Figure 1. Map showing the location of Ogbe-Ijoh Market in Delta State, Nigeria.

Crayfish samples were obtained from Ogbe-Ijoh Market (5.5053˚N, 5.9094˚E), a major commercial hub for dried crayfish trade within the Niger Delta region of Delta State, Nigeria. The market was selected because of its importance in regional seafood distribution and its relevance to consumer dietary exposure. The geographic location of the study area is shown in Figure 1. A total of twenty (20) dried crayfish samples were collected during the dry and wet seasons (10 samples per season). Each sample represented a vendor-linked crayfish consignment associated with a source community supplying the market. Thus, the ten “locations” referred to in this study represent distinct vendor sampling points linked to source communities, rather than arbitrary sampling points within the market. To support seasonal comparison, dry- and wet-season samples were obtained through repeated sampling of the same vendor locations during both periods. Samples were randomly purchased from the selected vendors, placed in clean polyethylene bags, and handled using disposable gloves to minimise contamination during collection and transportation. Upon arrival at the laboratory, samples were stored at 4˚C prior to processing.

Approximately 1.00 g of the homogenised crayfish sample was digested using aqua regia (9 mL HNO₃: 3 mL HCl) in 250 mL glass beakers. The mixture was partially covered with watch glass and heated on an electric hotplate at 100˚C for 2 h in a fume hood. After digestion, the samples were allowed to cool to room temperature and filtered through Whatman No. 41 filter paper into 50 mL volumetric flasks. The filtrates were diluted to the mark with deionised water and stored at 4˚C before analysis. The concentrations of lead (Pb), nickel (Ni), cadmium (Cd), and chromium (Cr) were determined using flame atomic absorption spectrophotometry (FAAS) (Buck Scientific Model 210). The instrument demonstrated the capability to detect trace concentrations of the analysed metals in the digest solutions, with Pb, Ni, and Cr detectable at approximately 0.010 mg/L, 0.009 mg/L, and 0.044 mg/L, respectively, while Cd was not detected under the analytical conditions employed. Concentrations reported as below detection/non-detect were treated as zero in subsequent exposure and risk calculations. Calibration curves were prepared using standard solutions of each metal and analyses were performed according to the manufacturer’s operational guidelines. Metal concentrations were expressed in mg·kg⁻¹ dry weight following established analytical procedures ([6]). The use of dry-weight concentrations was considered appropriate because the crayfish samples were purchased in commercially dried form, which reflects the actual marketed and consumed state of the product.

The analytical accuracy and precision were evaluated using spiked recovery and replicate analyses. For the recovery assessment, two aliquots of each homogenised crayfish sample were prepared: one portion was spiked with a known concentration of mixed metal standard solution, while the second portion remained unspiked and served as a control. Both portions were digested and analysed under identical conditions. The percentage recovery (%) was calculated as follows:

where:

S = heavy metal concentration in the spiked sample,

μ = heavy metal concentration in the unspiked sample, and

T = amount of metal added during spiking.

Reagent blanks were included in the analytical procedure to monitor for possible contamination. All samples were analysed in duplicate to evaluate precision, and mean concentrations were reported ([4]).

Human health risks associated with crayfish consumption were evaluated using established non-carcinogenic risk assessment models recommended by the United States Environmental Protection Agency (USEPA).

Estimated Daily Intake (EDI)

The estimated daily intake (EDI) of heavy metals through crayfish consumption was calculated as

where:

C = metal concentration in crayfish (mg·kg⁻¹),

IR = ingestion rate (0.02 kg·person⁻¹·day⁻¹) adopted from USEPA-based exposure assessment approaches and applied as a conservative screening-level estimate for both adults and children).

EF = exposure frequency (365 days·year⁻¹),

ED = exposure duration (70 years for adults),

BW = body weight (70 kg for adults; 16 kg for children),

AT = averaging time for non-carcinogens (ED × 365 days).

Although dried crayfish is typically consumed in relatively small quantities, the selected ingestion rate was retained as a protective assumption to avoid underestimation of dietary exposure.

