10.12834/VetIt.2464.17442.3 Accepted: 20.04.2022 | Available on line: 31.12.2022 Summary Environmental pollutants pose a health risk to animals and humans. We evaluated levels of some potentially toxic metals in environmental dust, blood, and hair samples of apparently healthy security dogs from a crude oil well drilling site (A) and liquefied natural gas production site (B) industrial environments in Nigeria. These samples were routinely digested and analyzed for lead, cadmium, nickel, chromium, and zinc using atomic absorption spectrophotometry assay. Mann‑ Whitney U test was used to compare concentrations of the metals in different samples. Dust samples contained a high amount of the metals considered. There was no significant difference between levels of heavy metals in blood and hair samples from dogs guarding both sites, except for blood (p=0.034) and hair (p=0.015) chromium which were higher in those securing site A compared with site B. Higher nickel (p=0.001) and zinc (p=0.001) with lower chromium (p=0.004) levels occurred in the hair samples than in the blood. Lead was not detected in blood and hair samples suggesting safety. There was no correlation between the same metal in blood and hair. Hair chromium and nickel levels were above the reference suggesting toxic exposure. There is a need for regular monitoring and decontamination of air pollutants within similar facilities for environmental safety. Please refer to the forthcoming article as: Gberindyer F.A. et al. 2022. Potentially toxic metals in dust, blood, and hair in exposed security dogs in an oil and gas industry. Vet Ital. doi: 10.12834/VetIt.2464.17442.3 Potentially toxic metals in dust, blood, and hairs from exposed security dogs in an oil and gas industry Introduction Heavy metals are defined as metals and metalloids having densities >5 gcm‑3 (Hawkes 1997). Heavy metals and metalloids are often used directly or indirectly at home, in agriculture, medicine, technology, and industries (Hawkes 1997). They are found naturally in the environment, certain foods, medicines, and water (Hawkes 1997; Zhuang et  al. 2009; Renner 2010; Vodyanitskii 2016; Nworu et al. 2019). Their wide distribution in the environment has potential effects on the environment, plant, animal, and human health (Slotnick et al. 2000; McBride et al. 2003; Zhuang et  al. 2009, Orisakwe et  al. 2012). Some heavy metals considered to be potentially toxic are required by the body in trace quantities for biochemical and physiological processes (Singh et al. 2018; Pourret and Hursthouse, 2019). However, at higher concentrations and depending on the dose, route of exposure, chemical speciation, age, gender, and nutritional status of the exposed animal or human being, they can have deleterious effects (Jaishankar et al. 2014; Pourret and Hursthouse, 2019). Animals and humans get exposed through ingestion of contaminated food, drinking of Levels of potentially toxic metals in occupationally exposed dogs Gberindyer et al. 406 Veterinaria Italiana 2022, 58 (4), 405-411. doi: 10.12834/VetIt.2464.17442.3 Materials and Methods Sampling and sample preparation This study was carried out on dogs used for security purposes in crude oil well drilling (site A) and liquified natural gas production (site B) in Nigeria. Blood (5 ml) and hair (2 g) samples opportunistically collected from 13 dogs (in July 2016) from blood used for health parameters evaluation, i.e., for routine clinical and laboratory screening were used for some heavy metal analysis. Technically, no animal was specifically sampled for this study. Ethical approval from the University of Ibadan Animal Care Use and Research Ethical Committee (UI‑ACUREC/ App/2015/027) was obtained for this study after reviewing the panel and the listed guidelines and principles of animal handling and care were strictly adhered to during and after