1 Introduction

Periwinkle, Tympanotonos fuscatus Linnaeus, is a marine gastropod commonly found in intertidal zones and shallow brackish water (Melatunan, 2012; Levakin et al., 2013). It is an edible small snail with low fat and calorie content. Periwinkle is consumed by many all over the world. It plays a vital role to many coastal dwellers in several parts of the world where it serves as food and a source of income (Chioma et al., 2021). Periwinkles are rich in protein, vitamins, and minerals (Ehigiator and Oterai, 2012; Elegbede et al., 2023). Specifically, the protein content of its soft body has been shown to range from 15 to 25% of the dry weight (Db et al., 2017; Johnnie et al., 2020). The populations of periwinkles and other aquatic animals are threatened by various human activities, during which the levels of toxic metals such as lead, mercury, cadmium, chromium, etc. might become elevated in the environment, thus exposing resident aquatic organisms to stress that may ultimately lead to death (Barone, 2013; Ekpo and Essien-Ibok, 2013; Baki et al., 2018). Aquatic molluscs including periwinkles have been employed as model organisms in ecotoxicological studies as bioindicators for toxic metal pollution; due to their ecological importance, relative abundance, and sensitivity to environmental changes (Otitoloju, 2002; Gupta and Singh, 2011; Krull and Newman, 2022).

Hexavalent chromium (Cr⁶⁺) is one of the four oxidation states of chromium that is toxic, water-soluble, and a pollutant of public health concern (Garg et al., 2016; Shahpiri et al., 2021, Aliu et al., 2023). It is widely used in industries for leather tanning, electroplating, dyeing of fabrics etc. (Nigussie et al., 2012; Coetzee et al., 2020; Musah et al., 2021), as such it is not uncommon to detect Cr⁶⁺ at elevated levels in water bodies (Arunachalam et al., 2013; Fagbohun, 2016). The toxic effects of hexavalent chromium on aquatic animals are well stated, they include alterations in the condition index and protein metabolism, histopathological damages, cytotoxicity, genotoxicity, and reproductive impairments (Neves et al., 2015; Aliu et al., 2023; Salami et al., 2023).

Bioaccumulation of heavy metals in different tissues and at different levels in aquatic animals have been reported by several researches (Yu et al., 2021; Salami et al., 2023). This accumulation is never without a consequence at organismal and suborganismal levels of the exposed level. Ni et al., (2020) reported an acute toxic effect of hexavalent chromium on the liver of marine medaka (Oryzias melastigma). The presence of toxic heavy metals above tolerable limits in aquatic habitats has been reported to cause significant redox imbalance in the resident organisms (Galli et al., 2005; Pujalté et al., 2011; Raeeszadeh et al., 2023). Oxidative stress, which is a condition characterized by an imbalance between the production of reactive oxygen species (ROS) and the ability of cells to scavenge the excess ROS results in damage to biomolecular structures like lipids, proteins, and DNA (Adeyemi et al., 2013; Adeyemi, 2014; Xu et al., 2018; Chen et al., 2020). However, there is a scarcity of information on the effects of hexavalent chromium on the redox homeostasis of periwinkles at sublethal concentrations, which is considered to be important for the ecological risk assessment of hexavalent chromium in aquatic environments. This study hypothesizes that exposure to sublethal concentrations of Cr⁶⁺ disrupts redox homeostasis in T. fuscatus. Therefore, the present study investigated the effects of exposure to hexavalent chromium on the activities of antioxidant enzymes and the levels of lipid peroxidation and reduced glutathione, which are important biomarkers of oxidative stress in aquatic animals.

2 Results

The activity of superoxide dismutase (SOD) in periwinkles exposed to sublethal concentrations of hexavalent chromium is revealed in Figure 1. The activity of superoxide dismutase was significantly higher, in a concentration-dependent manner in periwinkles that were exposed to Cr⁶⁺ compared to the control (F3, 28 = 39.93; p ˂ 0.0001). The SOD activity was significantly lower in the animals exposed to 0.42 mg/L Cr⁶⁺ compared to those exposed to 0.84 and 4.2 mg/L Cr⁶⁺ in which the SOD activity was statistically similar. The control experiment produced the least activity of SOD, signifying that the control animals were the least stressed when compared to the other animals exposed to different concentrations of Cr⁶⁺.

There was a significant difference in the catalase activity among the groups (F3, 28=74.72; p ˂ 0.0001). Catalase activity was significantly higher in periwinkles that were exposed to hexavalent chromium compared to the control (Figure 2). The catalase activity was lowest in the animals that were exposed to 0.42 mg/L while there was no significant difference in the catalase activity between those exposed to 0.84 and 4.2 mgl/L Cr⁶⁺ (Figure 2).