Target Hazard Quotient (THQ)

Non-carcinogenic health risks were assessed using the Target Hazard Quotient (THQ).

where RfD is the oral reference dose (mg·kg⁻¹·day⁻¹). The RfD values adopted were:

Cd = 0.001; Pb = 0.004; Ni = 0.02; Cr = 0.003.

A THQ value < 1 indicates negligible risk, whereas a THQ value ≥ 1 suggests a potential non-carcinogenic health risk ([29]).

Hazard Index (HI)

The cumulative noncarcinogenic risk from multiple metals was estimated using the Hazard Index (HI):

where THQ_i represents the individual hazard quotient for each metal. An HI value < 1 indicates an acceptable risk, whereas an HI value ≥ 1 suggests potential health concerns.

Pollution Index (PI)

The pollution index (PI) for each metal was calculated as follows:

where C_sample is the measured metal concentration in the crayfish and C_permissible is the corresponding benchmark concentration for each metal. A PI value > 1 indicates contamination ([5]). The PI approach adopted in this study was adapted from previous crayfish contamination assessments ([1]). Metal-specific benchmark values for Pb, Ni, and Cr were selected from commonly cited guideline values for fish and seafood in the literature. Cadmium was below the detection limit and was therefore excluded from PI interpretation.

Cancer Risk (CR)

The lifetime carcinogenic risk (CR) associated with dietary exposure to lead (Pb) through crayfish consumption was estimated using the cancer slope factor (CSF) approach. The CR was calculated using the following equation:

where CSF is the cancer slope factor (mg/kg/day), and the other parameters have been defined previously. An acceptable lifetime carcinogenic risk level of 10⁻⁵ was adopted based on established risk assessment guidelines ([17]; [22]). A CSF value of 0.0085 (mg/kg/day)⁻¹ was adopted for Pb for screening-level carcinogenic risk estimation, consistent with its application in previous food contamination and Pb exposure risk assessment studies ([16]; [1]). However, the use of an oral CSF for dietary Pb exposure is not uniformly harmonized across all regulatory frameworks and should therefore be interpreted with caution.

Statistical analyses were performed using R statistical software package ([21]). Descriptive statistics, including the mean, range, and standard deviation, were computed to summarise the distribution of heavy metal concentrations in crayfish samples. The pollution index (PI) was calculated to evaluate the contamination status of individual metals. Cluster analysis was performed to examine potential grouping patterns and similarities among the analysed metals. Pearson’s correlation analysis was conducted to assess the relationships between the heavy metals across the sampled matrices. Statistical significance was evaluated at a 95% confidence level (P < 0.05).

The accuracy of the analytical procedure was evaluated using spike recovery experiments. The percentage recoveries of Pb, Ni, Cr, and Cd ranged from 82.4% to 97.4%. These values fall within the generally accepted recovery range of 80% - 120% for elemental analysis in complex matrices, indicating satisfactory analytical accuracy and minimal matrix interference in the analysis. The recovery results are summarised in Table 1. This acceptance range is consistent with the method validation guidelines outlined in the U.S. Food and Drug Administration’s Elemental Analysis Manual ([8]), as well as the established analytical chemistry principles and international validation guidelines ([23]; [14]). Therefore, the analytical data generated in this study were considered reliable for subsequent risk assessments.

Table 1. Analytical results for percentage recovery (% R) of the analysed metals.

