Comprehensive sub-chronic toxicological profiling of illegal artisanal small-scale mining-polluted river bodies in four regions of Ghana

Du-Bois Asante; George Yaw Hadzi; Samuel Naaman; Michael Woode; Timothy Makwo; Benjamin Aboagye; Isaac Tabiri Henneh

doi:10.1016/j.toxrep.2026.102244

. 2026 Mar 24;16:102244. doi: 10.1016/j.toxrep.2026.102244

Comprehensive sub-chronic toxicological profiling of illegal artisanal small-scale mining-polluted river bodies in four regions of Ghana

Du-Bois Asante ^a,^c,^⁎, George Yaw Hadzi ^b, Samuel Naaman ^c, Michael Woode ^c, Timothy Makwo ^a, Benjamin Aboagye ^a, Isaac Tabiri Henneh ^d,^⁎⁎

PMCID: PMC13068583 PMID: 41972193

Abstract

Illegal artisanal mining, or ‘Galamsey’, in Ghana poses serious public health risks by contaminating water bodies with toxic heavy metals. This study assessed the sub-chronic toxicological effects of mining-polluted rivers in four Ghanaian regions using rats as an experimental model. A 90-day sub-chronic toxicity test was conducted using water samples from four regions associated with mining-polluted rivers: Bonsa, Birim, Nyam, and Subiri. Heavy metal analysis was conducted on the sampled water from these selected illegal mining-prone rivers. Sprague Dawley rats were divided into six groups: two controls (distilled water and pristine river) and four treatment groups (contaminated water from the four rivers). Rats were given 2 mL of water from each water source for 90 days. After 90 days, rats were anaesthetized. Blood samples were analyzed for hormonal, hematological, and several biochemical markers. Fasting blood sugar, urine, and semen analyses were also performed. Rats were euthanized, and organs were harvested for histopathological analysis. Lead was significantly (p < 0.05) elevated in all mining-polluted rivers when compared to the controls. Mercury, arsenic, cadmium, and zinc were also detected. Exposed groups showed significant (p < 0.05) weight loss, elevated serum creatinine kinase (CK-Nac), and the presence of WBC, RBC, crystals, and casts during urinalysis. Haematological profiles indicated erythrocytosis and immune variations. Serum biochemical parameters, such as AST, ALP, and ALT, were significantly elevated (p < 0.05), and sperm counts were significantly reduced (p < 0.05) in the exposed groups. Histopathology revealed non-caseating granulomas, necrosis, and leucocytosis in the liver and lungs, as well as pronounced hemosiderosis in the spleen. Histopathology of renal and testicular organs revealed necrotic tissue and degenerative cellular morphological changes. These results suggest that illegal mining-related water contamination in Ghana presents severe health risks, demonstrating its severe haematotoxic, nephrotoxic, hepatotoxic, pulmonotoxic, myotoxicity, and reproductive toxicity in the experimental rat model.

Keywords: Sub-chronic toxicity, Serum biochemistry, Histopathology, Hematology, Heavy metals

Graphical Abstract

Highlights

•
Lead was significantly elevated in all mining-polluted rivers when compared to the control samples.
•
Hematological profiles showed erythrocytosis and immune variations in treated groups compared to control groups.
•
Multiple serum biochemical parameters were significantly elevated in treated groups compared to control groups.
•
Degenerative cellular changes and pathological lesions were detected in tissues of polluted water treatment groups.

1. Introduction

The influence of the environment on human and animal health has been extensively studied over the past decades. Increasing modernization and the quest for sustainability have led people to engineer activities that affect various natural resources, which in turn cause detrimental effects on health in general [2], [1]. Within this context, artisanal small-scale mining, colloquially known as 'galamsey,' in Ghana, presents a grave concern in regions where it is prevalent [3]. Galamsey and unregulated small-scale mining operations, predominantly in sub-Saharan Africa, have been recognized as a source of substantial ecological disruption, with the contamination of water bodies being a paramount consequence of this unregulated mining practice [4]. These activities, characterized by the indiscriminate extraction of minerals such as gold, often entail the use of hazardous chemicals containing heavy metals, which are washed into nearby aquatic ecosystems [5]. These toxicants, once introduced into water bodies, undergo complex biogeochemical transformations, accumulating in the environment and ultimately impacting various trophic levels [6]. Consequently, these chemicals are ingested by aquatic organisms and subsequently enter the food chain, posing a pervasive threat not only to aquatic life but also to humans who depend on these resources [7]. More importantly, during the dry season, when water is scarce, several individuals within the rural communities where these polluted water bodies are found may use them as drinking water and for other domestic activities [8].

Globally, it is interesting to know that over 6 million miners globally suffer from moderate to chronic metallic mercury vapour intoxication [9]. Generally, intoxication of these heavy metals (lead, arsenic, cadmium, mercury etc.) can lead to a range of health problems in humans, from mild eye, nose, and skin irritations to severe symptoms, including headache, stomach ache, diarrhoea, blood vomiting, dizziness, and organ damage such as cirrhosis, necrosis, low blood pressure, high blood pressure, gastrointestinal issues [10], damage to the nervous system and the occurrence of birth-related disorders [11].

Although several studies in Ghana have assessed the soil and water quality at galamsey sites, with some estimating the varied concentrations of heavy metals and their potential health effects [12], [13], the systemic toxic effects of galamsey and unregulated small-scale mining-contaminated water on terrestrial organisms, particularly mammals, are yet to be investigated. Ultimately, these speculations further present the need to experimentally assess the potential toxic effects of galamsey and unregulated small-scale mining-contaminated rivers using rats as experimental models.

Herein, we seek to determine the sub-chronic toxicological effect of illegal artisanal small-scale mining-polluted river bodies in some selected regions in Ghana.

2. Methodology

2.1. Water sample preparation

2.1.1. Study area

The study area covered agricultural lands and some rivers/streams in the major mining areas in four regions of Ghana, with the land area covering between 138 and 2950 km². The rivers sampled were; Nyam river (Obuasi, Ashanti Region, 6°15'2.46"N and 1°41'29.60"W), Subri river (Kenyasi-Ntotroso, Brong Ahafo Region, 6°46'22.31"N, 2°46'38.33"W), river Bonsa (Tarkwa, Western region, 5°10'42.67"N, 2° 2′31.18"W) and Birim river (Kwabeng, Eastern region, 6°18'22.78"N, 0°35'50.66"W). Pristine water (pure and uncontaminated) was also taken from the Kakum River, Kakum National Park, Central region, 5°21'13"N, 1°23'0"W.

These areas were selected based on prior information from the Department of Geography, University of Cape Coast, the Council for Scientific and Industrial Research (CSIR), and the Ghana Forestry Commission (Wildlife Division). The selected areas were also influenced by widespread media reports of the contamination of those water bodies and a review of research articles that provide insight into the dynamics of toxic metals contamination and the physico-chemical parameters influencing metal mobility and transport of the areas. However, during the field study, the sampling plan was revised according to local conditions and situation.

2.1.2. Field methods

The fieldwork was conducted from January 2024 to February 2024 (dry season). Water samples were collected in the study districts with the help of the District Assemblies. Permission was sought from the district offices for support and escort when necessary.

2.1.3. Water sample collection and treatment

A total of sixteen composite water samples were collected from the rivers in the mining areas.

At each site, water samples (0–10 cm below the surface) were collected. For each river, four sampling points were marked 100 m apart along the river from downstream to upstream. At each sampling point, eight discrete water samples were collected and composited into one sample at that point of the river. Four composite water samples were collected from each river, giving a total of sixteen composite water samples for the four rivers.

The samples were collected in 1.5 L plastic bottles prewashed with detergent and a 1:1 concentrated nitric acid/distilled water solution and eventually rinsed with only distilled water.

The samples were stored in a refrigerator at 4 °C upon arrival in the laboratory for further analysis.

2.1.4. Digestion and analysis of water samples

Water samples were directly subjected to analysis by acidifying with 1 mL HNO₃ (70%), centrifuged at 3500 rpm for 15 min, and finally filtered through 0.45 µm pore size cellulose acetate filters. All samples were analyzed using an Agilent 8800 Quadrupole Inductively Coupled Plasma Emission Spectrometer (ICP-QMS) for toxic metals such as Lead (Pb), Cadmium (Cd), Mercury (Hg), Arsenic (As), and Zinc (Zn). To ensure reliability of the analytical method, quality control (QC) and blank samples were acid digested and subsequently analyzed for appropriate toxic metals through the same procedure. Measured concentrations were evaluated relative to their respective reporting limits, and values below RL were reported as BDL and treated accordingly in statistical analyses

2.1.5. Reproducibility and recovery studies

Reproducibility recovery studies were conducted using standard reference materials, with the methods of packaging, storage, and conveyance carefully controlled to maintain sample integrity throughout the analytical process. The recovery study evaluated how effectively the extraction method retrieves the target analyte from the sample matrix. The performance of the ICP—MS instrument was evaluated through a recovery study, and the percentages of the heavy metals analysed were within acceptable ranges. Spiked standards were used to evaluate the accuracy of the instrument. When known quantities of metals (standards) are added to a sample, the method’s ability to recover those known amounts reflects how well the instrument is performing.

2.1.6. Physico-chemical analysis of water

The physico-chemical measurements were conducted using the American Public Health Association (APHA, 2005) method for preparation and analysis of water samples and other standards. The pH was determined alongside the temperature using a pre-calibrated JENWAY 3310 and JENWAY 3510 pH meter. Conductivity was measured using a pre-calibrated PHYWE 13701.93 and WAGTECH 4510 conductivity meter. The turbidity was measured with a JENWAY turbidimeter.

2.2. Estimated daily metal intake

To facilitate toxicological interpretation and human exposure contextualisation, the estimated daily intake of each measured metal was calculated and expressed in mg/kg/day. Dose estimates were derived using the standard ingestion exposure equation:

Dose ((mg / kg) / day) = \frac{(C \times IR)}{BW}

where C represents measured metal concentration in water (mg/L), IR is the ingestion rate (0.002 L/day, corresponding to the administered 2 mL/day volume), and BW is the representative baseline body weight (0.173 kg), calculated from pooled group mean values. The baseline human weight used for the human dose calculations was 70 kg. This exposure modelling approach follows the water ingestion dose assessment framework described by the Agency for Toxic Substances and Disease Registry [14].

2.3. Experimental animal care and husbandry

Young healthy adult (thirteen weeks old) Sprague-Dawley rats of either sex with a weight range of 160–181 g were used for the acute and sub-chronic toxicity tests. The rats were randomly selected from the Animal House of the School of Biological Sciences, University of Cape Coast, for the study. The experimental rats were acclimatized for two weeks before the initiation of the experiment in standard aluminium cages (31.75 cm × 42.16 cm × 19.05 cm) with clean and soft wood shavings provided as bedding. They were maintained in a room with temperature and relative humidity maintained at 28 ± 2°C and 50 ± 5%, respectively, as well as a 12 h light/dark artificial light period. Feeding and water were done ad libitum. All animal experiments, procedures, and techniques used in this study were conducted in compliance with the National Institute of Health Guidelines for Care and Use of Laboratory Animals and also, guidelines in the use and care of experimental animals by the Organization for Economic Cooperation and Development [15]. Ethical approval was granted by the Council for Scientific and Industrial Research (CSIR) Institutional Review Board (IRB), Ghana, reference number: CSIR-IRB/RPN026/2024.

