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Oxidative stress responses in the aquatic macrophyte, Ceratophyllum Demersum L., as biomarkers of metal exposure
Metal pollution in aquatic environments is considered a major environmental concern because of variation in several abiotic factors that impose severe restrictions on organisms living in these areas. Ceratophyllum demersum L. (family Ceratophyllaceae), a hornwort or coontail, free floating rootless macrophyte has been suggested a suitable model for investigating metal stress and was used in the current study. This study assessed the use of selected biological responses, namely antioxidant responses and changes in chlorophyll concentration in Ceratophyllum demersum L., as biomarkers of metal exposure, and also investigated the field application of these responses in the Diep River. The ultimate aim was also to determine the usefulness of C. demersum as model of metal contamination and as phytoremediator after a pollution event. An investigation of metal bioaccumulation in this macrophyte exposed to different concentrations of a combination of metals over a five-week exposure period in a greenhouse, was undertaken, as well as a field study in the Diep River, Milnerton, Cape Town and a pond (reference site) at the Cape Peninsula University of Technology, Cape Town, to validate experimental results. In the laboratory study the water was contaminated once off at the beginning of the study, to simulate a pollution event. The metal concentrations in the water and plants were measured in the four treatments and the control every week over a five-week exposure period. The samples were acid-digested and analysed with an Inductively-Coupled Plasma-Mass Spectrophotometer (ICP-MS). The results showed that concentrations of the metals in the water varied in all treatments over time with no specific patterns amongst the treatment groups. This macrophyte proved highly effective in the bioaccumulation of these metals at all four exposure concentrations. The metals bioaccumulated rapidly in the plants after the water was spiked. The main focus of the study was to investigate the possible use of biochemical responses in C. demersum as possible biomarkers for metal exposure. A range of antioxidant/oxidative stress parameters were measured in the plant exposed to a combination of metals (Al, Cu, Fe, Zn) in four different treatments over the five week exposure period. Total antioxidant capacity (TAC) was measured using Total Polyphenols (TP), Ferric Reducing Antioxidant Power (FRAP) and Oxygen Radical Absorbance Capacity assay (ORAC), enzyme activity was determined using Catalase (CAT), Superoxide Dismutase (SOD), Ascorbate Acid (AsA) and Total Glutathione (GSHt) and lipid peroxidation was measured by using Thiobarbituric Acid Reactive Substances (TBARS) and Conjugated Dienes (CDs). The cocktail of the four metals induced significant changes in the antioxidant defence system of C. demersum, including the antioxidant enzyme activities. The different metal exposures disturbed the cellular redox status in the plant. The current study has demonstrated that this macrophyte shows tolerance to metal-induced oxidative stress and that it can survive under relatively high concentrations of these metals by adapting its antioxidant defence strategies. Chlorophyll was extracted in 80% chilled acetone in the dark and the absorbance values were determined using a spectrophotometer. Chlorophyll a (chl a), chlorophyll b (chl b) and total chlorophyll (chl t) contents were measured under different exposure concentrations of metals in the macrophyte. The results of this study indicated that chlorophyll contents were variable over the exposure period and no significant differences in chlorophyll concentrations were found between weeks. A field study in the Diep River and the pond located at the CPUT campus (reference site) was conducted to validate experimental results. Plants in a polluted section of the Diep River were shown to bioaccumulate metals to high concentrations. Bioaccumulation of metals in C. demersum might have induced oxidative stress, and other environmental factors such as temperature- and chemical stress might have caused chlorophyll degradation. The chlorophyll concentrations in the plants of the pond (reference site) might also have been affected by temperature and chemical stress of the water. Significantly higher AsA, CAT, ORAC, SOD and TBARS concentrations in the Diep River plants might be an indication that the plants in the river might be well adapted to the constant exposure to metals and that the plants might have developed a tolerance mechanism to cope with oxidative stress compared to those of the pond. The results show that metals are bioaccumulated quickly by C. demersum after the water is contaminated with metals, i.e. after the "pollution event". However, over time, metals are continuously exchanged between the plants and the water, accounting for the fluctuations in metal concentrations observed over time. This study has shown that C. demersum has phytoremediation potential because it was able to remove high concentrations of metals from the contaminated water. Therefore, C. demersum, can be applied as a model for metal contamination and a phytoremediator after a pollution event. The potential to antioxidant responses and chlorophyll content as biomarkers of metal exposure in C. demersum have been demonstrated.