The range and mean concentrations of the analysed metals (Pb, Ni, Cr, and Cd) in crayfish samples collected from oil-producing communities of the Niger Delta are presented in Table 2 and Table3. The results revealed noticeable variability in metal levels across sampling locations and seasons. During the dry season, the Pb concentrations ranged from 4.3 to 39.5 mg/kg, with a mean value of 18.2 ± 12.2 mg/kg. The Cr content ranged between 2.80 and 23.5 mg/kg (mean: 9.82 ± 7.47 mg/kg), whereas the Ni content varied from 1.4 to 14.2 mg/kg (mean: 3.46 ± 3.91 mg/kg). Cd levels were below the detection limit in all samples analysed. The coefficient of variation was the highest for Ni, indicating pronounced spatial heterogeneity across the sampling locations. Similarly, in the wet season, Pb remained the most abundant element, ranging from 3.7 to 55.4 mg/kg, with a mean value of 23.9 ± 16.3. Chromium ranged between 2.7 and 10.4 mg/kg (mean: 6.32 ± 2.85 mg/kg), whereas Ni varied from 0.5 to 5.9 mg/kg (mean: 2.26 ± 1.51 mg/kg). The Cd levels remained below the detection limit across all sampling sites. Across both seasons, the order of decreasing mean concentrations followed the pattern Pb > Cr > Ni > Cd. The dominance of Pb in both seasons suggests persistent anthropogenic inputs, which may be linked to oil exploration activities, pipeline corrosion, gas flaring emissions, and artisanal refining operations in the Niger Delta region. The relatively wide concentration ranges and elevated standard deviations, particularly for Pb and Cr, indicated localised contamination hotspots rather than a uniform distribution within the aquatic environment. Cadmium (Cd) was below the detection limit in all P. clarkii samples analysed in both seasons. This finding contrasts with previous studies conducted in the Niger Delta region, where detectable Cd accumulation has been reported in crayfish and other seafood species. For instance, [7] and [31] documented measurable Cd concentrations in crayfish and related aquatic organisms at impacted sites. Reported Cd levels in regional seafood have ranged from trace values to as high as 3.62 mg/kg in contaminated environments. The absence of detectable Cd in the present study may reflect a relatively lower Cd input at the sampled locations, reduced environmental bioavailability, or possible species-specific differences in uptake and accumulation patterns. These results suggest that Cd contamination is not currently a dominant concern in oil-producing communities compared with Pb and Cr. The total Cr concentrations in the crayfish samples exhibited substantial spatial variability across the oil-producing communities of the Niger Delta. During the dry season, Cr concentrations ranged from 2.8 to 23.5 mg/kg (mean: 9.82 ± 7.47 mg/kg), whereas wet-season concentrations ranged from 2.7 to 10.4 mg/kg (mean: 6.32 ± 2.85 mg/kg). Although higher mean values were observed during the dry season, paired statistical analysis indicated that seasonal differences were not statistically significant (p > 0.05), suggesting that localised contamination sources, rather than seasonal hydrological changes, predominantly influenced Cr distribution. When compared with the maximum permissible limit of 0.5 mg/kg recommended for chromium in fish by the FAO/WHO guidelines, the concentrations observed in this study markedly exceeded the regulatory thresholds across all sampling locations. Even the lowest recorded value exceeded the recommended limit several-fold, indicating potential environmental and public health concerns. Similar elevated Cr concentrations in crayfish from Delta State were reported by [10], who documented levels exceeding the recommended limits in selected towns. Similarly, [26] reported measurable Cr concentrations in seafood from the Niger Delta region. The elevated Cr levels observed in this study may reflect persistent anthropogenic inputs associated with oil exploration activities, industrial discharges, corrosion of petroleum infrastructure, and artisanal refining operations prevalent in the region. It should be noted that total chromium was quantified in this study, and the speciation between Cr(III) and the more toxic Cr(VI) forms was not determined. Nonetheless, the high total Cr concentration warrants continued monitoring and a detailed risk evaluation. Lead (Pb) was the most dominant metal detected in crayfish samples across both seasons. Concentrations ranged from 4.3 to 39.5 mg/kg (mean: 18.2 ± 12.2 mg/kg) in the dry season and from 3.7 to 55.4 mg/kg (mean: 23.9 ± 16.3 mg/kg) in the wet season. Although the seasonal differences were not statistically significant (p > 0.05), the magnitude of Pb accumulation was substantial. The concentrations recorded in this study were markedly higher than those reported in other Nigerian studies. [1] reported Pb levels ranging from 0.40 - 1.40 mg/kg (mean: 0.91 mg/kg) in Palaemon hastatus from Ile-Ife, Nigeria, with values below the WHO/USEPA permissible limits and associated with target hazard quotients less than one. Similarly, dried crayfish samples from Calabar markets in the Niger Delta contained Pb concentrations between 0.02 and 0.24 mg/kg (dry weight), with average values below the Codex maximum level of 0.2 mg/kg. Earlier Nigerian reports have documented Pb levels ranging from 0.002 - 2.04 mg/kg, which is substantially lower than the concentrations observed in the present study. In contrast, all the Pb concentrations recorded in this study exceeded the international guideline limits for fish, including the Codex maximum level of 0.2 mg/kg and the FAO/WHO permissible range of 0.3 - 0.5 mg/kg. The markedly elevated Pb levels observed may reflect localised contamination associated with oil exploration activities, pipeline corrosion, gas flaring emissions, artisanal refining, and other anthropogenic inputs prevalent in the oil-producing communities in the Niger Delta. These findings suggest a significantly higher contamination burden in the present study area than in previously reported Nigerian sites. To contextualise these findings within the broader scientific literature, previously reported concentrations of Pb, Cr, and Ni in crayfish from different geographical regions were compiled and are presented in Table 4. This comparison clearly indicates that the metal concentrations observed in the present study were substantially higher than those reported in most Nigerian and international studies, particularly for Pb and Cr.