2.4. Experimental design

2.4.1. Sub-chronic toxicity studies

A total of sixty rats were divided into six groups (1, 2, 3, 4, 5, and 6) with five males and five females in each group. Groups 1, 2, 3 and 4 were provided with 2 mL daily water samples from the four rivers (River Bonsa (RBA), River Birim (RBM), Nyam River (RNM), and Subri River (RSI)), for three months (90 days). Group 5, the sham control group, was given 2 mL of water from a pristine river, the Kakum River. Distilled water (2 mL) was also administered for the vehicle control group, Group 6. The 2 mL/day volume was selected to simulate repeated low-volume incidental ingestion. A similar study [16], but not in an illegal mining site in China, used 0.5 mL/day of mining-contaminated surface water and groundwater from a rare earth. We modified our samples to 2 mL/day, as the samples were taken from fast-flowing large rivers. Using this fixed low-volume dose, it ensured uniform exposure across animals and removed variability associated with ad libitum drinking patterns, thereby improving internal validity. This volume falls within accepted oral dosing limits for rats and is widely used in repeated-dose toxicology studies to avoid gastric overload, aspiration risk, and stress associated with larger bolus administrations. Also, the fixed volume was administered to ensure precise dose standardisation across experimental animals. This approach eliminated variability associated with voluntary drinking behaviour and allowed controlled delivery volumes of the mining-polluted water samples. Contaminated water did not constitute the primary hydration source. All animals had unrestricted access to clean drinking water ad libitum throughout the experimental period to maintain physiological hydration status. Routine cage-side observations were conducted to monitor general health and hydration, and no signs of dehydration were detected. Because exposure delivery was independent of voluntary intake, daily water consumption monitoring was not required for dose normalisation.

Physical signs of toxicity were observed continuously at 30 min, 2 h, 4 h, 6 h, 10 h, and 24 h, and then for a total of 90 days. Observations included changes in colour of the skin, fur, eyes, salivation, lacrimation, perspiration, piloerection, urinary incontinence, defecation, drowsiness, tremors, and convulsions. After 90 days, the rats were anesthetized with 2–3% isoflurane in oxygen, after which blood samples were collected via cardiac puncture for hematological and biochemical analyses. Subsequently, the animals were humanely euthanized by cervical dislocation, and vital organs such as the liver, kidneys, testes, lungs, and spleen were harvested for histopathological examination. Carcasses were disposed of in accordance with institutional ethical guidelines and the AVMA Guidelines for the Euthanasia of Animals [17] . All laboratory work took place at the Department of Biomedical Sciences Laboratory at the University of Cape Coast.

2.4.2. Animal weight determination

The body weight of each animal was measured and recorded at the following key time points during the 90-day study: Baseline (before the start of dosing), weekly throughout the ninety-day exposure period and at the end of the 90-day exposure period, before euthanasia. A clean rubber bowl was placed on the calibrated electronic balance (The Mettler Toledo X5 80LS, Mettler Toledo, Laboratory equipment and precision instruments, Columbus, Ohio, USA). The balance was then set to zero, effectively zeroing out the weight of the bowl. Rats from each group cage were gently transferred into the rubber bowl one at a time, and the weight of each rat was measured and recorded to the nearest gram.

2.4.3. Haematological analysis

For haematological analysis, whole blood was collected by cardiac puncture of the anaesthetized rat using a 10 mL syringe (Nipro Corporation, BSS140, Osaka, Japan), then transferred into 4 mL EDTA tubes (EDTA K2, Bioline, China). Blood parameters were estimated using an automated haematology analyser (Sysmex Apparatus, type 8999, Sysmex Corporation, Kobe, Japan).

2.4.4. Serum biochemical analysis

For biochemical analysis, 5 mL SSTs (Vacuum Tube SG BIOTECH) containing blood were centrifuged (Eppendorf 5702 R, Hamburg, Germany) at room temperature at 3000 rpm for 15 min to obtain sera for analysis. Biochemical parameters such as, gamma glutamyl transferase (GGT), total protein (TP), albumin (ALB), globulin (GLB), total bilirubin (TB), indirect bilirubin (IDB), direct bilirubin (DB), alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferases (AST) were analyzed using the automated chemistry analyser (Selectra Pro XL, ELITechGroup, Australia). Also, lipid profile such as total cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein (HDL) cholesterol, non-HDL cholesterol, very-low-density lipoprotein (VLDL), total triglycerides (TT), coronary risk, were evaluated. Kidney function tests such as creatinine, urea, and electrolytes (sodium, potassium, and chloride) were also analyzed using the automated chemistry analyzer.

2.4.5. Hormonal test

Serum samples were also used for hormone analysis using the Cobas^@ e 411 analyser (Roche Diagnostics, Rotkreuz, Switzerland). The hormones include Follicle-Stimulating Hormone (FSH), Luteinizing Hormone (LH), Progesterone, Thyroid-Stimulating Hormone (TSH), Thyroxine (T4), Triiodothyronine (T3), and Testosterone.

2.4.6. Urine test

Urine samples were collected as individual spot samples on the 89th/90th day of the exposure period. Animals were housed individually, and freshly voided urine was collected by gently placing each rat on a clean white board until spontaneous urination occurred. The urine was immediately transferred into clean containers for analysis. Urine samples were analyzed for glucose, bilirubin, ketone, specific gravity, pH, protein, urobilinogen, nitrite, leukocytes, and blood using commercial urine analysis strips (URIT 10 V urine reagent strips, URIT Medical Electronic Co. Ltd., Guilin, China). The remaining urine samples were then centrifuged at 3000 rpm for five minutes. The supernatants were discarded, and the residue was resuspended with PBS in the tube to allow even distribution of sediments. A drop of the resuspended sediments was transferred onto a glass slide and covered with a cover slip for examination under an Olympus CX23 binocular microscope (Tokyo, Japan), connected to a digital camera, Amscope (MD500, USA).

2.4.7. Sperm count test

The left caudal epididymis was separated from the testis, minced, and placed in 6 mL of pre-warmed (35–37°C) PBS with a pH of 6.8–7.4. From this sperm suspension, a 10 μL sample was pipetted and loaded onto an improved Neubauer hemocytometer (Bright-line Precolor, HBG, Germany). Photographs of four different fields of view on the hemocytometer were taken, using a microscope equipped with a 10x objective and a camera. The sperm count was calculated by determining the total number of sperm per 1 mL of the cauda epididymis suspension [18].

Sperm Count (mL) = \frac{(Average Total Count \times 1000)}{Dilution Factor} \times 1000

Dilution Factor = \frac{Semen Volume}{Saline Volume}

2.4.8. Fasting blood sugar test

Fasting blood glucose was measured at the end of the 90-day exposure period. Animals were fasted overnight for 12 h. Capillary blood was obtained via tail prick, and glucose levels were measured immediately using a portable glucometer (Safe-Accu 2 Blood Glucose Monitoring System, Changsha Sinocare Inc., China) according to the manufacturer’s instructions.

2.4.9. Organ weight determination

The internal organs (lungs, ovaries, testis, pancreas, liver, kidney, stomach, major salivary glands, thyroid, brain, and spleen) of the animals were individually weighed using an electronic balance (The Mettler Toledo X5 80LS, Mettler Toledo, Laboratory equipment and precision instruments, Columbus, Ohio, USA). Briefly, immediately after excision, organs were gently rinsed in phosphate-buffered saline (PBS) to remove excess blood and then blotted dry. It was then weighed. Following weighing, tissues were immediately fixed in 10% phosphate-buffered formalin for histological processing.

2.4.10. Gross pathology evaluation

Post-mortem examinations were carried out to investigate the gross pathology of harvested organs after the sub-chronic test. Harvested organs from treated rats and control groups were immediately weighed and recorded. Visual investigation of potential lesions from these organs was also documented.

2.4.11. Histopathological evaluations

Tissue specimens were fixed in 10% phosphate-buffered formalin before routine histological processing. Tissues from organs (liver, lungs, heart, pancreas, spleen, testis, ovaries, thyroid gland, major salivary glands, brain, and kidneys) were processed in an automatic tissue processor (Leica TP 1020, Germany). The tissues were finally embedded in paraffin, sectioned with a Rotary microtome (Leica RM2125, RTS, and United States of America) into thin slices (5 μm) and placed on glass slides. They were then stained with routine histological stains, hematoxylin and eosin (H&E), and subsequently mounted using DPX and a glass cover-slip. Afterwards, histopathological examinations were done to ascertain potential pathological lesions. In brief, an independent assessment of blinded histology samples from the H&E-stained slides of each organ from the groups was given a randomized identity code and assessed independently by two blinded pathologists: a senior medical pathologist and an experimental/veterinary pathologist, to improve accuracy and reduce bias in histopathological analysis. A semi-quantitative scoring/grading (Nil = –; Mild = +; Moderate = ++; Severe = +++) was further done to estimate the level of reported lesions identified within the experimental and control groups. Visualization of the tissue sections was done using an Olympus CX23 binocular microscope (Tokyo, Japan), connected to a digital camera, Amscope (MD500, USA).

2.5. Statistical analysis

All statistical analyses were performed using GraphPad Prism version 10.0 (GraphPad Software, San Diego, CA, USA). Values are expressed as mean ± standard deviation (SD) in both tables and graphical representations. Normality of data distribution was assessed using the Shapiro-Wilk and Kolmogorov-Smirnov tests, and no significant deviations from normality were observed for any endpoint (p > 0.05). Homogeneity of variances was evaluated using the Brown–Forsythe test. For datasets in which the assumption of equal variances was met, one-way analysis of variance (ANOVA) was performed, followed by Bonferroni post hoc multiple comparisons. When homogeneity of variance was violated, Welch’s one-way ANOVA was applied, followed by Dunnett’s T3 post hoc test, which does not assume equal variances. Statistical analyses were conducted separately for male and female animals. A p-value of < 0.05 was considered statistically significant. All graphs were generated using GraphPad Prism 10.0, and statistical significance was indicated in the figure legends. Qualitative analyses were descriptively represented in tables as categorical outcomes.

3. Results

3.1. Physico-chemical analysis

The physicochemical analysis of the water samples revealed slightly reduced pH levels, ranging from 6.31 to 6.62 (Table 1). EC values were generally high, with the RNM site recording the highest (818.5 ± 596.1 µS/cm). Turbidity showed marked variation among the sampling sites, with RBA (3350 ± 70.71 NTU) and RSI (2025 ± 1025 NTU) exhibiting extremely high values, while RBM (17.00 ± 8.49 NTU) and RNM (14.00 ± 4.95 NTU) recorded comparatively lower values.

Table 1.

Physico-chemical parameters of the water samples from sampling sites.

