Uranium accumulation and toxicokinetics in the crustacean Daphnia magna provide perspective to toxicodynamic responses.

The importance of incorporating kinetic approaches in order to gain information on underlying physiological processes explaining species sensitivity to environmental stressors has been highlighted in recent years. Uranium is present in the aquatic environment worldwide due to naturally occurring and anthropogenic sources, posing a potential risk to freshwater taxa in contaminated areas. Although literature shows that organisms vary widely with respect to susceptibility to U, information on toxicokinetics that may explain the variation in toxicodynamic responses is scarce. In the present work, Daphnia magna were exposed to a range of environmentally relevant U concentrations (0 - 200 µg L-1) followed by a 48 h depuration phase to obtain information on toxicokinetic parameters and toxic responses. Results showed time-dependent and concentration-dependent uptake of U in daphnia (ku = 1.2 - 3.8 L g-1 day-1) with bioconcentration factors (BCFs) ranging from 1,641 - 5,204 (L kg-1), a high depuration rate constant (ke = 0.75 day-1), the majority of U tightly bound to the exoskeleton (~ 50 - 60%) and maternal transfer of U (1 - 7%). Effects on growth, survivorship and major ion homeostasis strongly correlated with exposure (external or internal) and toxicokinetic parameters (uptake rates, ku, BCF), indicating that uptake and internalization drives U toxicity responses in D. magna. Interference from U with ion uptake pathways and homeostasis was highlighted by the alteration in whole-body ion concentrations, their ionic ratios (e.g., Ca:Mg and Na:K) and the increased expression in some ion regulating genes. Together, this work adds to the limited data examining U kinetics in freshwater taxa and, in addition, provides perspective on factors influencing stress, toxicity and adaptive response to environmental contaminants such as uranium.

Oligosaccharides derived from dragon fruit modulate gut microbiota, reduce oxidative stress and stimulate toll-pathway related gene expression in freshwater crustacean Daphnia magna.

Dragon fruit oligosaccharide (DFO) is an indigestible prebiotic. In this study, we aimed to investigate the effects of DFO on gut microbiota, oxidative stress and immune-related gene expression in Daphnia magna. The 10-day-old D. magna were treated with 0, 9, and 27 mg l-1 DFO for 85 h. The gut bacterial communities, superoxide dismutase (SOD) activity, lipid peroxidation and the expressions of genes in Toll signaling pathway were observed. The results showed that D. magna treated with 9 and 27 mg l-1 DFO altered gut microbiota composition by increasing Limnohabitans and Lactobacillus, and significantly increased SOD activity and reduced lipid peroxidation. Moreover, the expressions of Toll2, Toll3, Toll5, Toll7 and Pelle genes were significantly increased in D. magna treated with 9 and 27 mg l-1 DFO. Our results suggested that DFO changed the composition of the gut microbiota of D. magna by increasing the beneficial bacteria. DFO also had the ability to stimulate innate immunity in D. magna by increasing SOD activity, reducing lipid peroxidation, and increasing the expression of immune-related genes.

Biofilms grown in aquatic microcosms affect mercury and selenium accumulation in Daphnia.

Experiments examining mercury (Hg) toxicity in Daphnia are usually conducted in highly standardized conditions that prevent the formation of biofilm. Although such standardization has many advantages, extrapolation of results to natural conditions and inference of ecological effects is challenging. This is especially true since biofilms can accumulate metals/metalloids and play a key role in their transfer to higher trophic level organisms. In this study, we experimentally tested the effects of spontaneously appearing biofilm in Daphnia cultures on accumulation of Hg and its natural antagonist selenium (Se) in Daphnia magna. We added Hg (in the form of mercury (II) chloride) at two concentrations (0.2 µg/L and 2 µg/L) to experimental microcosms and measured the uptake of Hg and Se by D. magna in the presence and absence of biofilm. To test for consistent and replicable results, we ran two identical experimental sets one week apart. Biofilm presence significantly reduced the accumulation of Hg, while increasing the tissue Se content in D. magna, and these findings were reproducible across experimental sets. These findings indicate that highly standardized tests may not be adequate to predict the bioaccumulation and potential toxicity of metals/metalloids under natural conditions.

Transcriptome sequencing of a keystone aquatic herbivore yields insights on the temperature-dependent metabolism of essential lipids.