The activity of glutathione peroxidase (GPx) differed significantly among the groups (F3,28 = 6.55; p = 0.015). The GPx activity was significantly higher in the animals that were exposed to higher concentrations of Cr⁶⁺ (0.84 and 4.2 mg/L) compared to the control and those exposed to 0.42 mg/L Cr⁶⁺ in which the GPx activity was statistically similar. Also, the GPx activity was statistically similar between the 0.84 and 4.2 mg/L exposure groups (Figure 3).

The concentration of reduced glutathione (GSH) decreased significantly in the exposed groups compared to the control (F₃,₂₈ = 34.16; p ˂ 0.0001). However, the levels of GSH were not significantly different between the control and the animals exposed to 0.42 mg/L Cr⁶⁺ (Figure 4).

The levels of thiobarbituric acid reactive substances (TBARS) in the tissue homogenates of the periwinkles differed significantly (F_3,24 = 32.18; p ˂ 0.0001). The TBARS levels were significantly lower in the periwinkles exposed to 0.84 and 4.2 mg/L Cr⁶⁺ compared to the control, while the TBARS levels were significantly higher in the animals exposed to 0.42 mg/L Cr⁶⁺ compared to the control and the other exposure groups (0.84 and 4.2 mg/L) (Figure 5).

3 Discussion

Oxidative stress resulting from perturbation of redox homeostasis is a physiological disturbance that has been linked to exposure to toxic elements in several aquatic organisms (Lee et al., 2019; Nowicka, 2022; Saç and Yeltekin, 2023). The focus of the present study was oxidative stress effects in periwinkles due to exposure to sublethal concentrations of hexavalent chromium. The results obtained show a significant oxidative stress effect in the exposed periwinkles along concentration gradients, which are consistent with the findings of previous studies that have reported oxidative stress effects in aquatic organisms that were exposed to hexavalent chromium (Lushchak et al., 2009; Lushchak 2011; Aliu et al., 2023).

Changes in the activity of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase have been used as indices of oxidative stress in aquatic animals (Niki, 2008; Adeyemi, 2014; Aliu et al., 2023). The changes in the activity of antioxidant enzymes were due to the action of the enzymes to scavenge the reactive oxygen species produced in the cells in response to exposure to toxicants. Natural antioxidants level has also been reported to increase against reactive oxygen species produced by cadmium toxicity (Unsal et al., 2020; Huchzermeyer et al., 2022). Superoxide dismutase is an antioxidant enzyme involved in the conversion of toxic superoxide anions to hydrogen peroxide and molecular oxygen. Since hydrogen peroxide is equally toxic to cells, it is further acted upon by other enzymes such as catalase and glutathione peroxidase, which reduce it to water and molecular oxygen (Unsal et al., 2020; Huchzermeyer et al., 2022). The lack of significant difference in the activities of the antioxidant enzymes between the control and the animals exposed to 0.42 mg/L Cr⁶⁺ is an indication that the rate of production of ROS at this concentration was within the range that can be detoxified by the enzymes, thus preventing oxidative stress. On the other hand, the significantly higher activities of SOD, CAT, and GPx in the periwinkles exposed to 0.84 and 4.2 mg/L is an indication that the rate of production of ROS within these concentration ranges overwhelmed the scavenging activities of the antioxidant enzymes. This submission is similar to the submission of Schiebar and Chandel (2014) who reported that response to ROS is usually insignificant at low concentration of xenobiotic. Furthermore, hormesis can also set in a rear condition in which lower concentration of stressor will produce more oxidative stress than higher concentration of same stressor. Aliu et al., (2023) also reported a significant increase in SOD activity in hexavalent chromium exposed catfish along exposure concentration gradient.

Like the actions of antioxidant enzymes, reduced glutathione (GSH) played an important role in redox homeostasis of aquatic animals (Mello et al., 2015; Samuel et al., 2022). The thiol group of the GSH binds to ROS to form oxidized glutathione (GSSG), thus the cellular levels of GSH and the ratio of GSSG to GSH have been employed as biomarkers of oxidative stress in animals (Forman et al., 2009; RajRai et al., 2021). The significantly low levels of GSH in periwinkles exposed to higher concentrations of hexavalent chromium is an indication of oxidative stress in which GSH binds to ROS thus resulting in the depletion of cellular levels of GSH. This report is similar to the submission of Liu et al. (2022) who reported an imbalance in ROS/GSH in stressed organism and depletion in GSH level in an environment where ROS production is excessive.

