Comparative assessment of heavy metal bioaccumulation in skeletal muscles of softshell and hard-shell freshwater turtles

2.1 Study area and sampling

We collected dead specimens of turtle species (Kachuga smithii, Kachuga tecta and Lissemys punctata) to procure the body muscle samples from two headworks located on two major rivers of Pakistan i.e., River Ravi and River Chenab). The Balloki Headworks is constructed on River Ravi, while Trimmu Barrage is erected on River Chenab. (Fig. 1). For species identification, Minton’s (1966)

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Fig. 1

Water Storage Reservoirs and Barrages of Pakistan.

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Both study sites encircled on map manual was followed. The straight carapace length and width, and plastron length were measured using a digital calibrated tape, while body size was calculated using a measuring tape. Dead turtle body weight could not be recorded due to the loss of most body mass and damages due to decomposition or consumption by predators. Water samples were also collected from both sites to corroborate the corresponding levels of targeted metals analyses.

2.2 Samples preparation and metal content analysis

Skeletal muscles were collected after the dissection of dead turtles. Muscle tissues were washed with distilled water, wrapped in aluminum foil, and packed in polyethylene bags before storage at −20 °C before analysis. Samples were allowed to thaw at room temperature prior to examination. Acid-washed laboratory materials were used to prevent contamination of samples (Páez-Osuna et al., 2010). Muscles and water samples were prepared using a wet digestion protocol for metals analyses (Hseu, 2004; S.M.E.W.W., 1989). We targeted the evaluation of seven heavy metals viz. Cu, Zn, Cd, Co, Ni, Pb and Cr, and detection was carried out by AAS (atomic absorption spectrometry; Aurora AAS, Al-1200, Canada) by following the method described in AOAC (1990).

2.3 Statistical analysis

Before data analyses, the obtained dataset was subjected to a data normality check. Further, the data were statistically analyzed by one-way analysis of variance (ANOVA) method (Steel et al., 1996) followed by the Duncan’s Multiple Range (DMR) test to compare means in the species-wise and site-based levels of heavy metals concentrations in freshwater turtle species (P < 0.05). The student’s t-test was used for comparing metals concentration with standard values.

3.1 Body size comparisons

During the study period, we collected 35 dead freshwater turtles, out of which fifteen procured from Balloki Headworks (K. smithi = 4, K. tecta = 4 and L. punctate = 7) and twenty from Trimmu Barrage (K. smithi = 7, K. tecta = 10 and L. punctata = 3). Comparison of body size of deceased turtles from both study sites illustrated non-significant differences (Table 1). We collected the muscle samples from all dead turtle pieces from BH, whereas out of twenty dead turtles collected from TB, 18 muscle samples were collected. This discrepancy occurred due to the whole consumption of the turtle muscles of K. tecta by predators who also left their internal body organs almost entirely damaged and eaten away. Similar observations were reported by Akbar et al., (2006), that feral animals such as wandering dogs patrol extensively during the canal closure period and hunt for alive and dear turtles and trapped fish as their food items. Being slow-moving creatures, the freshwater turtles are highly prone to predation of feral terrestrial animals.

3.2 Heavy metals assessment

The concentrations of targeted heavy metals showed varying levels in water samples at the study sites. The presence levels of Zn, Cu and Cd were significantly lower, while Ni, Co, Pb and Cr displayed significantly higher loads at both localities in comparison with the given standards of WHO (2004) in riverine waters of Pakistan (Table 2). This alluded to a varying degree of anthropogenic activities in study areas as we estimated the concentrations of these heavy metals in water samples collected from both sites and compared using the student t-test.

Table 3 shows species-wise and locality-based concentrations of Cu, Zn and Ni in the freshwater turtle muscles sampled from BH and TB. The mean absorption of Cu was high in skeletal muscles of L. punctata (7.61 ± 1.88) mg Kg⁻¹ while lower in K. smithi (5.07 ± 1.12) and K. tecta (5.05 ± 0.52 mg Kg⁻¹). Species and the locality-based mean concentration of Cu showed non-significance (P > 0.05) statistically. The mean concentration of Cu in the muscles BH was lower than at TB i.e 5.03 ± 0.74 and 6.64 ± 1.19 mg Kg⁻¹, respectively. Species-wise mean levels of Zn varied highly significant (P < 0.01), whereas location-based mean concentration was non-significant (P > 0.05). The mean concentration of Zn was maximum in L. punctata (49.17 ± 4.11 mg Kg⁻¹) and minimum in K. smithi (29.98 ± 1.27 mg Kg⁻¹). The location-wise mean concentration of Zn in the freshwater turtle muscles at BH was 41.64 ± 4.28 mg Kg^−1, and was recorded as 30.75 ± 1.70 mg Kg⁻¹ at TB.