BACKGROUND: Nutritional quality of phytoplankton is a major determinant of the trophic transfer efficiency at the plant-herbivore interface in freshwater food webs. In particular, the phytoplankton's content of the essential polyunsaturated omega-3 fatty acid eicosapentaenoic acid (EPA) has been repeatedly shown to limit secondary production in the major zooplankton herbivore genus Daphnia. Despite extensive research efforts on the biological model organism Daphnia, and the availability of several Daphnia genomes, little is known regarding the molecular mechanisms underlying the limitations in Daphnia related to dietary EPA availability. RESULTS: We used RNA-seq to analyse the transcriptomic response of Daphnia magna which were fed with two different diets - each with or without supplementation of EPA - at two different temperature levels (15 and 20 °C). The transcripts were mapped to the D. magna genome assembly version 2.4, containing 26,646 translations. When D. magna fed on green alga, changing the temperature provoked a differential expression of 2001 transcripts, and in cyanobacteria-fed daphnia, 3385 transcripts were affected. The supplementation of EPA affected 1635 (on the green algal diet), or 175 transcripts (on the cyanobacterial diet), respectively. Combined effects for diet and temperature were also observed (669 for the green algal and 128 transcripts for the cyanobacterial diet). Searching for orthologous genes (COG-analysis) yielded a functional overview of the altered transcriptomes. Cross-matched transcript sets from both feed types were compiled to illuminate core responses to the factors temperature and EPA-supplementation. CONCLUSIONS: Our highly controlled eco-physiological experiments revealed an orchestrated response of genes involved in the transformation and signalling of essential fatty acids, including eicosanoid-signalling pathways with potential immune functions. We provide an overview of downstream-regulated genes, which contribute to enhance growth and reproductive output. We also identified numerous EPA-responsive candidate genes of yet unknown function, which constitute new targets for future studies on the molecular basis of EPA-dependent effects at the freshwater plant-herbivore interface.

Molecular characterisation of cytochrome P450 enzymes in waterflea (Daphnia pulex) and their expression regulation by polystyrene nanoplastics.

Cytochrome P450 (CYP) enzymes are one of the largest protein families, and they metabolise a wide range of lipophilic organic endogenous and exogenous compounds. Many cytochrome P450 genes have been cloned and characterised, and they are frequently used as biomarkers in environmental toxicology studies because of their sensitivity and inducibility. In the present study, the full-length cDNAs of DpCYP370B and DpCYP4 were cloned from Daphnia pulex for the first time. The sequence of DpCYP370B consisted of an ORF of 1515 bp that encoded a 504 amino acid polypeptide, while the sequence of DpCYP4 comprised an ORF of 1527 bp that encoded a 508 amino acid polypeptide. Homologous alignments revealed the presence of a conserved cysteine haeme-iron ligand signature, FxxGxxxCxG, located in the C-terminal portion. Both the proteins contained a sequence for a transmembrane region that was deduced to be located in the endoplasmic reticulum. Subsequently, the expression levels of DpCYP370B and DpCYP4, as well as those of CYP4AN1, CYP4C33, and CYP4C34, were investigated using quantitative real-time PCR after exposure to five polystyrene nanoplastic concentrations: 0 (control), 0.1, 0.5, 1, and 2 mg/L for 21 days. Except for DpCYP4, the highest mRNA expression was observed at 0.5 mg/L nanoplastics; next, the expression of three of the enzymes (DpCYP370B, CYP4AN1, CYP4C34,) decreased to that of the control level at 1 and 2 mg/L doses of nanoplastics. The expression of DpCYP4 did not significantly change compared with that of the control group. These results indicated that CYP genes might play an important role in protecting D. pulex against nanoplastic pollutants.

Nutrients mediate the effects of temperature on methylmercury concentrations in freshwater zooplankton.