Table 2. Heavy metal concentrations in crayfish during the dry season (mg/kg).

Table 3.Heavy metal concentrations in crayfish during the wet season (mg/kg).

Table 4. Comparison of mean heavy metal concentrations (mg/kg, dry weight) in crayfish—present study vs credible literature.

The seasonal variations in heavy metal concentrations across the ten sampling locations are presented in Table 5. All concentrations were expressed on a dry weight basis. Lead (Pb) exhibited higher mean concentrations during the wet season (23.9 ± 16.3 mg/kg) than during the dry season (18.2 ± 12.2 mg/kg). In contrast, chromium (Cr) and nickel (Ni) recorded relatively higher mean values during the dry season (Cr: 9.82 ± 7.47 mg/kg; Ni: 3.46 ± 3.91 mg/kg) than in the wet season (Cr: 6.32 ± 2.85 mg/kg; Ni: 2.26 ± 1.51 mg/kg). Cadmium (Cd) remained below the detection limit in both seasons. Despite observable fluctuations between seasons, paired statistical analysis revealed no significant seasonal differences in Pb (p = 0.400), Ni (p = 0.337), or Cr (p = 0.272) (Table 6). Seasonal comparisons were based on repeated sampling of the same vendor locations during the dry and wet seasons. These findings indicated that seasonal hydrological changes did not exert a statistically significant influence on heavy metal accumulation in crayfish across the sampled locations. Higher wet-season Pb concentrations may be associated with increased surface runoff and mobilisation of contaminants during rainfall events that are common in the Niger Delta region. Conversely, the relatively elevated dry-season Cr and Ni values may reflect reduced dilution effects during periods with lower water volumes. However, the lack of statistical significance suggests that spatial heterogeneity and localised contamination sources exert stronger control over heavy metal distribution than seasonal variation. As the concentrations were determined on a dry weight basis, caution should be exercised when comparing the values directly with international permissible limits, which are typically expressed on a wet weight basis. The relatively high standard deviations observed across locations further support the influence of spatial variability on metal distribution patterns.

Table 5. Seasonal variation of heavy metals in crayfish across sampling locations (mg/kg).

Values are presented as the mean ± standard deviation of triplicate measurements. BDL = below the detection limit.

Table 6. Seasonal summary statistics.

BDL = Below detection limit. p-values obtained using paired t-test (or Wilcoxon signed-rank test).

