Health risk assessment of toxic metals and DNA damage in somatic and germ cells by soil and groundwater of a major cement factory in Nigeria

Abstract

The waste generated from cement manufacturing is an important source of heavy metal contamination of groundwater and soil. This study investigated the concentration of toxic metals in the soil of a major cement factory and nearby groundwater. Ecological and carcinogenic risks of the metals were calculated. Potential reproductive toxicity and genotoxic effects of the samples were assessed in the sex and somatic cells of male mice using sperm abnormalities and bone marrow micronucleus (MN) assays, respectively. Also, the serum ALP, ALT, AST, total testosterone (TT), luteinizing hormone (LH), and follicle-stimulating hormone (FSH); and liver SOD and CAT activities were measured in the treated mice. Cr, Cu, Ni, Zn, Mn, Cd, and Pb levels in the soil and groundwater exceeded the allowable maximum standard. Ingestion and dermal contact were the most probable routes of human exposure with children having about 3 times higher probability of exposure to the metals than adults. Ni, Pb, and Cr presented carcinogenic risks in children and adults. In the MN result, nuclear abnormalities in the studied mice especially micronucleated polychromatic erythrocytes increased significantly (P < 0.05). Compared to the negative control, the ratio of PCE/NCE showed the cytotoxicity of the 2 samples. Data further showed a significant increase in the serum ALP, AST, and ALT while the liver CAT and SOD activities concomitantly decreased in the exposed mice. Sperm morphology results showed that the samples contained constituents capable of inducing reproductive toxicity in exposed organisms, with alterations to the concentrations of TT, LH, and FSH. Toxic metal constituents of the samples were believed to induce these reported reproductive toxicity and genotoxic effects. These results showed the environmental pollution caused by cement factories and the potential effects the pollutants might have on exposed eukaryotic organisms.

Cement manufacturing is one of the human activities that release high quantities of toxic metals into soil, surface/groundwater, and eventually the biosphere (Adejoh 2016). The processes involved in cement manufacturing can frequently result in severe contaminations as the suppression of the released dust is relatively difficult. Cement, which is an important binding agent in the construction industry, is produced in large quantities worldwide. This worldwide production is on the increase as cement is one of the most demanded products in the world. With the growing construction demands, the crushing of limestone as well as cement bagging and transportation have increased the environmental emission of particulate matter. Due to the fine particle size of the dust, cement dust can travel over long distances and spread over large areas carried by wind and rain, thus increasing the atmospheric total suspended particulate matter (Nwibo et al. 2012), and later accumulate on and in soils, plants, and water bodies (Ogunkunle and Fatoba 2013; Stanley et al. 2014). Contamination by the deposited dust particles may be harmful to the ecosystem because they contain various toxic chemicals with potential chemical and physical environmental effects (Lafta et al. 2013). If the topsoil accumulates cement dust there is the possibility of adverse effects on the soil chemistry, vegetation, ground and surface waters, and human and animal health.

Cement industries have been documented to release a wide range of pollutants such as gases, particulate matter, and different toxic metals which are dangerous to the biotic environment, especially the plants, aquatic and terrestrial fauna and flora (Akinci and Çaliskan 2010). Thus, cement industries are identified as probable sources of toxic metals like copper (Cu), zinc (Zn), cadmium (Cd), chromium (Cr), nickel (Ni), manganese (Mn), and lead (Pb) (Wufem et al. 2016; Yadegarnia Naeini et al. 2019). Most of these toxic metals from cement dust originated from the raw materials used for production and are known to have detrimental effects even at low concentrations in living organisms (Kabata-Pendias and Mukherjee 2007). The extent of their impact is majorly determined by their biological accumulation, geoaccumulation, persistence, biomagnification, and potential harmfulness (Bozkurt et al. 2000; Pekey et al. 2004). Many factors influence the distribution of toxic metals in cement dust and these include rain, wind (Alloway 2013), availability of calcium ions, and soil-pH which potentially determines the movement, retention, adsorption, and solubility of the toxic metals (De Matos et al. 2001). Humans, through dermal contact, direct ingestion, and inhalation of dust become exposed to these pollutants in cement-contaminated soil and water (Shi et al. 2011), causing severe public health effects such as skeletal and cardiovascular diseases, neurotoxicity, and infertility (Knight et al. 1997).

Nigeria has many companies that are producing cement with the West African Portland Cement Company (WAPCO) in the forefront of the industry producing per annum cement of about 1.6 million tons (Tijani et al. 2005). The cement produced by WAPCO is composed of 60% to 70% calcium oxide, 1% to 3% sulfur trioxide, 0.1% to 4% magnesium oxide, 17% to 25% silicon oxide, 0.5% to 0.6% iron oxide, and 3% to 8% aluminum oxide, (Ade-Ademilua and Umebese 2007). Due to the continuous increase in demand for housing and other infrastructure in developing countries like Nigeria, there has been a significant increase in the demand for cement in these areas (Imbabi et al. 2012). In 2012, cement estimated to be 2.18 billion tons was produced worldwide (Song et al. 2016) and 4.3 billion tons in 2014 (Adeniran et al. 2019). Recently in Nigeria, there has been an increase in the production of cement from 28 million tons in 2013 to about 30.75 million tons (Adeyanju and Okeke 2019).

