Article pubs.acs.org/est
Waterborne versus Dietary Zinc Accumulation and Toxicity in Daphnia magna: a Synchrotron Radiation Based X-ray Fluorescence Imaging Approach R. Evens,†,⊥,* K. A. C. De Schamphelaere,†,⊥ B. De Samber,‡ G. Silversmit,‡ T. Schoonjans,‡ B. Vekemans,‡ L. Balcaen,‡ F. Vanhaecke,‡ I. Szaloki,§ K. Rickers,∥ G. Falkenberg,∥ L. Vincze,‡ and C. R. Janssen† †
Laboratory of Environmental Toxicology, Ghent University, Ghent, Belgium Laboratory of Analytical Chemistry, Ghent University, Ghent, Belgium § Budapest University of Technology and Economics, Institute of Nuclear Techniques, Budapest, Hungary ∥ DESY, Hasylab, Hamburg, Germany ‡
S Supporting Information *
ABSTRACT: Recent studies have suggested that exposure of the freshwater invertebrate Daphnia magna to dietary Zn may selectively affect reproduction without an associated increase of whole body bioaccumulation of Zn. The aim of the current research was therefore to investigate the hypothesis that dietary Zn toxicity is the result of selective accumulation in tissues that are directly involved in reproduction. Since under field conditions simultaneous exposure to both waterborne and dietary Zn is likely to occur, it was also tested if accumulation and toxicity under combined waterborne and dietary Zn exposure is the result of interactive effects. To this purpose, D. magna was exposed during a 16-day reproduction assay to Zn following a 5 × 2 factorial design, comprising five waterborne concentrations (12, 65, 137, 207, and 281 μg Zn/L) and two dietary Zn levels (49.6 and 495.9 μg Zn/g dry wt.). Tissuespecific Zn distribution was quantified by synchrotron radiation based confocal X-ray fluorescence (XRF). It was observed that the occurrence of reproductive inhibition due to increasing waterborne Zn exposure (from 65 μg/L to 281 μg/L) was accompanied by a relative increase of the Zn burdens which was similar in all tissues considered (i.e., the carapax, eggs, thoracic appendages with gills and the cluster comprising gut epithelium, storage cells and ovaries). In contrast, the impairment of reproduction during dietary Zn exposure was accompanied by a clearly discernible Zn accumulation in the eggs only (at 65 μg/L of waterborne Zn). During simultaneous exposure, bioaccumulation and toxicity were the result of interaction, which implies that the tissue-specific bioaccumulation and toxicity following dietary Zn exposure are dependent on the Zn concentration in the water. Our findings emphasize that (i) effects of dietary Zn exposure should preferably not be investigated in isolation from waterborne Zn exposure, and that (ii) XRF enabled us to provide possible links between tissue-specific bioaccumulation and reproductive effects of Zn. reproduction was reduced.7 Thus, it has been suggested that dietary Zn specifically targets the reproductive physiology by hampering the conversion of energy reserves into offspring.7 Moreover, since no significant increase of the whole-body burden of Zn was detected at dietary exposure levels inhibiting reproduction, it is hypothesized that these effects are due to specific accumulation in tissues important for reproduction.7 These tissues include the storage cells where vitellogenin is produced, and the ovaries where vitellogenin is further transformed into lipovitellin.8,9 More recently, a gene
1. INTRODUCTION Considerable efforts have been made to investigate the mechanisms of zinc (Zn) toxicity to various freshwater species.1−3 Recent research with the model crustacean Daphnia magna suggested that different mechanisms of chronic toxicity are at play when daphnids are exposed via either water or diet. Waterborne Zn exposure of D. magna resulted in an increased whole-body Zn burden and a concomitant decrease of Ca levels,2 which is due to competition between Zn2+ and Ca2+ ions for ion transporters at ionoregulatory surfaces.2,4,5 It has been suggested that this reduced Ca body burden is a partial, but not the full, explanation for the observed lower feeding activity, reduced growth, and reproductive output of daphnids exposed via water.2,6 In contrast, the daphnids’ feeding activity and growth were not affected when exposed via diet; only © 2011 American Chemical Society
Received: Revised: Accepted: Published: 1178