Parameter	WHO	USEPA	Control 1 (dH₂O)	Control 2 (Pristine)	RBA	RBM	RNM	RSI
pH	6.5–8.5	6.5–8.5	7.00 ± 0.00	7.250 ± 0.0707	6.49 ± 0.0989	6.310 ± 0.509	6.595 ± 0.106	6.625 ± 0.0212
Electrical Conductivity (µS/cm)	500–1000	< 500	2.820 ± 0.0141	139.7 ± 0.566	281.8.0 ± 7.07	289.0 ± 74.25	818.5 ± 596.1	297.0 ± 62.93
Turbidity (NTU)	≤ 5	≤ 1	0.00	4.150 ± 0.0707	3350 ± 70.71	17.00 ± 8.485	14.00 ± 4.950	2025 ± 1025

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Values are presented as Mean ± SD (n = 3). RBA: River Bonsa; RBM: River Birim; RNM: River Nyam; RSI: River Subri; dH₂O: Distilled Water

3.2. Heavy metals analysis

Heavy metal analysis of the water samples from the contaminated rivers revealed the presence of Pb, Hg, As, Cd, and zinc at varying concentrations. Pb was significantly elevated (p < 0.05) across all sampled river bodies (RBA, RBM, RNM, and RSI). The highest concentrations were observed in RBM (1.754 ± 0.289 mg/L) and RBA (1.646 ± 0.0517 mg/L). Additionally, a notable elevation (0.0615 ± 0.0103 mg/L) in As levels was detected in RNM. Cd, although present in lower concentrations, was detected in RBA (0.00165 ± 0.0024 mg/L) and RSI (0.000125 ± 0.00019 mg/L). Similarly, Hg was detected in RNM (0.000175 ± 0.000206 mg/L) and RSI (0.0004 ± 0.000258 mg/L). Additionally, Zn levels were elevated in all water samples from the river bodies, with the highest concentration found in RBA (Table 2). The analytical performance of the ICP/MS instrument and the extraction method can be found in Supplementary Table 1.

Table 2.

Heavy metal analysis.

Parameter	Control 1 (dH₂O)	Control 2 (Pristine)	RBA	RBM	RNM	RSI	WHO
Cadmium (mg/L)	0.000 ±0.000	0.000 ±0.000	0.00165 ±0.0024	0.000 ±0.000	0.000 ±0.000	0.000125 ±0.00019	0.003
Arsenic (mg/L)	0.000 ±0.000	0.000 ±0.000	0.00123 ±0.015	0.000 ±0.000	0.0615 ±0.0103^{****††††}	0.00 ±0.000	0.01
Mercury (mg/L)	0.000 ±000	0.000 ±0.000	0.000025 ±0.012	0.000 ±0.000	0.000175 ±0.000206	0.0004 ±0.000258	0.001
Zinc (mg/L)	0.000 ±0.000	0.0041 ± 0.021	0.154 ±0.123	0.0848 ±0.00896	0.0923 ± 0.010	0.135 ± 0.179	3.0
Lead (mg/L)	0.000 ±0.000	0.00012 ±0.051	1.646 ±0.0517^{****††††}	1.754 ±0.289^{****††††}	1.119 ±0.0123^{****††††}	1.243 ±0.0395^{****††††}	0.01

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Values are presented as Mean ± SD (n = 5). RBA: River Bonsa; RBM: River Birim; RNM: River Nyam; RSI: River Subri. (*) =p < 0.05, (**) =p < 0.01, (***) =p < 0.001, (****) =p < 0.0001 were considered statistically significant when treatment groups were compared to control 1 (dH₂O: distilled water), while (†) =p < 0.05, (††) =p < 0.01, (†††) =p < 0.001, (††††) =p < 0.0001 were considered statistically significant when treatment groups were compared to control 2 (Pristine).

Furthermore, dose calculations demonstrated measurable exposure across all contaminated river groups, with marked variability by metal type and sampling location (Table 3). Lead exhibited the dominant exposure burden, with estimated animal intake ranging from 0.0129 to 0.0203 mg/kg/day across the four contaminated rivers. The highest exposure was observed in RBM, followed closely by RBA, while comparatively lower but still elevated doses were recorded in RNM and RSI. Also, arsenic exposure was notably elevated in RNM, yielding an estimated intake of approximately 0.00071 mg/kg/day, whereas concentrations in other rivers remained negligible. Cadmium and mercury exposures were detected at substantially lower concentrations, with intake estimates in the micro-dose range (≤0.00002 mg/kg/day), indicating trace-level environmental presence. Zinc intake values ranged from 0.00098 to 0.00178 mg/kg/day, reflecting moderate exposure but within nutritionally relevant thresholds. More importantly, comparative modelling of human ingestion exposure revealed similar order-of-magnitude dose relationships. Estimated human intake values were approximately two-fold higher than animal exposures due to greater assumed daily water consumption. Lead human exposure estimates ranged from 0.032 to 0.050 mg/kg/day, while arsenic exposure peaked at approximately 0.00176 mg/kg/day in RNM.

Table 3.

Estimated daily metal intake from mining-polluted water: animal exposure and human ingestion contextualisation.

Metal	River	Concentration (mg/L)	Animal Dose (mg/kg/day)	Human Dose (mg/kg/day)
Cadmium	RBA	0.00165	0.000019	0.0000471
	RBM	0.00	0.00	0.00
	RNM	0.00	0.00	0.00
	RSI	0.000125	0.0000014	0.0000036
Arsenic	RBA	0.00123	0.0000142	0.0000351
	RBM	0.00	0.00	0.00
	RNM	0.0615	0.00071	0.00176
	RSI	0.00	0.00	0.00
Mercury	RBA	0.000025	0.00000029	0.00000071
	RBM	0.00	0.00	0.00
	RNM	0.000175	0.00000202	0.000005
	RSI	0.0004	0.00000462	0.0000114
Zinc	RBA	0.154	0.00178	0.0044
	RBM	0.0848	0.00098	0.00242
	RNM	0.0923	0.00107	0.00264
	RSI	0.135	0.00156	0.00386
Lead	RBA	1.646	0.019	0.047
	RBM	1.754	0.0203	0.0501
	RNM	1.119	0.0129	0.0319
	RSI	1.243	0.0144	0.0355

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3.3. Weight of rat and serum creatinine kinase levels

The body weights of the rats were monitored over 90 days following treatment. A marked reduction (p < 0.05) in body weight was observed in all RBA, RBM, RNM, and RSI compared to the control groups. This decline was consistent across both male and female rats. The most pronounced weight suppression occurred between weeks 5 and 10. In parallel, CK-Nac levels were significantly elevated (p < 0.05) across all groups (RBA, RBM, RNM, and RSI) in comparison to both controls. The elevations were especially pronounced in the RBA, RBM, and RNM groups (Fig. 1).

Fig. 1 — Weight(g) of rat and their serum creatinine kinase levels.

3.4. Hematological analysis

Some haematological parameters in both female and male rats exhibited notable variations between the control and experimental groups. RBC counts and HGB levels were significantly (p < 0.05) decreased in the same experimental groups. HCT values also dropped significantly (p < 0.05) in the experimental groups compared to the controls. Additionally, although WBC levels (10^3/μL) were elevated in all experimental groups, only males (12.32 ± 4.199) and females (7.500 ± 0.212) in the RSI group reached significant levels (p < 0.05) when compared to the control groups (Table 4). This could be a result of significantly high neutrophil counts (p < 0.05) in this group (males: 5.910 ± 0.820, females:6.730 ± 0.537), as indicated by the neutrophil count (Neutrophil/10^3/μL). This suggests that the RSI group had acute inflammatory response activities within their organs, as a surge in neutrophilic counts marks it. On the contrary, lymphocytic count (LYMPH/ 10^3/μL) was significantly (p < 0.05) higher in the RBA (males: 4.705 ± 0.021, females: 3.325 ± 0.658) and the RNM (males: 4.615 ± 0.813, females:4.335 ± 0.813) groups, depicting a more advanced chronic inflammatory condition, marked by a surge of mononuclear WBCs and lymphocytes. However, percent neutrophil (%/%) and monocytes (%/%) were significantly (p < 0.05) high in both sexes across all experimental groups. Other parameters showed minor fluctuations and were not statistically significant (p > 0.05) (Table 4).

Table 4.

Haematological analysis.