Reports (Kim et al. 2015; Lee et al. 2016) have shown that residents in the neighborhood of cement industries had complaints about water, soil, and air pollution with cement dust reported in thick layers on the roadsides, parked cars, and verandas, making inhalation of fresh air nearly impossible. Unfortunately, these residents are in most cases unaware of the potential public health effects of the constituents of this cement dust that they are continuously exposed to in their environment. Different studies (Ogunkunle and Fatoba 2014; Kim et al. 2015; Lee et al. 2016; Kolo et al. 2018; El-Sherbiny et al. 2019) on the environmental impact of cement industries located in/close to residential communities have been carried out, however, studies on the potential DNA damage in both somatic and germ cells induced by soil and water contaminated with cement are still very limited. Therefore, the present study quantified the level of toxic metals in soil and groundwater located close to a major cement company in Nigeria and calculated the ecological and health risks of the carcinogenic and toxic metals and the DNA damage caused by the contaminated soil and groundwater in both somatic and germ cells.

Materials and methods

The study area

The study area is the West African Portland Cement Company (WAPCO), also called Lafarge–Cement WAPCO, located at Sagamu (6°50′7°00′N; 3°45′4°00′E) in southwestern Nigeria. It was in 1978 fully operational and the production capacity stands at 900,000 tons/yr (Lafarge Cement WAPCO PLC 2011). The area is a humid tropical region (Adamson 1996) which characteristically has high rainfall, relative humidity, evapotranspiration, and annual temperature (Akanni 1992). It is controlled by the tropical continental air masses and maritime (Ilalokhoin et al. 2013) with altitudes ranging between 30 and 61 m above sea level. In Sagamu, the soil type is ferruginous (Ilalokhoin et al. 2013) and ferralitic (Aweto 1981).

Soil and groundwater sampling

Soil samples were collected randomly in the eastern, western, northern, and southern vicinity of Lafarge–Cement WAPCO factory at a depth of 0 to 15 cm (5 samples per cardinal point) in clean polyethylene bags. The samples were carried to the laboratory and mixed to get a composite sample of the site before analysis. Drinking water samples were collected during the rainy season (July) from a major groundwater (well) used by the community and stored in bottles that were previously autoclaved to prevent contamination and stored at 4°C. The groundwater was about 650 m away from the factory. The control soil and groundwater samples were obtained from an area 26 km away from WAPCO and without any history of cement contamination.

Sample preparation and leachate simulation

Debris and coarse materials were removed from the soil samples, air-dried for 20 d before grinding with pestle and mortar, and sieved (2 mm sieve) before analysis. A composite sample made up of ten grams of soil from each cardinal point of the factory was used to simulate the leachate using the category-A extraction procedure of the American Society for Testing and Materials after slight modifications (Bakare et al. 2007). In brief, distilled water (1,000 ml) and the dried soil sample (250 g) were mixed in a beaker and put on a shaker at 28 ± 1°C for 24 h. Thereafter, Whatman No. 42 (5 mm filter paper) was used to filter the sample after settling down for 30 min. The leachate was kept at 4°C till all experiments were completed.

Analysis of toxic metals and physicochemical parameters

Some physicochemical parameters were analyzed in the simulated leachate and groundwater samples such as color, sulfates (SO₄), pH, total dissolved solids (TDS), biological oxygen demand (BOD), nitrates (NO₃), and chemical oxygen demand (COD). Also, 7 selected toxic metals namely Cr, Cd, Ni, Pb, Zn, Mn, and Cu were analyzed. Briefly, each soil sample (1 g) and groundwater (100 ml) were measured in separate 250 ml clean Kjeldahl digestion flasks and aqua regia (1:3 concentrated nitric acid and conc. hydrochloric acid; 20 ml) was added before heating the mixture to near dryness in a fume cupboard until it became a slightly clear color solution. It was then cooled, filtered (Whatman No. 42) into a standard flask (50 ml), and made up to mark using distilled water. An Atomic Absorption Spectrophotometer was used to analyze heavy metals in the digests (Chauhan 2014), while the soil pH, texture, and conductivity were determined with a pH meter, hydrometer method, and conductivity meter, respectively.

Model for assessment of potential health risk

Health risk assessment is based on the possibility that any hazard associated with pollution by toxic metals could be realized in any population exposed to the toxicants (USEPA 2004; Kolo et al. 2018). We adopted USEPA’s model for assessment of health risk for soil screening guidance to estimate the extent of adults' and children’s metal exposure from soil in the surrounding Lafarge–Cement WAPCO factory. Supplementary Material 1 shows the risk assessment parameters employed in the study, while Supplementary Material 2 shows the cancer potency factors (CPFs) and reference doses (RfDs). There are 3 principal pathways through which humans are exposed to metals in soil: dermal absorption from opened skin (DI_derm), direct ingestion (DI_ing), and inhalation (DI_inh). The extent of exposure and the toxicity of the pollutants dictate the health risks that will be associated with metal contamination (Schuhmacher et al. 2004). Through the individual exposure pathway, separate daily intake of individual probable toxic metals can be used to estimate human exposure. For each of the pathways, the daily intake (DI) can be calculated in mg kg⁻¹d⁻¹ using the following equation (USEPA 2001; Kolo et al. 2018; Alabi et al. 2024):

where R_inh and R_ing are the inhalation and ingestion rates, respectively, whereas AT, PEF, ED, BW, EF, ABF, SAF, SA, and C are the average time for the non-carcinogenic effect, particulate emission factor, exposure duration, body weight, exposure frequency, dermal absorption factor for all metals, skin adherence factor for soil, exposed skin surface area, and the concentration of each metal in the sample, respectively (Supplementary Material 1). The equations for the lifetime average daily dose (LADD) for the inhalation pathway in mg/kg⁻¹d⁻¹, which will be used to assess the carcinogenic risks for Ni, Pb, and Cr for both adults and children were calculated thus (Xu et al. 2015):

where C is the metal concentration of the soil samples (mgkg⁻¹). Supplementary Material 1 shows the other parameters used for the calculation.