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were determined following a standard protocol.13 These algae, representing the two dietary exposure levels, were used as food for D. magna in a 5 × 2 two-factorial exposure experiment. Tests were initiated with juveniles ( 0.05, Duncan). pH was 7.5 on average for all treatments and did not vary more than 0.2 units during the experiment. Brood sizes and total reproduction (i.e., after 16 days) as a function of combined waterborne and dietary Zn exposure are shown in Table 2 and Figure 2, respectively. Twoway ANOVA on all reproduction data from all treatments indicated significant main effects of waterborne and dietary exposure, as well as a significant interactive effect (p < 0.05). The same analysis demonstrated a significant interaction (p < 0.05) between both exposure routes in the range of optimal (i.e., 65 μg Zn/L) to toxic waterborne Zn concentrations. When the daphnids were fed with the control diet, reproduction was high for individuals exposed to 65 and 137 μg/L of waterborne Zn, and was significantly lower for organisms originating from the exposures to 12 (control, reduction by 39.0%), 208 (reduction by 26.2%), and 281 μg Zn/L (reduction by 30.4%) (p < 0.05; Tukey). The effects of dietary Zn exposure depended on the waterborne Zn concentration in the medium. In the control medium (12 μg Zn/L), no effects of dietary Zn exposure were observed (p > 0.05; t test). At all higher concentrations of Zn in the water, dietary Zn exposure resulted in a significant (p < 0.05; t test) reduction in reproduction by 28, 43, 33, and 25% for organisms exposed to 65, 137, 208, and 281 μg Zn/L, respectively. 3.2. Tissue Characterization and Tissue-Specific Zn Accumulation. The dorsoventral Zn distributions within D. magna after a 15-day exposure to waterborne and/or dietary Zn obtained by means of SR-based confocal micro-XRF are shown in Figure 3. Note that the results presented in this figure should be interpreted with caution as they are presented as volumetric concentrations (μg/cm3) (see scale bar at the right). Biomass based concentrations (i.e., Zn intensity normalized to Compton scattering) of the different clusters obtained from the elemental distributions in Figure 3 are presented in Figure 4 and these are used as the basis of our interpretation and discussion below. Also note that a clear structural distinction of the gut epithelium and ovaries was not always feasible, so that concentration differences between both tissues cannot be established. When the daphnids were fed with the control algae, only relatively small variations of tissue-specific Zn concentrations were observed between waterborne Zn exposures to 12 μg Zn/ L (i.e., control) and 65 μg Zn/L (i.e., maximal reproduction). Zn accumulation in all tissues of organisms exposed to 281 μg/ L was more obvious, that is, in the latter treatment relative
Figure 1. Schematic lateral overview of a female Daphnia magna. The virtual dorsoventral section that has been scanned by confocal XRF analysis has been outlined by the dotted line. (a) gills; (b) gut epithelium, storage cells and ovarium; (c) broodpouch with eggs/ embryos/juveniles.
measuring time at the synchrotron microprobe was limited, we limited our analyses to the organisms originating from nominal waterborne Zn exposures of 5 (control), 80 and 340 μg/L, and fed with the control and Zn-contaminated diet (thus a single individual from a total of six treatments). 2.3. Data Treatment and Statistics. Statistical comparisons were performed with Statistica 6.0 software (Statsoft, Tulsa, OK). All observations are reported as mean ± standard deviation. D. magna test solution chemistry was compared with Duncan’s multiple range test. All statistical comparisons regarding D. magna reproduction were carried out with analysis of variance (ANOVA), followed by a post hoc Tukey test for waterborne exposures (within the same dietary treatment). An independent samples t test was performed to investigate the effects of dietary Zn exposure (within the same waterborne treatment). Two-way ANOVA was performed to test for interaction between waterborne and dietary Zn toxicity. For the spectral deconvolution of the individual XRF point spectra, followed by building the elemental maps, the software packages AXIL and MICROXRF2 were used (for details, see the Supporting Information).16 For a more in-depth investigation on the elemental distributions within different tissues of D. magna, the K-means clustering algorithm within the MICROXRF2 software package was applied on the obtained elemental distributions.17 Before the actual clustering, a square root data pretreatment was performed to compensate for the Poisson counting statistics and a normalization was necessary to give equal weight to each element. Using the element maps of 1180
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All data are expressed as mean ± stdev. For each end point, treatments that are not significantly differing as a function of waterborne Zn exposure are grouped by identical characters. For each waterborne Zn concentration, data that are significantly different from their corresponding value of daphids exposed to dietary Zn, are indicated by *. All statistics considering the reproductive effects as a function of waterborne Zn exposure are performed with One way ANOVA (post hoc Tukey), whereas comparison of data in relation to dietary Zn exposure is performed with an Independent Samples t-test. Solution chemistry was statistically compared by One way ANOVA − Duncan’s multiple range test. Level of significance was accepted as p < 0.05.