sampling. The dogs sampled were all males, with a mean age of 9.7±1.4 years (range 8.0 ‑ 13.0), and a mean body weight of 33.2±5.3 kg (range 18.0 ‑ 38.0) which have spent at least 7 years in that environment. Also, environmental dust within the vicinity of the kennels (sites A and B) was collected for heavy metal analysis. Dust was collected manually by using clean dry cotton wool and plain paper to gather dust on windows and dry hard surfaces in the kennels. Dust samples were collected from each of the 16 and 10 kennel partitions from site A and site B, respectively. Subsequently, the dust samples from each site were pooled together into two sets before the levels of metals were quantified. All the samples collected were labeled appropriately and stored for further processing. Blood samples collected in heparinized bottles were stored at ‑20°C until heavy metals were quantified. The hair samples were repeatedly washed in the laboratory with metal‑free distilled water, properly rinsed and dried in a special drying oven, and kept in a humid‑free plastic container at room temperature before digestion and heavy metals analysis. The samples were digested following a standard procedure (Metals and Others, Method 971.21, Chapter 9) as described by the Association of Official Analytical Chemists (AOAC, 2000) in a laboratory shared by Veterinary Physiology, Biochemistry, Pharmacology, and Toxicology units of the Federal University of Agriculture, Makurdi, Nigeria. We measured 1ml of blood, 1g of hair, and 1g of the dust samples and digested them in three separate reactions with 2 ml of HNO 3 , HClO4 , and H 2 SO 4 mixture in a ratio of 3:1:1 (v/v/v), and microwaved in a closed container until fumes became clear. Subsequently, the volume was made up to 50 ml with deionized water and stored in clean plastic test tubes for metal analysis. contaminated water, inhalation of contaminated air, exposure to contaminated soils and industrial wastes, and absorption through the skin (Airey 1983; Alexander and Davidson 2006; Babalola et al. 2007; Renner 2010; Sharma et al. 2016). High blood levels of heavy metals imply high exposure levels, while lower levels of trace elements and essential minerals represent insufficient intake which may be a sign of nutritional deficiency (Flora et al. 2008). A whole blood level of heavy metals is representative of extracellular and intracellular metal concentrations, while plasma and serum concentrations depict extracellular concentrations (Flora et al. 2008). Heavy metal toxicity is primarily due to oxidative damage to the biological macromolecules following the binding of metals to the DNA and nuclear proteins (Flora et al. 2008). Consequently, this distorts the normal functions of the brain, lungs, kidneys, liver, and hematopoietic organs (Broun et al. 1990; Järup 2003; Dorne et  al. 2011). Some of the degenerative conditions associated with heavy metal toxicity are Parkinson’s and Alzheimer’s diseases among others (Flora et al. 2012). Chronic exposure to some of these potentially toxic metals could lead to varying types of cancers, while some metallic elements induce multiple organ damage even at lower levels of exposure (Aquino et al. 2012; Chervona et al. 2012). Some common heavy metals with potentially high occupational and environmental adverse effects are mercury (Hg), cadmium (Cd), lead (Pb), copper (Cu), vanadium (V), chromium (Cr), cobalt (Co), nickel (Ni), selenium (Se), arsenic (As), manganese (Mn), silver (Ag), zinc (Zn), and uranium (U) (Jaishankar et al. 2014; Vodyanitskii 2016). In Nigeria, crude oil and gas exploration, exploitation, and production, as well as gas flaring are common sources of environmental pollution (Nworu et al. 2016). Crude oil and gas pollution occurs in form of spillage from accidental discharges, corrosion of pipelines, oil well blowout, oil pipeline