Gene Coexpression Networks Drive and Predict Reproductive Effects in Daphnia in Response to Environmental Disturbances.

Increasing effects of anthropogenic stressors and those of natural origin on aquatic ecosystems have intensified the need for predictive and functional models of their effects. Here, we use gene expression patterns in combination with weighted gene coexpression networks and generalized additive models to predict effects on reproduction in the aquatic microcrustacean Daphnia. We developed models to predict effects on reproduction upon exposure to different cyanobacteria, different insecticides and binary mixtures of cyanobacteria and insecticides. Models developed specifically for groups of stressors (e.g., either cyanobacteria or insecticides) performed better than general models developed on all data. Furthermore, models developed using in silico generated mixture gene expression profiles from single stressor data were able to better predict effects on reproduction compared to models derived from the mixture exposures themselves. Our results highlight the potential of gene expression data to quantify effects of complex exposures at higher level organismal effects without prior mechanistic knowledge or complex exposure data.

Toxic dinoflagellates and Vibrio spp. act independently in bivalve larvae.

Harmful algal blooms (HABs) and marine pathogens - like Vibrio spp. - are increasingly common due to climate change. These stressors affect the growth, viability and development of bivalve larvae. Little is known, however, about the potential for interactions between these two concurrent stressors. While some mixed exposures have been performed with adult bivalves, no such work has been done with larvae which are generally more sensitive. This study examines whether dinoflagellates and bacteria may interactively affect the viability and immunological resilience of blue mussel Mytilus edulis larvae. Embryos were exposed to environmentally relevant concentrations (100, 500, 2500 & 12,500 cells ml(-1)) of a dinoflagellate (Alexandrium minutum, Alexandrium ostenfeldii, Karenia mikimotoi, Protoceratium reticulatum, Prorocentrum cordatum, P. lima or P. micans), a known pathogen (Vibrio coralliilyticus/neptunius-like isolate or Vibrio splendidus; 10(5) CFU ml(-1)), or both. After five days of exposure, significant (p < 0.05) adverse effects on larval viability and larval development were found for all dinoflagellates (except P. cordatum) and V. splendidus. Yet, despite the individual effect of each stressor, no significant interactions were found between the pathogens and harmful algae. The larval viability and the phenoloxidase innate immune system responded independently to each stressor. This independence may be related to a differential timing of the effects of HABs and pathogens.

Common European harmful algal blooms affect the viability and innate immune responses of Mytilus edulis larvae.

Like marine diseases, harmful algal blooms (HABs) are globally increasing in frequency, severity and geographical scale. As a result, bivalves will have to face the combined threat of toxic algae and marine pathogens more frequently in the (near) future. These stressors combined may further affect the recruitment of ecologically and economically important bivalve species as HABs can affect the growth, viability and development of their larvae. To date, little is known on the specific effects of HABs on the innate immune system of bivalve larvae. This study therefore investigates whether two common harmful algae can influence the larval viability, development and immunological resilience of the blue mussel Mytilus edulis. Embryos of this model organism were exposed (48 h) to five densities of Pseudo-nitzschia multiseries or Prorocentrum lima cells. In addition, the effect of six concentrations of their respective toxins: domoic acid (DA) and okadaic acid (OA) were assessed. OA was found to significantly reduce larval protein phosphatase activity (p < 0.001) and larval viability (p < 0.01) at concentrations as low as 37.8 µg l(-1). P. multiseries (1400 cells ml(-1)), P. lima (150 cells ml(-1)) and DA (dosed five times higher than typical environmental conditions i.e. 623.2 µg l(-1)) increased the phenoloxidase (PO) innate immune activity of the mussel larvae. These results suggest that the innate immune response of even the earliest life stages of bivalves is susceptible to the presence of HABs.

Hard X-ray nanoprobe investigations of the subtissue metal distributions within Daphnia magna.

