*3.4. Antioxidant Enzyme Activities*

Antioxidant enzymes such as CAT and SOD can be used to monitor changes in an environment through the measurement of their activities in an organism present in that same environment. The activities of CAT and SOD in this study are shown in Figure 3a,b respectively. Significant variation existed at *p* < 0.05 across the sites for the two antioxidants with sites S5 recording the highest activities for both CAT and SOD. However, CAT activities ranged from 6.67–72.46 Ug−<sup>1</sup> protein and SOD 2.70–12.49 Ug−<sup>1</sup> protein. The sequence for activities of CAT and SOD across the sites; S5 > S3 > S4 > S1 > S2 corresponded with that of heavy metals concentrations, *Igeo* and ErF.

**Figure 3.** Antioxidant enzymes activities in *Bellamya unicolor* across the sites for (**a**) catalase (CAT); (**b**) superoxide dismutase (SOD).

A significant positive correlation (*p* < 0.01) and (*p* < 0.05) existed between CAT, SOD and all the heavy metals except Fe (0.567) which did not correlate with CAT significantly.

#### **4. Discussion**

The high concentrations of heavy metals recorded in this study downstream (S5) and the significant variations across the sites both in sediment and freshwater molluscs can be as a result of anthropogenic activities from the nearby settlements and the industries sited close to the aquatic ecosystem under study [37,38]. However, even though reduced to non-anthropogenic activities were seen or recorded at site S1 which is located upstream, concentrations of heavy metals were still recorded. This can be due to the atmospheric deposition of heavy metals and the effect of runoff that ends up in the river [17,39]. The deposition of metals from the atmosphere, industries located at the catchment, effluents of agrochemical sources and domestic sewage discharge forms part of the input for metal pollutants of anthropogenic sources. Their effect is reflected in natural aquatic ecosystem sediments with negative consequences to benthic organisms and the general wellbeing of the ecosystem [40,41].

Several authors [4,6,14] report similar findings in ecological assessment of the aquatic ecosystem having variations in different sites, with maximum concentration at sites with industrial plants and more anthropogenic activities.

The concentrations of heavy metals such as Cu, Cd, Cr, Pb and Ni that fall between TEC and PEC values for 46.4, 20.8, 57.4, 100 and 61.2% respectively from sediment quality guidelines of the freshwater ecosystem may be an indication of occasionally serious negative effects of this metals upon the ecosystem under study [4]. However, 79.2% of samples with a concentration of Cd greater than PEL reflected the frequent occurrence of biological effect [42]. Comparison is made between metal concentrations with TEC and PEC values to identify if heavy metals present in the sediment has the possibility of threatening the aquatic life [14,42].

This site has industries sited close to it and receives effluents from the industries. Hence, serious attention might be required at this site due to high concentrations of metal pollutants in comparison with the other sites. Pollution as a result of industrial effluent and domestic waste discharge contributes to the rise in concentrations of metal pollutants leading to contamination of the ecosystem [4,43].

The *Igeo* of heavy metals in sediments were generally higher in site S5 and lower in sites S1 and S2. *Igeo* scoring of S1 and S2 into class zero [32] for Cd indicate no contamination with Cd. Cd is well known as one of the major pollutants with industrial effluents and domestic sewage as its source [43]. However, extreme pollution of the sites with heavy metals especially S5 with *Igeo* >5 having a score of 6 reflects on the source variations of the metal pollutants and the nature of catchments. Site S5 is downstream where the other stretch of the river ends up, receiving some of its contents apart from the effluents received from the nearby industrial plants. Downstream has been reported in several studies to have more pollutants affecting the natural wellbeing of that particular ecosystem setting [44–46].

Considerable ecological risk exhibited by the metals across the sites is linked with the level of geoaccumulation recorded. The ecological risk was consistent with *Igeo* for both metals and site variation as revealed by multi-metal ErF. Cd posed a high ecological risk in River Kaduna with an expectation of an adverse effect expected to occur suggesting that they are present in high concentrations, especially at site S5 with the maximum ErF value. This major ecological risk in surface sediment of the River Kaduna by Cd must have been seriously influenced by anthropogenic activities, leading to a high toxic response factor [6]. The low and moderate potential ecological risk in sites S1 and S2 may be a result of metals present in residual forms and minimal concentrations [31,47,48].

Source and migration of metals may be reflected by correlation analysis and PCA [49,50]. Non-significant correlation between Fe–Mn, Fe–Cr, Mn–Cu, Cu–Zn, Mn–Pb, Cu–Cr, Cu–Ni, Cr–Pb, and Pb–Co indicates different factors controlling the availability and concentrations of these metals [4,9]. This implies the metals might have originated from different sources. This was also revealed in the PCA; the groupings of metals, and component 1 and 2 accounting for 76.80% and 8.81%, respectively, with strong positive loading of Mn, Cu, Cd, Cr and Pb in the sediment and moderate positive loading of Cu and Pb in the sediment. This implies urban and industrial waste to be the source of Mn, Cu, Cd, Cr and Pb [51,52].