Risk characterization

The Hazard Quotient (HQ) is a dimensionless quantity of non-carcinogenic health risk used to express a systematic toxicity of individual metal in the soil by any exposed human being. Comparison of the reference dose (RfD) with the corresponding estimated daily intake of individual metal in a particular exposure pathway is usually used for assessing the probable risk of toxicity because of exposure to metals, and it is calculated thus:

The calculation of the human health risk caused by carcinogens is a dimensionless level of probability obtained by calculating “a person’s incremental probability of cancer development over a lifetime due to potential carcinogen exposure” (Gržetic and Ghariani 2008; Alabi 2024). It is usually expressed as:

where RfD is the chronic reference dose, CPF (mgkg⁻¹d⁻¹) is the cancer potency factor, and DI (mgkg⁻¹d⁻¹) is the daily intake obtained for each metal for a particular pathway using Equations (1)–(3) (Supplementary Material 2). During an individual’s lifetime, any value above the RfD means any daily human (together with sensitive subpopulations) exposure to any of the toxic metals through any of the exposure pathways might lead to adverse risk (Gržetic and Ghariani 2008; Du et al. 2013). Therefore, if any DI value through any exposure pathway for a given pollutant is greater than its corresponding RfD, which means that HQ > 1, then, there is a possibility that there will be a deleterious health effect that is non-carcinogenic through that exposure route (USEPA, 1986, 1993). However, CPF relates the probability of incurring any carcinogenic effect to exposure (Gold et al. 1995; Schuhmacher et al. 2004). All risks are additive whether carcinogenic or noncarcinogenic. The addition of HQ values of different pollutants and/or multiple exposure routes is the chronic noncarcinogenic hazard index (HI) which is calculated thus (Zheng et al. 2010; Xu et al. 2015):

where RfD_i, DI_i, and HQ_i are the RfD, DI, and HQ values for the ith metal, respectively. An HI > 1 signifies the possibility that noncarcinogenic effects will occur, and this possibility increases as the HI increases. However, an HI < 1 indicates an occurrence of negligible risk of non-carcinogenic health effects (USEPA 2001). Similarly, the possibility of a person having cancer from co-exposure to different carcinogenic pollutants described by the cumulative carcinogenic risk is calculated thus (Gržectic and Ghariani 2008):

where CPF_i and Di_i are the cancer potency factor and daily intake for the ith pollutant, respectively. 10⁻⁴ to 10⁻⁶ is the precautionary range provided for regulatory purposes for carcinogenic risk (USEPA, 1990, 1991). For the assessment of the human cumulative health risks from exposure to toxic metals in soil samples around the studied cement factory through the 3 exposure pathways, cancer risk methods and hazard index were used. For all the calculations made in the present study, it was assumed that all the site-base parameters were constant for all metals (Supplementary Material 1), and 70 years was used as the lifetime of an individual’s exposure in estimating the risks. Risk assessment models are indispensable tools used to establish the relationship between metal toxicity and human health in other to quantify the carcinogenic and non-carcinogenic effects through any route of exposure despite the many uncertainties with the models (Shi et al. 2011; Kolo et al. 2018).

Animal

Male Mus musculus (Swiss albino mice) with an average age of 4 to 5 and 11 to 13 wk old were acclimatized at 22°C ± 2°C under a relative humidity of 50% to 60%, and 12-h dark/12-h light cycle for 2 wk with drinking water and pelleted feed supplied ad libitum. Mice averagely weighing 30 g (6 to 7 wk old) and 43 g (13 to 15 weeks old) were grouped and used for the micronucleus (MN) and sperm morphology tests, respectively. The Animal Care and Use in Research Ethics Committee (ACUREC) of our university approved the experiment (FUTA/App/2023/0168) and standard guidelines of “ARRIVE” were followed in caring for the mice.

Micronucleus test

Twelve groups (5 mice per group) of 50%, 25%, 10%, 5%, and 1% (leachate: distilled water; v/v) of the simulated leachate; 100%, 50%, 25%, 10%, and 5% (groundwater: distilled water; v/v) of the groundwater sample, cyclophosphamide (positive control), and distilled water (negative control) were used in the MN test. In each group, each mouse was intraperitoneally administered 0.3 ml of their respective concentration per day for 5 consecutive days. This study favors the intraperitoneal route because of its efficient and fast delivery of the test agent into the lab mice (Alabi et al. 2022). Cervical dislocation was the technique used to sacrifice the mice after 24 h of the last injection and MN assessment was carried out by the preparation of bone marrow cells using the methods described by Schmid (1976) and modified by Alabi et al. (2014). In brief, the femurs of each mouse were flushed using fetal bovine serum to release the bone marrow followed by centrifugation of the cells for 5 min at 600 × g. Slides were prepared and treated with May-Grunwald and Giemsa stains, sequentially. In each mouse, 1,000 erythrocytes were scored for nuclear abnormalities including MN in normochromatic (MNNCE) and polychromatic (MNPCE) erythrocytes at ×1,000.

Biochemical analysis

Mice used for the MN test were used for this analysis. Blood was obtained by cardiac puncture into EDTA bottles and commercially available kits were used for the determination of alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) activities in the serum (Labtest Diagnostics SA, Lagoa Santa, MG, Brazil) according to the manufacturer’s instructions (technical semi-automated biochemical analyzer Thermo Plate1 Analyzer).