One consequence of elevated production of reactive oxygen species in living cells is the oxidative damage to biomolecules e.g. peroxidation of lipid molecules (Dorts et al., 2009). Lipid peroxidation has been quantified in aquatic organisms by measuring the tissue levels of thiobarbituric acid reactive substances, which is a red- or pink-coloured final product of lipid peroxidation (Samuel et al., 2022; Osagie and Morayo, 2023). The significantly higher level of TBARS in periwinkles exposed to 0.42 mg/L Cr⁶⁺ is evidence of oxidative stress at the low exposure concentrattion. Notwitstanding, the activities of antioxidant enzymes did not support oxidative stress at the exposure concentration. However, the levels of TBARS were significantly low at higher exposure concentrations (0.84 and 4.2 mg/L) in this study. This could be as a result of increased activity of antioxidant enzymes at high exposure concentrations, which helped to scavenge the deleterious ROS, thus minimizing lipid peroxidation (Barata et al., 2005).  

4 Conclusion

The findings from the study indicate that the exposure to sublethal concentrations of hexavalent chromium resulted in oxidative stress in periwinkles as demonstrated by significant high activity of antioxidant enzymes and depletion of GSH level, especially at higher exposure concentrations (0.84 and 4.2 mg/L Cr⁶⁺). Although, the activity of the antioxidant enzymes did not suggest the incidence of oxidative stress in periwinkles exposed to a low concentration of Cr⁶⁺ (0.42 mg/L), however, the results of the lipid peroxidation revealed chromium-induced oxidative stress. This could be an indication that measuring the level of thiobarbituric acid reactive substances is a sensitive biomarker of oxidative stress at low exposure concentrations. The proper understanding of the oxidative stress patterns in aquatic animals like periwinkles can be used as biomarkers for early warning signals of environmental stress and potential ecological harm, even before visible effects like mortality or population decline play out.

5 Materials and Method

5.1 Collection of periwinkles

The experimental periwinkles were got from marine water in Abealala, Ilaje Local Government, Ondo State, Nigeria (6^o21′18″ N 4^o66′10″ E). The ambient salinity of the marine water at the collection point was found to be 7.9 ppt. The periwinkles collected where for this experiment were of table size of shell lengths between 4 and 5 cm and the mean body weight of 4.2 ± 0.8 g. After collecting water and sufficient periwinkles from the location, the samples were transported to the Postgraduate Research Laboratory of the Department of Biology, Federal University of Technology, Akure, Nigeria, in pristine plastic containers. Before the exposure of the periwinkles to hexavalent chromium, the organisms were given fourteen days to acclimate to the laboratory conditions and were fed water leaves (Talinum triangulare) during the period of laboratory acclimation. Using atomic absorption spectrophotometry, the total chromium concentration of the brackish water samples taken from the periwinkles collection point was found to be 0.3 mgl^-1.

5.2 Periwinkle exposure to hexavalent chromium

The laboratory-acclimatized periwinkles were exposed to three sublethal concentrations; 0.42, 0.84, and 4.2 mg/L being ¹/₂₀, ¹/₁₀, and ¹/₂ of the 96 h LC₅₀ of Cr⁶⁺, respectively for thirty (30) days. The LC₅₀ (median lethal concentration) was previously determined in an earlier experiment (Salami et al., 2023). Throughout the experiment, the exposure medium was changed out every four days to avoid excretory product buildup in the experimental medium. Three replicates were established for each treatment, and the animals were exposed to Cr⁶⁺ in plastic containers of 150 × 80 × 50 mm, with twenty animals per container. The water used to maintain the control animals was free of Cr⁶⁺ contamination. The plastic containers were shielded with muslin cloth to allow for entree to oxygen but barred the animals from evasion during experiments. Control groups of periwinkles were also maintained under identical conditions without Cr⁶⁺ exposure. Following the exposure, representative animal samples were crushed, the soft tissues were removed, and the samples were frozen at −20 oC for later determination of activities of antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Likewise, the levels of reduced glutathione (GSH) and lipid peroxidation (LPO) were quantified.

5.3 Preparation of crude homogenate

The soft bodies were homogenized by macerating in an ice-cold potassium phosphate buffer (0.05 M; pH 7.5) in a ceramic mortar. The homogenates were centrifuged at 10,000 ×g for 15 min at 4 °C. The obtained supernatants were kept at -20 °C for the determination of activities of antioxidant enzymes and the levels of reduced glutathione while the lipid peroxidation levels were determined using the whole homogenates.