Similarity and dissimilarity in groups are effectively represented using cluster analysis. Three cluster formations for sites under study revealed a close relationship between these sites. Grouping of S3, S4, and S5 together at a similarity level of 89.09 indicates possible similarities in human activities, site morphology and the pristine background types of sources [53]. The significant relationship revealed by cluster analysis at 99.57 similarity level for the relationship between the metals, grouping Mn, Cr, Cu, Cd, and Pb together makes it possible that they have the same natural and anthropogenic sources [54,55]. This is also a link to the multiple effects of these metals on benthic organisms. For example, *Bellamya unicolor* collected during our sampling for this study. Our result agrees with the finding of Peijnenburg et al. [56]; Li et al. [55]; Chung et al. [54]. These authors assess and monitor heavy metal sources and their risk in different aquatic environments.

CAT and SOD activities were used in this study as biomarkers to measure the extent of stress in the River Kaduna as reflected in *Bellamya unicolor* sampled from the same environment at different sites. CAT and SOD are a type of antioxidant enzyme that scavenges reactive oxygen species (ROS) (H2O2, OH, O2, etc.) produced by organisms as a result of oxidative stress caused as a result of unfavorable environmental conditions such as metal pollution [1]. Significant variation in CAT and SOD activities across the sites with high activities in site S5 may be a result of different concentrations of ROS production at the sites with high concentrations at site S5 due to high metal pollution [57]. The scavenging ability of antioxidants increases through their activities in response to increased ROS to reduce or prevent membrane lipid peroxidation by ROS and to improve membrane stability of the cell [58,59].

The significant positive correlation between the antioxidants and the metals showed that the organism *Bellamya unicolor* is under stress as a result of an increase in metal contamination. Bakshi et al. [1] report similar findings for the biological response of an aquatic organism to metal contamination through antioxidant activities. They revealed an increase in antioxidants with a spatial and temporal increase in heavy metal concentrations.

Our results in this study tend to bridge the gap in knowledge for metal pollution risk assessment, and the use of CAT and SOD as primary biomarkers in benthic organisms of a tropical ecosystem. There has so far been no report, to the best of our knowledge, assessing risk assessment using ecological indices, antioxidant enzymes and *Bellamya unicolor* as a biomonitor. Our study will provide data to be used as a baseline for studies in the River Kaduna and other related tropical rivers in the same region.

#### **5. Conclusions**

The results from this study provide information on the contamination of heavy metals Fe, Mn, Cu, Zn, Cd, Cr, Pb, Ni, and Co in surface sediment of the River Kaduna and their bioavailability in the freshwater mollusc *Bellamya unicolor* sampled from the same ecosystem.

At a 95% confidence interval, significant variation existed in the concentration of metals in surface sediment and freshwater molluscs across the sampling sites with the site S5 (downstream) having the maximum metal concentrations.

Cd concentration in sediment was greater than PEL, which implies likely frequent occurrence of biological effect by Cd in the River Kaduna with the possibility of threatening the aquatic life. This was reflected in Cd ErF values that are higher than the values of ErF for other metals. *Igeo* and ErF values follow the same sequence with maximum values at site S5 revealing very high ecological risk with *Igeo* > 5 and ErF > 320. Mn, Cu, Cd, Cr, and Pb originate mainly from urban and industrial waste as revealed by PC1 and PC2 analysis while S3, S2, and S3 have similar anthropogenic activities and natural metal inputs as shown in cluster analysis. Significant variations existed in CAT and SOD activities in *Bellamya unicolor* across the sites with maximum activities recorded at sites S5 for both antioxidants. This also coincides with the high values of *Igeo* and ErF at site S5. However, there is a significant positive correlation between the antioxidants and metal concentrations.

Important approaches and policies should be put in place to prevent the discharge of untreated industrial and domestic waste into the River Kaduna. The approach should involve the prevention of irrigation farming close to the river and, in that way, non-point sources of pollution can be abated and there can be a decrease in ecological risk associated with metal pollutants.

**Author Contributions:** M.O.A., and A.B.A. conceived the idea and performed the laboratory analysis. All of the authors contributed to give the manuscript its present shape. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant number D-024-130-1441. The authors, therefore, gratefully acknowledge DSR technical and financial support.

**Acknowledgments:** The authors acknowledge the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah for their technical and financial support.

**Conflicts of Interest:** The authors declare there is no conflict of interest.
