Assessment of the toxicity of pharmaceuticals in the microalgae Pseudokirchneriella subcapitata and Dunaliella tertiolecta based on cell viability and oxidative stress biomarkers

Abstract

The detection of pharmaceuticals in the environment and their impact on the ecosystems have been receiving much interest. Not all the excess pharmaceuticals disposed of by consumers are fully eliminated in the waste water treatment system plants. The final discharge to the environment may contain residual pharmaceuticals which contribute to the burden of chemical pollutants in the receiving waters. The contaminants may have an adverse impact on micro algae, which form the basis of the aquatic food chain. The aim of this study was to assess the toxicity of four pharmaceuticals, namely triclosan, tetracycline, ibuprofen and paracetamol in two microalgae, namely Pseudokirchneriella subcapitata and Dunaliella tertiolecta based on their growth response and cell viability assays, as well as oxidative stress biomarkers such as ROS levels, antioxidant capacity, antioxidant enzymes’ activities and carotenoids. Preliminary growth inhibition tests showed that the sensitivities of both microalgae to the pharmaecuticals were as follows: triclosan > tetracycline > ibuprofen > paracetamol. Triclosan was the most toxic pharmceutical tested, with 96 h EC50 of 42.49 μg/L for P. subcapitata and 10.52 μg/L for D. tertiolecta. The least toxic drug was paracetamol, with 96 h EC50 of 1.96 g/L and 3.33 g/L for P. subcapitata and D. tertiolecta respectively. Triclosan and ibuprofen were chosen for cell viability and oxidative stress biomarker analyses to compare the effect of different classes of drugs in the same microalgae. Cell viability assessed by flowcytometric measurement of cellular fluorescence after staining with fluorescein diacetate (FDA) revealed that the cell viability of P. subcapitata and D. tertiolecta was reduced by approximately 60-90% compared to the control after exposure to ibuprofen, while the cell viability of both microalgae was slightly decreased (5-30%) after exposure to triclosan. The presence of reactive oxygen species (ROS) determined by flowcytometric measurement of cellular fluorescence after staining with 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) showed that the percentage of H2DCFDA positive cells increased by 20% when P. subcapitata was exposed to ibuprofen. However, D. tertiolecta did not show any increase in intracellular ROS after being exposed to both toxicants. The total antioxidant capacity of the microalgae determined using DPPH free radical scavenging assay showed that the total antioxidant capacity was significantly increased for D. tertiolecta exposed to triclosan and ibuprofen, while no significant changes were observed for P. subcapitata exposed to both toxicants. Photosynthetic pigments in the microalgae were also affected by pharmaceutical exposure. The carotenoid content in D. tertiolecta increased from 0.4 to 0.5 pg/cell after exposure to ibuprofen, while in P. subcapitata, the carotenoid content decreased from 0.15 to 0.05 after exposure to ibuprofen. Both pharmaceuticals did not cause significant changes in the chlorophyll-a content of D. tertiolecta, while ibuprofen caused severe reduction of chlorophyll-a content in P. subcapitata. Analyses on the antioxidant enzymes’ activities showed that SOD and CAT activities were significantly increased (p < 0.05) in P. subcapitata after exposure to triclosan and ibuprofen. No changes were observed for the SOD enzyme activity in D. tertiolecta exposed to triclosan and ibuprofen, while CAT activity in D. tertiolecta was inhibited by triclosan exposure but enhanced by ibuprofen exposure. In conclusion, the findings suggested that pharmaceuticals could adversely affect the growth and survival of the microalgae tested. Pharmaceuticals could also disrupt the oxidative balance of microalgae and trigger changes in the oxidative stress biomarkers. However, these effects varied between different pharmaceuticals and species of microalgae.