5.4 Superoxide dismutase activity assay

The Beauchamp and Fridovich (1971) method was used to measure the superoxide dismutase activity. This involved adding 0.25 mL of the supernatant to 1.5 mL of SOD reagent, which contained 1.17 mM ribofavin, 0.1 M methionine, 20 mM potassium thiocyanide, and 56 mM nitro blue tetrazolium. After that, the mixture was incubated at ambient temperature for one hour. A blank was also simultaneously prepared, which contained distilled water in the place of the supernatant. After the period of incubation, the absorbance was read at 560 nm, by means of a UV/Vis spectrophotometer (BK-UV 1900 model). The SOD activity was expressed as μmoles min-1mg protein-1.

5.5 Catalase activity assay

Using Ellerby and Bredesen's (2000) approach, spectrophotometric quantification of catalase activity was conducted. The pace at which catalase in the supernatant breaks down hydrogen peroxide determines the course of the reaction. The amount of enzyme needed to break down one µmol of hydrogen peroxide in one minute at 25 ℃ is known as one unit of the enzyme. In brief, 0.3 mL of a 0.03 M hydrogen peroxide solution was introduced to 0.6 mL of potassium phosphate buffer (0.01 M; pH 7.5) in a cuvette. After that, 20 µL of the supernatant was added, and after a minute, the change in absorbance at 240 nm wavelength was measured. Catalase activity was expressed as µmol min-1 mg protein-1 using a molar extinction coefficient of 39.6 M^-1 cm^-1.

5.6 Glutathione peroxidase (GPx) activity

Glutathione peroxidase activity was determined by the procedure of Paglia and Valentine (1967). First, 100 µL of the supernatant was added to 10 µL of 200 mM GSH in test tubes. Thereafter, 100 µL of H₂O₂ (2 mM) was then added to the mixture to initiate the reaction. The reaction was observed at 340 nm for 3 min using the UV-Visible spectrophotometer, and the activity of GPx was expressed as µmol NADPH min^-1 mg^-1 protein.

5.7 Reduced glutathione concentration

The concentration of reduced glutathione in the supernatant was measured by adopting the method of Ellman (1959), with minor changes. Ellman’s reagent also known as 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB) was used as the substrate. The procedure involved mixing 10 µL of the supernatant with an equivalent volume of 10 mM DTNB in 0.1 M potassium phosphate (pH 7.5) that contained 17.5 mmol/L of EDTA. The samples were centrifuged at 2000 ×g for 10 minutes, and the resulting supernatants were added to cuvettes containing 10 µL of 0.5 U ml-1 glutathione reductase (GR) in 0.1 M potassium phosphate (pH 7.5). The cuvettes were then incubated at room temperature for one minute. The reaction assay was started by adding 220 nM of reduced nicotinamide adenine dinucleotide phosphate in 0.1 M potassium phosphate (pH 7.5) containing 5 mM EDTA in a concluding volume of 1 ml. The rate of reduction of DTNB was observed and recorded spectrophotometrically at a wavelength of 412 nm using a UV/Vis spectrophotometer. Using a standard curve generated from known concentrations of GSH in potassium phosphate buffer solution of pH 7.5, the total GSH concentration was determined.

5.8 Lipid peroxidation assay

The levels of lipid peroxidation in the tissue homogenates were determined following the procedures reported by (Devasagayam et al. 2003). 1 ml of the tissue homogenate was mixed with 1ml of 10% trichloroacetic acid (TCA) solution. Two to three drops of butylated hydroxytoluene (BHT) solution were added to the mixture, vortexed, carefully positioned on ice for 10 min, and centrifuged at 10,000 ×g for 15 min at 4 °C. To the supernatant was added same volume of thiobarbituric acid (TBA), and the mixture was incubated in a boiling water bath for 45 min to allow for the color to develop. After incubation, the sample was cooled in an ice bath and then centrifuged at 2000 rpm for 10 min at 4 °C to eliminate any precipitates. The absorbance of the supernatant was then measured at 532 nm wavelength using a UV-spectrophotometer. Level of lipid peroxidation was expressed as μM TBARS/mg protein using a molar extinction coefficient of 156,000 M^-1/cm^-1.

5.9 Protein estimation

Using bovine serum albumin as a reference, the Bradford test (Bradford, 1976) was used to measure the protein content of the tissue homogenate.

5.10 Data analysis

One-way analysis of variance was used to examine the data obtained (since the group are independent) in order to find the differences among the means of the various values obtained from the treatment groups. The means were then separated by Tukey’s multiple comparison tests to check significant difference among the means. IBM SPSS version 21 was used for all statistical calculations. For reporting purposes, data were expressed as mean ± standard deviation, and statistical significance was assumed at p = 0.05.

Acknowledgments

The authors appreciate Mrs. Alade Toyin of the Department of Biology, Federal University of Technology, Akure for assistance with some assay procedures.

Conflict of Interest Disclosure

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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