The unique potential of nanoscale elemental imaging of major/minor and trace-level elemental distributions within thin biological tissue sections of the ecotoxicological model organism Daphnia magna is demonstrated by synchrotron radiation nano-X-ray fluorescence (nano-XRF). The applied highly specialized sample preparation method, coupled with the high spatial resolution (∼180 nm) and high X-ray photon flux (6 × 10(11) photons/s) available at the European Synchrotron Radiation Facility (ESRF) ID22NI beamline proved to be critical for the high-quality visualization of (trace-)metal distributions on the submicron level within the target structures of interest. These include the branchial sacs on the thoracic appendages (epipodites) of D. magna, which are osmoregulatory regions where ion exchange occurs. For the main element of interest (Zn), detection limits of 0.7 ppm (3 ag) was reached in fast-scanning mode using an acquisition time of 0.3 s/pixel. As demonstrated, synchrotron radiation nano-XRF revealed the elemental distributions of Ca, Fe, and Zn within this osmoregulatory region on the submicron scale, aiding the exploration of possible detoxification mechanisms of Zn within D. magna at the subtissue level.

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.

The potential for adaptation in a natural Daphnia magna population: broad and narrow-sense heritability of net reproductive rate under Cd stress at two temperatures.

The existence of genetic variability is a key element of the adaptive potential of a natural population to stress. In this study we estimated the additive and non-additive components of the genetic variability of net reproductive rate (R(0)) in a natural Daphnia magna population exposed to Cd stress at two different temperatures. To this end, life-table experiments were conducted with 20 parental and 39 offspring clonal lineages following a 2 × 2 design with Cd concentration (control vs. 3.7 µg Cd/L) and temperature (20 vs. 24 °C) as factors. Offspring lineages were obtained through inter-clonal crossing of the different parental lineages. The population mean, additive and non-additive genetic components of variation in each treatment were estimated by fitting an Animal Model to the observed R(0) values using restricted maximum likelihood estimation. From those estimates broad-sense heritabilities (H(2)), narrow-sense heritabilities (h(2)), total (CV(G)) and additive genetic coefficients of variation (CV(A)) of R(0) were calculated. The exposure to Cd imposed a considerable level of stress to the population, as shown by the fact that the population mean of R(0) exposed to Cd was significantly lower than in the control at the corresponding temperature, i.e. by 23 % at 20 °C and by 88 % at 24 °C. The latter difference indicates that increasing temperature increased the stress level imposed by Cd. The H² and CV(G) were significantly greater than 0 in all treatments, suggesting that there is a considerable degree of genetic determination of R(0) in this population and that clonal selection could rapidly lead to increasing population mean fitness under all investigated conditions. More specifically, the H² was 0.392 at 20 °C+Cd and 0.563 at 24 °C+Cd; the CV(G) was 30.0 % at 20 °C+Cd and was significantly higher (147.6 %) in the 24 °C+Cd treatment. Significant values of h(2) (= 0.23) and CV(A) (= 89.7 %) were only found in the 24 °C+Cd treatment, suggesting that the ability to produce more offspring under this stressful condition may be inherited across sexual generations. In contrast, in the less stressful 20 °C+Cd treatment the h(2) (0.06) and CV(A) (7.0 %) were very low and not significantly higher than zero. Collectively our data indicate that both the asexual and sexual reproduction phases in cyclic parthenogenetic D. magna populations may play a role in the long-term adaptive potential of Daphnia populations to chemical stress (with Cd as the current example) and that environmental variables which influence the stress level of that chemical may influence this adaptive potential (with temperature as current example).

Toxicological availability of nickel to the benthic oligochaete Lumbriculus variegatus.

It is generally accepted that the bioavailability of metals in sediments is influenced by the presence of acid volatile sulfides (AVS). The pore water hypothesis predicts that, if the molar concentration of simultaneously extracted metals (SEM) in a sediment is smaller than the molar concentration of AVS, the free metal ion activity in the pore water is very small and that consequently no metal toxicity in short-term toxicity tests is observed. In this study we examined (1) if this concept can be extended to predict the absence of chronic Ni toxicity to the oligochaete deposit-feeding worm Lumbriculus variegatus and (2) if the organic carbon normalized excess SEM; i.e. [SEM-AVS]/f(OC) predicts the magnitude of Ni toxicity to L. variegatus. A 28-day toxicity experiment was performed in which biomass production of L. variegatus was determined in two natural sediments with different [AVS] and f(OC), spiked at different Ni concentrations. The absence of toxicity is predicted correctly by the [SEM-AVS]<0 criterion when only the 0-1 cm surface layer of the sediment is considered, but not when the whole bulk sediment is considered (0-3 cm). In both sediments, the same [SEM-AVS]/f(OC) at the surface corresponds with a similar decrease in L. variegatus biomass. Thus, [SEM-AVS]/f(OC) in the surface layer accurately predicts the magnitude of toxicity. This measure is therefore a good estimator of toxicologically available Ni. On the other hand, the free Ni(2+) ion activity in the overlying water appeared to be an equally good predictor of the magnitude of toxicity. Consequently, it was not possible to determine the relative importance of the overlying water and pore water exposure route with the semi-static laboratory experiments.