Murine sperm morphology test

In this test, 7 mice in each concentration and the same concentrations as the MN test were used. A 0.4-ml intraperitoneal injection of the different concentrations was administered for 5 consecutive days to the respective mice and left for an extra 30 d. In mice, the process of spermatogenesis requires around 34.5 d for completion, therefore, on the 35th day from the first exposure (Bartke et al. 1974), the mice were subjected to cervical dislocation and their testes dissected. The caudal epididymides were stained with eosin Y (1%) for 45 min after thoroughly mincing it in normal saline using a plastic pipette. One drop of the stained suspension was smeared on a grease-free clean slide and 6 slides per mouse were prepared and used for microscopic examination after air-drying. One thousand sperm cells per mouse were morphologically assessed for abnormalities.

Sperm count

Sperm cells from mice used in the sperm morphology test were counted by surgically removing the caput epididymis in the testes before mincing thoroughly in normal saline and the suspension obtained was filtered to remove tissue debris. The filtrate was then stained for 30 min with eosin Y (1%) before it was slowly sucked into a hemocytometer to the 0.5 mark with more dilution using Phosphate buffered saline up to the 11 mark after thorough mixing. The diluted sperm suspension was counted at 400× in the Neubauer counting chamber. The sperm count of all animals in each group was pooled and expressed as the mean sperm count per ml of suspension.

Total testosterone, luteinizing hormone, and follicle stimulating hormone determination

Serum obtained from the blood in mice used for the sperm morphology test was centrifuged for 5 min at 3,000 g before the determination of total testosterone (TT), follicle stimulating hormone (FSH), and luteinizing hormone (LH) concentrations using chemiluminescent immunoassay with an automated Unicel Dlx 800 Access Immunoassay System and kits that are available commercially (Beckman Coulter, Inc., USA).

Statistical analysis

The 2007 Microsoft Excel and 17.0 version of the Statistical Package for the Social Sciences (SPSS) were used for the analysis of all data. The obtained data were expressed as percentage frequency and mean ± standard error. Significance at various concentrations was tested using one-way ANOVA and Duncan’s new multiple range test (DMRT) test. The difference between the individual treatment group and the negative control group was analyzed at the 0.05 probability level.

Results

Analysis of toxic metals and physicochemical parameters

Physicochemical parameters and the toxic metals analyzed in the samples and controls are presented in Table 1. The samples have a pH range within the allowable limits (NESREA 2009; USEPA 2009). In the soil sample, varying levels of Cu, Ni, Cr, Cd, Zn, and Pb were recorded at higher concentrations than the control soil and in comparison with the WHO/FAO permissible limit. Pb has the highest concentration ranging from 404.36 to 656.38 mg/kg, while Ni has the lowest concentration varying from 70.04 to 89.21 mg/kg. Pb > Cu > Mn > Cd > Cr > Zn > Ni was the order of the concentration of toxic metals in the soil sample. In the groundwater sample, COD, BOD, TDS, alkalinity, hardness, ammonia, sulfate, and nitrate were recorded to have higher levels than the maximum allowable level in drinking water. Furthermore, the levels of the toxic metals in the cement-contaminated groundwater were higher than the control groundwater and maximum allowable level in drinkable water (NESREA 2009; USEPA 2009). Zn was the most abundant while Ni was the least. Zn > Pb > Cu > Cd > Mn > Cr > Ni was the order of the concentration of toxic metals in the groundwater. Table 2 compares reports in the literature with the present study using different instruments for the analysis of toxic metals.

Model for assessment of potential health risk

Results of the LADD and DI of toxic metals from soil samples surrounding a major cement factory for both adults and children through the 3 exposure pathways are shown in Table 3. The total maximum DI of Ni, Pb, Cr, Cu, Cd, Mn, and Zn were 7.3 × 10⁻³, 5.0 × 10⁻², 1.4 × 10⁻², 4.6 × 10⁻², 2.6 × 10⁻², 2.8 × 10⁻², and 1.5 × 10⁻²mg/kg/d; and 4.0 × 10⁻³, 2.8 × 10⁻², 7.6 × 10⁻³, 2.5 × 10⁻², 1.2 × 10⁻², 1.5 × 10⁻², and 8.1 × 10⁻³mg/kg/d, for children and adults, respectively. Generally, the DI of the toxic metals through the ingestion pathway was at least twice higher than for the dermal and inhalation routes from the children’s perspective. Similarly, the DI of the toxic metals in adults was 2 to 4 times higher for the ingestion route compared to dermal contacts and inhalation. In adults, the order of highest mean DI for the metals is Pb > Cu > Mn > Cd > Zn > Cr > Ni while for children is Cu > Pb > Mn > Cd > Zn > Cr > Ni. For carcinogenic metals, the LADD values ranged from 1.2 × 10⁻⁷mg/g/d (Cu) to 1.1 × 10⁻⁶mg/g/d (Pb) in children, and 5.0 × 10⁻⁷mg/g/d (Ni) to 3.2 × 10⁻⁶mg/g/d (Cu) in adults.

Risk characterization

The results obtained for HQ and HI for the 3 routes of exposure are in Table 4. The HI and HQ for non-carcinogenic risk for both adults and children showed a similar increasing trend of Pb > Cr > Cu > Mn > Zn > Ni. Furthermore, the HQ values obtained for the exposure routes increased in the order of ingestion > dermal contact > inhalation, except for Mn which showed an increasing order of ingestion > inhalation > dermal contact for both adults and children.

