Significantly enhanced effects of heavy metals on the toxicity, bioconcentration and biomagnification under combined exposure.

Heavy metal (HM) co-contamination is prevalent in the aquatic ecosystems and often induces complex combined effects such as synergism or antagonism, bioconcentration and biomagnification on the food-chain organisms, which is threatening the survival of living creatures and even to human health. However, the combined effects of HMs under combined exposure on the aquatic food chains still remain poorly understood. Therefore, toxic responses, bioconcentration and biomagnification of four typical HMs, lead (Pb), cadmium (Cd), nickel (Ni) and zinc (Zn), were systematically investigated under different combined exposure conditions. Results demonstrated that combined toxicity significantly exceeded single-metal effects and increased with component number, following the order: quaternary > ternary > binary > single. The CdNi combination exhibited the strongest toxicity at 96 h (pEC50 = 4.15), confirming significant synergistic. Further analysis of the reactive oxygen species (ROS) production, membrane integrity, and chlorophyll a and b contents demonstrated that HM interactions disrupt redox balance and photosynthetic function, amplifying toxicity. Under the combined exposure, HMs were readily bioconcentrated in Chlorella pyrenoidosa and transferred to Daphnia magna. Notably, Cd showed clear biomagnification under single-metal exposure. As the number and complexity of metal components increased, biomagnification factor values for both Cd and Ni further increased. Cd and Ni exhibited stronger trophic magnification effects, indicating that Cd and Ni may be the key drivers of combined toxicity and biomagnification. All the findings in this study will provide the basic reference for the ecological risk assessment of compound contamination of multiple HMs.