Influence of water hardness on zinc toxicity in Daphnia magna

Abstract

Zinc is an essential trace metal required for the maintenance of multiple physiological functions. Due to this, organisms can experience both zinc deficiency and toxicity. Hardness is recognized as one of the main modifying physiochemical factors regulating zinc bioavailability. Therefore, the present study analyzed the effect of hardness on zinc toxicity using Daphnia magna. Endpoint parameters were acute-toxicity, development, reproduction, and expression data for genes involved in metal regulation and oxidative stress. In addition, the temporal expression profiles of genes during the initiation of reproduction and molting were investigated. Water hardness influenced the survival in response to exposures to zinc. A zinc concentration of 50 μg/l in soft water (50 mg CaCO₃ /L) caused 73% mortality after 96 h exposure, whereas the same zinc concentration in the hardest water did not cause any significant mortality. Moreover, increasing water hardness from 100 to 200 mg CaCO₃ /L resulted in a reduced number of offspring. Fecundity was higher at first brood for groups exposed to higher Zn concentrations. The survival data were used to assess the precision of the bioavailability models (Bio-met) and the geochemical model (Visual MINTEQ). As the Bio-met risk predictions overestimated the Zn toxicity, a competition-based model to describe the effects of hardness on zinc toxicity is proposed. This approach can be used to minimize differences in setting environmental quality standards. Moreover, gene expression data showed that using the toxicogenomic approach was more sensitive than the physiological endpoints. Therefore, data presented in the study can be used to improve risk assessment for zinc toxicity.

Keywords: BLM; bioavailability; gene expression; risk assessment; toxicogenomics.

FIGURE 1

Twenty Daphnia magna neonates (<24 h old) per well were exposed to varying Zn concentrations for 96 h, and the survival rate was recorded for soft (A), medium (B), and hard water (C). Dose–response curves were generated for each water hardness by plotting mortality rates at the end of 96 h exposure (D–F). Mean ± SEM. n = 4

FIGURE 2

Free ion concentrations were calculated using Visual MINTEQ. The sum of Ca²⁺ and Mg²⁺ concentrations was divided by the Zn concentrations and plotted against the mortality rates of daphnids at the end of 96 h exposure. Mean ± SEM. n = 4

FIGURE 3

Daphnia magna neonates (<24 h old) were kept individually in 40 ml exposure water for 21 days, and progeny numbers were recorded during the exposure. Total number of offspring at the end of the exposure period is shown (A–C). Number of cumulative progenies produced during first four brood release is shown (D–F). Mean ± SEM. n = 6

FIGURE 4

Fourteen 72 h old daphnids were exposed to varying zinc concentrations for 24 h in different water hardness for 24 h. qRT‐PCR was performed to analyze the gene expression profiles for (A) mt‐a, (B) cat, and (C) gst. The 25 μg/L Zn concentration at all three water hardness was selected as a control for one‐way ANOVA followed by Dunnett posttest to determine statistical significance (^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001). Mean ± SEM. n = 8

FIGURE 5

PCA bipolt (A) and hierarcichal clustering of exposure groups (B) were generated using principal component analysis. In addition, temporal expreesion level of genes related to reproduction was analyzed using qRT‐PCR. Twenty newly hatched daphnids were exposed to soft, medium, and hard water for 112 h. Expression levels are shown relative to 24 h in soft water. (C) jhe, (D) vtg‐1, and (E) vtg‐2