Wild birds as biomonitors of mining-induced heavy metal pollutionABSTRACT
Heavy metals released by mining activities are persistent environmental contaminants that can accumulate in ecosystems and pose risks to wildlife. Despite active mining operations in District Mardan, Pakistan, information on heavy metal bioaccumulation in local bird communities remains scarce. This study provides a preliminary assessment of heavy metal contamination in a mining-affected area by evaluating the concentrations of Fe, Pb, Mn, Cd, Cu, Cr, Ni, Co, and Zn in liver and kidney tissues of wild birds and in associated soils. A total of 24 birds representing four species-Acridotheres tristis (Linnaeus, 1766); Corvus splendens Vieillot, 1817; Argya caudata (Dumont, 1823); and Halcyon smyrnensis (Linnaeus, 1758)-were collected from a mining area (Babuzai) and a reference area (Mian Khan), and tissue samples were analyzed using inductively coupled plasma optical emission spectrometry (ICP-OES). Histopathological alterations were assessed using hematoxylin and eosin staining. Heavy metal concentrations were significantly higher (p < 0.05) in birds and soils from the mining area than in the reference area. Across species, kidneys generally exhibited higher metal concentrations than livers, indicating tissue-specific accumulation patterns. Soil contamination factors ranged from 19.8 for Cu to 550.6 for Pb, demonstrating substantial metal enrichment in the mining-affected area. Histopathological examination revealed necrosis, vacuolization, and cellular degeneration in liver and kidney tissues of birds from the mining area-findings consistent with chronic exposure to environmental contaminants. These results provide evidence of elevated heavy metal contamination associated with mining activities in District Mardan and support the potential use of wild birds as biomonitors of environmental pollution in the region.
KEYWORDS:
Bioaccumulation; ecotoxicology; histopathology; kidney; liver; soil contamination; trace elements
INTRODUCTION
Mining activities are among the most important anthropogenic sources of environmental metal contamination worldwide. The extraction and processing of mineral resources release potentially toxic elements into soils, water bodies, and the atmosphere, often resulting in localized contamination hotspots that may persist long after mining operations have ceased (Anju and Banerjee 2012, Ding et al. 2017, Chakraborty et al. 2021, Rehman et al. 2023). Elevated concentrations of heavy metals in mining-affected environments can pose risks to ecosystems because these elements are persistent, non-degradable, and capable of accumulating in biological tissues (Han et al. 2023, Maftouh et al. 2024, Rahman et al. 2026).
Wild birds are widely recognized as useful biomonitors of environmental contamination because they occupy different trophic levels, exploit diverse habitats, and may integrate contaminant exposure over broad spatial scales (Movalli 2000, Kitowski et al. 2017, Grúz et al. 2019). Heavy metals can enter avian food webs through contaminated soils, plants, invertebrates, and vertebrate prey, resulting in their accumulation in internal organs, particularly the liver and kidneys. These tissues are commonly used in biomonitoring studies because they reflect both exposure and physiological processing of contaminants (Plessl et al. 2017, Squadrone et al. 2019).
Several studies have documented elevated heavy metal concentrations in birds inhabiting mining and industrial areas, demonstrating the value of avian species as indicators of environmental quality and potential ecological risk (Movalli 2000, Kitowski et al. 2017, Grúz et al. 2019). However, information on heavy metal contamination in wild birds remains scarce for many regions where mining activities are expanding, particularly in parts of South and Central Asia.
District Mardan, located in Khyber Pakhtunkhwa Province, Pakistan, hosts active mining operations that may contribute to environmental contamination by heavy metals. Despite the ecological importance of the region, data on heavy metal accumulation in local avifauna are currently lacking. Consequently, baseline information is needed to assess the potential environmental consequences of mining activities and to support future monitoring efforts.
