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.

For the past decades, anthropogenic impacts on natural systems are of growing concern as the human population expands and global biological diversity declines. Agricultural practices often involve the use of fertilisers and pesticides to improve the yield of crops. Agricultural runoff with excess nitrogen (N) and phosphorus (P) could stimulate the excessive growth of algae in the aquatic ecosystems, including lakes and coastal regions, resulting in eutrophication. Besides fertilisers, pesticides are also used in large amounts for pest controls. Microalgae have been frequently used as bio-assay organisms to assess the toxicity of pesticides. However, most of the reports were on the effects of single or mixed pesticides without considering the possible influence of nutrients such as N and P on its toxicity. Therefore, our study aims to assess the changes of various biomarkers in microalgae after exposure to pesticides under nutrient-limited and nutrient-enriched conditions. In this study, two commonly used pesticides, atrazine and endosulfan were chosen. Atrazine, a herbicide of the triazine family, is one of the most frequently used herbicides worldwide while endosulfan is an organochlorine insecticide that causes acute neurotoxic to insects and mammals. A total of 10 isolates from Tasik Jaya and Cameron Highlands were selected to determine their sensitivities to atrazine and endosulfan. The EC50 for atrazine ranged from 43.07 µg/L to 1313.90 µ/L, while the EC50 for endosulfan ranged from 1.51 mg/L to > 50 mg/L after 96 h of exposure. Two of the microalgae, namely Scenedesmus arcuatus (highly sensitive) and Chlorella sp. (highly resistant) were selected for further studies, and compared with the model species, Pseudokirchneriella subcapitata. These three microalgae were also cultured in mediums with different levels of N (in the form of sodium nitrate, ammonium chloride and urea) and P (in the form of potassium phosphate) to determine the levels of N and P that limit/enhance the growth of the microalgae. The N-limited and N-excess levels were determined as 0.0117 mM and 3mM respectively while the P-limited and P-excess levels were 0.0002 mM and 1.7210mM respectively. To determine the effects of different nutrient conditions to the toxicity of atrazine and endosulfan, the three chosen microalgae were exposed to EC10 and EC50 of atrazine and endosulfan, being determined earlier in the dose-response assay in four different culture media: N- and P-limited (LNLP); N-limited, P-excess (LNHP); N-excess, P-limited (HNLP); as well as N- and P-excess (HNHP). In general,all three microalgae showed better tolerance to atrazine and endosulfan when they were cultured in nutrient-limited conditions as compared to nutrient-enriched conditions based on their growth response. In addition to growth response, the effects of different nutrient conditions to the toxicity of atrazine and endosulfan were also assessed using different biomarkers such as oxidative stress biomarkers, photosynthetic biomarkers and morphological biomarkers. The oxidative stress biomarkers of microalgae showed different changes when exposed to atrazine and endosulfan at different nutrient conditions. Reactive oxygen species (ROS) production and lipid peroxidation were induced significantly when microalgae were exposed to atrazine and endosulfan in nutrient-excess conditions, while superoxide dismutase (SOD) levels were generally higher in microalgae grown in nutrient-limited conditions. At N-limited conditions, exposure to atrazine and endosulfan has caused inhibition of SOD in P. subcapitata. The catalase (CAT) enzyme did not show many changes after exposure to atrazine and the response varied in different microalgae in different nutrient conditions after exposure to endosulfan. The photosynthetic biomarkers also showed different changes when microalgae were exposed to atrazine and endosulfan at different nutrient conditions. The maximum potential quantum efficiency of photosystem II (Fv/Fm) was significantly reduced with decreasing nutrient levels, while exposure to atrazine and endosulfan has stimulated the Fv/Fm regardless of the nutrient conditions. Similar trends were observed for the photosynthetic pigments. However, alpha and rETRmax were generally being reduced after exposure to atrazine and endosulfan in various nutrient conditions, except for Chlorella sp. 5 that showed significant elevation in alpha and rETRmax after exposure to endosulfan in nutrient-limited conditions. The presence of atrazine and endosulfan in nutrient-limited conditions has caused more severe damage to the algal cellular structure compared to those in nutrient-excess conditions, such as cell wall disruption and chloroplast damage. On the other hand, increased number of lipid bodies and starch granules were observed in nutrient-limited conditions, together with more severe damage to the cell wall and chloroplast of the microalgae compared to nutrient excess conditions. In conclusion, both atrazine and endosulfan caused significantly different adverse effects to different algal strains, based on their effects on various biomarkers assessed. In addition, nutrient conditions do affect the toxicity of pesticides.