Since Eu-PS/ZnO NP coexposure with Cd
2+ could change the bioavailability and distribution of Cd, the adverse effects caused by the coexposure on zebrafish larvae were investigated. Cd can induce oxidative stress and cause toxicity through the production of reactive oxygen species (ROS) [
31]. Excessive production of ROS can cause lipid and protein peroxidation, leading to death and pathological damage [
32]. Nonetheless, fish also have a suite of defensive mechanisms to cope with increasing oxidative stress. Therefore, we investigated the levels of ROS and antioxidative stress in zebrafish larvae (
Fig. 4). From
Fig. 4A, the exposure of Cd
2+ would increase the ROS level significantly. On the contrary, Eu-PS/ZnO NPs exposure would not affect the ROS level. When Eu-PS NPs co-exposed with Cd
2+, the ROS level increased with the increase of Cd
2+ concentration, and the degree was equivalent to that of Cd
2+ exposed alone. Co-exposure of ZnO NPs and Cd
2+ would cause the ROS level to rise sharply compared with the exposure of Cd
2+. Excessive ROS lead to lipid peroxidation, which can lead to the formation of malondialdehyde (MDA) [
33]. Thus, from
Fig. 4B, the ZnO NPs and 100 µg/L Cd
2+ co-exposure group caused the highest MDA content. However, the MDA content barely change for ZnO NPs exposure group, Eu-PS exposure group and the ZnO NPs/Eu-PS + Cd
2+ co-exposure group with low concentration of Cd
2+. Under these exposure conditions, the ROS level might not cause significant lipid peroxidation, therefore, the MDA content did not change. Cd
2+ exposure could also affect the activity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) in zebrafish [
34]. As can be seen from
Figs. 4C and D, under the exposure of Cd
2+ alone, the SOD and CAT activity increased with the increase of Cd
2+ concentration, indicating that the zebrafish larvae produced an anti-oxidative stress response to resist the ROS. ZnO NPs exposure alone would increase SOD activity. Adult zebrafish exposed to a certain concentration of Zn
2+ showed increased antioxidant enzyme activity to address the adverse effects of Cd
2+ [
35]. Therefore, when ZnO NPs were exposed with low concentrations of Cd
2+, the larvae absorbed more Zn and Cd at the same time (
Fig. 2), and the SOD activity was higher than that of ZnO NPs or Cd
2+ exposure alone. More Zn can alleviate the adverse effects of Cd, which is consistent with the fact that the MDA content does not increase significantly under this exposure condition. When high concentration Cd
2+ was exposed with ZnO NPs, the SOD and CAT activity was equivalent to that of 100 µg/L Cd
2+ exposure. Considering that more Cd has been absorbed by larvae than 100 µg/L Cd
2+exposure alone at this time, it may not be sufficient for the regulation of oxidative stress, resulting in an increase in ROS and a decrease in survival rate (Fig. S2 in Supporting information). However, for Eu-PS NPs, SOD and CAT activity only increased when coexposed to 100 µg/L Cd
2+. Cd remained in the abdomen where Eu-PS NPs were located (
Fig. 1), which indicated that the "fixed" Cd failed to attack other systems and did not cause an increase in ROS levels. Both Cd
2+ and Zn
2+ exposure could enhance the expression of SOD and CAT through the Nrf2 pathway to resist oxidative stress in fish [
36]. The experimental results in
Figs. 4E and F showed similar results to changes in antioxidant enzymes. Oxidative stress could also induce immunotoxicity. IL-8 and TNF
α have been used as markers to activate the inflammatory response in zebrafish embryos [
37]. Similar to the results of oxidative stress, coexposure to ZnO and 100 µg/L Cd
2+ caused the highest inflammatory response (Fig. S3 in Supporting information).