Parameters	Female						Male
Parameters	Control 1 (dH₂O)	Control 2 (Pristine)	RBA	RBM	RNM	RSI	Control 1 (dH₂O)	Control 2 (Pristine)	RBA	RBM	RNM	RSI
WBC/ 10^3/μL	5.445 ±0.488	5.995 ±0.262	6.010 ±0.127	6.655 ±0.276	6.195 ±0.375	7.500 ±0.212^**†	6.130 ±0.002	5.480 ±0.008	6.920 ±0.035	8.555 ±0.728	6.580 ±2.319	12.32 ±4.199^*††
RBC/ 10^6/μL	8.885 ±0.092	8.330 ±0.014	6.020 ±0.537*	6.010 ±0.268*	4.405 ±1.336^**††	6.205 ±0.077*	8.530 ±0.028	8.240 ±0.070	5.930 ±0.085^**††	6.125 ±0.770^**††	5.795 ± 0.148^**††	6.430 ± 0.594^*†
HGB/ g/dL	14.560 ±0.085	14.470 ±0.184	11.280 ±0.388^**††	11.350 ±0.490^**††	10.310 ±0.410^***†††	12.580 ±0.944^*†	14.440 ±0.289	14.170 ±0.268	9.750 ±0.354 ^{****††††}	11.100 ±0.141^***†††	12.100 ±0.707^**††	12.950 ±0.212^*†
HCT/ %	48.355 ±0.354	48.923 ±1.252	39.206 ±1.273^**††	41.003 ±2.828^*†	39.100 ±0.424^**††	43.952 ±1.909	48.403 ±0.328	49.700 ±0.427	34.700 ±0.566^**††	37.751 ±1.202^**††	32.901 ±0.566^**††	40.800 ±3.536^*†
MCV/ fL	72.903 ±0.210	71.902 ±1.022	61.406 ±2.263	70.050 ±36.270	57.850 ±1.202	62.900 ±0.141	75.400 ±0.001	74.514 ±0.033	57.912 ±0.566^**††	55.551 ±0.495 ^**††	56.252 ±2.899 ^**††	57.253 ±0.212 ^**††
MCH/ pg	19.203 ±0.334	18.706 ±0.980	17.755 ±0.778	16.550 ±1.061	17.250 ±0.495	18.350 ±0.212	17.900 ±0.642	18.200 ±0.517	16.700 ±0.424	16.300 ±0.141	16.950 ±0.354	16.450 ±0.778
MCHC/ g/dL	30.300 ±0.450	29.300 ±0.340	28.900 ±0.283	29.100 ±1.273	29.800 ±0.283	29.100 ±0.283	31.800 ±0.346	30.001 ±0.149	28.800 ±0.707	29.400 ±0.566	29.700 ±0.849	28.750 ±1.485
PLT/ 10^3/μL	520.001 ±0.380	527.002 ±0.240	611.500 ±16.26	532.500 ±30.41	688.500 ±112.4	612.001 ±86.27	624.002 ±5.678	629.002 ±6.245	872.001 ±1.414	812.450 ±19.170	787.510 ±176.100	878.340 ±57.280
RDW-SD/ fL	34.400 ±0.007	32.800 ±0.066	32.600 ±2.970	33.400 ±2.404	26.650 ±1.626	29.950 ±1.626	31.900 ±0.071	31.800 ±0.085	32.900 ±0.707	32.700 ±1.414	28.500 ±1.562	27.850 ±3.182
RDW-CV/ %	21.700 ±0.845	20.200 ±0.739	16.550 ±1.909	16.350 ±1.768	16.100 ±0.707	14.150 ±0.919	20.500 ±0.537	20.700 ±0.804	16.500 ±0.707	18.950 ±0.354	16.900 ±1.980	16.001 ±0.141
PDW/ fL	6.900 ±0.348	6.900 ±0.215	7.550 ±0.212	7.150 ±1.061	7.800 ±0.849	7.400 ±0.849	6.900 ±0.057	7.300 ±1.740	6.100 ±0.283	7.200 ±0.283	6.700 ±0.141	8.000 ±1.697
MPV/ fL	7.700 ±0.342	7.200 ±0.537	7.400 ±0.654	7.400 ±0.707	7.800 ±0.212	7.450 ±0.689	7.100 ±0.649	7.200 ±0.318	6.800 ±0.212	7.300 ±0.141	6.900 ±0.187	7.750 ±1.061
P-LCR/ %	7.300 ±0.254	7.01 ±0.364	5.900 ±0.141	3.700 ±0.283^**	5.850 ±0.354	4.800 ±0.566*	6.900 ±0.684	6.300 ±0.425	2.750 ±0.354	5.001 ±0.707	3.950 ±0.212	9.300 ±6.930
PCT/ %	0.320 ±0.098	0.620 ±0.011	0.455 ±0.071	0.420 ±0.438	0.530 ±0.042	0.460 ±0.099	0.440 ±0.017	0.640 ±0.074	0.580 ±0.014	0.580 ±0.255	0.685 ±0.120	0.820 ±0.424
NEUTROPHIL/ 10^3/μL	1.260 ±0.014	1.440 ±0.063	1.535 ±0.233	1.985 ±0.289	2.060 ±0.141	5.910 ±0.820^**††	1.990 ±0.556	1.730 ±0.478	1.170 ±0.099	3.770 ±0.608	1.805 ±0.021	6.730 ±0.537^**††
LYMPH/ 10^3/μL	1.550 ±0.071	2.150 ±0.212	3.325 ±0.658*	2.590 ±0.269	4.335 ±0.813^**†	2.545 ±0.361	1.495 ±0.092	1.517 ±0.443	4.705 ±0.021^*†	3.660 ±0.622	4.615 ± 0.813^*†	3.755 ±0.901
MONOCYTES/ 10^3/μL	0.380 ±0.007	0.330 ±0.012	0.390 ±0.297	0.185 ±0.120	0.940 ±0.198	0.605 ±0.163	0.420 ±0.009	0.440 ±0.089	0.360 ±0.014	0.400 ±0.410	0.855 ±0.0212	1.885 ±1.054
EOSINOPHILS/ 10^3/μL	0.240 ±0.081	0.290 ±0.036	0.120 ±0.057	0.120 ±0.127	0.265 ±0.078	0.170 ±0.042	0.260 ±0.084	0.220 ±0.001	0.0650 ±0.007	0.205 ±0.191	0.550 ±0.382	0.400 ±0.113
BASOPHILS/ 10^3/μL	0.028 ±0.016	0.030 ±0.072	0.010 ±0.041	0.025 ±0.021	0.015 ±0.007	0.010 ±0.051	0.030 ±0.007	0.027 ±0.008	0.015 ±0.007	0.020 ±0.014	0.040 ±0.014	0.050 ±0.057
NEUTROPHIL%/ %	17.203 ±0.654	19.816 ±1.237	39.551 ±2.192^{****†††}	47.207 ±1.414 ^{****††††}	41.908 ±0.989 ^{****††††}	46.252 ±1.485 ^{****††††}	17.205 ±0.852	17.300 ±0.870	46.105 ±0.283^{****††††}	47.044 ±0.424^{****††††}	45.356 ±0.778^{****††††}	48.502 ±0.424^{****††††}
LYMPH%/ %	50.018 ± 0.552	52.815 ± 2.333	55.185 ± 3.182	54.725 ± 1.626	68.705 ± 1.485^**††	60.651 ± 2.445	51.306 ± 1.838	54.109 ± 2.970	64.853 ± 0.778*	46.010 ± 2.940^†	62.250 ± 1.273*	45.436 ± 1.047
MONOCYTES%/ %	7.400 ±0.312	7.800 ±0.143	15.090 ±0.304^**	15.150 ±0.354^**	18.100 ±0.283^**†	12.750 ±3.041*	7.900 ±0.211	8.001 ±0.138	15.330 ±0.169^*††	18.001 ±0.566^**††	16.300 ±0.989^*††	15.350 ±3.606*
EOSINOPHILS%/ %	3.100 ±0.089	3.700 ±0.036	5.250 ±0.636	3.700 ±0.141	5.200 ±0.989	3.200 ±0.566	4.10 ±0.071	4.00 ±0.026	5.250 ±0.212	5.600 ±0.225	4.100 ±0.283	3.450 ±0.212
BASOPHILS%/ %	0.300 ±0.008	0.400 ±0.010	0.400 ±0.141	1.200 ±0.248	0.350 ±0.070	0.2500 ±0.071	0.400 ±0.005	0.200 ±0.005	0.300 ±0.141	0.300 ±0.283	0.600 ±0.138	0.350 ±0.354
IG%/ %	0.200 ±0.009	0.190 ±0.063	0.150 ±0.212	0.350 ±0.212	0.150 ±0.212	0.100 ±0.141	0.200 ±0.018	0.200 ±0.045	0.200 ±0.058	0.150 ±0.071	0.200 ±0.001	0.350 ±0.071
MICROR/ %	65.400 ±0.066	66.900 ±0.566	71.950 ±2.616	59.500 ±0.707	72.550 ±4.031	51.250 ±0.071	59.400 ±0.024	68.500 ±0.085	69.250 ±0.212	72.850 ±3.606	75.150 ±3.748	63.200 ±2.263
MACROR/ %	1.200 ±0.107	1.200 ±0.125	1.700 ±0.840	1.400 ±0.142	0.900 ±0.251	1.950 ±0.081	0.900 ±0.205	0.900 ±0.123	0.750 ±0.131	0.750 ±0.212	0.950 ±0.354	0.750 ±0.071
NRBC%/ %	0.200 ±0.065	0.180 ±0.012	0.400 0.141	1.300 ±0.283	1.650 ±0.212	1.001 ±0.566	0.100 ±0.053	0.100 ±0.089	0.750 ±0.071	0.650 ±0.072	0.500 ±0.141	0.150 ±0.064

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Values are presented as the Mean ± SD (n = 5). RBA: River Bonsa; RBM: River Birim; RNM: River Nyam; RSI: River Subri. WBC: White Blood Cells; RBC: Red Blood Cells; HGB: Haemoglobin; HCT: Haematocrit; MCV: Mean Corpuscular Volume; MCH: Mean Corpuscular Haemoglobin; MCHC: Mean Corpuscular Haemoglobin Concentration; PLT: Platelets; RDW-SD: Red Cell Distribution Width – Standard Deviation; RDW-CV: Red Cell Distribution Width – Coefficient of Variation; PDW: Platelet Distribution Width; MPV: Mean Platelet Volume; P-LCR: Platelet-Large Cell Ratio; PCT: Plateletcrit; IG%: Immature Granulocyte percentage; MICROR: Microcytic Red Cells; MACROR: Macrocytic Red Cells; NRBC%: Nucleated Red Blood Cell percentage. (*) =p < 0.05, (**) =p < 0.01, (***) =p < 0.001, (****) =p < 0.0001 were considered statistically significant when treatment groups were compared to control 1 (dH2O: distilled water), while (†) =p < 0.05, (††) =p < 0.01, (†††) =p < 0.001, (††††) =p < 0.0001 were considered statistically significant when treatment groups were compared to control 2 (Pristine).

3.5. Liver function test & lipid profile

There were significant reductions (p < 0.05) in total protein and globulin across all the experimental groups. Significant changes (p < 0.05) in albumin were also observed, predominantly in the male group across all experimental groups (Table 5). Elevated (p < 0.05) levels of AST, ALP & ALT were noted in both male and female subjects in groups, with the most notable increase seen in RBA and RNM (Fig. 2).

Table 5.

Serum liver function test & lipid profile analysis.

Parameters	Female						Male
Parameters	Control 1 (dH₂O)	Control 2 (Pristine)	RBA	RBM	RNM	RSI	Control 1 (dH₂O)	Control 2 (Pristine)	RBA	RBM	RNM	RSI
Albumin (g/l)	33.650 ±1.294	32.300 ±0.283	27.850 ±0.608*	27.330 ±2.680*	27.740 ±0.608*	30.640 ±0.0283	33.250 ±0.21	33.050 ±0.354	27.690 ±1.824^**††	26.150 ±0.849^**††	24.250 ±1.351^***†††	30.340 ±0.141
Total protein (g/l)	75.001 ±1.414	72.200 ±1.412	54.500 ±0.707^***†††	60.800 ±2.970^***†††	61.650 ±1.626^**††	69.350 ±1.344	74.250 ±1.920	73.930 ±0.520	54.500 ±0.707^{****††††}	61.200 ±0.989^{****††††}	60.400 ±1.131^{****††††}	70.450 ±0.071
Globulin (g/l)	44.050 ±0.226	43.890 ±0.778	36.080 ±1.838^***††	42.480 ±0.289	36.920 ±2.666^**††	40.220 ±0.799	43.720 ±0.511	43.530 ±0.545	36.760 ±0.300^***†††	40.050 ±1.040^**†	36.160 ±0.179 ^***†††	39.610 ±0.635 ^**††
Bilirubin Total (μmol/l)	1.820 ±0.0848	1.895 ±0.0212	2.055 ±0.0919	1.680 ±0.141	2.020 ±0.0566	2.020 ±0.0989	1.500 ±0.0566	1.530 ±0.0424	1.420 ±0.212	2.210 ±0.594	1.660 ±0.255	1.720 ±0.085
Bilirubin Direct (μmol/l)	0.425 ±0.064	0.535 ±0.035	0.585 ±0.163	0.735 ±0.431	0.890 ±0.665	0.96 ±0.184	0.450 ±0.057	0.460 ±0.028	0.565 ±0.134	1.210 ±0.891	0.345 ±0.035	1.155 ±0.021
Bilirubin Indirect (μmol/l)	1.385 ±0.092	1.290 ±0.028	1.475 ±0.078	0.945 ±0.573	1.130 ±0.608	1.060 ±0.085	1.055 ±0.021	1.055 ±0.007	0.870 ±0.368	1.001 ±0.297	1.315 ±0.289	0.570 ±0.071
Cholesterol (mmol/l)	0.8900 ±.028	1.040 ±0.042	1.335 ±0.276	1.425 ±0.417	1.100 ±0.424	1.540 ±0.085	1.560 ±0.141	1.185 ±0.092	1.310 ±0.184	1.370 ±0.184	1.220 ±0.113	1.370 ±0.255
HDL CHOL (mmol/l)	0.390 ±0.028	0.420 ±0.099	0.580 ±0.240	0.695 ±0.289	0.505 ±0.417	0.570 ±0.071	0.595 ±0.021	0.5850 ±0.021	0.580 ±0.014	0.615 ±0.262	0.580 ±0.014	0.725 ±0.078
Triglycerides (mmol/l)	0.390 ±0.028	0.420 ±0.099	0.580 ±0.240	0.695 ±0.289	0.505 ±0.417	0.570 ±0.071	0.655 ±0.007	0.905 ±0.021	0.855 ±0.163	0.920 ±0.057	0.820 ±0.453	0.665 ±0.078
LDL CHOL (mmol/l)	0.900 ±0.042	0.105 ±0.007	0.270 ±0.042	0.390 ±0.028	0.290 ±0.014	0.425 ±0.092	0.240 ±0.014	0.210 ±0.028	0.340 ±0.099	0.335 ±0.007	0.265 ±0.149	0.280 ±0.127
NON-HDL CHOL (mmol/L)	0.635 ±0.035	0.640 ±0.014	0.755 ±0.035	0.730 ±0.127	0.595 ±0.007	0.720 ±0.057	0.620 ±0.042	0.610 ±0.057	0.730 ±0.169	0.755 ±0.035	0.640 ±0.057	0.645 ±0.177
VLDL (mmol/l)	0.405 ±0.021	0.330 ±0.028	0.480 ±0.071	0.485 ±0.092	0.305 ±0.021	0.450 ±0.042	0.280 ±0.028	0.400 ±0.028	0.390 ±0.071	0.420 ±0.028	0.375 ±0.205	0.305 ±0.035
Coronary Risk	1.600 ±0.014	1.680 ±0.042	2.410 ±0.523	2.110 ±0.283	2.780 ±1.457	2.213 ±0.205	1.535 ±0.120	1.49 ±0.042	2.255 ±0.262	2.330 ±0.523	2.105 ±0.007	1.885 ±0.149

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Values are presented as the Mean ± SD (n = 5). RBA: River Bonsa; RBM: River Birim; RNM: River Nyam; RSI: River Subri. Total protein: TP; Globulin: GLB; Low Density Lipoprotein: LDL, Non-High-Density Lipoprotein CHOL: NON-HDL; VLDL: Very Low-Density lipoprotein; AST: Aspartate Aminotransferase; ALP: Alkaline Phosphate; ALT: Alanine Aminotransferase. (*) =p < 0.05, (**) =p < 0.01, (***) =p < 0.001, (****) =p < 0.0001 were considered statistically significant when treatment groups were compared to control 1 (dH2O: distilled water), while (†) =p < 0.05, (††) =p < 0.01, (†††) =p < 0.001, (††††) =p < 0.0001 were considered statistically significant when treatment groups were compared to control 2 (Pristine).

