Roel Evens

Comparison of laser ablation-inductively coupled plasma-mass spectrometry and micro-X-ray fluorescence spectrometry for elemental imaging in Daphnia magna

Visualization of elemental distributions in thin sections of biological tissue is gaining importance in many disciplines of biological and medical research. Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and scanning micro-X-ray fluorescence spectrometry (micro-XRF) are two widely used microanalytical techniques for elemental mapping. This article compares the capabilities of the two techniques for imaging the distribution of selected elements in the model organism Daphnia magna in terms of detection power and spatial resolution. Sections with a thickness of 10 and 20 microm of the fresh water crustacean Daphnia magna were subjected to LA-ICP-MS and micro-XRF analysis. The elemental distributions obtained for Ca, P, S and Zn allow element-to-tissue correlation. LA-ICP-MS and micro-XRF offer similar limits of detection for the elements Ca and P and thus, allow a cross-validation of the imaging results. LA-ICP-MS was particularly sensitive for determining Zn (LOD 20 microg g(-1), 15 microm spot size) in Daphnia magna, while the detection power of micro-XRF was insufficient in this context. However, LA-ICP-MS was inadequate for the measurement of the S distributions, which could be better visualized with micro-XRF (LOD 160 microg g(-1), 5 s live time). Both techniques are thus complementary in providing an exhaustive chemical profiling of tissue samples.

The effects of dietary nickel exposure on growth and reproduction of Daphnia magna.

Although there is growing evidence that dietborne metals can be toxic to various aquatic species, there is still insufficient knowledge to integrate this information in environmental risk assessment procedures. In this study, we investigated the effects of a 21-day exposure of Daphnia magna to a control diet (i.e. the green alga Pseudokirchneriella subcapitata containing <4.0microgNi/g dry wt) and five diets with elevated Ni concentrations (i.e. the same alga contaminated with Ni burdens between 33.7 and 837microgNi/g dry wt). A significant accumulation of dietborne Ni in D. magna, i.e. between 49.6 and 72.5microgNi/g dry wt, was observed when they were fed with diets containing between 85.6 and 837microgNi/g dry wt. This was paralleled by a significant reduction of reproduction (by 33.1%), measured as the total number of juvenile offspring per female and growth (by 9.1%), measured as the carapax length of 21-day-old females. Life-history analysis showed that the time to first brood of Ni exposed organisms was between 7.8 and 8.2 days, and occurred 0.7-1.1 days earlier than for the control organisms (time to first brood=8.9 days). The number of offspring in the first brood was significantly reduced (by 21-33% compared to the control) in all dietary treatments. Longer exposure (> or =8.9 days, i.e. from the second brood onwards) led to a reduction of brood size only when given diets containing 85.6 and 837microgNi/g dry wt. The results suggest that a variety of mechanisms may be involved in the effects of dietary Ni exposure, including altered resource allocation or targeted reproductive inhibition. While Ni exposure clearly altered the quality of the diet (measured as essential omega3 polyunsaturated fatty acid content and C:P ratio), we found no conclusive evidence that these diet quality shifts could have affected growth or total reproductive output. More research is required to fully understand the mechanisms of Ni toxicity associated with the dietary exposure route.

Waterborne versus dietary zinc accumulation and toxicity in Daphnia magna: A synchrotron radiation based X-ray fluorescence imaging approach

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.). Tissue-specific 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.

Liposomes as an alternative delivery system for investigating dietary metal toxicity to Daphnia magna

Dietary metal toxicity studies with invertebrates such as Daphnia magna are often performed using metal-contaminated algae as a food source. A drawback of this approach is that it is difficult to distinguish between the direct toxicity of the metal and indirect effects caused by a reduced essential nutrient content in the metal-contaminated diet, due to prior exposure of the algae to the metal. This hampers the study of the mechanisms of dietary metal toxicity in filter-feeding freshwater invertebrates. The aim of the present study was to develop a technique for producing metal-contaminated liposomes as an alternative delivery system of dietary metals. These liposomes are not vulnerable to metal-induced shifts in nutrient quality. Liposomes were prepared by the hydration of phosphatidylcholine in media containing either 0 (control) or 50mg Ni/L. The liposomes had average diameters of 19.31 (control) and 10.48 μm (Ni-laden), i.e., a size appropriate for ingestion by D. magna. The liposome particles were then mixed with uncontaminated green algae in a 1/10 ratio (on a dry wt. basis) to make up two diets that differed in Ni content (i.e., 2.0 μg Ni/g dry wt. in the control and 144.2 μg Ni/g dry wt. in the Ni-contaminated diet, respectively). This diet was then fed to D. magna during a 21-day chronic bioassay. The experiment showed that the Ni content and the size distribution of the liposomes were stable for at least 7 days. Also the use of phosphatidylcholine as a liposome component did not affect the reproduction of the daphnids. Exposure to increased level of dietary Ni resulted in 100% mortality after 14 days of exposure and in an increased whole-body Ni concentration in D. magna of 14.9 and 20.4 μg Ni/g dry wt. after 7 and 14 days of exposure, respectively. The Ni-exposed daphnids also exhibited a reduced size (i.e., 30% smaller than the control) after 7 days and a completely halted growth between day 7 and day 14. In terms of reproduction, the size of the first brood (number of juveniles) of the Ni-exposed daphnids was significantly reduced (by 85%) compared to the control. None of the Ni-exposed individuals were able to produce a second brood before dying. The algal ingestion rate - after correction for the indirect effect of a reduced size - was increased (by 68%) by dietary Ni after 6 days of exposure compared to the control, but was severely reduced (by 80% compared to the control) after 13 days. These data suggest that an inhibition of the ingestion process may have contributed to the observed effects of dietary Ni on growth and reproduction beyond 6 days of exposure, although the involvement of other mechanisms cannot be excluded. The mechanism(s) which led to the reduced growth during the first week of exposure remain unclear, although inhibition of the ingestion process can likely be excluded here as an explanation. Overall, this paper demonstrates, using this new method of delivering dietary Ni via liposome carriers and thus excluding potential diet quality shifts, that dietary Ni can indeed induce toxic effects in D. magna. This method may therefore be a promising tool to help further elucidate the mechanisms of dietary metal toxicity to filter-feeding invertebrates.