This study provides a preliminary assessment of heavy metal contamination in a mining-affected area of District Mardan using wild birds as biomonitors. Specifically, we compared concentrations of Fe, Pb, Mn, Cd, Cu, Cr, Ni, Co, and Zn in the liver and kidney tissues of birds collected from a mining area and from a reference site with lower anthropogenic influence. In addition, we evaluated histopathological alterations in these organs to investigate whether birds from the mining-affected area exhibited tissue changes associated with increased contaminant exposure. We hypothesized that birds from the mining area would show higher heavy metal concentrations and a greater frequency of histopathological alterations than birds from the reference area.
MATERIAL AND METHODS
Study area
This study was conducted in the northeastern region of Mardan District, Khyber Pakhtunkhwa, Pakistan. The district spans 1,632 km², bordered by Malakand (north/northwest), Swabi (east/south), Nowshera (south/west), and Buner (north/east), with geographic coordinates 34.833333°N, 72.133333°E. The Babuzai Garhi Sakra Range Wildlife Division, adjacent to mining sites, serves as the primary habitat for local fauna (Rehman et al. 2024). The study was conducted at two distinct locations within District Mardan, each selected to represent different levels of human disturbance.
The primary site, Babuzai (34.496653°N, 72.288318°E), is a mining-intensive area known for the extraction of limestone, bentonite, lignite, and iron ore. Open-cast mining operations in this region involve heavy machinery and frequent blasting, which have significantly disrupted the local habitat and introduced potential sources of heavy metal contamination into the environment. In contrast, the control site was located approximately 11-12 kilometers away in the Gharray rest house area (34.442947°N, 72.187289°E), within the Mian Khan designated wildlife-protected zone, characterized by minimal anthropogenic activity (Fig. 1). This site served as a baseline for comparison due to its relatively undisturbed environment and ecological similarity to the mining area. Both sites, Mian Khan and Babuzai, share comparable terrain, climate, and vegetation, making them ideal for assessing the environmental impacts of mining on avian populations.
Map of the study area showing the mining site (Babuzai) and the control site (Gharray rest house).
Data collection
Bird carcasses were collected opportunistically between March and October 2024 from the mining-affected site (Babuzai) and the reference site (Mian Khan) in District Mardan, Khyber Pakhtunkhwa, Pakistan (Grúz et al. 2019, Ashraf et al. 2022). Specimens were obtained through systematic field surveys and notifications from local residents and hunters. Only freshly dead birds showing no signs of advanced decomposition, scavenging damage, or autolysis were included, to minimize post-mortem effects on tissue integrity and metal analyses. Species were identified using standard regional field guides, assigned unique identification codes, and necropsied. No birds were intentionally captured or euthanized for this study (Biswas and Chinthala 2023, Grúz et al. 2019).
A total of 24 carcasses representing four species-common myna, Acridotheres tristis (Linnaeus, 1766); house crow, Corvus splendens Vieillot, 1817; common babbler, Argya caudata (Dumont, 1823); and white-throated kingfisher, Halcyon smyrnensis (Linnaeus, 1758)-were examined. For each species, three individuals were collected from the mining site and three from the reference site, resulting in 12 birds per site (N = 24). These species were selected because of their abundance, sedentary habits, and suitability for reflecting local contaminant exposure (Lim et al. 2003, Yosef et al. 2019, BirdLife International 2025). The two sites share similar ecological characteristics, minimizing environmental differences unrelated to mining activities.
The use of opportunistically obtained carcasses, rather than intentionally collected specimens, also conforms to the principles of Replacement, Reduction, and Refinement (3Rs) by avoiding the sacrifice of wild birds (Zemanova 2021). Implications of this sampling approach for sample size and inference are addressed in Study limitations.
Liver and kidney tissues were carefully excised from collected carcasses, stored in pre-labeled tubes, and frozen at -20 °C until further analysis. Approximately 0.25 g of each sample (wet weight) was oven-dried at 60 °C for 24 hours, then digested using aqua regia (HCl:HNO₃, 3:1) in a microwave digestion system (CEM MARS 6), following established protocols (Kachenko and Singh 2006, US EPA 2007). Digested samples were diluted to 50 mL with deionized water and filtered through 0.45 µm syringe filters before analysis.