Chronic toxicity of dietary copper to Daphnia magna.

There is a growing concern that dietborne metal toxicity might be important in aquatic ecosystems. However, the science behind this matter is insufficiently developed to explicitly and accurately account for this in metal regulation or risk assessment. We investigated the effects of a chronic exposure of Daphnia magna to an elevated level of Cu (3000 microg Cu/g dry wt) in their diet (the green alga Pseudokirchneriella subcapitata). Compared to daphnids fed with P. subcapitata containing a background of 10.6 microg Cu/g dry wt, daphnids fed for 21 days with this Cu-contaminated food accumulated a total copper body burden of 325 microg Cu/g dry wt, which is about 30-fold higher than the control body burden of 12.1 microg/g dry wt. The exposed daphnids experienced a 38% reduction of growth (measured as final dry body weight), a 50% reduction of reproduction (total number of juveniles produced per daphnid), and only produced three broods versus four broods by the control daphnids. Unlike most other studies, we were able to demonstrate that these effects were most likely not due to a reduced nutritional quality of the food, based on C:P ratios and fatty acid content and composition of the Cu-contaminated algae. Life-history analysis showed that time to first brood was not affected by dietary Cu, while the second and third broods were significantly delayed by 0.7 and 1.5 days, respectively. On the other hand, brood sizes of all three broods were significantly lower in Cu exposed daphnids, i.e. by 32-55%. The variety of effects observed suggest the possible, and perhaps simultaneous, involvement of several toxicity mechanisms such as increased metabolic cost, reduced energy acquisition (potentially via inhibition of digestive enzyme activity), targeted inhibition of reproduction (potentially via inhibition of vitellogenesis), and/or direct inhibition of molting. Further research is needed to differentiate between these postulated mechanisms of dietary Cu toxicity and to determine whether they act separately or in concert.

Development and validation of a terrestrial biotic ligand model predicting the effect of cobalt on root growth of barley (Hordeum vulgare).

A Biotic Ligand Model was developed predicting the effect of cobalt on root growth of barley (Hordeum vulgare) in nutrient solutions. The extent to which Ca(2+), Mg(2+), Na(+), K(+) ions and pH independently affect cobalt toxicity to barley was studied. With increasing activities of Mg(2+), and to a lesser extent also K(+), the 4-d EC50(Co2+) increased linearly, while Ca(2+), Na(+) and H(+) activities did not affect Co(2+) toxicity. Stability constants for the binding of Co(2+), Mg(2+) and K(+) to the biotic ligand were obtained: logK(CoBL)=5.14, logK(MgBL)=3.86 and logK(KBL)=2.50. Limited validation of the model with one standard artificial soil and one standard field soil showed that the 4-d EC50(Co2+) could only be predicted within a factor of four from the observed values, indicating further refinement of the BLM is needed.

An ecosystem modelling approach for deriving water quality criteria.