Methylmercury (MeHg) bioaccumulation in freshwater aquatic systems is impacted by anthropogenic stressors, including climate change and nutrient enrichment. The goal of this study was to determine how warmer water temperatures and excess nutrients would alter zooplankton communities and phytoplankton concentrations, and whether those changes would in turn increase or decrease MeHg concentrations in freshwater zooplankton. To test this, we employed a 2 × 2 factorial experimental design with nutrient and temperature treatments. Mesocosms were filled with ambient water and plankton from Cottage Grove Reservoir, Oregon, U.S.A., a waterbody that has experienced decades of elevated MeHg concentrations and corresponding fish consumption advisories due to run-off from Black Butte Mine tailings, located within the watershed. Treatment combinations of warmer temperature (increased by 0.7 °C), nutrient addition (a single pulse of 10× ambient concentrations of nitrogen and phosphorous), control, and a combination of temperature and nutrients were applied to mesocosms. The individual treatments altered phytoplankton densities and community structure, but alone the effects on MeHg concentrations were muted. Importantly, we found a significant interactive effect of nutrients and temperature: the nutrient addition appeared to buffer against increased MeHg concentrations associated with elevated temperature. However, there was variability in this response, which seems to be related to the abundance of Daphnia and edible phytoplankton. Nutrients at low temperature were associated with marginal increases (1.1×) in zooplankton MeHg. Our findings suggest that global change drivers that influence community composition and ecosystem energetics of both zooplankton and phytoplankton can alter MeHg pathways through food webs.

The threshold elemental ratio of carbon and phosphorus of Daphnia magna and its connection to animal growth.

The growth of animal consumers is affected by the balance of elements in their diet with the transition between limitation by one element to another known as the threshold elemental ratio (TER). Precise estimates of TERs with known levels of uncertainty have yet to be generated for most zooplankton consumers. We determined the TER for carbon (C) and phosphorus (P) in for a common lake zooplankter, Daphnia magna, using experimental measurements and theoretical considerations. Daphnia growth responses to food C:P ratios across a relatively narrow range (80-350) generated an empirical estimate of TERC:P of 155 ± 14. While this TER matched our modelled estimate of TERC:P (155 ± 16), it was lower than previous estimates of this dietary transition point. No threshold was found when we examined daphnid body C:N or C:P ratios in response to changing food C:P ratios, which indicates P-limitation at even lower food C:P ratios. Our results provide strong evidence that D. magna is likely to experience acute P-limitation when food C:P ratios exceed even relatively low ratios (~155). Our model further demonstrated that while physiological adjustments may reduce the likelihood of P-limitation or reduce its intensity, these changes in animal material processing would be accompanied by reduced maximum growth rates.

The pH-dependent toxicity of triclosan to five aquatic organisms (Daphnia magna, Photobacterium phosphoreum, Danio rerio, Limnodrilus hoffmeisteri, and Carassius auratus).

Triclosan (TCS) is an antibacterial and antifungal agent widely used in personal care products, and it has been frequently detected in the aquatic environment. In the present study, the acute toxicity of TCS to Daphnia magna, Photobacterium phosphoreum, Danio rerio, and Limnodrilus hoffmeisteri was assessed under different pH conditions. Generally, TCS was more toxic to the four aquatic organisms in acidic medium. The LC50 values for D. magna and D. rerio were smaller among the selected species, suggesting that D. magna and D. rerio were more sensitive to TCS. In addition, the oxidative stress-inducing potential of TCS was evaluated in Carassius auratus at three pH values. Changes of superoxide dismutase (SOD) and catalase (CAT) activity, glutathione (GSH) level, and malondialdehyde (MDA) content were commonly observed in all TCS exposure groups, indicating the occurrence of oxidative stress in the liver of C. auratus. The integrated biomarker response (IBR) index revealed that a high concentration of TCS induced great oxidative stress in goldfish under acidic condition. This work supplements the presently available data on the toxicity data of TCS, which would provide some useful information for the environmental risk assessment of this compound.

Bi-directional effects of vitamin B12 and methotrexate on Daphnia magna fitness and genomic methylation.

Here we interrogated, using three separate but complementary experimental approaches, the impact of vitamin B12 availability and methotrexate exposure on Daphnia magna, which we hypothesised should have an opposite effect on One carbon metabolism (OCM). OCM is a vital biological process supporting a variety of physiological processes, including DNA methylation. Contrary to mammalian models, this process remains largely unexplored in invertebrates. The purpose of this study was to elucidate the impact of OCM short-term alteration on the fitness and epigenome of the keystone species, Daphnia. We used maternal age at reproduction, brood size and survival rates in combination with DNA methylation sensitive comet assay to determine the effects of vitamin B12 or MTX on fitness and the epigenome. Vitamin B12 had a positive influence on Daphnia fitness and we provide evidence demonstrating that this may be associated with an increased level of genome-wide DNA methylation. Conversely, exposing D. magna to MTX negatively influenced the fitness of the animals and was associated with loss of global DNA methylation, translating in decreased fitness. These results highlight the potential importance of OCM in invertebrates, providing novel evidence supporting a potential role for epigenetic modifications to the genome in D. magna environmental adaptability.