Soil samples
At each study site, three sampling locations, separated by approximately 300 m, were selected to capture within-site spatial variability. At each location, three subsamples of topsoil (0-15 cm depth) were collected within a 5-m radius using a stainless-steel auger and pooled to generate one composite sample. This procedure yielded three independent composite samples per site (n = 3), resulting in six composite soil samples overall. Topsoil was targeted because it represents the most biologically active fraction and is most susceptible to contamination from atmospheric deposition and mining-related surface disturbances (Liu et al. 2020, ACIAR 2021). Collected samples were immediately placed into clean polyethylene bags, labeled, and transported to the laboratory. In the laboratory, soil samples were oven-dried at 60 °C for 24 hours to constant weight, finely ground, and sieved through a 2 mm mesh. Digestion of soil samples followed the same standardized aqua regia (3:1 HCl:HNO₃) microwave digestion procedure as described for tissue samples (Bettinelli 2000, Nawab et al. 2015).
Quality assurance and quality control
Digested liver, kidney, and soil samples were analyzed for Fe, Pb, Mn, Cd, Cu, Cr, Ni, Co, and Zn using inductively coupled plasma optical emission spectrometry (ICP-OES; Perkin-Elmer Model 700). All analyses were conducted at the Pathology Laboratory, College of Veterinary Sciences and Animal Husbandry, Abdul Wali Khan University, Mardan, Pakistan. The instrument was calibrated using multi-element standard solutions prepared according to the manufacturer’s instructions, and calibration checks were performed throughout analytical runs to verify instrument stability and measurement accuracy. All concentrations were expressed in parts per million (ppm).
Quality assurance and quality control (QA/QC) procedures were implemented throughout sample preparation and analysis to minimize contamination and ensure analytical reliability. All glassware, digestion vessels, and laboratory equipment were thoroughly cleaned and rinsed with deionized water prior to use. Tissue and soil samples were processed using the same digestion and analytical procedures under standardized laboratory conditions. Analytical data were checked for consistency and completeness before statistical analysis.
Histopathological analysis
Liver and kidney tissues from each bird specimen were processed for histopathological examination following established protocols. Tissue samples were fixed immediately upon collection in 10% neutral buffered formalin for 24-48 hours to ensure optimal preservation of cellular architecture. Following fixation, tissues were dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin wax using an automated tissue processor. Serial sections (3-5 µm thick) were prepared using a rotary microtome, mounted on glass slides, and stained with hematoxylin and eosin (H&E) following standard protocols (Suvarna et al. 2019). Stained sections were systematically examined under a light microscope at magnifications ranging from 100× to 400×.
The histopathological assessment focused on identifying lesions commonly reported in avian tissues exposed to heavy metals (Aljohani 2023, Scheuhammer 1987, Riaz et al. 2020). Hepatic evaluation focused on hepatocellular integrity, cytoplasmic changes (eosinophilia and vacuolization), nuclear morphology, lobular organization, and inflammatory infiltration (Scheuhammer 1987, Riaz et al. 2020). Renal assessment examined glomerular and tubular architecture, epithelial degeneration, cytoplasmic vacuolization, necrosis, and interstitial inflammation (Scheuhammer 1987, Riaz et al. 2020, Shaffique et al. 2024). Histological sections were evaluated qualitatively for the presence or absence of pathological alterations, including necrosis, cytoplasmic vacuolization, inflammatory cell infiltration, and tissue degeneration. Only lesions consistently observed in birds from the mining-affected area, and absent in birds from the reference area, were considered biologically significant (Gibson-Corley et al. 2013, Costa et al. 2009).