The Pollution Index (PI) values for Pb, Ni, and Cr in crayfish samples across the dry and wet seasons are presented in Table 7. The PI was calculated using metal-specific benchmark values, and a PI value > 1 was taken to indicate contamination. The results showed that PI values for all three metals exceeded unity at multiple sampling locations in both seasons, confirming contamination of the marketed crayfish samples. Lead (Pb) contamination was evident across most sampling locations, with dry-season PI values ranging from 2.15 to 19.8 and wet-season values ranging from 1.85 to 27.8. Although Pb contamination remained widespread, the magnitude of contamination was generally lower than that observed for Ni and Cr at several locations. Elevated Pb PI values during the wet season at some locations suggest possible seasonal redistribution of metal contaminants, potentially influenced by runoff and environmental mobilisation. Nickel (Ni) exhibited pronounced contamination at several sampling points, with PI values ranging from 2.80 to 28.4 in the dry season and 1.00 to 11.8 in the wet season. The highest Ni contamination was recorded at Location 2 during the dry season, indicating substantial accumulation at that point. The consistently elevated Ni PI values across many locations suggest that Ni represents an important contamination concern in the marketed crayfish samples. Chromium (Cr) also showed marked contamination, with dry-season PI values ranging from 2.80 to 23.5 and wet-season values ranging from 2.70 to 10.4. Particularly high Cr contamination was observed at Locations 3, 5, and 6 during the dry season, indicating notable spatial variability in Cr accumulation. Compared with the wet season, the higher dry-season Cr PI values at several locations may reflect localised concentration effects and reduced dilution. Across the dataset, the contamination pattern did not follow a single uniform order at all locations; however, Ni and Cr generally exhibited stronger contamination intensity than Pb at several sampling points. The widespread occurrence of PI values above unity indicates persistent metal contamination in the analysed crayfish and suggests potential public health concern, especially considering the frequent dietary use of crayfish as a food ingredient in the study area.

Table 7.Pollution Index (PI) of heavy metals in crayfish during dry and wet seasons.

Note: PI = C_sample/C_permissible, where C_permissible represents the benchmark value adopted for each metal from commonly cited fish/seafood guideline values in the literature. PI > 1 indicates contamination.

3.5.1. Estimated Daily Intake (EDI)

The estimated daily intake (EDI) values of Pb, Ni, and Cr for adults and children across the ten source locations are presented in Supplementary Tables S1-S4. For adults, during the dry season, the Pb EDI values ranged from 0.00123 to 0.01129 mg/kg/day, whereas those of Cr ranged from 0.0008 to 0.0067 mg/kg/day. Nickel showed comparatively lower exposure levels (0.0004 - 0.0041 mg/kg/day). Similar exposure patterns were observed during the wet season, with Pb exhibiting the highest intake value across most locations. In all cases, children recorded substantially higher EDI values than adults due to their lower body weight, reflecting greater susceptibility to dietary metal exposure.

3.5.2. Target Hazard Quotient (THQ) and Hazard Index (HI)

The calculated Target Hazard Quotient (THQ) and cumulative Hazard Index (HI) values are presented in Tables 8-11. A THQ value of less than 1 indicates negligible non-carcinogenic risk, whereas THQ ≥ 1 suggests potential health concerns. For adults during the dry season, the Pb THQ values exceeded unity at seven of the ten sampling locations, with the highest value observed at Location 6 (THQ = 2.82). Chromium also exceeded the safety threshold at Locations 3, 5, and 6, with a maximum THQ of 2.24 at Location 5. In contrast, the Ni THQ values remained below 1 at all locations, indicating a relatively low noncarcinogenic risk of nickel exposure. The cumulative risk, expressed as HI, exceeded 1 at nine of the ten locations, with the highest value recorded at Location 6 (HI = 4.37). These results indicate the potential combined health risks associated with simultaneous exposure to multiple metals at several source locations. For children, THQ and HI values were markedly higher across all sampling locations. All sites recorded HI values substantially greater than unity, with the highest cumulative risk observed at location 6 (HI = 19.12). Elevated THQ values for both Pb and Cr were widespread, whereas Ni values remained below the risk threshold. The consistently higher indices in children reflect their increased vulnerability, resulting from their lower body weight and proportionally greater intake. Across all locations, the contribution to non-carcinogenic risk followed the order of Pb > Cr > Ni.