vandalism, and gas flaring. Dogs are used to support securing many of the crude oil and gas production facilities in Nigeria and are exposed to the environmental pollutants associated with these anthropogenic activities just as human beings working or living within those locations. Also, the pathophysiology of the adverse effects of these toxic metals in animals and human beings are similar (Airey 1983; Jaishankar et al. 2014; Santin et al. 2005). Animals are valuable sentinel for environmental contamination; however, they are often ignored. Consequently, this study was designed to assess the levels of some potentially toxic heavy metal elements in environmental dust, blood, and hair of dogs used for securing a crude oil well drilling and liquefied natural gas production facility, generally regarded as one of the environments at higher risk of elemental pollutants. Gberindyer et al. Levels of potentially toxic metals in occupationally exposed dogs Veterinaria Italiana 2022, 58 (4), 405-411. doi: 10.12834/VetIt.2464.17442.3 407 tissue samples to check if any relationship existed between blood and hair metals concentrations was applied. A statistical test of significance was set at p<0.05. Results Results (Table I) showed a higher level of Pb, Cr, and Ni in the dust samples from the kennel located within site B compared to site A where crude oil well drilling activities take place. On the other hand, dust samples from the kennel in site A contained a higher level of Cd and Zn as compared with dust samples from site B. Furthermore, there was no significant difference between the mean amount of heavy metals in blood and hair samples from dogs securing sites A and B, except for blood (p=0.034) and hair (p=0.015) Cr levels which were significantly higher in dogs guarding site A compared with those in site B. The level of Cr in the hair and blood of the dogs guarding site A were 1.6‑ and 1.7‑folds, respectively higher than the mean level in those operating within site B. Regarding the body tissues, significantly higher levels of Ni (p=0.001) and Zn (p=0.001) were observed in the hair than in the blood samples. No amount of Pb was detected in the hair and blood samples from the investigated dogs. Correlation analysis between blood and hair metal levels showed no connection between the same metal in blood and hair. However, some interesting correlations observed were that between blood Cr and hair Cd (r= 0.64; p=0.045); and that between Cr and Zn in the blood (r= 0.57; p=0.041). Analyses of heavy metal concentrations in the samples The levels of Pb, Cd, Cr, Ni, and Zn in the dust, hair, and blood samples were determined employing atomic absorption spectrophotometer (Buck Scientific‑210VGP AAS, Norwalk, CT, United States) in the Department of Soil Science, University of Ibadan, Nigeria. Standard salt preparations for each metal were used to calibrate the AAS machine. Limits of detection for the analyzed samples, expressed as a wet weight (w/w) were ‑ Cd 0.00024 mg/kg, Cr 0.0015 mg/kg, Pb 0.013 mg/kg, Ni 0.004 mg/kg, and Zn 0.014 mg/kg for both hair and dust. Those of blood were ‑ Cd 0.00013 mg/L, Cr 0.0021 mg/L, Pb 0.002mg/L, Ni 0.0041mg/L, and Zn 0.053mg/L. Data analysis The reading obtained from the AAS was multiplied by the dilution factor (50) to obtain the actual amount of heavy metal in each of the samples. Statistical analysis was performed with SPSS v20 Program. The normality of data was assessed by applying the Kolmogorov‑Smirnov test. As the data were not normally distributed, non‑parametric statistics were applied. Mann‑Whitney U test statistic by ranks on blood and hair results of the sampling sites (A and B) was considered. Because the data were not normally distributed, the geometric mean of the quantified metals was computed to take care of outliers in order to avoid overestimating the means or