increases of tissue Zn concentrations by 200, 183, 216, and 249% were observed in the gills, gut epithelium/ovaries, eggs and the carapax, respectively (when compared with the 65 μg/L treatment). With regard to dietary Zn exposure, no distinct accumulation was observed when the waterborne Zn concentration was 12 μg Zn/L. However, in the treatment fed with the Zn contaminated diet at 65 μg Zn/L, we observed a relative increase of the Zn content by 15.5, 30.1, 158, and 16.5% in the gills, gut epithelium/ovaries, eggs and carapax, respectively (compared to the treatment at 65 μg/L fed with the control diet). Finally, the daphnids suffering from waterborne Zn toxicity (i.e., in 281 μg Zn/L) did not exhibit any accumulation of Zn via the diet (Figure 4, Table 2).
4. DISCUSSION 4.1. Waterborne Zinc Toxicity. The observation of an optimal waterborne Zn concentration (here: 65 μg/L), that is, resulting in higher reproductive output compared with lower (12 μg/L) and higher (281) Zn concentrations, corroborates the results of previous experiments.2,18 An optimal concentration range of essential elements like Zn in D. magna could be distinguished. It has been suggested that lower reproduction at low waterborne Zn concentrations results from Zn deficiency.2,18 However, our Zn distribution data indicate similar Zn concentrations in all tissues at 12 and 65 μg/L, which does not suggest a deficiency at 12 μg/L. It is possible, however, that tissues other than those investigated here may be involved. Another possibility is that daphnids living at these low zinc concentrations have to invest energy, at the expense of their reproductive output, in order to achieve tissue Zn concentrations comparable to those observed in the optimal concentration range.18 Regarding the occurrence of toxicity at concentrations higher than this optimum zinc range (here at 207 and 281 μg/L), it was suggested that a disturbed Ca balance is probably the first cause of waterborne Zn toxicity to D. magna.2 Although a clear relation between reduced Ca supplies and reduced algal ingestion rates (i.e., suppressed food uptake) has been described,2 it was recently shown that hypocalcaemia is most likely not the only mechanism involved in waterborne Zn toxicity.6 Zn exposure has also been found to be responsible for the down-regulation of several genes involved in the production of digestion enzymes in D. magna.19 Since the gut epithelium is responsible for the holocrine secretion of digestive enzymes,20 it is possible that the observed Zn accumulation in the gut epithelium is responsible for a lower digestive enzyme activity, resulting in a potential reduction of nutrient absorption, ultimately leading to reduced reproduction. Further research is required to establish the relative importance of hypocalcaemia and inhibition of digestive enzyme activity in reproductive toxicity following waterborne exposure. Zn contents in the eggs and carapax increased with the same magnitude as in the gills and gut/ovaria (by 216 and 249%, respectively) when comparing optimal (i.e., 65 μg Zn/L) and toxic (i.e., 281 μg/L) exposures. However, until now no supporting physiological data are available to relate accumulation in these tissues to reproductive toxicity. Nonetheless, these similar accumulations among all tissues investigated suggest that waterborne Zn exposure is not accompanied by an accumulation preference for specific tissues. The observation of Zn accumulation in the carapax is in line with several studies that have shown that molting is an important elimination route for metals in D. magna.18,21−23
a
170 137.1 ± 12.4 c 7.54 5.2 ± 1.0 a, d * 6.8 ± 2.1 b, c * 7.9 ± 2.4 b * 20.8 ± 2.9 b * 80 65.2 ± 7.5 b 7.54 6.2 ± 1.4 a, c * 9.6 ± 2.2 a * 12.3 ± 1.5 a * 27.7 ± 1.9 a * control (5) 11.7 ± 1.6 a 7.54 6.7 ± 1.9 a * 10 ± 2.7 a 12.4 ± 4.2 a 28.3 ± 8.0 a 340 281.6 ± 13.0 e 7.51 6.2 ± 2.8 a 10.3 ± 3.0 a, c * 12.5 ± 3.4 a, c * 26.8 ± 6.2 a * 250 207.4 ± 15.2 d 7.52 5.4 ± 2.2 a * 9.8 ± 2.5 a, c * 12.4 ± 3.1 a, c * 28.4 ± 5.7 a * 170 137.2 ± 12.0 c 7.52 12.4 ± 7.7 b* 13.8 ± 2.2 b * 17.5 ± 2.4 b, c * 36.3 ± 3.3 b * nominal conc. (μg Zn/L) experimental conc. (16 day average) average pH first brood size second brood size third brood size (after day 14) total 16 day reproduction
