Chlorella vulgaris (C.vulgaris), a fast-growing unicellular micro-alga, is commonly used as a supplement of food for human and animal consumption.As C.vulgaris readily absorbs metals, this micro-algae has also been employed as an important indicator for monitoring the pollution and toxicity of metals in aquatic environments.Due to both natural geological sources as well as anthropogenic sources, the escalating contamination of aquatic environments by metals such as zinc (Zn) is becoming of increasing concern.A limited concentration of Zn is essential for growth of C.vulgaris, but limited knowledge exists concerning toxic impacts of Zn on the alga.The present study investigated various biochemical variables including biomass, the activities of antioxidant enzymes including superoxide dismutase (SOD) and peroxidase (POD), the intracellular Zn assimilated, and the inducement of Zn-MT-like proteins in C.vulgaris when the alga was exposed to various Zn²⁺ concentrations.Results showed that the growth of C.vulgaris was inhibited by exposure to Zn²⁺ at concentrations ranging from 5-100 μmol l/L.The algal biomass rapidly decreased with increased Zn²⁺ exposure concentration.Algal biomass also decreased with increased exposure time to concentrations of Zn²⁺> 10 μmol/L.The results indicated that the growth of the alga was significantly inhibited by exposure to Zn²⁺,especially at higher exposure concentrations.The activities of SOD increased with the increase of Zn²⁺ exposure concentrations within the ranges of 0-50 μmol/L, but slight decreases in SOD activity occurred with exposure concentrations of Zn²⁺> 50 μmol/L.The highest activity of SOD was evident when the alga was exposed to 50 μmol/L of Zn²⁺, in which the value of SOD activity was two times more than that of the control group.However, the activities of POD within the alga significantly decreased (P < 0.05) with the increase of Zn²⁺ exposure concentrations.For example, the activity of POD was 7.4 times less than that of the control group when C.vulgaris was exposed to 10 μmol/L of Zn²⁺.The results also showed that C.vulgaris has a strong ability to assimilate waterborne Zn²⁺.For example, the concentrations of intracellular Zn were about 17 and 34 times more than that of the control group when the alga were exposed to 5 μmol/L and 10 μmol/L of Zn²⁺, respectively.However, the concentrations of intracellular Zn did not increase when the exposure concentrations of Zn > 10 μmol/L.A slight decrease of intracellular Zn was observed when the alga were exposed to 100 μmol/L of Zn²⁺.After being homogenized and centrifuged, the cell extracts of C.vulgaris after exposure to Zn²⁺ were separated with a size-exclusion high-performance liquid chromatography coupled with the detector of inductively coupled plasma mass spectrometry (ICP-MS).Two Zn-binding species were observed within the cells of C.vulgaris, in which the compounds of the Zn-binding fraction at the retention time (t_R) of 6.3 min were referred to as the Zn-MT-like proteins because the retention time (t_R) was found to be similar to that (t_R 6.1 min) of the standard Zn-MTs derived from rabbit liver.In addition, the amounts of Zn-MT-like proteins induced in the alga were found to increase with the increase of the Zn²⁺ exposure concentrations.The results indicate that the Zn-MT-like proteins induced in C.vulgaris can be used as the indicator to reflect the pollution of Zn²⁺in aquatic environments.