Dual detection X-ray fluorescence cryotomography and mapping on the model organism Daphnia magna

Micro X-ray fluorescence (-XRF) is a rapidly evolving analytical technique which allows visualising the trace level metal distributions within a specimen in an essentially non-destructive manner. At second generation synchrotron radiation sources, detection limits at the sub-ppm level can be obtained with micrometer resolution, while at third generation sources the spatial resolution can be better than 100 nm. Consequently, the analysis of metals within biological systems using micro and nano X-ray fluorescence imaging is a quickly developing field of research. Since X-ray fluorescence is a scanning technique, the elemental distribution within the sample should not change during analysis. Biological samples pose challenges in this context due to their high water content. A dehydration procedure is commonly used for sample preparation, enabling an analysis of the sample under ambient temperature conditions. Unfortunately, a potential change of elemental redistribution during the sample preparation is difficult to verify experimentally and therefore can not be excluded completely. Creating a cryogenic sample environment allowing an analysis of the sample under cryogenic condition is an attractive alternative, but not available on a routine basis. In this article, we make a comparison between the elemental distributions obtained by micro SR-XRF within a chemically fixed and a cryogenically frozen Daphnia magna, a model organism to study the environmental impact of metals. In what follows, we explore the potential of a dual detector set-up for investigating a full ecotoxicological experiment. Next to conventional 2D analysis, dual detector X-ray fluorescence cryotomography is illustrated and the potential of its coupling with laboratory absorption micro-CT for investigating the tissue specific elemental distributions within this model organism is highlighted.

The effects of Zn-contaminated diets on Daphnia magna reproduction may be related to Zn-induced changes of the dietary P content rather than to the dietary Zn content itself

The effect of dietary zinc (Zn) exposure to Daphnia magna fed living algae remains unsure as existing experimental data exhibit considerable inconsistency. In this study, we examined if Zn-induced changes in nutritional quality (i.e., the molar carbon to phosphorus ratio (C:P) and concentrations of essential omega-3-poly-unsaturated fatty acids (ω3-PUFA)) may contribute to the reproductive effects of dietary Zn exposure to D. magna. We prepared 8 different algal diets differing in Zn content, C:P ratio and ω3-PUFA, by varying the culture conditions (i.e., exposure duration) and culture medium (i.e., Zn concentration and mineral composition). These diets were representative for the diets typically used in published dietary metal toxicity bioassays. The algal diets were offered to D. magna during a standard chronic bioassay, using reproduction as endpoint. A generalized linear model (GLM) was used to determine which algal characteristics significantly explained the observed variability in D. magna reproduction. The most parsimonious GLM resulting in the best prediction of the first brood size had the molar C:P ratio as the sole predictor. The 21-day reproduction was also predicted best by the molar C:P ratio, whereas the contribution of other variables (notably Zn and ω3-PUFA content of the diet) to enhanced predictability was only marginal. In addition, our GLM, which only uses C:P as a predictor, could accurately predict reproduction in an independent (previously published) chronic bioassay with dietary Zn and D. magna. Furthermore, this GLM also accurately predicted the observed effects of algal C:P ratio shifts on D. magna reproduction as reported in ecological literature. Our analysis highlights that the reproductive effects of dietary Zn exposure in D. magna, as observed in previous studies, are probably not caused by direct toxicity of Zn in the diet, but may rather be related to Zn-induced shifts of the dietary C:P ratio. Our study thus seems to resolve inconsistencies among results from different previous studies and has important implications for the experimental design of future dietary metal toxicity research.

F-39 A TOP-DOWN APPROACH USING X-RAY IMAGING TECHNIQUES: INSTRUMENTAL DEVELOPMENTS AND APPLICATIONS IN LIFE SCIENCE

Synchrotron radiation X-ray fluorescence micro- and nanobeam techniques at second- and third generation SR sources offer the potential of non-destructive multi-element analysis down to trace concentration levels with unrivalled spatial resolution among X-ray based analytical techniques. At these sources, relative detection limits at the sub-ppm (fg/ng) level can be achieved. With respect to absolute detection limits (DL), sub-micron sized X-ray beams can offer DLs below 1 ag for the most efficiently excited transition elements, with a potential lateral resolution level better than 100 nm. These characteristics of micro/nanobeam SR-XRF allow spatially resolved multi-element determination of major, minor and trace constituents in microscopic sub areas and volumes within biological specimens in an essentially non-destructive/non-invasive manner. However, the complexity of performing such an experiment is often quite considerable, involving dedicated sample preparation, transportation towards and experimenting at the synchrotron facility, installing an appropriate experimental set-up and performing a thorough data analysis on large amounts of spectral data. The ecotoxicological research on Daphnia magna, a frequently used model organism for investigating the mechanisms of toxicity of metals, has often been difficult because many analytical techniques are not able to investigate trace metal distributions in a spatially resolved manner at a (sub)microscopic resolution. As illustrated by this presentation, SR-XRF microanalysis allows to fill this gap and moreover, due to the variety in sizes of X-ray beams available, this research can be performed from the organism level towards the tissue and cellular level, representing a top-down approach.