Species-wise mean concentrations of Ni differed significantly (P < 0.05), whereas location-wise- mean concentration did not show significant (P > 0.05) loadings. The mean concentration of nickel was maximum in the muscle of L. punctata (2.77 ± 0.41 mg Kg⁻¹), while the minimum records noted in K. tecta (1.41 ± 0.30 mg Kg⁻¹). The mean concentration of nickel in muscle of K. smithi was 1.65 ± 0.31 mg Kg⁻¹. The mean concentration of nickel in the muscle tissues at TB was greater than those from BH. The present study's findings are in line with Nisa et al. (2015) who analyzed metals in the blood samples of three different freshwater turtle species and reported the highest bioaccumulation of Cu, Zn, Ni, Cd and Cr in L. punctata than other species.

Table 4 shows species and location-wise concentration of Cd, Co, Pb and Cr in freshwater turtle’s muscle collected from BH and TB. Species-wise and the locality-wise mean concentrations of Cd did not differ significantly (P > 0.05). The mean concentration of cadmium was higher in L. punctata (0.21 ± 0.02 mg Kg⁻¹) and lower in K. smithi 0.15 ± 0.02 mg Kg⁻¹. The mean concentration in the muscle of K. tecta was (0.17 ± 0.03 mg Kg⁻¹). The mean concentration of cadmium in the muscle of freshwater turtles at BH was greater than TB i.e. 0.19 ± 0.02 mg Kg⁻¹ and 0.16 ± 0.02 mg Kg⁻¹, respectively. Species-wise and locality-wise mean concentrations of Co were non-significant (P > 0.05). The mean Co concentrations were lower in K. smithi (1.68 ± 0.70 mg Kg⁻¹) and higher in K. tecta (3.33 ± 0.47 mg Kg⁻¹). However, the mean cobalt concentration recorded in the muscle tissues of L. punctata was intermediate between two hard shell species i.e. 3.31 ± 0.43 mg Kg⁻¹. The mean concentration of cobalt in the muscle of freshwater turtles at BH was 2.49 ± 0.48 mg Kg⁻¹ and TB was 3.37 ± 0.69 mg Kg⁻¹. The present study results are parallel to the findings of Nisa et al. (2015), Nisa et al. (2019), who concluded maximum bioaccumulation of Ni, Cd, Co and Cr in the liver of L. Punctata than other species of freshwater turtles (Mehana et al., 2020).

Species-wise and locality-wise Pb concentrations in the muscle tissues of freshwater turtle showed non-significant (P > 0.05) variations among the study sites. The Pb level was higher in L. punctata (4.70 ± 0.62 mg Kg⁻¹) and lower in K. tecta (2.58 ± 0.66 mg Kg⁻¹). The mean concentration of Pb in the muscle tissue samples of K. smithi was (3.94 ± 0.67 mg Kg⁻¹). However, the mean concentration of Pb in the muscle of freshwater turtles at BH was 3.66 ± 0.59 mg Kg⁻¹ and at TB, it was recorded as 4.04 ± 0.54 mg Kg⁻¹. Nisa et al. (2015), Nisa et al. (2019) reported the estimated Pb level in blood and liver of freshwater turtle at TB compared to BH.

The locality-wise concentration of Cr in the skeletal muscle tissues of the freshwater turtle was highly significant (P < 0.01), whereas according to the turtle species, it was statistically non-significant (P > 0.05). Cr's mean concentration was higher in L. punctata (6.09 ± 0.39) and lower in K. smithi (5.51 ± 0.281 mg Kg⁻¹). The mean concentration of Cr in the muscle of K. tecta was (5.92 ± 0.29). Overall, the maximum mean concentration of Cr detected in the freshwater turtle's skeletal muscle tissues was higher (6.21 ± 0.29 mg Kg⁻¹) at TB than recorded at BH (5.38 ± 0.12 mg Kg⁻¹).