Fig. 2 — AST, ALP & ALT levels in both males & females (A, D & G), males (C, F & I) and females (B, E & H) respectively.

3.6. Hormone analysis

A mark reduction (p < 0.05) in TS was observed in only males in the experimental groups. Similarly, a significant (p < 0.05) decrease in PG was observed in only females of the experimental groups. E2 and LH, on the other hand, were significantly (p < 0.05) elevated in both males and females of some of the experimental groups (Table 6) compared to the controls. Conversely, TSH, FT3 and FT4 levels were not significantly different (p > 0.05) when the experimental groups were compared with the controls (Table 6).

Table 6.

Hormone analysis.

Parameters	Female						Male
Parameters	Control 1 (dH₂O)	Control 2 (Pristine)	RBA	RBM	RNM	RSI	Control 1 (dH₂O)	Control 2 (Pristine)	RBA	RBM	RNM	RSI
E2 (pg/mL)	64.980 ±0.127	62.340 ±0.792	73.450 ±1.195^†	74.150 ±3.649^*†	81.150 ±0.361^**††	71.001 ±4.525	42.21 ±0.968	44.360 ±1.273	54.250 ±2.333*	51.970 ±3.062	55.400 ±3.677^*†	56.750 ±3.606^**†
LH (mIU/mL)	0.321 ±0.074	0.281 ±0.013	0.123 ±0.033^**†	0.108 ±0.011^**†	0.214 ±0.002	0.234 0.016	0.216 ±0.007	0.239 ±0.028	0.110 ±0.014^**	0.115 ±0.021^**	0.207 ±0.018	0.216 ±0.240
PG (ng/mL)	47.150 ±0.495	50.020 ±0.459	22.100 ±8.648^**††	20.590 ±3.309^**††	22.330 ±0.262^**††	25.970 ±6.074^*††	11.940 ±0.0989	11.680 ±1.732	4.7350 ±0.658	5.440 ±0.778	5.805 ±1.732	7.255 ±4.264
TS (ng/mL)	1.145 ±0.064	1.655 ±0.049	0.755 ±0.700	1.411 ±0.622	0.512 ±0.028	0.613 ±0.311	4.790 ±0.240	5.430 ±0.438	1.625 ±0.191^**††	2.845 ±0.163^†	0.517 ±0.105^**†††	1.483 ±1.212^**††
TSH (μIU/mL)	0.005 ±0.007	0.005 ±0.009	0.015 ±0.014	0.005 ±0.049	0.049 ±0.062	0.024 ±0.026	0.005 ±0.003	0.005 ±0.001	0.005 ±0.098	0.011 ±0.008	0.005 ±0.001	0.005 ±0.001
FT3 (pmol/L)	28.060 ±0.749	37.100 ±0.842	31.640 ±3.345	22.290 ±0.707	37.930 ±17.070	29.410 ±4.702	28.460 ±0.085	31.660 ±0.509	26.810 ±1.174	26.030 ±0.948	30.001 ±0.001	28.070 ±0.587
FT4 (pmol/L)	99.800 ±0.023	99.560 ±0.042	98.540 ±0.057	96.870 ±1.336	99.800 ±0.520	89.150 ±1.534	99.700 ±0.446	99.670 ±2.334	96.120 ±1.120	97.080 ±0.456	99.200 ±0.045	98.020 ±0.234

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Values are presented as Mean ± SD (n = 5). RBA: River Bonsa; RBM: River Birim; RNM: River Nyam; RSI: River Subri. E2: Estradiol; LH: Luteinizing Hormone; PG: Progesterone; TS: Testosterone; FT3: Triiodothyronine; FT4: Thyroxine; TSH: Thyroid-Stimulating Hormone. (*) =p < 0.05, (**) =p < 0.01, (***) =p < 0.001, (****) =p < 0.0001 were considered statistically significant when treatment groups were compared to control 1 (dH2O: distilled water), while (†) =p < 0.05, (††) =p < 0.01, (†††) =p < 0.001, (††††) =p < 0.0001 were considered statistically significant when treatment groups were compared to control 2 (Pristine).

3.7. Kidney function test & urinalysis

Observed results showed significant changes (p < 0.05) in the creatinine, urea, and chloride levels in the experimental groups compared to the control groups (Table 7). Moreover, urine in experimental groups showed notable changes compared to controls in the urine-strip test analysis (Table 8). Urobilinogen and bilirubin were consistently elevated in all test groups (up to +2 and 0.5 mg/dL, respectively). Proteinuria was observed in all experimental groups, ranging from + + (RBM & RSI, 100 mg/dL) to + ++ (RBA & RNM, 300 mg/dL). Similarly, traces of leucocytes and RBCs were identified in RBA, RBM and RNM but not RSI. RSI showed trace ketones (±5 mg/dL). Specific gravity was also high in RBA and RNM (1.030). Hematuria (±, 10 cells/μL) was detected in RNM and RBA. No significant changes were observed with the glucose test (Table 8).

Table 7.

Serum kidney function test.

Parameters	Female						Male
Parameters	Control 1 (dH₂O)	Control 2 (Pristine)	RBA	RBM	RNM	RSI	Control 1 (dH₂O)	Control 2 (Pristine)	RBA	RBM	RNM	RSI
Creatinine (μmol/L)	55.860 ±0.707	54.390 ±1.414	67.130 ±2.369^†	58.960 ±4.632	70.010 ±5.317^*†	61.190 ±3.012	53.160 ±1.414	54.100 ±0.707	68.910 ±7.679^*†	57.420 ±0.261	64.28 ±1.768	58.980 ±2.234
Urea (mmol/L)	2.550 ±0.141	3.750 ±0.071	6.975 ±0.586^**†	5.950 ±1.768*	5.090 ±0.523	7.280 ±1.386^***††	2.670 ±0.141	3.425 ±0.176	6.825 ±0.572^**†	5.085 ±1.068	4.980 ±0.523	5.870 ±1.174*
Sodium (mmol/L)	142.300 ±0.883	141.600 ±0.141	142.700 ±2.192	143.300 ±1.273	141.700 ±1.061	142.001 ±0.424	144.001 ±0.353	141.900 ±0.707	144.600 ±4.172	142.100 ±0.282	143.002 ±0.141	142.300 ±0.060
Potassium (mmol/L)	8.395 ±0.233	9.040 ±0.141	7.975 ±1.025	5.630 ±0.240^††	7.065 ±0.360	5.800 ±0.707^††	8.130 ±0.282	8.175 ±0.247	6.390 ±0.905	7.335 ±1.789	7.185 ±2.312	5.420 ±0.028
Chloride (mmol/L)	108.6 ±1.414	103.4 ±1.414	104.2 ±0.495	100.5 ±2.899*	103.0 ±0.495	106.1 ±3.606	114.9 ±0.0707	106.1 ±0.353	107.0 ±4.525*	104.5 ±0.141^**	103.2 ±0.848^**	103.9 ±0.071^**

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Values are presented as Mean ± SD (n = 5). RBA: River Bonsa; RBM: River Birim; RNM: River Nyam; RSI: River Subri. (*) =p < 0.05, (**) =p < 0.01, (***) =p < 0.001, (****) =p < 0.0001 were considered statistically significant when treatment groups were compared to control 1 (dH2O: distilled water), while (†) =p < 0.05, (††) =p < 0.01, (†††) =p < 0.001, (††††) =p < 0.0001 were considered statistically significant when treatment groups were compared to control 2 (Pristine).

Table 8.

Urine strip test.

	Control 1 (dH₂O)	Control 2 (Pristine)	RBA	RBM	RNM	RSI
Glucose/mg/dL(mmol/L)	- (0)	- (0)	- (0)	- (0)	- (0)	- (0)
Urobilinogen/mg/dL(μmol/L)	Normal	Normal	+ 2(33)	+ 2(33)	+ 2(33)	+ 2(33)
Bilirubin/mg/dL(μmol/L)	- (0)	_- (0)	+ 0.5(8.6)	+ 0.5(8.6)	+ 0.5(8.6)	+ 0.5(8.6)
Leukocyte/ CELL/μL	- (0)	- (0)	± (15)	± (15)	± (15)	- (0)
Nitrite	-	-	-	-	-	-
Ketone/mg/dL(mmol/L)	- (0)	- (0)	- (0)	- (0)	- (0)	± 5(0.5)
Protein/mg/dL(g/L)	- (0)	- (0)	+ ++ 300(3.0)	+ + 100(3.0)	+ ++ 300(3.0)	+ + 100(1.0)
Specific Gravity	1.005	1.010	1.030	1.025	1.030	1.020
pH	6.5	7.0	6.0	6.5	6.5	6.0
Blood/ CELL/μL	- (0)	- (0)	± (10)	± (0)	± 10	- (0)

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Values are represented as semi-quantitative for all groups (Males plus females of the same group; n = 10). RBA: River Bonsa; RBM: River Birim; RNM: River Nyam; RSI: River Subri

3.8. Semen concentration and fasting blood sugar analysis

High levels (p < 0.05) of fasting blood sugar (FBS) were observed in RBA and RNM. These differences were mostly observed in the female group as compared to the male group (Fig. 3). With the semen concentration, there was a significant reduction (p < 0.05) in sperm concentration observed across all experimental groups (RBA, RBM, RNM, RSI) compared to both controls. This marked decline was evident in the RNM and RBA groups, where sperm counts dropped to nearly a quarter of the levels compared to the control groups. Moreover, in the RBM and RSI groups, while they showed markedly reduced sperm concentrations, they retained slightly higher values than RBA and RNM (Fig. 3).

Fig. 3 — Semen concentration and fasting blood sugar (FBS) levels in both males and females.