skewness of the mean values towards higher values. Furthermore, a correlation test within the Table I. Heavy metals mean values ± SD (ppm) in kennel dust, hair and blood of dogs from crude oil well drilling (site A) and liquified natural gas production (site B). Different letters in the same column means statistical difference {a,b(p=.015), a,c(p=.034) for site; a,d(p=.001), a,e(p=.004), a,f(p=.001)}. Statistical comparison was not applied to the dust samples since they were pooled together before elements quantification; LOD denotes limit of detection. Site Mean + SD Pb Cd Ni Cr Zn Dust (ppm) A 109.0 1.7 89.3 128.6 4620.0 B 300.0 0.1 304.3 248.1 3095.0 Hair (ppm) A (n = 7) < LOD 0.05) difference in the levels of heavy metals in blood and hair samples of dogs from the two sites investigated, except for Cr which was observed to be significantly higher in the hair (p=0.015) and blood (p=0.034) of dogs guarding site A compared with those in site B. The high levels of Cr in the hair and blood of dogs from site A could be attributed to the higher exposure level due to anthropogenic or crude oil well drilling activities which release these pollutants into the atmosphere and consequently settle as dust and precipitations. Naturally, Cr is found in high concentrations in activities involving the burning of oil, coal, and crude oil well drilling (Jaishankar et al. 2014). This result also revealed a significant difference in the levels of Ni (p=0.001), Cr (p=0.004), and Zn (p=0.001) (all trace elements) in hair and blood samples from dogs kept in the two units of the company. The levels of Ni and Zn were about 1.8‑ and 2.2‑fold, Discussion Heavy metals are environmental pollutants that cause harmful effects in animals, and humans following absorption into the body through soft tissues above the specified permissible levels (Flora et al. 2012; WHO, 2020). The most common metals that the human body could absorb in toxic amounts are Hg, V, As, Pb, and Cd (Jaishankar et al. 2014; Renner 2010; Flora et al. 2012). High levels of exposure to these heavy metals could be from contaminated food, air, water, medicine, food containers with improper coating, and industrial exposures (Zhao et al. 2009; Zhuang et al. 2009; Renner 2010; Flora et al. 2012; Wei et al. 2015; Sharma et al. 2016; Singh et al. 2018). In this study, we evaluated levels of Cd, Cr, Ni, Pb, and Zn in the dust from the kennels as well as blood and hairs of some dogs used for security purposes in the oil and gas industry which are zones with potentially high levels of elemental pollutions. This study revealed that dust, hair, and whole blood samples obtained from the dog kennels and dogs guarding the crude oil drilling and liquified natural gas‑producing areas contained varying amounts of Cd, Ni, Cr, and Zn. However, Pb was not detected in any of the blood and hair samples that were analyzed, except for dust samples. The high enrichment of dust with heavy metals observed in this study could be explained by the anthropogenic activities, gas flaring, and non‑biodegradability of these metals in the dust medium (Wei 2015; Nworu et al. 2016). There is no reported safe or beneficial level of Pb in blood and other biological tissues for animals or humans (Renner 2010; WHO 2020; Flora et al. 2012). Therefore, the non‑detection of Pb in the blood and hair of the investigated dogs also suggests that they could be safe from Pb poisoning and perhaps those working in that environment at the period within which the samples were obtained. The hair tissue mineral analysis showed that the amount of Cr (3.9 ± 1.5 ppm), Ni (20.6 ± 1.5 ppm), and Zn (110.3 ± 1.9 ppm) recovered from the blood and hairs of the dogs investigated were well above the reference ranges of 0.02 ‑ 0.08 ppm, 0 ‑ 0.1 ppm, and 10 ‑ 21 ppm, respectively considering animal and human reference values (Puls 