The effect of copper on behaviour, memory, and associative learning ability of zebrafish (Danio rerio).

Copper is an essential element in many biological processes, but may exert toxic effects at levels surplus to metabolic requirements. Herein we assess the effect of copper on zebrafish behaviour using two assays, namely the novel tank diving test and a T-maze test with food reward. Novel tank diving tests were conducted on days 0, 4, and 10 of a 10 day Cu exposure (at concentrations of 0.77 µM (25% of the 240 h LC50) and 1.52 µM (50% of the 240 h LC50) to assess the alterations of behavioural responses in repeating novel tank diving assays and the effect of Cu on these patterns. Results demonstrate habituation to novelty, which is an indicator of spatial memory. Copper exposure had no effect on the latency of entry into the upper zones of the tank, nor on the total time spent therein, but did cause a greater number of freezing bouts in comparison to the control group. Additionally, Cu exposure had no effect on the habituation responses of zebrafish. Using the T-maze assay, we tested the effect of prior exposure to Cu for 10 days on subsequent behavioural trainings. The T-maze protocol was based on associative learning, where a visual stimulus (colour) was linked with a natural stimulus (food). Results of the control group showed that zebrafish are able to perform associative learning tasks. Moreover, Cu was found to negatively affect the associative learning capabilities. Specifically, while zebrafish in the control group achieved a significant number of correct choices (leading to food reward) throughout the T-maze training, such a trend was not observed for Cu exposed fish. Thus at the exposure concentrations and exposure times considered herein, Cu has no determinative impact on instinctual behavioural responses of zebrafish in repeated novel tank diving assays but does limit the associative learning capabilities.

The interactive effect of copper(II) and conspecific alarm substances on behavioural responses of zebrafish (Danio rerio).

Environmental contaminants such as metal ions can have detrimental effects on aquatic organisms at the molecular, organismal and population levels. In the present work, we examined the interactive effect of Cu(II) and conspecific alarm substance on zebrafish behavioural responses utilizing the novel tank diving assay. To this end, 3 novel tank diving tests (on day 0, 3 and 10 of the experimental phase) were conducted on zebrafish in 4 experimental groups: (1) control: no Cu(II) and no alarm substance, (2) Cu(II) only: exposed to 0.78 µM Cu(II) (25 % of the 240 h LC50) in the home tank for 10 days, (3) alarm substance only: exposed to alarm substance for 6 min concomitant with behavioural testing, and (4) Cu(II) + alarm substance: exposed to 0.78 µM Cu(II) in the home tank for 10 days and treated with alarm substance for 6 min during the behavioural testing. Results showed robust habituation response of zebrafish. Exposure to Cu(II) did not affect the behavioural phenotypes of zebrafish in the novel tank diving test or habituation responses. Alarm substance treatment evoked strong anxiety-like behaviour. Finally, zebrafish in the Cu(II) + alarm substance group lost their sensitivity to alarm substance in repeated novel tank assays throughout the concomitant Cu(II) exposure; this observation is tentatively ascribed to Cu(II)-induced olfactory impairment.

The synergistic toxicity of Cd(II) and Cu(II) to zebrafish (Danio rerio): Effect of water hardness.

We have evaluated the interactive toxicity of Cu(II) and Cd(II) in water with different hardness levels using adult zebrafish (Danio rerio). Zebrafish were exposed to Cd(II) (0.2-22 µM) or Cu(II) (0.1-8 µM) in single or binary exposures in very soft, moderately hard or very hard water. The whole body burdens of Cd(II) and Cu(II) reflect the net effect of biouptake and elimination, mortality was the indicator of toxicity, and whole body major ion content was measured to assess ion regulatory functions. Cu(II) was found to be more toxic than Cd(II) for zebrafish, and Cu(II) and Cd(II) exhibited a significant synergistic effect. The toxicity of metal ions increased upon decreasing the ionic strength of the exposure medium, probably due to elevated competition between metal ions with other cations in hard water and increased activity of Ca2+ pathways in soft water treatments. Whole body metal accumulation and the accumulation rate of both Cu and Cd increased as the metal ion concentration in the exposure medium increased. Nevertheless, neither parameter explained the observed synergistic effect on mortality. Finally, we observed a significant loss of whole body Na+ in fish which died during the metal exposure compared to surviving fish, irrespective of exposure conditions. Such an effect was not observed for other major cations (K+, Ca2+ and Mg2+). This observation suggests that, under the applied exposure conditions, survival was correlated to the capacity of the organism to maintain Na+ homeostasis.

The effect of thermal pre-incubation and exposure on sensitivity of zebrafish (Danio rerio) to copper and cadmium single and binary exposures.

Zebrafish (Danio rerio) is a prominent model organism in a wide range of biological studies including toxicology. However, toxicological studies are often performed at species specific optimum temperature, and knowledge on the effect of different temperature regimes on the toxicity of metal ions is rather limited. To address this knowledge gap, present study investigates the effect of various thermal scenarios (simultaneous and sequential; acute and chronic) on the toxicity of Cu and Cd in zebrafish. For this purpose we assessed mortality and whole body metal burdens as indicators of toxicity and bioavailability, respectively, and whole body electrolyte concentrations and body condition as the indicators of physiological condition. Thermal pre-incubations (for 12 or 96 h or 28 days) and subsequent metal ion exposures (for 10 days) were conducted at 17, 22, 25, 28, 32 and 34 °C. The metal exposures were performed at Cu concentrations of 1.2 µM and Cd concentrations of 0.2 µM, both singly and in binary mixtures. Irrespective of thermal treatments, Cu exposures resulted in greater mortality than Cd exposures at the given concentrations. Moreover, the Cu and Cd mixture indicated a synergistic effect. While acute pre-incubation for 12 or 96 h at elevated temperatures increased mortality in the subsequent metal exposure at the optimum temperature (28 °C), pre-incubation at cold temperatures in this scenario appeared to increase tolerance towards the subsequent metal exposure. Chronic thermal pre-incubation of zebrafish to a range of temperatures for 28 days moderated the effect of temperature fluctuations on subsequent metal toxicity at the optimum temperature. Chronic thermal pre-incubation at a range of temperatures followed by metal exposure at the same temperature showed that environmental temperature variations (higher or lower than optimal temperature) coupled with metal exposure, led to increased mortality, furthermore, the highest whole body metal burdens were measured in this scenario. Nevertheless, neither the whole body burden of metals, nor the metal accumulation rate, were predictors of mortality, i.e. these two values were not higher in dead fish in comparison to those that survived the exposures. Finally, we observed a significant decrease in the whole body Na+ level of dead fish in comparison to fish which survived the exposure conditions, suggesting that survival depends on maintaining Na+ homeostasis under the applied multi-stress conditions. Overall, our results show that thermal pre-history and ambient temperature play an important role in determining the tolerance of zebrafish towards metal ion stress.