EFFECTS OF THE HEAVY METALS COPPER, CADMIUM AND NICKEL ON THE INDUCTION OF CELL DEATH AND OXIDATIVE STRESS IN DUNALIELLA TERTIOLECTA AND PAVLOVA GYRANS

Abstract

Copper (Cu), cadmium (Cd) and nickel (Ni) are three major heavy metal contaminants in the aquatic ecosystem. Such contaminants can cause adverse effects on microalgae, which may in turn affect organisms at the higher trophic levels in the food chain of the aquatic ecosystem. While there have been many studies on the toxicity of heavy metals in microalgae, there has been no report on the mode of cell death in microalgae in response to heavy metal toxicity. Cell death in microalgae could be resulted from oxidative stress caused by heavy metal toxicity. The aim of this study was to assess the effects of Cu, Cd and Ni on cell death and oxidative stress in microalgae in response to heavy metal toxicity in two marine microalgae, namely Dunaliella tertiolecta and Pavlova gyrans. Toxicity testing (96 h) of the three heavy metals based on OD620 revealed that Cu was the most toxic heavy metal to both D. tertiolecta (EC50 = 8.422 mg/L) and P. gyrans (EC50 = 3.986 mg/L). Overall, P. gyrans was more sensitive to the three heavy metals than D. tertiolecta. Chlorophyll a and carotenoid concentrations of D. tertiolecta and P. gyrans decreased significantly at increasing levels of heavy metals. The ratios of carotenoids to chlorophyll a were generally higher in P. gyrans (0.60 – 0.95) compared to D. tertiolecta (0.47 – 0.59). The carotenoids to chlorophyll a ratios in both microalgae iii increased when exposed to increased Cd concentration. The algal cells were exposed to the heavy metals for 0, 1, 2, 4, 6 and 24 h at three concentrations for further investigations on cell death and reactive oxygen production: control, EC50 and highest concentration used in the preliminary toxicity testing. Light microscopic examination revealed that Cu-treated D. tertiolecta formed aplanospores after 2 h exposure to the Cu whereas Ni-treated D. tertiolecta formed aplanospores after 1 h exposure to Ni. Many lysed cells of D. tertiolecta were observed when the alga was exposed to Cd, especially after 4 h exposure to the highest concentration. Cell size of D. tertiolecta and P. gyrans increased after exposure to the heavy metals. Heavy metals induced ultrastructural changes, including vacuolisation, distortion of thylakoids structures, slight condensation of chromatin, and cell lysis in both microalgae. Blebbing and change of structure of the pyrenoid and its starch granules in D. tertiolecta were observed after exposed to heavy metals for 24 h. Increased amounts of electron-opaque deposits in vacuoles and deformation of mitochondria and Golgi apparatus were observed in P. gyrans after exposed to heavy metals. Flow cytometric analysis showed that percentage cell viability of D. tertiolecta decreased by 13.6 % and 51.6 % after exposure to 20.48 mg/L Cu for 4 and 6 h respectively, compared to the control. Pavlova gyrans showed a marked decrease in cell viability (87%) after exposure to 20.48 mg/L Cu for 2 h. In comparison, the percentage cell viability of the cultures exposed to Cd and Ni remained similar to the control cultures throughout the growth period. Cell cycle analysis showed that D. tertiolecta underwent longer duration of G0/G1 phase and shorter duration of G2 phase at 6 h after exposure to 20.48 mg/L Cu. In comparison, at 4 h after exposure to 20.48 mg/L Cu, there was an increase on the percentage of cells of P. gyrans in S and G2 phases. Flow cytometric analysis showed that ROS production increased in both species iv when treated with Cu. Percentage of ROS production in D. tertiolecta increased after 1 h exposure to the highest Cu concentration, while in P. gyrans it increased after 4 h exposure to the highest Cu concentration. Neither DNA laddering nor smearing was detected in algae exposed to heavy metals for up to 72 h based on gel electrophoresis analysis. Study on Annexin/PI staining showed that most of the Cu-treated cells underwent necrosis, with a small percentage of cells underwent apoptosis. Analysis of heavy metal contents using inductively coupled plasma mass spectrometry (ICP-MS) showed that the percentage of Cu and Ni that remained in the medium decreased significantly for both microalgae. In summary, the findings revealed that Cu, Cd and Ni caused oxidative stress and morphological changes, and induced necrosis-like cell death in D. tertiolecta and P. gyrans within the short period (24 h) of exposure to the heavy metals.