3.9. Organ weight and gross pathology

Significant reductions (p < 0.05) in kidney weights were observed in female rats exposed to RBA, RBM, RNM, and RSI, with the most profound decrease noted in the RNM (0.280 ± 0.014 g) group, followed closely by RBA (0.255 ± 0.007 g) and RBM (0.350 ± 0.028 g). A similar pattern was also observed in male rats, particularly in RNM (0.255 ± 0.421 g) and RBA (0.320 ± 0.014 g) (Table 9). Likewise, the ovaries of females in RNM (0.555 ± 0.049 g) and RBA (0.540 ± 0.014 g) had significantly higher weights (p < 0.05) compared with the controls. This could be due to the presence of the ovarian cysts (Supplementary Figure 3B) identified exclusively in these groups (Supplementary Table 2).

Table 9.

Organ weight.

Parameters (g)	Female						Male
Parameters (g)	Control 1 (dH₂O)	Control 2 (Pristine)	RBA	RBM	RNM	RSI	Control 1 (dH₂O)	Control 2 (Pristine)	RBA	RBM	RNM	RSI
Lungs	1.290 ±0.356	1.310 ±0.940	1.470 ±0.223	1.760 ±0.198	1.400 ±0.780	1.620 ±0.023	1.310 ±0.014	1.355 ±0.021	1.815 ±0.035	1.505 ±0.007	1.640 ±0.014	2.120 ±1.342
Ovaries/Testis	0.070 ±0.120	0.050 ±0.640	0.540 ±0.014^***†††	0.135 ±0.021	0.555 ±0.049^***†††	0.120 ±0.014	1.145 ±0.049	1.435 ±0.035	0.800 ±0.014	1.445 ±0.064	0.850 ±0.424	0.865 ±0.205
Thyroid	0.016 ±0.367	0.018 ±0.853	0.012 ± 0.243	0.014 ± 0.854	0.013 ± 0.675	0.013 ± 0.350	0.015 ± ±0.235	0.018 ± ±0.960	0.011 ± 0.025	0.012 ± 0.564	0.010 ± 0.580	0.013 ± 0.782
Pancreas	0.920 ±0.146	0.840 ±0.056	0.873 ±0.255	1.145 ±0.134	0.900 ±0.587	1.110 ±0.255	1.105 ±0.107	1.205 ±0.021	0.980 ±0.028	1.340 ±0.060	1.140 ±0.212	0.980 ±0.028^†
Liver	6.370 ±0.022	5.950 ±0.760	6.620 ±0.318	6.575 ±0.117	5.850 ±0.016	5.560 ±1.018	6.460 ± 0.113	6.415 ±0.012	6.715 ±0.512	7.805 ±0.065	8.215 ±0.790^***†††	7.045 ±1.546
Kidney	0.670 ±0.060	0.620 ±0.890	0.255 ± 0.007^*** ^†††	0.350 ± 0.028^*** ^†††	0.280 ±0.014^**†††	0.465 ±0.007^**††	0.650 ±0.013	0.680 ±0.024	0.320 ±0.014^***†††	0.435 ±0.021^***†††	0.255 ± 0.421^***†††	0.620 ±0.450^†
Submandibular & Sublingual glands	0.70 ±0.340	0.680 ±0.850	0.707 ±0.166	0.691 ±0.023	0.630 ±0.001	0.680 ±0.127	0.650 ±0.014	0.730 ±0.028	0.660 ±0.028	0.700 ±0.141	0.725 ±0.007	1.030 ±0.651
Brain	1.750 ±0.001	1.820 ±0.001	1.603 ±0.379	1.745 ±0.007	1.670 ±0.000	1.700 ±0.057	1.965 ±0.021	1.935 ±0.021	1.935 ±0.021	1.925 ±0.021	1.690 ±0.099	1.795 ±0.134
Spleen	0.620 ±0.001	0.600 ±0.001	0.733 ±0.090	0.555 ±0.177	0.511 ±0.010	0.585 ±0.045	0.635 ±0.212	0.645 ±0.021	0.560 ±0.014	0.645 ±0.021	0.515 ±0.134	0.620 ± 0.010

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Values are presented as the Mean ± SD (n = 5). g: grams; RBA: River Bonsa; RBM: River Birim; RNM: River Nyam; RSI: River Subri. (*) =p < 0.05, (**) =p < 0.01, (***) =p < 0.001, (****) =p < 0.0001 were considered statistically significant when treatment groups were compared to control 1 (dH2O: distilled water), while (†) =p < 0.05, (††) =p < 0.01, (†††) =p < 0.001, (††††) =p < 0.0001 were considered statistically significant when treatment groups were compared to control 2 (Pristine).

Additionally, cervical lymph node enlargement (Supplementary Figure 3A) and hind limb oedema were present in the RBA, RBM, and RNM groups, indicating possible systemic inflammation and lymphatic involvement (Supplementary Table 2). Dispersed and focal pale patches on the serosal surfaces of the liver and lungs were also observed, suggesting localised tissue damage. Additionally, piloerection and focal facial hair loss were consistently noted across all experimental groups (RBA, RBM, RNM & RSI) (Supplementary Table 2).

3.10. Histopathological evaluation

Histopathological examination of the liver (Fig. 4A-F) from the normal control (Fig. 4A) showed normal polygonal-shaped hepatocytes, containing basophilic central rounded nuclei, devoid of structural changes and cellular injuries. Liver sections from the pristine control (Fig. 4B) were similar to Fig. 4A, but with a few leucocytic cell infiltrates in sinusoids. In contrast, sections from the RBA group (Fig. 4C) showed the presence of periportal and centrilobular leucocytosis, accompanied by spotty necrosis within the liver lobule. Hepatocytes manifested diﬀerent stages (pyknosis, karyorrhexis, and karyolysis) of necrosis, with pyknosis being more evident than the other two (karyorrhexis and karyolysis). There is also Kupffer cell activation and mild edema. Liver sections also showed the presence of multiple well-circumscribed non-caseating granulomatous inflammatory lesions. These granulomatous lesions depict signs of chronic inflammatory reaction towards non-degradable agents, such as heavy metals. Likewise, the RBM group had similar changes compared with the RBA group, but with few non-caseating granulomas, and with marked leucocytosis, predominantly seen around the periportal regions (Fig. 4D). Liver section from the RNM group also revealed the presence of multiple well-circumscribed non-caseating granulomas (similar to RBA). Sinusoidal congestion and leucocytosis, accompanied by multiple necrotic nuclear degenerative changes (Fig. 4E). Similarly, sections from the RSI (Fig. 4F) group showed the presence of non-caseating granulomas at their early formative stages (Thus, not well circumscribed), accompanied by leucocytosis, sinusoidal congestion, and edema. Spotty necrosis of hepatocytes is present within liver lobules in sections from RBA, RBM, and RNM.

Analyzed sections of kidney tissue from the normal control (Fig. 5A) revealed a normal shape of the renal corpuscle and convoluted tubules. Similarly, the pristine control (Fig. 5B) showed normal renal corpuscles and tubules devoid of any cellular degeneration or necrotic activity. Conversely, there were focal glomerular atrophy and degenerative cellular changes observed within some corpuscles, with increased capsular space, accompanied by distortion and collapse of the convoluted tubule structure in the RBA group (Fig. 5C). Necrosis was also identified in some tubules. The collapse and distortion of the convoluted tubules, along with necrosis in some tubular cells, suggest acute tubular injury. These morphological alterations indicate compromised glomerular filtration and tubular reabsorption processes. In the RBM group (Fig. 5D), convoluted tubules displayed normal structure, but some corpuscles showed a focal arrangement of highly eosinophilic, degenerative, and atrophied glomeruli, along with increased capsular space. Additionally, necrotizing cells within glomeruli, as well as increased capsular spaces, were observed. Some tubule structures were collapsed and distorted, with hydropic changes in the RNM group (Fig. 5E). However, a few degenerative and distorted tubules, as well as focal atrophied and degenerative glomeruli, were noted in the RSI group (Fig. 5F).

Observations of tissue sections of the spleen from the normal control group (Fig. 6A) revealed a white and red pulp, showing normal cellularity of parenchymal cells devoid of obvious pathological insults. Likewise, the pristine control group (Fig. 6B) displayed splenic architecture and parenchymal integrity comparable to the normal control. However, the RBA group (Fig. 6C) showed a significant reduction in cellularity in red and white pulp, with notable stromal fibrosis and vacuolization. Mild hemosiderosis was also observed. Moreover, very high hemosiderosis and mild fibrosis within the red pulp were observed in the RBM group (Fig. 6D). Moreover, the RNM group (Fig. 6E) showed a reduction in cellularity in red and white pulp, with marked fibrosis and vacuolization within stroma. Haemosiderosis is also high in this group. In contrast, sections from the RSI group revealed high haemosiderosis and mild stromal fibrosis within red pulp, similar to the RBM group. (Fig. 6F).

Lung tissue section from the normal group (Fig. 7A) had thin alveolar septa lined with simple squamous epithelium, and that of the pristine control (Fig. 7B) had slightly thickened alveolar septa. Presence of fibrotic alveolitis (potential granuloma formation), seen as very thick alveolar septa marked by leukocytosis in the RBA group (Fig. 7C), which indicates a chronic inflammatory response to persistent irritants due to the heavy metal intoxication. The RBM group (Fig. 7D) showed alveolar septal thickening, leukocytosis, and oedema. The presence of oedema indicates increased capillary permeability and vascular damage, reflecting fluid leakage into the alveolar spaces, leading to the observed thickening. Furthermore, the RNM group (Fig. 7E) also exhibited fibrosis, alveolar septal thickening, leukocytosis, and oedema formation. There was marked alveolar septal thickening, with complete disruption of normal alveolar structure, along with haemorrhagic oedema within the lumen of the alveoli.

Lastly, the histological section of the testis from the normal and pristine groups (Fig. 8A and B) revealed normal histology of seminiferous tubule germ (spermatogonium, spermatocytes, spermatids, and spermatozoa) and sustentacular (Sertoli) cells, and also the surrounding wall of laminar propria and germinal epithelia. Interstitial/stromal layer surrounding the tubules has normal thickness, with the presence of interstitial Leydig cells and intact blood vessels in the interstitium. In the treated groups (RBA, RBM, RNM and RSI), sections (Fig. 8C-F) showed pathological insults such as hemorrhagic edema, leucocytic cell infiltrates, and separation of lamina propria (tubule wall) from adjacent seminiferous tubules within the interstitial area. Also, Leydig cells that produce testosterone are found within this interstitial area and thus, their cellularity and function could be impaired. There is also reduced cellularity and disorganized cellular arrangement within tubules. More importantly, the germ and sustentacular cells are seen ‘pulling off’ from the inner germinal wall of the tubules in these groups. This is clear evidence of loss of tight junctions and potential disruption of the blood-testis barrier (BTB) between adjacent Sertoli cells at the base of the tubules. Also, evidently, interstitial/stromal layer surrounding tubules is thicker, especially in the RNM group (Fig. 8E), compared to the normal and pristine groups. This could be as a result of stromal and immune reactivity, causing increased connective tissue septa (early signs of fibrosis).

Overall, although sections from all the treated groups (RBA, RBM, RNM, and RSI) showed presence of cellular derangement and degenerative changes in the parenchyma of organs compared with the controls, generally, sections from RBA and RNM had myriad and more advanced stage pathological lesions/disease compared to RBM and RSI.

