Cadmium uptake rates in freshly hatched zebrafish (Danio rerio) larvae in the presence of two dissolved organic matter (DOM) isolates

Cadmium (Cd2+), reputed for its toxicity, has become widely distributed in the aquatic environment as an industrial waste. Environmental parameters alter the uptake of Cd2+ by aquatic organisms, complexation by organic ligands (dissolved organic matter, DOM) being one major modulator (Meinelt et al., 2001; Burnison et al., 2006). DOM has a high ion-exchange and complexation capacity (Perdue, 1998). In this study, the effect of DOM with contrasting physio-chemical properties on Cd2+ uptake rates in newly hatched zebrafish (Danio rerio) larvae was studied. Zebrafish are often used in laboratory tests because of their convenience: short generation interval, short spawning interval, and embryonic development completed in 96 h at 26°C. The aim of this study was to find out if DOM qualities have an impact on the uptake rate of Cd2+, as from other tests with DOM it is well understood that not only the quantity, but particularly the DOM quality, controls the effects (Paul et al., 2003; Meinelt et al., 2007). Production of eggs and rearing of embryos to the larval state is described in Burnison et al. (2006). Fifty freshly hatched larvae were placed in a Petri dish (Falcon Tight-Fit Lid, 50 mm, Fisher Scientific) containing 151.2 nm109Cd (specific activity, 1.92 MBq μg−1, Amersham Biosciences) in 6 ml of either reconstituted water (Burnison et al., 2006) containing no added carbon (control), or with 16.9 mg L−1 C of either LM-DOM or SP-DOM. After 4- or 24-h exposures, the larvae were rinsed three times with reconstituted water to remove free 109Cd. Three milliliters of reconstituted water were added to the rinsed larvae and 100 μl of this solution was monitored for the removal of radioactivity. Ten milliliters of the scintillation cocktail (Ultima Gold, Packard Instrument Co.) were added to the larvae to determine their 109Cd radioactivity in a liquid scintillation counter (Tri-Carb 4430, Packard Instrument Co.). The experiments were run in triplicates. Dissolved organic matter (DOM) was isolated from two natural Canadian waters of different trophic status and with different natural dissolved organic carbon (DOC) concentrations: Luther Marsh (LM) and Sanctuary Pond (SP). Sampling procedure and characterization of the lakes are provided by Richards et al. (2001). DOM fractionation was done according to Sachse et al. (2005) with the three fractions achieved: high-molecular substances, humic substance (HS), and low-molecular substances. Statistical comparisons between the triplicates were made by means of anova (spss version 11.5). The Tukey-HSD post hoc test was applied in cases of significant differences between the groups. Variance homogeneity was tested with the help of the Levene test. Significance level was set at P < 0.05. The main chemical features of the exposed DOM are given in Table 1. The two DOM sources differ mainly in their HS content: 90% of the dissolved organic carbon in the LM isolate is comprised of HS, compared to less than 70% in SP. Consequently, the SUVA of LM DOM is twice that of SP DOM. After 4-h exposure, a significantly reduced Cd uptake rate was observed in the presence of both DOM isolates, LM-DOM 36.7% and SP-DOM 46.1% of the control. Although the reduction by the DOM isolates did not differ statistically, there was a tendency of LM to be more effective. After 24 h, the modulation of the Cd uptake rates was diverse. In the LM exposed larvae the rate was lower, and in the SP-DOM exposed larvae higher, than in the controls (LM-DOM 56.5% and SP-DOM 127.6%). However, these differences were not significant (P = 0.808). At both exposure times, the Levene test supported the assumption of equal variances in all three groups (Fig 1). Cadmium uptake rates after 4 h and 24 h exposure in freshly hatched zebrafish larvae in Luther Marsh and Sanctuary Pond DOM. (* = statistically significant different from control, P < 0.05) Over a period of 4 h, both DOM sources effectively reduced Cd2+uptake rates in the newly hatched larvae. For this reduction, several mechanisms may apply. According to Playle (1998), parts of the exposed free Cd2+ ions are bound to the functional groups of the DOM, particularly of the HS, thus reducing the Cd2+uptake of the larvae. It appears that LM-DOM with the higher HS content tends to be more effective. The chemical mechanism will be elucidated below. This holds true with LM-DOM even in the 24-h exposure: the rate tends to be further reduced. Yet, in the SP-DOM exposure, the rate lay above the control, the mechanism behind this being somewhat obscure. There is some evidence that the bioavailability of metals at low concentrations in natural waters can be enhanced, if HS are co-exposed (Stackhouse and Benson, 1989). Most likely, different DOM physio-chemical properties affect the cadmium uptake rate differently. Changes in the net-uptake may also be a result of modulated simultaneous depuration, for instance by the multixenobiotic resistance (MXR) mechanism (Bard, 2000). However, it is questionable whether or not this mechanism applies to Cd, because Matz et al. (2007) found a recovery from Cd body burden in zebrafish larvae only following high short-term acutely toxic exposures which lay three orders of magnitude above our experimental approach. However, those authors did not check co-exposure to DOM and its potentially modulating effects. At environmentally realistic DOM concentrations, the MXR activity may be reduced by bioaccumulated HS (Timofeyev et al., 2007). Again, we assume that different DOM qualities have a different impact on this depuration. We attribute the slight differences in the Cd uptake rates between the two co-exposed DOM isolates to the differences in their chemical features (Table 1). For instance, LM-DOM has a higher content of HS and lower contents in nitrogen and sulfur than does SP-DOM. At natural concentrations of HS, the metal binding partners are particularly N atoms (other than amino acids) and reduced S atoms, rather than oxygen-containing functional groups (Frenkel et al., 2000). In sum, the apparent uptake rates are trade-offs of potentially competing mechanisms; their controlling factors represent a fascinating issue for future studies.

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