1994; Renner 2010; Flora et al. 2012). Natural sources of Cr are the burning of oil and coal as well as crude oil well drilling activities (Jaishankar et al. 2014). The later natural source is one of the major activities in the study area which probably explains the basis for the high level of Cr recorded in the hair samples. Two forms of Cr exist, Cr (III) and Cr (VI) also referred to as hexavalent Cr which is highly soluble, extremely toxic to animals and humans and predominates in the environment (Cervantes et al. 2001). Even though Cr is essential for many biological functions, when the concentrations in the body are above the safe level, it reacts with the Gberindyer et al. Levels of potentially toxic metals in occupationally exposed dogs Veterinaria Italiana 2022, 58 (4), 405-411. doi: 10.12834/VetIt.2464.17442.3 409 of these binding sites following chronic exposure, leading to a plateau in metals concentrations and consequently the lack of correlation with blood levels (Patra et al. 2007). This study was limited by small sample size as only 13 dogs were kept in these facilities. Conclusions This study showed that non‑invasive analysis of Ni and Zn using hair samples from animals could be used to predict previous exposures, environmental pollution, and the nutritional metabolic activity that has occurred within a given period. Non‑detection of Pb and a negligible amount of Cd in hair and blood samples from dogs in the study area suggest environmental safety from these metals in this location within the period considered. The observed higher levels of Cr and Ni in hair samples above the reference values instead suggest the high level of exposure of security dogs and by extension, workers in this industrial area to these environmental pollutants. Consequently, there is a need for routine monitoring and decontamination of potentially toxic environmental metal pollutants for the safety of aquatic lives, security dogs, and workers within the vicinity of related industries. Acknowledgements The authors appreciate Gan‑Rovet Animal Hospital, Warri for the opportunity given to one of the leading authors that resulted in this study. respectively higher in the hair samples than what was obtained in the blood samples. Inversely, no significant difference (p>.05) was observed for Cd levels in blood and hair samples. Mineral and heavy metal contents of the hair are considered as the spillover from what is in the body. Essential elements and toxic heavy metals are sequestered into the hair from the follicular cells and their blood supply as part of the detoxification mechanism to prevent the expression of their adverse biological effect, thus implying that hair element analysis is a valid means for screening mineral deficiencies and toxic element exposures (Airey 1983). Correlation analysis between blood and hair metal levels showed no connection between the same metal in blood and hair. This could suggest differences in the kinetics of the metals considered (Zaccaroni et al. 2014). Also, it is established that mineral and heavy metal contents of the hair are the spillover from what is in the body (Airey 1983). The existing study recorded a correlation between the same metal (Ni and Cr) in hair and blood (Zaccaroni et al. 2014). Two remarkable correlations were that between blood Cr and hair Cd (r=0.64, p=0.045), and that between Cr and Zn in the blood (r=0.57, p=0.041). However, the explanation for these connections remains elusive and warrants further investigation. The lack of correlation between blood and hair Cd and Pb is consistent with existing reports (Gyori et al. 2005; Patra et al. 2007; Zaccaroni et al. 2014). Hair is a keratin‑rich tissue, with abundant sulfhydryl groups which bind divalent cations such as Pb and Cd, leading to their persistence in hair (Hasan et al. 2004). This lack of correlation between blood and hair Pb and Cd may be linked to the saturation Levels of potentially toxic metals in occupationally exposed dogs Gberindyer et al. 410 Veterinaria Italiana 2022, 58 (4), 405-411. doi: 10.12834/VetIt.2464.17442.3 Airey, D., 1983. Mercury in human hair due to environment and diet: a review. Environ Health Perspect, 52, 303‑316. https://doi.org/10.1289/ ehp.8352303. Alexander, T.H. & Davidson, T.M. 2006. Intranasal Zinc and Anosmia: The Zinc‐Induced Anosmia Syndrome. The Laryngoscope 116, 217‑220. https:// doi.org/10.1097/01.mlg.0000191549.17796.13. 