control (5) 11.6 ± 4.3 a 7.53 4.4 ± 1.5 a* 9.3 ± 1.3 a 10.5 ± 1.5 a 23.5 ± 1.4 a
80 64.6 ± 8.0 b 7.54 7.5 ± 1.1 a,b* 12.8 ± 1.8 b, c * 18.4 ± 2.7 b * 38.5 ± 2.7 b *
control diet waterborne Zn
Table 2. Experimental Details of Daphnia magna Chronically Exposed to a Combined Waterborne and Dietary Zn Treatmenta
Zn contaminated diet
250 208.4 ± 14.1 d 7.53 3.7 ± 0.8 b, d * 7.3 ± 1.7 a, c * 7.8 ± 2.6 a, b * 19.0 ± 3.7 b *
340 281.0 ± 14.4 e 7.52 4.9 ± 1.1 b, c, d 7.7 ± 1.2 a, c * 7.4 ± 1.8 b * 20.0 ± 1.9 b *
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Figure 2. Reproduction of D. magna after 16 days of exposure to waterborne Zn and fed with control algae (blue dots) and Zn contaminated algae (pink dots). Reproduction data which are not significantly different as a function of waterborne Zn exposure are grouped by identical characters, for both the organisms fed with the control and Zn-contaminated diet. P-values indicate the significance level for comparison of reproduction for daphnids fed with the control and Zn contaminated diet at the same waterborne Zn concentration.
reproduction (by 28%). Due to the absence of significant accumulation of dietary metals at the organism level (previous study), it was suggested that a targeted accumulation of elements occurs in tissues where vitellogenin is produced (i.e., storage cells scattered along the digestive tract), or where vitellogenin is further processed to yolk protein (i.e., oocytes in the ovaries).7,24 Here, we did not observe significant accumulation of Zn in the cluster containing ovaries and storage tissue (and gut epithelium). Unfortunately, the spatial resolution did not allow a distinction between these different tissues within that cluster, such that differential accumulation among these tissues might have been masked. However, when comparing the tissue-specific Zn contents in organisms exposed to 65 μg Zn/L and fed with the control diet with the tissuespecific Zn contents in the organisms exposed to 65 μg Zn/L and fed with the Zn-contaminated diet, the very pronounced increase of Zn accumulation in the eggs (i.e., by 158%) does suggest that significant accumulation of dietary Zn into the oocytes occurred. It is most likely that this accumulation from dietary Zn occurred when these eggs were still present in the ovaries. Indeed, Zn uptake in eggs could in theory also have occurred via waterborne exposure in the brood pouch, but we can exclude this possibility because this waterborne exposure level (65 μg/L) is the same as in the daphnids fed with the control diet. Our hypothesis that dietary Zn selectively accumulates in the reproductive tissues thus seems to hold at a Zn concentration in the water that is optimal for reproduction of the daphnids. These results also reinforce the importance of distinguishing between the accumulation of metals in various tissues and that the application of XRF-mediated metal localization in small specimens like daphnids is in this context very useful. For example, since the total egg mass of D. magna is on average not higher than 10% of the total body mass,25 the 158% increase of the Zn content in their eggs would likely not have resulted in a detectable increase of the whole body burden (e.g., quantified by classical acid digestion). The distinct accumulation into the eggs following dietary Zn exposure is supported by other studies,26 wherein neonatal release was an important route for organismal Zn depuration.
Figure 3. Accumulation of Zn at the tissue specific level in D. magna after 15 days of a combined exposure to waterborne and dietary Zn. Data are expressed as μg/cm3 (note scale bar), but not normalized to location specific biomass density. (a) exoskeleton, (b) thoracic appendages with gills, (c) ovarium, (d) gut epithelium with storage cells, (e) egg/embryo.