Ecological effects of chemicals on ecosystems are the result of direct effects of the chemical, determined in single-species toxicity testing, and indirect effects due to ecological interactions between species. Current experimental methods to account for such interactions are expensive. Hence, mathematical models of ecosystems have been proposed as an alternative. The use of these models often requires extensive calibration, which hampers their use as a general tool in ecological effect assessments. Here we present a novel ecosystem modelling approach which assesses effects of chemicals on ecosystems by integrating single-species toxicity test results and ecological interactions, without the need for calibration on case-specific data. The methodology is validated by comparing predicted ecological effects of copper in a freshwater planktonic ecosystem with an experimental ecosystem data set. Two main effects reflected by this data set (a decrease of cladocerans and an increase of small phytoplankton) which were unpredictable from single-species toxicity test results alone, were predicted accurately by the developed model. Effects on populations which don't interact directly with other populations, were predicted equally well by single-species toxicity test results as by the ecosystem model. The small amount of required data and the high predictive capacity can make this ecosystem modelling approach an efficient tool in water quality criteria derivation for chemicals.

Zinc toxicity to the alga Pseudokirchneriella subcapitata decreases under phosphate limiting growth conditions.

Previous studies have suggested that phosphorus (P) deficiency can increase the sensitivity of microalgae to toxic trace metals, potentially due to reduced metal detoxification at low cell P quota. The existing evidence is, however, inconsistent. This study was set up to determine the combined effects of zinc (Zn) and P supplies on Zn and P bioaccumulation and growth of the green microalgae Pseudokirchneriella subcapitata. Zinc toxicity was investigated in (i) a 24h growth rate assay with cells varying in initial cell P quota (0.5-1.7% P on cell dry weight) with no supplemental P during Zn exposure (Expt. 1) and in (ii) a 48h growth assay initiated with cells at the end of a 14-days steady state culture at three P addition rates (RARs) between 0.8 and 1.6day(-1) (Expt.2). The solution Zn concentrations required to reduce final cell density by 10% relative to control (EbC10) were 5-fold (Expt.1) or 2-fold (Expt.2) lower at the highest P supply than at the lowest P supply, i.e. Zn was more toxic at higher P supply, in contrast with the suggestions from previous studies. Cell P quota increased with increasing Zn in the exposure solution (Expt.2), thereby partially overcoming P deficiency under moderate Zn toxicity compared to low Zn exposure. Similarly, cell Zn increased with increasing P supply, potentially induced by Zn-P complexation or precipitation inside the cell. A dynamic growth model accounting for effects of external Zn and internal P on the specific growth rate was calibrated to all data. This model shows that the effect of solution Zn on specific growth rate (ErC50) was statistically unaffected by cell P quota. In contrast, this model predicts that the EbC10 (i.e. EC10 based on cell numbers) varies with P supply because cell P depends on external P and Zn. Moreover, scenario analysis predicts even contrasting trends of the EbC10 with increasing P supply depending on the duration of the growth assay and the P supply scenario. Our data at two experimental scenarios and the prediction under various relevant scenarios suggest a weaker effect of secondary stress factor (Zn) when nutrient deficiency (first stress factor) is prevailing.

Reverse osmosis sampling does not affect the protective effect of dissolved organic matter on copper and zinc toxicity to freshwater organisms.

Dissolved organic matter (DOM) plays a significant role in protecting freshwater organisms against metal toxicity. To study this, reverse osmosis (RO) has been widely used as a highly efficient method for rapid collection of large quantities of DOM from natural surface waters. The objective of this study was to examine the potential impact of the RO isolation technique on the protective effects of DOM on the toxicity of copper and zinc to the cladoceran Daphnia magna and the green alga Pseudokirchneriella subcapitata. DOM was concentrated from a natural surface water using RO and at the same time a natural (unconcentrated) surface water was taken. The concentrated DOM was rediluted to the level of the natural water to obtain the so-called reconstituted water. Chemical analyses and toxicity tests were performed with both the natural surface water and the reconstituted water. First, most chemical parameters were not significantly changed by the RO sampling. For both copper and zinc, no significant differences were observed in 48 h-EC50s for D. magna and in 72 h-EC50s for P. subcapitata between the reconstituted water and the natural water. Hence, it may be concluded that reverse osmosis does not significantly affect the protective effect of natural DOM against copper and zinc toxicity.

Environmental risk assessment of metals: tools for incorporating bioavailability.