Photomicrographs of the cardiac sections revealed varying degrees of myocardial alterations across treatment groups (Fig. 9). Sections from normal and pristine groups (Figure A&B) displayed normal myocardial morphology with well-organized cardiac muscle fibers, intact striations, and centrally located nuclei. In contrast, the heart sections from treated rats exhibited lesions of varying severity. RBA group showed marked pathological alterations characterized by extensive leucocytic cell infiltrates, interstitial fibrosis, myocardial edema, and multifocal areas of myocardial necrosis. Also, degenerating myofibers exhibited homogeneous eosinophilic sarcoplasm with pyknotic nuclei (Fig. 9C). Likewise, the RBM group exhibited mild pathological changes, including few leucocytic infiltrates, focal hemorrhage, and discrete myocardial degenerations (Fig. 9D), whereas RNM demonstrated minimal myocardial degeneration and vacuolation of cardiomyocytes with little inflammatory response (Fig. 9E). RSI group revealed focal areas of myocardial hemorrhage associated with mild inflammatory cell infiltrates and scattered degenerating myofibers (Fig. 9F). Across all examined organs, the control groups exhibited absent to minimal histopathological alterations, while the exposed groups showed a distinct, progressive increase in the severity of lesions (Table 10). In the liver, treatment groups exhibited predominantly moderate to severe cellular degeneration, granuloma formation, leucocytosis, congestion, and oedema, with the highest scores generally recorded in RBA, RBM, and RNM. Renal lesions were characterized by moderate to severe structural distortion, glomerular atrophy, and hydropic change. Also, the spleen showed consistent moderate to severe vacuolization, stromal fibrosis, hemosiderosis, and reduced cellularity across treatment groups. Likewise, the lungs and testes displayed largely moderate to severe inflammatory and degenerative changes.

Table 10.

Evaluation of the histopathological parameters in the organs.

Organ	Histopathological Parameters	Control 1 (dH₂O)	Control 2 (Pristine)	RBA	RBM	RNM	RSI
Liver	Cellular degeneration	–	–	+ ++	+ ++	+ ++	+
	Granuloma	–	–	+ +	+ +	+ ++	+
	Leucocytosis	–	+	+ ++	+ +	+ ++	+ +
	Congestion	–	–	+ +	+	+ +	+
	Oedema	–	–	+ +	+ +	+	+ +
Kidney	Cellular degeneration	–	–	+ +	+	+ +	+
	Leucocytosis	–	–	+ +	+	+ +	+ +
	Structural distortion	–	–	+ ++	+	+ ++	+
	Glomerular atrophy	–	–	+ ++	+ +	+ ++	+
	Hydropic change	–	–	+ ++	+ +	+ ++	+ +
Spleen	Vacuolization	–	–	+ ++	+	+ +	+
	Stromal fibrosis	–	–	+ ++	+	+ ++	+
	Hemosiderosis	–	–	+ +	+	+ +	+
	Reduced cellularity	–	–	+ +	+	+ +	+
Lungs	Thickened alveolar septa	–	+	+ ++	+ ++	+ ++	+ ++
	Granuloma	–	–	+ ++	+	+ +	-
	Leucocytosis	–	+	+ ++	+ ++	+ ++	+ +
	Haemorrhagic oedema	–	+	+ ++	+ +	+ ++	+ +
	Structural distortion	–	–	+ +	+ +	+ +	+ +
Testis	Cellular degeneration	–	–	+ ++	+ +	+ ++	+ +
	Stromal fibrosis	–	–	+ +	+ +	+ ++	+ +
	Leucocytosis	–	–	+ ++	+ +	+ +	+ +
	Haemorrhagic oedema	–	–	+ +	+	+ +	+ +
	Reduced cellularity	–	–	+ +	+	+ +	+

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Grading scale: “ – ” = Nil; “+ ” = Mild; “+ +” = Moderate; “+ + + ” = Severe

4. Discussion

Illegal mining activities, commonly known as ‘galamsey’, are conducted by untrained and poorly equipped laborers who often use toxic heavy metals such as Hg, Pb, and As for mineral extraction, which ends up polluting and contaminating nearby river bodies (Obiri-Yeboah et al., 2021). These mining-polluted river bodies serve as a source of water for domestic purposes for nearby communities, and thus, may have detrimental effects on individuals or livestock that use them [19].

Findings from this study revealed significant Pb contamination in the sampled rivers, especially in RBA and RBM. Similarly, a survey [13] reported high concentrations of lead in RBA, which exceeded the WHO and Ghana EPA standard values. Likewise, a significant increase in As levels was observed in RNM. Cd and Hg were present at lower concentrations in the RBA, RNM, and RSI. These traces of heavy metals in mining-polluted water bodies pose a significant health concern due to their toxic effects, as mentioned above [6].

In terms of weight evaluation, substantial changes in the body weight of exposed groups compared to the control group serve as a key indicator of toxicity and disease progression [20]. Multiple studies have alluded that heavy metals induce oxidative stress in muscle tissues, causing muscle damage [21]. This confirms the significantly elevated creatinine kinase levels in the experimental rats' serum, further indicating muscle wasting. In this study, elevated serum creatinine kinase levels correlated with the extent of body weight loss in all the exposed groups. This implies that prolonged consumption or exposure to heavy metal-contaminated water bodies can lead to significant muscle wasting that can manifest as weight loss [21]

Due to the numerous functions of blood within the body, the full blood count gives a detailed report on the body’s state of health. Hematological tests are vital for diagnosing diseases and assessing blood damage due to toxic chemicals [22]. Our research findings revealed no significant alterations within MCH, MCHC, PLT, RDW-SD, RDW-CV, PDW, MPV, PCT, IG, Micro R, Macro R, Eosinophil, and Basophil across all groups when compared to the two control groups. However, the levels of RBC, HGB, and MCV significantly decreased among both males and females in all experimental groups, indicating the presence of microcytic anaemia [23]. Notably, the significant reduction in RBC and HGB levels confirms anaemia, which can be confirmed by the marked hemosiderosis in the parenchyma of the spleen of the treated groups. By contrast, the levels of WBCs increased significantly in the treated groups. The significantly high neutrophilic levels observed in the RSI group suggest that they had acute inflammatory response activities within their organs [24]. On the contrary, lymphocytic count was significantly higher in the RBA and the RNM groups, depicting a more advanced chronic inflammatory condition, marked by a surge of mononuclear lymphocytes [25]. These inflammatory lymphocytosis and neutrophilia have been associated with heavy metal intoxication [26]. The liver function tests and lipid profile provide an ideal interpretation of the liver health and function. A report by Yan & Allen [27] revealed that heavy metals, especially Cd, interfere with liver protein synthesis, resulting in decreased total protein and globulin levels. This is consistent with the current study.

Three key liver function tests, AST, ALP, and ALT, are used to assess overall hepatic health [28]. All these three parameters were observed to be significantly elevated across both males and females of all treatment groups when compared to both control groups. The elevation of ALP, which is a marker for bile duct and gallbladder dysfunction, indicates the extent of the heavy metals’ toxic effects on the biliary system [29]. The elevation of AST and ALT is a result of heavy metals interfering with normal hepatocyte function [21], resulting in the immune response of their elevation in the bloodstream. This reveals that there is overall impairment of liver function.

Chronic exposure to heavy metals such as Cd and Pb has been shown to cause damage to renal tubules and impair glomerular filtration [30]. Moreover, it has also been reported that exposure to Cd results in renal dysfunction characterised by an increase in serum creatinine and urea levels [27]. This concurs with our findings, which showed significant changes in creatinine, urea and chloride levels in the exposed groups compared to the control groups. Hence, long-term exposure to mining-polluted water can cause kidney damage or dysfunction [31]. Furthermore, the urine strip test revealed proteinuria in the exposed groups compared to the control groups. Normally, proteins are not present in high amounts in urine because healthy glomeruli and renal tubules prevent significant protein loss. Thus, elevated urinary protein levels indicate glomerular or tubular damage, either from impaired filtration or poor reabsorption by the renal tubules. This is in line with other studies that reported that Pd [32] and Cd [33] accumulation in the kidneys led to proteinuria due to damage to renal tubules. Additionally, though bilirubinuria is not a primary marker of renal tubular damage, its presence alongside proteinuria and urobilinogen supports a systemic toxic effect, possibly involving both hepatic and renal impairment [34], [35].

Hormones play crucial roles in nutritional metabolism, growth and development as well as reproduction. Impairment in the metabolism of hormones and their subsequent release due to heavy metal intoxication results in several complications, including diseases and organ malfunction [36]. Notably, in the current study, the levels of TS and PG were significantly lowered among males and females, respectively, in the experimental groups, indicating potential impairment to their release via the reproductive organs that produce them [37]. Our findings are therefore in agreement with previous studies, which demonstrated that Pb and Cd interfere with the hypothalamic-pituitary-gonadal axis, leading to lower levels of testosterone and estrogen in males and females, respectively [37], [38]. Intoxicants such as heavy metals in water have been reported to reduce testicular production of testosterone through the destruction of the Leydig cells in males [39]. Similarly, E2 and LH, on the other hand, were significantly elevated in both males and females of some of the experimental groups, confirming the potential disruptive activity of heavy metals on the reproductive endocrine system [36]. Overall, the significant reduction in these hormones provides a clue that heavy metals have a direct effect on the endocrine system and the physiological processes they facilitate within the body.

Additionally, a significant reduction in sperm count was observed among all experimental groups compared to control groups. This reduced level can partly be attributed to the reduced TS levels in the experimental male rats and the direct tissue injuries within the seminiferous tubules. This confirms earlier findings that associated heavy metals such as Pb, Cd and Hg with reduced spermatogenesis [40].

Furthermore, our research revealed an increasing trend of FBS elevation among the treatment groups, compared with controls. The disruptions of blood sugar indicate potential metabolic toxicity and or organ dysfunction [41]. This suggests that the long-term consumption of water from mining-polluted water bodies can negatively affect the pancreas, leading to a diabetic condition [42]. Thus, consumption of water polluted with heavy metals may potentially increase the chance of developing diabetes.

Also, assessment of organ weights serves as another crucial indicator in toxicological evaluation [43]. In this study, significant weight changes were observed in the kidneys of experimental rats (RBA, RBM, RNM, and RSI). This aligns with previous studies documenting the nephrotoxicity of heavy metals [31], including their effects on organ weight [44]. Another important finding from this study was the presence of ovarian cysts in female rats from the RBA and RNM groups. This correlates with the significant changes observed in the hormone analysis in this study, and it is in agreement with an earlier report, which associated polycystic ovarian syndrome with heavy metals [45]. Likewise, other gross pathological changes were seen exclusively amongst the experimental groups (RBA, RBM, RNM, and RSI), which include enlargement of the cervical lymph nodes, oedema in the hind limbs, and dispersed focal pale patches on the serosal surfaces of the liver and lungs. All these are indicative of localised tissue damage and associated chronic inflammation [46]. Thus, the gross pathological observations highlight the extent of health risks associated with prolonged exposure to heavy metals from a mining-polluted river.

For histopathological analysis, liver sections from the groups dosed with contaminated water from RBA, RBM, RNM, and RSI showed evidence of leucocytosis, oedema, activated Kupffer cells, necrosis (pyknotic, karyorrhetic, and karyolitic degenerative changes), and non-caseating granulomatous inflammatory changes, potentially due to inflammatory activities against potentially non-degradable substances such as heavy metals in water bodies. This concurs with a previous study done using rat models [16]. Similar findings have also been reported earlier in liver tissues of tilapia species (Oreochromis niloticus) in a heavy metal-contaminated water body [47]. Likewise, an in vitro study has shown that Cd can cause apoptosis and necrosis of hepatocytes (HEB3B cells), which is characterized by nuclear condensation and fragmentation [48]. Also, the observed spotty, periportal, and centrilobular necrotic changes seen in this current study indicate generalized degenerative changes in the liver parenchyma [49] and thus demonstrate the potential hepatotoxic activity of these heavy metal-contaminated water bodies.

