Antagonistic and Additive Toxicity Assessment of Quinary Mixtures of Metal and 2,4-D Combinations on Algal Phosphatase Function

The present study comprehensively examined the impact of combined metal and pesticide mixtures on the activity of the hydrolytic phosphatase enzyme in the freshwater alga Chlorella vulgaris. Toxicological assessments entailed evaluating inhibition of phosphatase-mediated conversion of p-nitrophenylphosphate to p-nitrophenol, monitored spectrophotometrically at 410 nm. Both unary and quinary mixtures of Copper (Cu²⁺), Zinc (Zn²⁺), Lead (Pb²⁺), Chromium (Cr²⁺), Cadmium (Cd²⁺), and 2,4-dichlorophenoxyacetic acid (2,4-D) were investigated at predetermined fixed molar ratios. Experimental and model-predicted dose-response analyses revealed that all tested heavy metals elicited concentration-dependent inhibitory effects on phosphatase activity. Notably, Cr²⁺ and Cu²⁺ demonstrated the highest toxicities with IC50 values of 0.19 ± 0.01 mM and 0.28 ± 0.00 mM, respectively, whereas 2,4-D manifested comparatively low inhibition, inducing approximately 20% enzymatic suppression. Quinary mixtures formulated at fixed ratios of 20:20:20:20:20 and 16:21:21:21:21 exhibited differential toxicological profiles wherein the 2,4-D/Pb²⁺/Zn²⁺/Cr²⁺/Cd²⁺ mixture consistently presented the highest toxicity. Across the different mixtures, a general decline in toxicity was observed, yet toxicity saturation was reached, indicating uniform toxic effects at higher concentrations. Toxicity index evaluations predominantly signalled antagonistic interactions (TI >>1), aside from one mixture in the 20:20:20:20:20 ratio displaying additive interactions. The modulation of toxicity by 2,4-D within these cocktails notably attenuated metal toxicity yet concurrently amplified its own toxic effects. This dualistic modulation likely arises from differential modes of action, temporal exposure variances, and receptor site interactions among the toxicants. Collectively, the findings underscore the ecological risk posed by metal and pesticide co-exposures to freshwater microalgae, mediated through complex antagonistic and additive enzymatic inhibition mechanisms.