21. AOAC (Association of Official Analytical Chemists), 2000. Official Methods of Analytical Chemists., Metals and Others, Method 971.21, Chapter 9, p.35 18th ed., Washing D.C. Aquino, N.B., Sevigny, M.B., Sabangan, J. & Louie, M.C., 2012. The role of cadmium and nickel in estrogen receptor signaling and breast cancer: metalloestrogens or not? J Environ Sci Health C Environ Carcinog Ecotoxicol Rev 30, 189‑224. https:// doi.org/10.1080%2F10590501.2012.705159. Babalola, O.O., Adekunle, I.M., Okonji, R.E., Ejim‑Eze E.E. & Terebo, O. 2007. Selected Heavy Metals in Blood of Male Nigerian Smokers. Pak J Biol Sci 10, 3730‑3733. Broun, E.R, Greist, A., Tricot G. & Hoffman, R 1990. Excessive zinc ingestion. A reversible case of sideroblastic anemia and bone marrow depression. J Am Med Assoc 264, 1441‑1443. Cervantes, C., Campos‐Garcia, J., Devars, S., Gutierrez‐ Corona, F., Loza‐Tavera, H., Torres‐Guzman, J.C. & Moreno‐Sanchez, R., 2001. Interactions of chromium with microorganisms and plants. FEMS Microbiol Rev 25, 335‑347. https://doi. org/10.1111/j.1574‑6976.2001.tb00581.x. Chervona, Y., Arita, A. & Costa, M., 2012. Carcinogenic metals and the epigenome: understanding the effect of nickel, arsenic, and chromium. Metallomics 4, 619‑27. https://doi.org/10.1039/ c2mt20033c. Das, K.K., Das, S.N. & Dhundasi, S.A., 2008. Nickel, its adverse health effects and oxidative stress. Indian J Med Res 128, 412‑425. PMID: 19106437. Dorne, J.L.C.M., Kass, G.E.N., Bordajandi, L.R., Amzal, B., Bertelsen, U., Castoldi, A.F., Heppner, C., Eskola, M., Fabiansson, S., Ferrari, P., Scaravelli, E., Dogliotti, E., Fuerst, P., Boobis, A.R. & Verger, P. 2011. Human risk assessment of heavy metals: principles and applications. In: Sigel, A., Sigel, H. & Sigel, R.K. (eds) Metal ions in life sciences. Royal Society of Chemistry, pp 27‑60. Flora, G., Gupta, D. & Tiwari, A. 2012. Toxicity of lead: A review with recent updates. Interdiscip Toxicol 5, 47‑58. https://doi.org/10.2478%2 References Fv10102‑012‑0009‑2. Flora, S.J.S., Mittal, M. & Mehta, A. 2008. Heavy metal induced oxidative stress and its possible reversal by chelation therapy. Indian J Med Res 128, 501‑ 523. PMID: 19106443. Girodon, F., Galan, P., Monget, A., Boutron‑Ruault, M., Brunet‑Lecomte, P., Preziosi, P., Arnaud, J., Manuguerra, J. & Hercberg, S.; the MIN.VIT. AOX. geriatric network, 1999. Impact of trace elements and vitamin supplementation on immunity and infections in institutionalized elderly patients: A randomized controlled trial. Arch Intern Med 159, 748‑754. https://doi.org/10.1001/ archinte.159.7.748. Gyori, Z., Kovacs, B., Daniels, P., Szabo, P. & Phillips, C. 2005. Cadmium and lead in Hungarian porcine products and tissues. J Sci Food Agric 85, 1049‑ 1054. Hasan, M.Y., Kosanovic, M., Fahim, M.A., Adem, A. & Petroianu, G. 2004. Trace metal profiles in hair samples from children in urban and rural region of the United Arab Emirates. Vet Hum Toxicol 46, 119121. Hawkes, J.S., 1997. What is a “heavy metal”. J Chem Educ 74, 1374. https://doi.org/10.1021/ ed074p1374. Jaishankar, M., Tseten, T., Anbalagan, N., Mathew, B.B. & Beeregowda, K.N., 2014. Toxicity, mechanism and health effects of some heavy metals. Interdiscip Toxicol 7, 60‑72. https://doi. org/10.2478/intox‑2014‑0009. Järup L., 2003. Hazards of heavy metal