Furthermore, the HI values for Cr and Pb for adults and Cu, Cr, and Pb for children were observed to be higher than one which is the safety limit. The cumulative HI as a result of metal exposure through the 3 studied routes of exposure were 10.61 and 17.59 for adults and children, respectively (Table 4), of which Pb ingestion is the most contributor. The carcinogenic risk results from Ni, Pb, and Cr exposure in the soil sample studied revealed that the level of cancer risk for Ni, Pb, and Cr were 2.10 × 10⁻⁵, 1.34 × 10⁻⁶, and 1.43 × 10⁻⁶; and 7.56 × 10⁻⁶, 4.62 × 10⁻⁷, and 2.52 × 10⁻⁷ for adults and children, respectively. Also, 2.76 × 10⁻⁶ (children) and 7.92 × 10⁻⁶ (adults) were recorded for the average cumulative cancer risks from Ni, Pb, and Cr exposure in the soil samples studied. The levels of cancer risk of Ni for children (7.56 × 10⁻⁶) and adults (2.10 × 10⁻⁵) exceeded 1 × 10⁻⁶ set by USEPA (1990) as the lowest precautionary limit. Ni ingestion was responsible for most of the total risk for cancer.

Micronucleus test

Table 5 shows a significantly (P < 0.05) induced MN recorded in the bone marrow of exposed mice by the different concentrations of the simulated leachate and the groundwater. This induction was concentration-dependent with the highest concentrations of each sample inducing the highest MN frequency. The induction of MN by the leachate sample was higher than the groundwater at all the tested concentrations. A similar pattern to the MN data was observed in the total nuclear abnormalities in the mice exposed to both samples. Significantly (P < 0.05) increased total nuclear abnormalities were recorded at all the tested concentrations in the 2 samples. This, in comparison to the negative control, was concentration-dependent with the highest concentrations inducing the highest total nuclear abnormalities. Different nuclear abnormalities observed include helmet cell, oval PCE, Cabot’s ring, teardrop, microcytes, cone NCE, and burr macrocytes (Fig. 1). After 5 wk of exposure, PCE to NCE ratio reduced significantly at 5% to 50% concentrations of the leachate and 10% to 100% of the groundwater compared to the negative control.

Biochemical assay

The effects of leachate and groundwater exposure on liver injury and biomarkers of oxidative stress in mice are reported in Table 6. At 5% to 50% of the leachate sample, the liver CAT and SOD decreased significantly (P < 0.05) with a concomitant rise in the serum activities of ALT, ALP, and AST of the exposed mice when compared with the negative control group. A similar result showing a significant (P < 0.05) rise in the activities of serum ALT, ALP, and AST was observed at 25% to 100% concentrations of the groundwater sample, while a reduction in the activities of liver SOD and CAT was only observed at 50% and 100% concentrations.

Murine sperm morphology test

The results of the sperm abnormalities (percentage frequency and mean) observed in mice after treatment with various concentrations of leachate and groundwater are presented in Table 7. Compared with the negative control, the mean sperm abnormalities were concentration-dependent and increased significantly (P < 0.05) from 5% to 50% of the simulated leachate and 25% to 100% of the groundwater. The simulated leachate at concentrations of 1%, 5%, 10%, 25%, and 50% induced mean sperm abnormalities of 132.06, 183.41, 217.51, 287.43, and 302.31, respectively, while the groundwater at 5%, 10%, 25%, 50%, and 100% concentration induced 127.33, 142.38, 187.43, 221.05, and 258.39, respectively. A 441.02 mean sperm abnormalities were recorded in the positive control while the negative control had 115.02 mean abnormalities.

Furthermore, the % frequency of sperm abnormality significantly (P < 0.05) increased at 1%, 5%, 10%, 25%, and 50% concentrations of the leachate inducing 14.16%, 19.67%, 23.00%, 28.92%, and 32.18% sperm abnormalities, respectively. Also, 12.79%, 14.57%. 20.24%, 23.08%, and 25.40% sperm abnormalities were induced by the groundwater at 5%, 10%, 25%, 50%, and 100% concentrations, respectively, when compared with the negative control of 11.17%. Generally, the simulated leachate induced a significant reduction in the percentage frequency and mean of abnormal sperm cells in comparison to the groundwater.

The observed morphological abnormalities in sperm cells of exposed mice include no hook, long hook at the wrong angle, projecting filament from midpiece, amorphous head, long hook, wrong tail attachment, pinhead, double tail, fused neck with tail, double head, folded sperm, looped tail, and folded sperm with distal droplet (Fig. 2). Amorphous head sperms were observed most and sperm cells with double heads occurred the least.

Sperm count

Table 8 shows the mean sperm count recorded after exposure to the simulated leachate and groundwater in mice. Animals of the negative control had a mean sperm count of 6.05 × 10⁶ while the positive control recorded a mean of 9.75 × 10⁴. Mice exposed to 5% to 50% and 25% to 100% concentrations of the simulated leachate and groundwater, respectively, showed concentration-dependent, significant (P < 0.05) decrease in mean sperm count, with the lowest sperm counts recorded at the highest concentrations (50% and 100%) of the 2 samples.

Total testosterone, luteinizing hormone, and follicle-stimulating hormone determination

Table 9 presents the data recorded for the concentration of TT, FSH, and LH in the serum of mice after exposure to various concentrations of the simulated leachate and groundwater. The concentrations of TT and FSH significantly (P < 0.05) increased in the mice from 5% to 50% of the simulated leachate and 25% to 100% of the groundwater groups in comparison to the negative control group, however, the concentration of LH significantly (P < 0.05) reduced at the same concentrations.

Discussion

Activities of cement industries in Nigeria are causing environmental pollution and there is a need for constant environmental monitoring of this contamination and the potential adverse health effects on the local populace. This study analyzed the presence of toxic metals in groundwater and soil from a major cement factory and the potential genotoxicity of these samples in both somatic and germ cells. The health risks associated with these metals were also calculated.