3.5.3. Cancer Risk (CR)

The lifetime carcinogenic risk (CR) associated with Pb exposure was estimated using the cancer slope factor approach. It should be noted that the carcinogenic risk estimates for Pb presented in this study are based on a screening-level application of a literature-derived cancer slope factor and should therefore be interpreted with caution. For adults, the CR values at several sampling locations exceeded the acceptable benchmark level of 10⁻⁵, indicating potential carcinogenic concerns associated with long-term consumption. The highest CR values corresponded to locations with elevated Pb concentrations, particularly Locations 6 and 5. For children, the CR values were substantially higher across most sampling locations owing to elevated EDI levels, exceeding the acceptable risk level at most sites. These findings suggest that prolonged consumption of crayfish sourced from certain communities may pose long-term carcinogenic risks, particularly for younger consumers. Although minor numerical differences were observed between the dry and wet seasons, statistical analysis indicated no significant seasonal variation (p > 0.05). Therefore, spatial variability among source locations appears to exert a stronger influence on health risk levels than seasonal variability. Generally, Pb was identified as the dominant contributor to both non-carcinogenic and carcinogenic risks, Cr contributed moderately at selected locations, and Ni posed a comparatively minimal risk. The markedly elevated HI and CR values observed in children underscore their heightened susceptibility to heavy metal exposure through dietary intake of crayfish from contaminated sources.

Table 8. Target Hazard Quotient (THQ) and Hazard Index (HI) of heavy metals in crayfish (Children-Dry Season).

Table 9. Target Hazard Quotient (THQ) and Hazard Index (HI) of heavy metals in crayfish (Adult-Dry Season).

Table 10.Target Hazard Quotient (THQ) and Hazard Index (HI) of heavy metals in crayfish (Adult-Wet Season).

Table 11.Target Hazard Quotient (THQ) and Hazard Index (HI) of heavy metals in crayfish (Children-Wet Season).

Pearson’s correlation analysis was performed to evaluate the relationships among the concentrations of Pb, Ni, and Cr in crayfish samples across seasons (Table S5, Supplementary Material). In the wet season, Pb exhibited a weak negative correlation with Ni (r = −0.26) and a weak positive correlation with Cr (r = 0.27). Notably, Ni showed a moderate positive correlation with Cr (r = 0.45), suggesting potential co-mobilisation and similar geochemical behaviours. This association may be linked to rainfall-induced redox fluctuations that promote the dissolution of Fe-Mn oxyhydroxides in the sediments. As both Ni and Cr are known to be strongly associated with Fe-Mn oxide phases, their concurrent release into the water column during periods of increased runoff and sediment disturbance could enhance their bioavailability and subsequent accumulation in crayfish. During the dry season, Pb showed weak negative correlations with Ni (r = −0.20) and Cr (r = −0.15) concentrations. A moderate positive correlation was observed between Pb and Ni (r = 0.61), possibly reflecting concentration effects under reduced dilution and more stable sediment conditions. Conversely, Cr demonstrated weak to moderate negative correlations with Pb (r = −0.32) and Ni (r = −0.29), indicating differences in seasonal mobility patterns or competitive uptake mechanisms. Seasonal variability in the correlation structure suggests that hydrological dynamics, redox-driven sediment processes, and changes in metal bioavailability play important roles in governing heavy metal accumulation in crayfish.

Hierarchical cluster analysis (HCA) based on Euclidean distance was performed to examine the similarity patterns of heavy metals in crayfish samples. The Euclidean distance matrix is presented in Table S6 (Supplementary Material), The clustering pattern is illustrated in Figure 2. The dendrogram revealed two distinct clusters. Ni and Cr were grouped closely together, exhibiting a very small Euclidean distance (7.55), indicating a strong similarity in their distribution patterns and possible shared geochemical controls or common environmental sources. In contrast, Pb formed a separate cluster, reflecting its distinct accumulation behaviour relative to that of Ni and Cr. This clustering pattern is consistent with the correlation analysis and further supports the likelihood of similar mobility pathways for Ni and Cr, which are potentially associated with Fe-Mn oxide dynamics and sediment interactions during seasonal hydrological fluctuations. The separation of Pb suggests differences in its source contributions, transport mechanisms, and bioavailability in aquatic environments.

Figure 2. Hierichical Cluster Analysis of Heavy metals concentration of cray fish.