contamination. Br Med Bull 68, 167‑182. McBride, K., Slotnick, B. & Margolis, F.K. 2003. Does intranasal application of zinc sulfate produce anosmia in the mouse? An olfactometric and anatomical study. Chem Senses, 28, 659‑670. https://doi.org/10.1093/chemse/bjg053. Nworu, J.S., Aniche, D.C., Ogbolu, B.O. & Olajide, A.J. 2019. Levels of heavy metals from selected Soils in crude oil mining residences of Niger Delta. Int J Sci Eng Res, 10, 692‑701. Orisakwe, O.E., Nduka, J.K., Amadi, C.N., Dike, D.O. & Bede, O. 2012. Heavy metals health risk assessment for population via consumption of food crops and fruits in Owerri, South Eastern, Nigeria. Chem Cent J 6, 77‑83. Patra, R.C., Swarup, D., Naresh, R., Kumar, P., Nandi, D., Shekhar, P., Roy, S. & Ali, S.L. 2007. Tail hair as an indicator of environmental exposure of cows to lead and cadmium in different industrial areas. Gberindyer et al. Levels of potentially toxic metals in occupationally exposed dogs Veterinaria Italiana 2022, 58 (4), 405-411. doi: 10.12834/VetIt.2464.17442.3 411 Ecotoxicol Environ Saf 66, 127‑131. Pourret, O. & Hursthouse, A., 2019. It’s time to replace the term “heavy metals” with “potentially toxic elements” when reporting environmental research. Int J Environ Res Public Health 16, 44‑46. https://doi.org/10.3390/ijerph16224446. Puls, R., 1994. Mineral Levels in Animal Health. second ed. Clearbrook, British Columbia. Renner, R., 2010. Exposure on tap: Drinking water as an overlooked source of lead. Environ Health Perspect 118, A68‑A74. https://doi. org/10.1289%2Fehp.118‑a68. Santin, F., Stelletta, C. & Morgante, M. 2005. Utilizzo degli animali domestici nella valutazione dei rischi di inquinamento ambientale: indagini epidemiologiche e studi sperimentali. Le Point Veterinaire 412, 416. Sharma. A., Katnoria, J.K. & Nagpal, A.K. 2016. Heavy metals in  vegetables: screening health risks involved in  cultivation along  wastewater drain and  irrigating with  wastewater. SpringerPlus 5, 488. DOI 10.1186/s40064‑016‑2129‑1 Singh, H., Kushwaha, A. & Shukla, D.N. 2018. Assessment of eco‑environmental geochemistry of heavy metals pollution of the river Gandak, a major tributary of the river Ganga in Northern India. AIP Conf Proc 1952, 020038. https://doi.org/ 10.1063/1.5032000. Slotnick, B., Glover, P. & Bodyak, N. 2000. Does intranasal application of zinc sulfate produce anosmia in the rat? Behav Neurosci 114, 814‑829. PMID: 10959540. Stohs, S.J. & Bagchi, D. 1995. Oxidative mechanisms in the toxicity of metal ions. Free Radic Biol Med 18, 321,336. https://doi.org/10.1016/0891‑ 5849(94)00159‑h. Vodyanitskii, Y.N., 2016. Standards for the contents of heavy metals in soils of some states. Ann Agrar Sci 14, 257‑263. https://doi.org/10.1016/j. aasci.2016.08.011. Wei, X., Gao, B., Wang, P., Zhou, H. & Lu, J., 2015. Pollution characteristics and health risk assessment of heavy metals in street dusts from different functional areas in Beijing, China. Ecotoxicol Environ Saf, 112, 186‑192. D https://doi. org/10.1016/j.ecoenv.2014.11.005. WHO (World Health Organization), 2020. Lead poisoning and health. https://www.who.int/ news‑room/fact‑sheets/detail/lead‑poisoning‑ and‑health; accessed on 23/05/2020 [accessed 25 May 2020]. Zaccaroni, A., Corteggio A., Altamura, G., Silvi, M., Di Vaia, R., Formigaro C. & Borzacchiello, G. 2014. Elements levels in dogs from ‘‘triangle of death’’ and different areas of Campania region (Italy). Chemosphere 108, 62‑69. Zhao, J., Shi, X., Castranova, V. & Ding, M., 2009. Occupational toxicology of nickel and nickel compounds. J Environ Pathol Toxicol Oncol 28, 177‑208. https://doi.org/10.1615/ jenvironpatholtoxicoloncol.v28.i3.10. Zhuang, P., Zou, B., Li, N.Y. & Li, Z.A. 2009. Heavy metal contamination in soils and food crops around Dabaoshan mine in Guangdong, China: implication for human health. Environ Geochem Health 31, 707‑715.