The heavy metal analysis data showed that the levels of analyzed metals in the groundwater and soil around the cement factory were higher than in control areas when compared with other similar studies (Ogunkunle and Fatoba 2014; Kolo et al. 2018; El-Sherbiny et al. 2019) and the recommendation by WHO on the concentration thresholds of potentially toxic metals (Silva et al. 2021). The metals observed in this study include Cr, Cd, Mn, Cu, Zn, Pb, and Ni. Observing the same type of metals in both the soil and the groundwater in this area indicates a common source of contamination which is the cement factory, as these metals have been reported as raw materials in the production of cement (Scoullos 2001; Al-Khashman and Shawabkeh 2006). The presence of these metals in the groundwater suggests possible percolation of the metals through the soil or by direct emission into the well dug for drinking by the people in the neighborhood. Metals like Cd, Cu, and Pb have been documented as part of the toxic metals released when cement is produced (Kakareka and Kukharchyk 2011). The high level of these metals that ordinarily should be low or absent in soils is majorly due to the cement production activities of WAPCO. A similar study in Riyadh City by Al-Oud et al. (2011) reported a significantly high concentration of Cd, Cu, and Pb in the soils of the vicinity of a cement factory, an indication of the release of the metals along the cement production line. The data showing that Pb, Cu, and other metals were significantly high in the soil of this study is similar to the report of Bi et al. (2006), Wu et al. (2010) and Fikadu and Mekassa (2022) in their respective studies of similar sites. Toxic metal levels reported in the present study are the third highest reported concentrations of toxic metals in soil samples around cement companies. However, the present level of toxic metals is lower compared to the concentration reported by Ogunkunle and Fatoba (2014) in the same Sagamu, Nigeria.

Toxic metals in the soil can cause adverse effects on plants growing on it and subsequently indirectly or directly affect animals and humans who consume the plants (Cao et al. 2009). Because the toxic metals reported in this study are active metals, the tendency for them to bioaccumulate in plants growing on polluted soil is high (Kang et al. 2003) and they can be translocated to the parts of the plant above-ground where they can be easily available for transfer up the food chain (Liang et al. 2011).

The toxicity of the metals detected in this study has been documented. Lead is classically a cumulative or chronic poison. Depending on the duration and level of exposure in humans, Pb induces adverse effects in the system ranging from cardiovascular, behavioral, renal, and neurological to hematological effects. Children are often more vulnerable than adults to the effect of Pb because of their rapid rate of absorption. Chromium is absorbed primarily in the jejunum after entering the gastrointestinal tract. The mechanism of carcinogenicity and toxicity of Cr is a complex process which includes DNA lesions, free radicals production, and higher redox potential (Krawic and Zhitkovich 2023). Due to the easy accumulation of Cu in the body, chronic low-level intake is detrimental to animals and humans because they do not possess any good mechanism for eliminating it from their systems. Oral intake of Cu is a likely cause of kidney and hepatic disease such as Wilson’s disease which is related to the accumulation of Cu in organs rather than its excretion by the bile (Wan et al. 2020). Increase in the body burden of Cu causes such as gum diseases, scoliosis, migraine, osteoporosis, cancer, fatigue, heart seizures, heart disease, insomnia, arthritis, skin and hair problems (Schuhmacher et al. 1993; Conti and Carcea 2000). Zn has the least toxicity among all the heavy metals and it is essential in the human diet for the maintenance of immune system functions (Kudirat and Funmilayo 2011). Zn in salt form is more toxic than in elemental form. Human exposure to high concentrations of Zn can elicit acute responses which include nephrotoxicity, brain’s enzymatic and morphological changes, lowered food utilization, anemia, neurotoxicity, weakness, loss of hair, alterations in serum and liver enzyme concentrations, diminished growth, functional and histological changes in the kidney, pulmonary toxicity, anorexia, vomiting and gastrointestinal irritation (diarrhea, cramps, nausea) (Conti and Carcea 2000; CAC 2003).

An association between cancer and environmental exposure to Cd through inhalation has been documented (Nawrot et al. 2006). There is no recorded biological function of Cd in humans, however, it has been shown to interfere with certain Zn essential functions including the inhibition of nutrient utilization and enzyme reactions. Cadmium also causes tissue damage by generating free radicals after catalyzing oxidation reactions (WHO 1992). Ni toxicity in humans is not yet fully documented because the human body does not absorb it readily (Mottet 1980; Adejoh 2016). However, the toxicity of Cd, Pb, Ni, and Cu can cause enzyme inactivation especially those responsible for DNA repair and synthesis and cellular energy pathways (Onder et al. 2007).

The DIs were compared with their respective RfDs for ingestion and the data showed that for both subpopulations the values for DI were higher than the values for the respective RfD for Cu, Pb, Mn in children and Pb, Cu, and Mn in adults. Also, the values for dermal DI were higher than the respective RfD values for Cu and Pb in children and Pb and Cr in adults. Furthermore, when the inhalation DIs were compared with their respective RfDs for all the studied metals, the data showed that for both subpopulations, the values for DIs were lower than the values for their respective RfD. This shows that dermal contact and ingestion are the 2 major routes of human (adults and children) exposure to these metals. These routes of exposure were further confirmed by the result of the HQs. Data comparing the rate of exposure between the 2 subpopulations indicated that exposure to the soil metals is higher in children than in adults which might be as a result of their regular hand-to-mouth dietary habits (Kolo et al. 2018; Alabi et al. 2024).

Data on the risk of developing cancer after exposure to the toxic metals by adults and children in the soil showed that the possibility of cancer risk for Ni, Pb, and Cr is high with children having more than 3 times the risk of developing cancer from these metals than the adults. The risk of having cancer from Ni for adults and children exceeded the USEPA’s lowest precautionary limit (USEPA 1990). Ni ingestion is majorly responsible for the total risk of cancer in the study area. This calls for concern as the study area was seen with many children and 2 primary schools were located within the neighborhood. Although the species of the Cr in this study was not analyzed, however, the data of the carcinogenic risks suggest that the Cr present is likely the carcinogenic Cr(VI) form which is known to be present in cement. Also, the nontoxic Cr(III) is poorly water-soluble and therefore, cement-derived Cr(VI) predominates in water and soil extracts (Zhitkovich 2011).