Toxicity identification and evaluation for the effluent from a nonmetallic mineral mining facility in Korea using D. magna.

Industrial wastewater has attracted increasing attention in recent years because of its impact on ecosystems and human health. Whole-effluent tests are generally used to monitor toxicities of unknown chemicals and conventional pollutants from industrial effluent discharges. This study described identification evaluation (TIE) procedures to determine the acute toxicity of a nonmetallic mineral mining facility effluent that was toxic to Daphnia magna. In the characterization step (TIE phase I), toxic effects of heavy metals, organic compounds, oxidants, volatile organic compounds, suspended solids, and ammonia were screened. Results revealed that the source of toxicity was beyond these toxicants. Chemical analysis (TIE phase II) of total dissolved solid showed that the concentration of chloride ion (15,302.5 mg/L) was substantially higher than the predicted EC50 value for D. magna. Chemical analysis for heavy metal and ionic materials used ion chromatography and induced coupled plasma-optic emission spectroscopy. In the confirmation step (TIE phase III), using spiking and deletion approaches, it was demonstrated that chloride ion was the main toxicant in this effluent. Concentrations of potassium (317.5 mg/L), magnesium (970.5 mg/L), sodium (8595.3 mg/L), and sulfate (2854.3 mg/L) were not high enough to cause toxicity to D. magna. Finally, we concluded that chloride was the main toxicant in the nonmetallic mineral mining facility effluent. Based on these results, advanced treatment processes such as ion exchange and reverse osmosis technology are recommended to treat wastewater in this and similar situations. Further research is needed to provide technical support for toxin identification and evaluation of various types of wastewater treatment plant discharge.

Exchange of polar lipids from adults to neonates in Daphnia magna: Perturbations in sphingomyelin allocation by dietary lipids and environmental toxicants.

Because xenosensing nuclear receptors are also lipid sensors that regulate lipid allocation, we hypothesized that toxicant-induced modulation of HR96 activity would alter lipid profiles and the balance between adult survival and neonate production following exposure in Daphnia magna. Adult daphnids were exposed to unsaturated fatty acid- and toxicant- activators or inhibitors of HR96 and later starved to test whether chemical exposure altered allocation toward survival or reproduction. The HR96 activators, linoleic acid and atrazine, decreased reproduction as expected with concomitant changes in the expression of HR96 regulated genes such as magro. The HR96 inhibitors, docosahexaenoic acid (DHA) and triclosan, increased reproduction or neonate starvation survival, respectively. However, pre-exposure to triclosan increased in neonate survival at the expense of reproductive maturation. Lipidomic analysis revealed that sphingomyelins (SM) are predominantly found in neonates and therefore we propose are important in development. DHA and triclosan increased neonatal SM, consistent with HR96's regulation of Niemann-Pick genes. While DHA altered expression of magro, Niemann-Pick 1b, mannosidase, and other HR96-regulated genes as expected, triclosan primarily perturbed sphingomyelinase and mannosidase expression indicating different but potentially overlapping mechanisms for perturbing SM. Overall, SM appears to be a key lipid in Daphnia maturation and further support was provided by carmofur, which inhibits sphingomyelin/ceramide metabolism and in turn severely represses Daphnia maturation and initial brood production. In conclusion, toxicants can perturb lipid allocation and in turn impair development and reproduction.

Interactive effects of genotype and food quality on consumer growth rate and elemental content.

Consumer body stoichiometry is a key trait that links organismal physiology to population and ecosystem-level dynamics. However, as elemental composition has traditionally been considered to be constrained within a species, the ecological and evolutionary factors shaping consumer elemental composition have not been clearly resolved. To this end, we examined the causes and extent of variation in the body phosphorus (P) content and the expression of P-linked traits, mass specific growth rate (MSGR), and P use efficiency (PUE) of the keystone aquatic consumer Daphnia using lake surveys and common garden experiments. While daphnid body %P was relatively constrained in field assemblages sampled across an environmental P gradient, unique genotypes isolated from these lakes showed highly variable phenotypic responses when raised across dietary P gradients in the laboratory. Specifically, we observed substantial inter- and intra-specific variation and differences in daphnid responses within and among our study lakes. While variation in Daphnia body %P was mostly due to plastic phenotypic changes, we documented considerable genetic differences in daphnid MSGR and PUE, and relationships between MSGR and body P content were highly variable among genotypes. Overall, our study found that consumer responses to food quality may differ considerably among genotypes and that relationships between organismal life-history traits and body stoichiometry may be strongly influenced by genetic and environmental variation in natural assemblages.