Data analysis
To compare metal concentrations between the two sites, the Wilcoxon rank-sum test was applied for each metal-tissue combination, as the data were non-normally distributed. Non-normality was assessed through visual inspection of Q-Q plots and confirmed by the Shapiro-Wilk test. All analyses were conducted in R (v4.3.0), and significance was set at p < 0.05. Boxplots were generated using ggplot2 to visualize the distribution of concentrations by site, metal, and tissue type.
Soil heavy metal concentrations from triplicate samples per site were analyzed using the Wilcoxon rank-sum test (Mann-Whitney U test), appropriate for small sample sizes and non-normally distributed data. Statistical significance was set at p < 0.05.
In addition, the contamination factor (CF) was calculated to assess the degree of heavy metal contamination in mining-affected soils relative to background (control) soils (Hakanson 1980, Liu et al. 2020, Akoto et al. 2023). The CF was calculated as: CF = Concentration in mining-affected soil/Concentration in control soil. This index provides a quantitative measure of contamination intensity and was used as a descriptive indicator rather than as a test of statistical significance.
Study limitations
This study was based on opportunistically obtained bird carcasses collected from mining-affected and reference areas in District Mardan. Consequently, biological variables such as age, sex, reproductive status, diet, and movement patterns could not be standardized among individuals. The relatively small sample size reflects the limited availability of suitable specimens and should be considered when interpreting interspecific comparisons and population-level inferences. In addition, the assessment of mining-related effects was restricted to measurements of heavy metal concentrations in liver and kidney tissues, and routine histopathological examination. Therefore, the study was designed to provide an initial assessment of heavy metal bioaccumulation and associated tissue alterations rather than a comprehensive evaluation of the physiological mechanisms underlying metal toxicity.
RESULTS
Heavy metal concentrations in avian tissues
Heavy metals were detected at substantially higher concentrations in the liver and kidney tissues of birds collected from the mining-affected area than in those from the reference site. Across the four species examined, kidneys generally exhibited higher concentrations than livers, indicating a greater tendency for metal accumulation in renal tissues (Fig. 2).
Heavy metal accumulation (ppm) in visceral organs (kidneys and livers) of wild birds from the mining-affected area in District Mardan, Khyber Pakhtunkhwa, Pakistan.
Birds from the mining area showed detectable concentrations of all analyzed metals, whereas birds from the reference site exhibited only trace concentrations of Fe and Zn, with the remaining metals below detection limits (Fig. 3). Among the metals detected, Cd, Fe, and Zn were present at the highest concentrations in several samples from the mining area.
Heavy metal accumulation (ppm) in visceral organs (kidneys and livers) of wild birds from the control area in District Mardan, Khyber Pakhtunkhwa, Pakistan.
The Wilcoxon rank-sum test indicated significantly higher concentrations (p < 0.05) of all analyzed metals (Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, and Zn) in birds collected from the mining-affected site compared with those from the reference site, for both liver and kidney tissues. This pattern is consistent with elevated environmental exposure to heavy metals in the mining area.
Boxplots further illustrate the marked separation between sites, with birds from the mining-affected area consistently exhibiting higher concentrations across all metals and tissue types (Fig. 4).
Boxplots showing concentrations of nine heavy metals (Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, and Zn) grouped by tissue type: kidney (left panel) and liver (right panel). The mining-affected site is represented by blue-green boxes, and the control site by orange boxes. Each box shows the median, interquartile range (IQR, 25th to 75th percentiles), and data range (whiskers); individual points indicate outliers.
Heavy metal concentrations in soil
Soil samples collected from the mining-affected area exhibited substantially higher concentrations of all analyzed metals than those from the reference site (Table 1). The magnitude of enrichment varied among metals, with Pb showing the highest increase relative to the control area, followed by Co and Cd. Elevated concentrations were also observed for Cr, Zn, Ni, Mn, Cu, and Fe.
Heavy metal concentrations in soils from control and mining-affected sites (n = 3 per site). All metals differed significantly between sites (Wilcoxon rank-sum test, p < 0.05).