4.2. Dietary Zinc Toxicity. Total reproduction was not significantly affected by dietary Zn in the control waters (12 μg/L), which is in accordance with the accumulation profiles, where no significant enrichment of Zn in any of the tissues was observed. Our data are, however, in contrast with another study,7 where reproductive toxicity occurred in an identical exposure medium as used here. As yet, we have no straightforward explanation for this difference. One could attribute this irregularity to the varying conditions, in terms of Zn concentration and exposure time, in which the algae have been cultured in both studies. More research should be performed to examine the effects of varying algal culture conditions on the outcome of dietary metal toxicity studies using living diets as metal vector. In contrast, at the optimal waterborne concentration (65 μg/ L), dietary Zn exposure resulted in a significant reduction of 1182
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Figure 4. Zn distribution in cluster-based defined body compartments in Daphnia magna, in relation to waterborne exposure to 12 (control), 65 (optimal) and 281 (toxic) μg Zn/L, and fed with control (left) and Zn-contaminated (right) diets. Tissue specific Zn content is expressed as relative Zn fluorescence intensity (Zn counts/Compton counts). Error bars represent the standard deviation of the relative Zn intensity (i.e., the Zn fluorescence signal of the cluster specific sumspectrum divided by the respective Compton scatter signal). (*) denotes the tissues where Zn accumulates when D. magna was subjected to waterborne Zn, compared to the exposure to 65 μg Zn/L with control diet. (**) denotes the tissues where Zn accumulates when D. magna was subjected to dietary Zn, compared to the exposure to 65 μg Zn/L with control diet.
interaction between both exposure routes occurs implies that, if dietary effects are to be incorporated in risk assessment, they need to be treated together with waterborne concentrations. More research is however required to understand the implications of our results for the mechanistic framework of biotic ligand models (BLM) and their application in risk assessments. This study showed that XRF can offer an opportunity to link tissue-specific accumulation to effects and can in this regard help formulating models, similar to the BLM for waterborne only exposure, that incorporate dietary exposure.
Furthermore, as it has been shown that dietary Zn exposure is not accompanied by differential expression of the Vtg gene itself,10 this accumulation may suggest that reduced reproduction is possibly due to a disturbance of the conversion of vitellogenin into yolk proteins. Further research is required to test this hypothesis. 4.3. Waterborne Versus Dietary Zn Exposure: Interactive Effects. When considering the organisms having optimal reproduction (i.e., exposed to 65 μg/L waterborne Zn and fed the control diet) as a reference, the degree of toxic effects in these organisms under combined exposure to additional waterborne Zn and a Zn-contaminated diet is also the result of interaction (p < 0.05, 2-way ANOVA). The adverse effect of an additional exposure to dietary Zn was the highest at waterborne concentrations of 137 μg Zn/L (43% reduction of reproduction) and decreased to 33% and 25% at waterborne exposures to 207 and 282 μg Zn/L, respectively. Thus, the contribution of dietary Zn to reproductive inhibition gradually decreased with increasing waterborne Zn concentrations. The most likely explanation for this observation is the occurrence of lower filtration and ingestion rates in organisms suffering from waterborne Zn toxicity, so that Zn exposure via the diet is proportionally decreasing (because less Zn is ingested). Although this end point was not verified in this experiment, a decrease in filtration rate as a function of similar waterborne Zn concentrations was observed in another study.2 The absence of dietary Zn accumulation in the organisms from the 281 μg Zn/L exposure supports this assumption. It is important to note here, however, that the interaction between both exposure routes, which was only observed for one dietary Zn concentration, cannot be generalized so that this observation may be different when different dietary Zn burdens would have been tested. Further research should elucidate this. 4.4. Implications for Risk Assessment. On the basis of the data described in this paper it can be inferred that tissue specific accumulation patterns in D. magna give more insight in the toxicity of waterborne and dietary Zn. The observation that
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ASSOCIATED CONTENT
* Supporting Information S
Figure S1. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions ⊥
Joint first authorship: these authors contributed equally to this manuscript
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ACKNOWLEDGMENTS R.E. is supported by a Ph.D. grant of the Institute for the promotion of Innovation through Science and Technology in Flanders (IWT Vlaanderen). We thank Gisèle Bockstael and Leen Van Imp for technical assistance.
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