Extended anaerobic conditions in the biological wastewater treatment: Higher reduction of toxicity compared to target organic micropollutants.

Extended anaerobic conditions during biological wastewater treatment may enhance the biodegradation of micropollutants. To explore this, we combined iron-reducing or substrate-limited anaerobic conditions and aerobic pilot-scale reactors directly at a wastewater treatment plant. To investigate the detoxification by these processes, we applied two in vitro bioassays for baseline toxicity (Microtox) and reactive toxicity (AREc32) as well as in vivo bioassays with aquatic model species in two laboratory experiments (Desmodesmus subspicatus, Daphnia magna) and two on-site, flow-through experiments (Potamopyrgus antipodarum, Lumbriculus variegatus). Moreover, we analyzed 31 commonly occurring micropollutants and 10 metabolites. The baseline toxicity of raw wastewater was effectively removed in full-scale and reactor scale activated sludge treatment (>85%), while the oxidative stress response was only partially removed (>61%). A combination of an anaerobic pre-treatment under iron reducing conditions and an aerobic nitrification significantly further reduced the residual in vitro toxicities by 46-60% and outperformed the second combination consisting of an aerobic pre-treatment and an anaerobic post-treatment under substrate-limiting conditions (27-43%). Exposure to effluents of the activated sludge treatment did not induce adverse in vivo effects in aquatic invertebrates. Accordingly, no further improvement in water quality could be observed. Compared to that, the removal of persistent micropollutants was increased. However, this observation was restricted to a limited number of compounds and the removal of the sum concentration of all target micropollutants was relative low (14-17%). In conclusion, combinations of strictly anaerobic and aerobic processes significantly enhanced the removal of specific and non-specific in vitro toxicities. Thus, an optimization of biological wastewater treatment can lead to a substantially improved detoxification. These otherwise hidden capacities of a treatment technology can only be uncovered by a complementary biological analysis.

Does infection tilt the scales? Disease effects on the mass balance of an invertebrate nutrient recycler.

While parasites are increasingly recognized as important components of ecosystems, we currently know little about how they alter ecosystem nutrient availability via host-mediated nutrient cycling. We examined whether infection alters the flow of nutrients through hosts and whether such effects depend upon host diet quality. To do so, we compared the mass specific nutrient (i.e., nitrogen and phosphorus) release rates, ingestion rates, and elemental composition of uninfected Daphnia to those infected with a bacterial parasite, P. ramosa. N and P release rates were increased by infection when Daphnia were fed P-poor diets, but we found no effect of infection on the nutrient release of individuals fed P-rich diets. Calculations based on the first law of thermodynamics indicated that infection should increase the nutrient release rates of Daphnia by decreasing nutrient accumulation rates in host tissues. Although we found reduced nutrient accumulation rates in infected Daphnia fed all diets, this reduction did not increase the nutrient release rates of Daphnia fed the P-rich diet because infected Daphnia fed this diet ingested nutrients more slowly than uninfected hosts. Our results thus indicate that parasites can significantly alter the nutrient use of animal consumers, which could affect the availability of nutrients in heavily parasitized environments.

Functional genomics of intraspecific variation in carbon and phosphorus kinetics in Daphnia.

Understanding how the genome interacts with the environment to produce a diversity of phenotypes is a central challenge in biology. However, we know little about how traits involved in nutrient processing interact with key ecological parameters, such as the supply of mineral nutrients, particularly in animals. The framework of ecological stoichiometry uses information on the content of key elements such as carbon (C) and phosphorus (P) in individuals to predict the success of species. Nevertheless, intraspecific variation in content and the underlying mechanisms that generate such variation has been poorly explored. We studied two genotypes (G1 and G2) of Daphnia pulex that exhibit striking genotype × environment (G × E) interaction in response to shifts in dietary stoichiometry (C:P). G1 had higher fitness under C:P ∼ 100 diet, while G2 performed better in C:P ∼ 800. Dual (14) C/(33) P radiotracer assays show that G1 was more efficient in C processing, while G2 was more efficient in P use. Microarrays revealed that after 3 days of incubation, the genotypes differentially expressed ∼ 25% (7,224) of the total genes on the array under C:P ∼ 100 diet, and ∼ 30% (8,880) of genes under C:P ∼ 800. These results indicate large differences in C and P use between two coexisting genotypes. Importantly, such physiological differences can arise via differential expression of the genome due to alterations in dietary stoichiometry. Basic frameworks such as ecological stoichiometry enable integration of physiological and transcriptomic data, and represent initial steps toward understanding the interplay between fundamental ecological parameters such as nutrient supply and important evolutionary processes such as G × E interactions.