More concerning was the presence of multiple focal well well-circumscribed, non-caseating-granulomatous inflammatory lesions, seen within the liver sections. Heavy metal toxicity in the liver has been associated with potential granuloma formation [50], and it has been associated with chronic inflammation and fibrosis [51]. Histologically, it is evident by the presence of mononuclear immune infiltrates such as macrophages [51], which differentiate into epithelioid cells and form giant cells in response to non-degradable agents or irritants such as heavy metals [52]. These lesions are associated with activation of fibroblasts, reduced hepatic cellularity and function [53], and also a precursor to the deadly liver cirrhosis [54].

Observed tissue sections of the kidney cortex, revealed a bigger capsular space of renal corpuscle due to atrophied glomerulus, glomerulonephritis, necrosis, and oedema, alongside tubular degeneration. This aligns with the previous report, where exposure of rats to heavy metal-contaminated waters resulted in glomerular and tubular degeneration [55]. Cell degeneration, which is a hallmark of cellular necrosis and apoptosis, has been reported to occur as a result of exposure to toxic metals like Pb, Hg, As, and Cd [56]. Alteration to the structure of the nephron, as seen in the kidneys sections in rats from RBA, RBM, RNM, and RSI groups, could potentially lead to progressive loss of healthy nephrons by necrosis, one of the characteristics of chronic kidney disease (CKD) [57], [58]. Heavy metals such as Pd, Cd, As, Hg, and Zn have been associated with CKD [59]. More specifically, increased capsular space and atrophy of the glomerulus, due to cell loss and degenerative changes, may be induced by heavy metals [60]. This suggests that end-stage kidney failure can potentially occur [61], with long-term use of these contaminated water sources in both humans and domestic animals.

A recent report has shown that heavy metals such as Cd can induce anoikis in the spleen, causing loss of contact between cells and also the extracellular matrix, leading to apoptosis [62]. Treated groups showed disruption of splenic architecture as demonstrated by the breakdown of the boundary between red and white pulp. Decreased lymphocytes as a result of reduced cellularity and stromal fibrosis. This is in accordance with earlier reports [62], [63]. These results suggest that chronic ingestion of heavy metal-contaminated water or food can lead to splenic damage. Hemolytic anaemia, with iron pigment deposition, hemosiderin, can be evident as hemosiderosis in organs such as the spleen [64]. In the current study, hemosiderosis was evident in the rats dosed with water from RBA, RBM, RNM, and RSI, depicting signs of hemolytic anaemia as confirmed by reduced RBCs from these groups.

Furthermore, the histopathological analysis of the lungs revealed thickening of alveoli septa, increased inflammatory cells, granuloma formation, and congestion in the groups (RBA, RBM, RNM and RSI) treated with the contaminated water compared to the control groups, which had moderate to no thickening of alveoli septa only. This agrees with previous studies done earlier, where the lungs of experimental rabbits given feed mixed with Pb showed congestion, inflammatory cells, and thickening of alveolar septa [65] . Also, the presence of fibrosis suggests an immune and inflammatory response triggered by heavy metal exposure [66]. The thickening of alveoli septa indicates a gradual change of lung architecture, damage of epithelial lining and oedema of the septa [67], which leads to reduced surface area for gaseous exchange, leading to respiratory stress [68]. These pathological features observed are indicative of potential respiratory complications, such as chronic obstructive pulmonary disease [69]. Also, the presence of the granulomatous inflammation in some of the groups may have resulted from oxidative stress by heavy metals like Cd, Pd, As and Hg that can activate fibrogenic cells like fibroblast and myofibroblast, leading to cellular damage and the accumulation of fibrotic tissue [70]. This is indicative of chronic lung inflammation and mimics sarcoidosis [71]. It is in agreement with an earlier report [72]. These pathological features may cause a reduction in lung function and increased airway limitations [73].

Histopathological evaluations of the testis revealed leakage of blood vessels due to the disruption of BTB, which is highly susceptible to heavy metal toxicity [74], leading to oedema and haemorrhage. Again, visible loss of germ cells (spermatogonia and spermatocytes) is observed, revealing that heavy metal toxicity can induce necrosis within the testis [75], [74], reducing the cellularity in the tubules.

Lastly, histopathological alterations observed in the cardiac tissues, including leucocytic infiltrates, myocardial necrosis, vacuolation, interstitial fibrosis, and hemorrhage, are consistent with previously described degenerative myocardial lesions in rodents exposed to toxic agents [76]. Thus, chemically induced myocardial degeneration typically manifests as diffuse sarcoplasmic vacuolation, interstitial cell proliferation, and variable inflammatory responses, which may progress to fibrosis in severe or prolonged cases. Findings from this present study, particularly in the treated groups (RBA, RBM, RNM & RSI), correspond to this reported morphological pattern. The pattern observed in this study is in agreement with cardiotoxicity induced by heavy metals such as Pb, as reported in an earlier study [77]. Overall, the microscopic changes observed in the testis may have contributed to the reduced sperm count and testosterone levels in the male rats from the experimental groups.

In general, heavy metals from waterbodies within the vicinity of unregulated mining sites are therefore able to induce toxicity in the lungs, liver, spleen, kidneys, heart, and testes, causing multiple pathological insults in the parenchyma cells of these organs. Suggesting multiple organ-specific vulnerabilities to exposure to these heavy metals [78]

We acknowledge that the current study had a couple of limitations. Firstly, we did not monitor for normal water intake within and among the groups. Secondly, we screened for only the well-known and important heavy metals that were associated with the illegal-mining activities. Other heavy metals should have been detected. Lastly, though the reduced number of animals (five for both males and females in each group), improves study efficiency and cost effectiveness, it lowers/reduces statistical power, and can potentially fail to detect important but less frequent toxicological effects.

5. Conclusion

These results demonstrate that chronic exposure from illegal or unregulated mining sites may partly be responsible for the hematotoxic, nephrotoxic, hepatotoxic, pulmonotoxic, myotoxicity, and endocrine/reproductive toxic effects seen in this current study. The presence of extensive granuloma formations, cellular degeneration, and inflammatory conditions can set the grounds for pre-neoplastic lesions. Thus, analyzing serum biomarkers, performing immunohistochemical staining, and evaluating genetic alterations for potential cancer risk assessment is warranted for similar studies in the near future. Moreover, studies evaluating the teratogenic effects of this illegal-mining-contaminated water bodies are necessary, as fluctuations in hormonal levels and testicular tissue derangement were observed in this study. Finally, a cognitive impairment (CI) assessment could be carried out in a similar study due to the relatively high levels of Pd in the contaminated rivers, as intoxication of this heavy metal is associated with CI.

Implementation of stringent policies is therefore needed as this does not only affect systemic toxicology but also carries profound implications for the broader fields of environmental toxicology and public health. By bridging the gap between environmental degradation and its ramifications on terrestrial organisms, including humans, this current study underscores the urgent need for effective environmental management and regulation in galamsey and unregulated small-scale mining-prone regions, while contributing to the collective effort to safeguard ecosystems and human health globally. This, therefore, calls for strict regulatory measures to be implemented regarding the practice of illegal mining and cessation of the spillage of heavy metals into nearby water bodies.

CRediT authorship contribution statement

Du-Bois Asante: Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. George Yaw Hadzi: Writing – review & editing, Supervision, Funding acquisition, Data curation, Conceptualization. Samuel Naaman: Writing – review & editing, Data curation. Michael Woode: Writing – review & editing, Visualization, Formal analysis, Data curation. Timothy Makwo: Writing – review & editing, Data curation. Benjamin Aboagye: Writing – review & editing, Supervision. Isaac Tabiri Henneh: Writing – review & editing, Funding acquisition, Conceptualization.

Ethical clearance

The experiments were conducted according to internationally accepted standard guidelines on the care and handling of animals, as obtained in specifications provided by the Organization for Economic Cooperation and Development (OECD) (Test No. 425, 2022). Moreover, ethical approval was granted by the Council for Scientific and Industrial Research (CSIR) Institutional Review Board (IRB), reference number: CSIR-IRB/RPN026/2024.

Funding

Du-Bois Asante, George Yaw Hadzi & Isaac Tabiri Henneh were awarded a grant from The University of Cape Coast Directorate of Research, Innovation and Consultancy (DRIC). Research Support Grant No.: DRIC-RSG-INT-23–001.

Declaration of Competing 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 paper.

Acknowledgement

The authors wish to acknowledge Adobe Illustrator and Servier Medical Art de Servier for using their icons in the creation of the graphical abstract

Handling Editor: Prof. L.H. Lash

Footnotes

^{Appendix A}

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.toxrep.2026.102244.

Contributor Information

Du-Bois Asante, Email: duasante@ucc.edu.gh.

Isaac Tabiri Henneh, Email: isaac.henneh@ucc.edu.gh.

Appendix A. Supplementary material

Supplementary material

mmc1.docx^{(13MB, docx)}

Data availability

Data will be made available on request.

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Associated Data

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Supplementary Materials

Supplementary material

mmc1.docx^{(13MB, docx)}

Data Availability Statement

Data will be made available on request.

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PERMALINK

Comprehensive sub-chronic toxicological profiling of illegal artisanal small-scale mining-polluted river bodies in four regions of Ghana

Du-Bois Asante

George Yaw Hadzi

Samuel Naaman

Michael Woode

Timothy Makwo

Benjamin Aboagye

Isaac Tabiri Henneh

Abstract

Graphical Abstract

Highlights

1. Introduction

2. Methodology

2.1. Water sample preparation

2.1.1. Study area

2.1.2. Field methods

2.1.3. Water sample collection and treatment

2.1.4. Digestion and analysis of water samples

2.1.5. Reproducibility and recovery studies

2.1.6. Physico-chemical analysis of water

2.2. Estimated daily metal intake

2.3. Experimental animal care and husbandry

2.4. Experimental design

2.4.1. Sub-chronic toxicity studies

2.4.2. Animal weight determination

2.4.3. Haematological analysis

2.4.4. Serum biochemical analysis

2.4.5. Hormonal test

2.4.6. Urine test

2.4.7. Sperm count test

2.4.8. Fasting blood sugar test

2.4.9. Organ weight determination

2.4.10. Gross pathology evaluation

2.4.11. Histopathological evaluations

2.5. Statistical analysis

3. Results

3.1. Physico-chemical analysis

Table 1.

3.2. Heavy metals analysis

Table 2.

Table 3.

3.3. Weight of rat and serum creatinine kinase levels

Fig. 1.

3.4. Hematological analysis

Table 4.

3.5. Liver function test & lipid profile

Table 5.

Fig. 2.

3.6. Hormone analysis

Table 6.

3.7. Kidney function test & urinalysis

Table 7.

Table 8.

3.8. Semen concentration and fasting blood sugar analysis

Fig. 3.

3.9. Organ weight and gross pathology

Table 9.

3.10. Histopathological evaluation

Fig. 4.

Fig. 5.

Fig. 6.

Fig. 7.

Fig. 8.

Fig. 9.

Table 10.

4. Discussion

5. Conclusion

CRediT authorship contribution statement

Ethical clearance

Funding

Declaration of Competing Interest

Acknowledgement

Footnotes

Contributor Information

Appendix A. Supplementary material

Data availability

References

Associated Data

Supplementary Materials