Toxic metals present in the tested soil and groundwater samples are documented mutagens, hence, detecting them in the present samples at high concentrations necessitated our study of their cytogenotoxicity in animal models (germ and somatic cells) to confirm this suspicion. In the MN test, the data revealed the aneugenic and/or clastogenic potential of the soil and groundwater sample constituents on the erythrocytes from mouse bone marrow because this test measures indirectly the induction of structural and numerical chromosome aberrations. The aneugenic and/or clastogenic effects recorded in this study are likely due to the constituents of the samples interacting during cell division with the mitotic spindle components, directly with the DNA or chromosome segregation proteins (Alabi and Bakare 2011). Furthermore, the ratio of PCE-to-NCE recorded in this study indicated that the samples tested were cytotoxic to murine bone marrow (Krishna and Hayashi 2000) and possibly suppressing the proliferation of the bone marrow. Also, the data showed that the bone marrow nuclear abnormalities increased significantly in mice exposed to the 2 samples. These nuclear abnormalities indicate a variety of anemia which include megaloblastic, uremia, myelophthisic, and megaloblastic anemia as presented by the presence of basophilic stippling, burr cells, teardrop, and oval macrocytes, respectively. Cabot’s ring, on the other hand, indicated severe anemia, as these are the remains of the nuclear membrane (Adewoyin et al. 2019).

The result further revealed that serum ALP, ALT, and AST activities significantly increased in the simulated leachate and groundwater-exposed mice. Activities of serum ALP, AST, and ALT have been commonly used as markers of hepatocyte necrosis as they are regarded as sensitive hepatic injury indicators (Friedman et al. 1996), and cell membrane leakage and damage (Kaplan 1993). Data obtained in this study presented systemic damage by the constituents of the 2 samples in mice. It’s a general belief that the generation of oxidative stress is the mechanism of cytogenotoxicity of many chemical agents in biological systems. Hence, this study assessed in the exposed mice the potential modulation of biomarkers of oxidative stress. The data confirmed that indeed the samples modulated the concentrations and activities of the assessed biomarkers. Increased levels of MDA which is liver lipid peroxidation indicated oxidative damage suggesting a formation of reactive oxyradicals with the capability of damaging cellular macromolecules including lipids, DNA, and protein (Pandey et al. 2003; Magdolenova et al. 2014). There was increased activities of CAT and SOD by the 2 samples. Against any oxidative stress, the first line of defense is the antioxidant system of CAT-SOD. SOD act as the catalyst for superoxide radicals to be converted to hydrogen peroxides and water before CAT detoxifies the hydrogen peroxides to harmless compounds. The significant increase of CAT in this study indicates a probable increase in hydrogen peroxide generation in the exposed mice, hence, more CAT was produced to degrade it.

The data on sperm abnormalities indicated that the samples of the soil and groundwater from the surroundings of the cement factory are capable of causing reproductive toxicity in treated mice. The data indicated the spermatotoxicity of the 2 samples as a significant increase in sperms with morphological abnormalities was obtained at all the concentrations tested. The structure and morphology of sperm cells are very important indicators in the determination of sperm quality and as a result of their rapid cell division, this cell type is highly susceptible to cytotoxins (Alabi et al. 2017; 2022). To assess a potential genotoxin, morphological abnormality in sperm cells is one of the reliable biological indicators in a short-term study (Ieradi et al. 2003). These morphological abnormalities are the consequences of variations in chromosomes and point mutations due to genotoxin exposure (Narayana et al. 2005). The morphology of the sperm indicates its functional capability as well as the quality of the DNA in the sperm’s head. Hence, any morphological abnormality is most possibly due to changes in the content of its DNA (Sun et al. 2006). An association exists between male infertility, sterility, and sperm cell abnormalities; and the structure of the sperm is responsible significantly for fertilization and other pregnancy outcomes (Saacke 2001). Studies by Mendoza-Lujambio et al. (2002) and Alabi et al. (2022) proposed small deletions, abnormal chromosomes, and point mutations as likely mechanisms of such abnormalities during the packaging of the DNA in the sperm head. Furthermore, morphological abnormalities in sperm cells could be due to errors in spermatogenesis during the process of spermatozoa differentiation (Wyrobek et al. 1983).

Different morphological abnormalities were observed in the sperm cells after exposure to the 2 samples. Pinhead sperm cell has little or no paternal DNA content, while sperm cells having distal droplets and folded sperms are cells that have damaged tails and therefore are immotile (LLUCF 2021). No hook or knobbed hook sperm cells might be incapable of oocyte attachment for fertilization (Cooper 2005), and amorphous head sperm cells usually contain damaged paternal DNA (LLUCF 2021). Short-tailed sperm cells also called Dysplasia of Fibrous Sheath (DFS) often have low or no motility (LLUCF 2021). The morphological abnormalities in sperm cells induced by the studied samples are important to public health because spermatozoa with morphological abnormalities may contribute altered genomes to the oocytes leading to serious deleterious effects on fertilization, fetal, embryonic, and postnatal development (Lewis and Aitken 2005).