Retroconversion of docosapentaenoic acid (n-6): an alternative pathway for biosynthesis of arachidonic acid in Daphnia magna.

The aim of this study was to assess metabolic pathways for arachidonic acid (20:4n-6) biosynthesis in Daphnia magna. Neonates of D. magna were maintained on [(13)C] enriched Scenedesmus obliquus and supplemented with liposomes that contained separate treatments of unlabeled docosapentaenoic acid (22:5n-6), 20:4n-6, linoleic acid (18:2n-6) or oleic acid (18:1n-9). Daphnia in the control treatment, without any supplementary fatty acids (FA) containing only trace amounts of 20:4n-6 (~0.3% of all FA). As expected, the highest proportion of 20:4n-6 (~6.3%) was detected in Daphnia that received liposomes supplemented with this FA. Higher availability of 18:2n-6 in the diet increased the proportion of 18:2n-6 in Daphnia, but the proportion of 20:4n-6 was not affected. Daphnia supplemented with 22:5n-6 contained ~3.5% 20:4n-6 in the lipids and FA specific stable isotope analyses validated that the increase in the proportion of 20:4n-6 was due to retroconversion of unlabeled 22:5n-6. These results suggest that chain shortening of 22:5n-6 is a more efficient pathway to synthesize 20:4n-6 in D. magna than elongation and desaturation of 18:2n-6. These results may at least partially explain the discrepancies noticed between phytoplankton FA composition and the expected FA composition in freshwater cladocerans. Finally, retroconversion of dietary 22:5n-6 to 20:4n-6 indicates Daphnia efficiently retain long chain n-6 FA in lake food webs, which might be important for the nutritional ecology of fish.

Differing Daphnia magna assimilation efficiencies for terrestrial, bacterial, and algal carbon and fatty acids.

There is considerable interest in the pathways by which carbon and growth-limiting elemental and biochemical nutrients are supplied to upper trophic levels. Fatty acids and sterols are among the most important molecules transferred across the plant-animal interface of food webs. In lake ecosystems, in addition to phytoplankton, bacteria and terrestrial organic matter are potential trophic resources for zooplankton, especially in those receiving high terrestrial organic matter inputs. We therefore tested carbon, nitrogen, and fatty acid assimilation by the crustacean Daphnia magna when consuming these resources. We fed Daphnia with monospecific diets of high-quality (Cryptomonas marssonii) and intermediate-quality (Chlamydomonas sp. and Scenedesmus gracilis) phytoplankton species, two heterotrophic bacterial strains, and particles from the globally dispersed riparian grass, Phragmites australis, representing terrestrial particulate organic carbon (t-POC). We also fed Daphnia with various mixed diets, and compared Daphnia fatty acid, carbon, and nitrogen assimilation across treatments. Our results suggest that bacteria were nutritionally inadequate diets because they lacked sterols and polyunsaturated omega-3 and omega-6 (omega-3 and omega-6) fatty acids (PUFAs). However, Daphnia were able to effectively use carbon and nitrogen from Actinobacteria, if their basal needs for essential fatty acids and sterols were met by phytoplankton. In contrast to bacteria, t-POC contained sterols and omega-6 and omega-3 fatty acids, but only at 22%, 1.4%, and 0.2% of phytoplankton levels, respectively, which indicated that t-POC food quality was especially restricted with regard to omega-3 PUFAs. Our results also showed higher assimilation of carbon than fatty acids from t-POC and bacteria into Daphnia, based on stable-isotope and fatty acids analysis, respectively. A relatively high (>20%) assimilation of carbon and fatty acids from t-POC was observed only when the proportion of t-POC was >60%, but due to low PUFA to carbon ratio, these conditions yielded poor Daphnia growth. Because of lower assimilation for carbon, nitrogen, and fatty acids from t-POC relative to diets of bacteria mixed with phytoplankton, we conclude that the microbial food web, supported by phytoplankton, and not direct t-POC consumption, may support zooplankton production. Our results suggest that terrestrial particulate organic carbon poorly supports upper trophic levels of the lakes.

