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.