Ratio-Dependent Inhibition of Alkaline Phosphatase by Heavy Metals and 2,4-D in Freshwater Microalgae

This study examined the combined toxic effects of heavy metals and the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) on alkaline hydrolytic phosphatase activity in the freshwater microalga Chlorella vulgaris. Toxicity was assessed by measuring inhibition of the enzyme's ability to convert p-nitrophenyl phosphate to p-nitrophenol at an optical density of 410 nm. Unary and senary mixtures of Copper (Cu²⁺), Zinc (Zn²⁺), Lead (Pb²⁺), Chromium (Cr²⁺), Cadmium (Cd²⁺), and 2,4-D were tested at fixed molar ratios. All single metal ions caused concentration-dependent inhibition, with toxicity ranking as Cr²⁺ > Cu²⁺ > Zn²⁺ > Pb²⁺ > Cd²⁺ > 2,4-D. The most potent inhibitors were Cr²⁺ and Cu²⁺, were the most toxic, with IC50 values of 0.19 ± 0.01 mM and 0.28 ± 0.00 mM, respectively. In contrast, 2,4-D alone showed a relatively low inhibitory effect, with only 20% suppression of enzyme activity. Senary mixtures prepared at two fixed molar ratios (10:18:18: 18:18:18 and 50:10:10: 10:10:10 for 2,4-D, Cu²⁺, Pb²⁺, Zn²⁺, Cr²⁺, Cd²⁺) revealed variable toxicity, with the first ratio showing higher inhibition concentration indicating ratio-dependent combinatorial effects. The mixtures exhibited mainly additive and antagonistic toxicity interactions. Toxicity index values above 1 indicate antagonism, meaning the combined toxicity was less than expected. The presence of 2,4-D reduced the heavy metals' toxicity but increased its own toxicity, demonstrating complex modulation effects within the mixture. This suggests distinct molecular pathways by which metals and 2,4-D interact with biological targets, influencing overall toxic outcomes. This is likely due to complex interactions affecting modes of action and target receptors. Overall, the study shows that combined exposure can pose ecological risks to freshwater microalgae in a mixture composition ratio.