In this paper, some of the main processes and parameters which affect metal bioavailability and toxicity in the aquatic environment and its implications for metal risk assessment procedures will be discussed. It has become clear that, besides chemical processes (speciation, complexation), attention should also be given to physiological aspects for predicting metal toxicity. The development of biotic ligand models (BLMs), which combine speciation models with more biologically oriented models (e.g. GSIM), has offered an answer to this need. The various BLMs which have been developed and/or refined for a number of metals (e.g. Cu, Ag, Zn) and species (algae, crustaceans, fish) are discussed here. Finally, the potential of the BLM approach is illustrated through a theoretical exercise in which chronic zinc toxicity to Daphnia magna is predicted in three regions, taking the physico-chemical characteristics of these areas into account.

Reproductive toxicity of dietary zinc to Daphnia magna.

Regulatory assessments of metals in freshwaters are mostly based on dissolved metal concentrations, assuming that toxicity is caused by waterborne metal only. Little attention has been directed to the toxicity of dietary metals to freshwater invertebrates. In this study the chronic toxicity of dietary zinc to Daphnia magna was investigated. The green alga Pseudokirchneriella subcapitata was exposed for 64 h to a control and three dissolved zinc concentrations, i.e. 23, 28 and 61 microg L(-1), resulting in internal zinc burdens in the algae of 130, 200, 320 and 490 microg g(-1) dry weight, respectively. These algae were used as a food source in chronic, 21-day bioassays with D. magna in a test medium to which no dissolved zinc was added. None of the treatments resulted in effects on feeding rates or somatic growth of D. magna. In contrast, a significant 40% decrease of total reproduction (number of juveniles per adult) was observed in the 28 and 61 microg L(-1) treatments. Time to first brood was not affected, whereas the mean brood size and the fraction of reproducing parent daphnids were reduced from the second brood onwards and the magnitude of these reductions increased with each subsequent brood. The reduced reproduction was accompanied with an elevated zinc accumulation in the 61 microg L(-1) treatment only, suggesting that total body burden is no good indicator of dietary zinc toxicity. Overall our data suggest that dietary zinc specifically targets reproduction in D. magna through accumulation in particular target sites, possibly cells or tissues where vitellogenin synthesis or processing occur. Further, our data illustrate that the potential importance of the dietary exposure route should be carefully considered and interpreted in regulatory assessments of zinc.

Toxicity of lead (Pb) to freshwater green algae: development and validation of a bioavailability model and inter-species sensitivity comparison.

Scientifically sound risk assessment and derivation of environmental quality standards for lead (Pb) in the freshwater environment are hampered by insufficient data on chronic toxicity and bioavailability to unicellular green algae. Here, we first performed comparative chronic (72-h) toxicity tests with three algal species in medium at pH 6, containing 4 mg fulvic acid (FA)/L and containing organic phosphorous (P), i.e. glycerol-2-phosphate, instead of PO4(3-) to prevent lead-phosphate mineral precipitation. Pseudokirchneriella subcapitata was 4-fold more sensitive to Pb than Chlorella kesslerii, with Chlamydomonas reinhardtii in the middle. The influence of medium physico-chemistry was therefore investigated in detail with P. subcapitata. In synthetic test media, higher concentrations of fulvic acid or lower pH protected against toxicity of (filtered) Pb to P. subcapitata, while effects of increased Ca or Mg on Pb toxicity were less clear. When toxicity was expressed on a free Pb(2+) ion activity basis, a log-linear, 260-fold increase of toxicity was observed between pH 6.0 and 7.6. Effects of fulvic acid were calculated to be much more limited (1.9-fold) and were probably even non-existent (depending on the affinity constant for Pb binding to fulvic acid that was used for calculating speciation). A relatively simple bioavailability model, consisting of a log-linear pH effect on Pb(2+) ion toxicity linked to the geochemical speciation model Visual Minteq (with the default NICA-Donnan description of metal and proton binding to fulvic acid), provided relatively accurate toxicity predictions. While toxicity of (filtered) Pb varied 13.7-fold across 14 different test media (including four Pb-spiked natural waters) with widely varying physico-chemistry (72h-EC50s between 26.6 and 364 µg/L), this bioavailability model displayed mean and maximum prediction errors of only 1.4 and 2.2-fold, respectively, thus indicating the potential usefulness of this bioavailability model to reduce uncertainty in site-specific risk assessment. A model-based comparison with other species indicated that the sensitivity difference between P. subcapitata and two of the most chronically Pb-sensitive aquatic invertebrates (the crustacean Ceriodaphnia dubia and the snail Lymnaea stagnalis) is strongly pH dependent, with P. subcapitata becoming the most sensitive of the three at pH > 7.4. This indicates that inter-species differences in Pb bioavailability relationships should be accounted for in risk assessment and in the derivation of water quality criteria or environmental quality standards for Pb. The chronic toxicity data with three algae species and the bioavailability model presented here will help to provide a stronger scientific basis for evaluating ecological effects of Pb in the freshwater environment.

