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.

The use of liposomes to differentiate between the effects of nickel accumulation and altered food quality in Daphnia magna exposed to dietary nickel.

A potential drawback of traditional dietary metal toxicity studies is that it is difficult to distinguish between the direct toxicity of the metal and indirect effects caused by altered concentrations of essential nutrients in the metal-contaminated diet. In previous studies it has become clear that this can hamper the study of the real impact of dietary metal exposure and also complicates the analysis of the mechanisms of dietary metal toxicity in filter-feeding freshwater invertebrates like Daphnia magna. This problem has been partly circumvented by the production of liposomes, since these vectors are invulnerable to metal-induced food quality shifts and as such can be applied to study the mechanisms of dietary metal toxicity without the confounding effect of nutritional quality shifts. The aim of current study was to evaluate if there is relevance for dietary Ni toxicity under natural exposures, i.e., when D. magna is exposed to dietary Ni via living algae, and secondly, to quantify how nutritional quality shifts contribute to the toxic effects that are observed when algae are used as contaminated food vectors. For this aim, liposomes were prepared by the hydration of phosphatidylcholine in media containing 0 (control), 10, 50, 100 and 500 mg Ni/L. The liposome particles were then mixed with uncontaminated green algae in a 1/10 ratio (on a dry wt basis) to make up diets with constant nutrient quality and varying Ni contents (i.e., 1.2 µg Ni/g dry wt in the control and 18.7, 140.3, 165.0 and 501.6 µg Ni/g dry wt in the Ni-contaminated diet, respectively). A second food type was prepared on the basis of a 1/10 mixture (on a dry weight basis) of control liposomes and Ni-contaminated algae, representing a diet that differed in Ni content (i.e., 1.2, 26.8, 84.7, 262.3 and 742.7 µg Ni/g dry wt) and concentrations of essential nutrients (in terms of P and omega 3 poly-unsaturated fatty acids like eicosapentaenoic acid and α-linolenic acid). Both diets were then simultaneously fed to D. magna during a 21-day chronic bioassay, using reproduction, growth, survival, ingestion rate and Ni bioaccumulation as endpoints. Ni delivered by liposomes caused a significant inhibition of reproduction and growth when the metal accumulated to minimum levels of 11.9 and 20.0 µg Ni/g dry wt after 7 and 14 days, respectively. Using algae as Ni vector, similar effects of dietary Ni exposure occurred when algae had been pre-exposed to concentrations of at least 133 µg/L of bioavailable Ni (i.e., Ni2+), which is similar to the reproductive EC50 of waterborne Ni exposure for D. magna (115 µg Ni2+/L). While this may have some consequences for predicting chronic Ni toxicity in this range of Ni concentrations with the biotic ligand model--which could be further improved by including the dietary toxicity pathway in this model, the occurrence of such high concentrations in the field is very rare. Hence, there seems to be very little environmental relevance for dietary Ni toxicity to D. magna. Finally, besides the direct effects of Ni there was no evidence that nutritional quality shifts could have affected daphnids' growth, but it is very likely that the impairment of reproduction at toxic exposure levels of Ni was also partly the result of reduced fatty acid levels.

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.

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.