Contamination factor (CF) values indicated pronounced enrichment of several metals in soils from the mining area, particularly Pb, Co, and Cd, while the remaining metals also showed elevated contamination levels relative to the reference site. The Wilcoxon rank-sum test confirmed significant differences (p < 0.05) between sites for all analyzed metals.
Overall, the soil data demonstrate substantial heavy metal enrichment in the mining-affected area, supporting its use as a contaminated site for evaluating environmental exposure in wild birds.
Histopathological evaluation
Liver
Liver tissues from birds collected in the reference area showed normal histological architecture, with no evident pathological alterations (Fig. 5A). In contrast, liver samples from birds collected in the mining-affected area exhibited histopathological alterations, including necrosis and inflammatory cell infiltration (Fig. 5B, C).
Histopathological examination of liver and kidney tissues. Liver: (A) section from the control group (A. caudata) stained with hematoxylin and eosin (H&E), showing no signs of degeneration or infiltration; (B) section from the study group (exposed to high metal levels) stained with H&E, exhibiting necrosis and degeneration in epithelial cells; (C) section from the study group stained with H&E, showing infiltration of immune cells. Kidney: (D) section from the control group (A. tristis) stained with hematoxylin and eosin (H&E), showing no signs of degeneration or infiltration; (E) section from the study group (exposed to high metal levels) stained with H&E, exhibiting infiltration of immune cells. Scale bars: 10 µm.
Kidney
Kidney tissues from birds collected in the reference area displayed preserved tissue architecture and no evident histopathological alterations (Fig. 5D). In contrast, kidney samples from birds inhabiting the mining-affected area exhibited histopathological changes, including necrosis, cytoplasmic vacuolization, and inflammatory cell infiltration (Fig. 5E).
DISCUSSION
Soils and avian tissues from the mining-affected area consistently showed higher concentrations of all analyzed metals than those from the reference site, indicating substantial environmental enrichment associated with mining activities in District Mardan.
Across the sampled birds, kidneys generally contained higher metal concentrations than livers. This pattern is consistent with the recognized role of renal tissues in the accumulation and excretion of several metals and has been reported in previous studies of birds inhabiting contaminated environments (Plessl et al. 2017, Grúz et al. 2019). However, given the limited sample size and exploratory nature of this study, apparent differences among species should be interpreted with caution.
The pronounced enrichment observed in soils from the mining area supports the hypothesis that mining activities constitute an important source of environmental contamination in the study region. Particularly notable were the elevated concentrations of Pb, Co, and Cd, which exhibited the highest contamination factors relative to the reference site. The correspondence between soil contamination and elevated tissue concentrations in birds suggests that local wildlife is being exposed to increased levels of heavy metals in the mining-affected area.
Histopathological alterations were observed only in birds collected from the mining area. Liver tissues exhibited necrosis and inflammatory cell infiltration, whereas kidney tissues showed necrosis, vacuolization, and inflammatory infiltration. Similar lesions have been reported in birds exposed to environmental contaminants, including heavy metals (Cobbina et al. 2015, Riaz et al. 2020, Shaffique et al. 2024). Nevertheless, because multiple environmental stressors may contribute to tissue alterations in wild animals, these results should be interpreted as evidence of an association between elevated metal exposure and histopathological changes rather than direct proof of causation.
These findings should be interpreted in light of the methodological limitations described in Study limitations.
Despite these limitations, the study provides the first evidence of elevated heavy metal concentrations in soils and wild birds from a mining-affected area of District Mardan. The results support the value of avian tissues as indicators of environmental contamination and establish baseline information for future monitoring efforts in the region. Further studies incorporating larger sample sizes, additional species, and complementary biomarkers would help clarify exposure pathways and the long-term ecological consequences of mining-related contamination.
ACKNOWLEDGMENTS
We thank King Saud University, Riyadh, Saudi Arabia, for funding this work through the Ongoing Research Funding Program (ORF-2026-1721).
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