Furthermore, present data revealed that the mean sperm count reduced significantly as the concentration of the 2 samples increased, indicating a possible decrease in sperm cell production due to exposure to the test agents. Results of the morphological abnormalities in sperm cells suggest potential interactions between the sample constituents and the genetic processes in mice spermatogenesis which was further buttressed by the significant reduction of the mean sperm count in exposed mice in comparison with negative control. This indicated that the constituents of the samples were capable of altering spermatogenesis and at the same time reduced the production and viability of sperm cells. Reduced sperm count significantly affects the chances of fertilization, thus resulting in sexual function problems including erectile dysfunction, lump in the testicle area, swelling, pain, and low sex drive (Alabi et al. 2017; 2022).

To understand the mechanism involved in the production of the observed morphologically abnormal sperm cells in exposed mice, this study measured the concentrations of some gonadotropic hormones responsible for mice spermatogenesis. Results revealed a reduction in FSH and LH levels in the exposed mice. Literature (Jensen et al. 1997; Mahmoud et al. 1998) indicates that in men, an association exists between quality parameters in the semen and the levels of certain reproductive hormones circulating in the system. In the production of spermatozoids, LH, FSH, and testosterone are required with LH stimulating Leydig cells for the production of testosterone, and spermatogenesis stimulating the binding of FSH to Sertoli cells. Present data of this study suggest an active participation of TT, LH, and FSH in sperm morphology development in the studied mice, with the reduction of LH and FSH levels and a concomitant increase in TT levels producing a significant reduction in the number of normal murine sperm cells. The observed low FSH and LH levels in this study signify androgen deficiency (Weinbauer and Nieschlag 1995) since these pituitary gonadotropins are essential spermatogenesis regulators. A low FSH level always leads to a 30% to 45% significant decrease in Sertoli cell number when compared with normal testicular development (Sharpe et al. 2003). This is medically important as the population of Sertoli cells determines the quantity of sperm produced since each Sertoli cell can only support a specific maximum number of sperm cells (Sharpe et al. 2003; Abel et al. 2008). Thus, the reported low FSH level in this study possibly means low sperm production in mice exposed to the samples. Failure of the pituitary to secret LH and FSH could disrupt the normal function of the testes leading to infertility (Sultan et al. 1985). The paracrine agent, testosterone, is responsible for facilitating spermatogenesis in Sertoli cells and providing feedback for the secretion of LH, which suggests that sperm morphological abnormalities herein reported are related to the disruptions in the hypothalamus-pituitary-gonadal axis and compensatory mechanisms (Meeker et al. 2007). It is therefore believed that the tested samples’ ability to cause reproductive toxicity and modulate enzyme activities was due to their various chemical constituents, of which some were analyzed in the study.

We believe that the observed cytogenotoxicity and oxidative damage in the germ and somatic cells by soil and groundwater samples in this study is induced by the toxic metal constituents. Exposure to harmful chemicals such as heavy metals through drinking water could lead to the transformation and prognosis of reproductive abnormalities, decreased cell survival, and eventually cancer (Shugart et al. 1992). Individually, these metals can induce mutations, cancer, and other adverse health effects in living cells (Elinder and Jarup 1996; Banu et al. 2001). In vitro study reported that Cr caused micronuclei strand breaks and chromosomal aberrations (Wise et al. 2002) while Cd is carcinogenic (Elinder and Jarup 1996). Cd was documented to alter semen quality, hormonal imbalance, or testicular function of various animal species (Wang et al. 2017). Findings on Cr showed the increased prevalence of sperm abnormalities in an exposed animal model (Danadevi et al. 2003). Cu has been shown to significantly reduce the concentration, motility, and viability of spermatozoa (Radetski et al. 2004). Some experimental studies have reported that As intoxication can be spermatotoxic (Waalkes et al. 2000), inhibit testicular steroidogenesis, and reduce testicular weight and the weight of other accessory sex organs (Sarkar et al. 2003). Long-term exposure to Cd, Cr, As, Cd, and Cu through soil and water can lead to cancer and toxicity in organ systems such as skeletal, urinary, cardiovascular, reproductive, and respiratory systems (Alabi et al. 2021; Rahinetu 2021). Therefore, high concentrations of these metals in the samples are believed to be responsible for the observed reproductive toxicity in the exposed mice. Also, metal-to-metal interactions including potentiation, synergy, and additivity in the mixture and with the DNA in the sex and somatic cells of mice might have contributed to our observation.

Conclusion

The cement factory has contaminated the soil and groundwater in the study area as evidenced by the increased concentration of heavy metals, and other physicochemical parameters. The high levels of Pb, Cu, Mn, Cd, Cr, Zn, and Ni and some physicochemical parameters led to the observed somatic and reproductive abnormalities in mice. The samples also modulated the activities of AST, ALT, ALP, CAT, and SOD. Altered concentrations of gonadotropic hormones (FSH, LH) and testosterone were also observed. Results of the potential ecological and health risks showed that Pb is the major contributor and ingestion is the most likely route of exposure. There is a need for the government of Nigeria to prevent further environmental contamination in the area and employ effective remediation techniques to reclaim the already contaminated soil. The ecotoxicological study aimed at in situ assessment of plants, animals, and residents of this area is highly encouraged so that steps can be taken to treat the potential somatic and reproductive effects of this contamination on the exposed populace.

Author contribution

Okunola Adenrele Alabi, Funmilayo E. Ayeni and Tomiwa Amos Afolabi: contributed to the study conception and design. Okunola Adenrele Alabi and Funmilayo E. Ayeni: performed material preparation, data collection, and analysis. Okunola Adenrele Alabi and Tomiwa Amos Afolabi: wrote the first draft of the manuscript and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Supplementary material

Supplementary material is available at Toxicological Sciences online.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

Arrive statement

The Animal Care and Use in Research Ethics Committee (ACUREC) of our university approved the experiment (FUTA/App/2023/0168) and standard guidelines of “arrive” were followed in caring for the mice.

References