Microscopic investigation of single-wall carbon nanotube uptake by Daphnia magna.

The objectives of this study were to determine the extent of absorption of functionalized single-wall carbon nanotubes (SWCNTs) across the gut epithelial cells in Daphnia magna. Several microscopic techniques were utilized, including micro-Raman spectroscopy, high-resolution transmission electron microscopy (HRTEM) and selective area diffraction (SAD). In an effort to examine the variation in uptake due to surface properties, four groups of differently functionalized SWCNTs were used: hydroxylated (OH-SWCNTs), silicon dioxide (SiO2-SWCNTs), poly aminobenzenesulfonic acid (PABS-SWCNTs) and polyethylene glycol (PEG-SWCNTs). Raman spectroscopy was able to detect OH-SWCNTs within the gut, but lacked the spatial resolution that is needed to identify lower concentrations of SWCNTs that may have been absorbed by body tissues. Initially, low-magnification imaging of exposed D. magna sections in the TEM revealed several features, which suggested absorption of SWCNTs. However, subsequent analysis with additional techniques (HRTEM, X-ray energy-dispersive spectroscopy and SAD) indicated that these features were either artifacts produced via the specimen staining process or consisted of non-graphitic, organic structures. This latter observation emphasizes the inherent difficulty in resolving SWCNTs embedded within a complex, organic matrix, as well as the care with which imaging results must be interpreted and supplemented with other, more analytical techniques.

Biodegradability, toxicity and mutagenicity of detergents: Integrated experimental evaluations.

The widespread use of detergents has raised concern with regard to the environmental pollution caused by their active ingredients, which are biorefractory, toxic and persistent. Since detergents are complex mixtures of different substances, in which synergistic effects may occur, we aimed to assess the mutagenicity of different detergent formulations, taking into account aquatic toxicity and ready biodegradability. We performed a ready biodegradability test (OECD 301 F), Daphnia magna and Vibrio fischeri toxicity tests, and mutagenicity tests (Salmonella/microsome test, Allium cepa test and comet assay). Six detergent formulations were examined, 3 pre-manufacture and 3 commercially available. All detergents presented ready biodegradability. EC50 values varied for all products, according to the marker organism used, but were always higher than the more stringent value considered for aquatic toxicity assessment (V. fischeri 10-60 mg/L; D. magna 25-300 mg/L; A. cepa 250-2000 mg/L). None of the detergents caused mutations in bacteria. However, one commercial ecolabelled product induced an increase in micronucleus frequency in A. cepa root cells. All pre-manufacture detergents and one commercial one, which gave negative results in the Ames and A. cepa tests, induced DNA damage in human leukocytes. A more accurate evaluation of the environmental impact of complex mixtures such as detergents requires a battery of tests to describe degradation, as well as toxicological and mutagenic features.

The potential of dietary polyunsaturated fatty acids to modulate eicosanoid synthesis and reproduction in Daphnia magna: a gene expression approach.

Nutritional ecology of the aquatic model genus Daphnia has received much attention in past years in particular with regard to dietary polyunsaturated fatty acids (PUFAs) which are crucial for growth and reproduction. Besides their significant role as membrane components, C20 PUFAs serve as precursors for eicosanoids, hormone-like mediators of reproduction, immunity and ion transport physiology. In the present study we investigate transcriptomic changes in Daphnia magna in response to different algal food organisms substantially differing in their PUFA composition using quantitative real-time PCR and relate them to concomitantly documented life history data. The selection of target genes includes representatives that have previously been shown to be responsive to the eicosanoid biosynthesis inhibitor ibuprofen. The beneficial effect of C20 PUFA-rich food on reproduction and population growth rates was accompanied by an increased vitellogenin (DmagVtg1) gene expression in D. magna. Additionally, genes involved in eicosanoid signaling were particularly influenced by dietary C20 PUFA availability. For example, the cyclooxygenase gene (Cox), coding for a central enzyme in the eicosanoid pathway, was highly responsive to the food treatments. Our results suggest that dietary PUFAs are fundamental in D. magna physiology as substrate for eicosanoid synthesis and that these eicosanoids are important for D. magna reproduction.