Ecotoxicity of binary mixtures of Microcystis aeruginosa and insecticides to Daphnia pulex.

In aquatic ecosystems, mixtures of chemical and natural stressors can occur which may significantly complicate risk assessment approaches. Here, we show that effects of binary combinations of four different insecticides and Microcystis aeruginosa, a toxic cyanobacteria, on Daphnia pulex exhibited distinct interaction patterns. Combinations with chlorpyrifos and tetradifon caused non-interactive effects, tebufenpyrad caused an antagonistic interaction and fenoyxcarb yielded patterns that depended on the reference model used (i.e. synergistic with independent action, additive with concentration addition). Our results demonstrate that interactive effects cannot be generalised across different insecticides, not even for those targeting the same biological pathway (i.e. tebufenpyrad and tetradifon both target oxidative phosphorylation). Also, the concentration addition reference model provided conservative predictions of effects in all investigated combinations for risk assessment. These predictions could, in absence of a full mechanistic understanding, provide a meaningful solution for managing water quality in systems impacted by both insecticides and cyanobacterial blooms.

Ecotoxicity and uptake of polymer coated gold nanoparticles.

Bioconjugated gold nanoparticles (Au NPs) are a promising tool for pharmaceutical applications. However, the ecotoxicity of these types of NPs has hardly been studied. We investigated the ecotoxicity and uptake of 4-5 nm Au NPs to which two types of polymer coatings were attached. One coating was an amphiphilic polymer only and the other an amphiphilic coating to which 10 kDa polyethylene glycol chains were attached. In both 72 h algal growth inhibition tests with the alga Pseudokirchneriella subcapitata and in 24 h resazurin cytotoxicity tests with the rainbow trout gill cell line RTGill-W1, the pegylated Au NPs were found less toxic compared to the amphiphilic coated particles. No uptake or direct interaction between particles and algal cells was observed. However, uptake/adsorption in fish gill cells reached up to >10(6) particles/cell after 1 h and particles were eliminated for ≥96% after 24 h depuration. Both particle types were found within membrane enclosed vesicles in the cytoplasm of RTgill-W1 cells.

Functional characterization of four metallothionein genes in Daphnia pulex exposed to environmental stressors.

We characterized the metallothionein genes (Mt1, Mt2, Mt3, and Mt4) in Daphnia pulex on both molecular and ecotoxicological level. We therefore conducted a bioinformatical analysis of the gene location and predicted protein sequence, and screened the upstream flanking region for regulatory elements. The number of these elements and their positions relative to the start codon varied strongly among the four genes and even among two gene duplicates (Mt1A and Mt1B), suggesting different roles of the four proteins in the organisms' response to stress. We subsequently conducted a chronic 16-day exposure of D. pulex to different environmental stressors (at sublethal levels causing approximately 50% reduction in reproduction). Based on prior knowledge, we exposed them to the metals Cd, Cu, and Ni, the moulting hormone hydroxyecdysone (20E), and the oxidative stressors cyanobacteria (Microcystis aeruginosa), and paraquat (Pq). We then compared mRNA expression levels of the four Mt genes under these stress conditions with control conditions in "The Chosen One" clone (TCO), for which the full genome was sequenced and annotated. All together, the mRNA expression results under the different stress regimes indicate that different Mt genes may play different and various roles in the response of D. pulex to stress and that some (but not all) of the differences among the four genes could be related to the pattern of regulatory elements in their upstream flanking region.

The chronic toxicity of molybdate to freshwater organisms. I. Generating reliable effects data.

The European Union regulation on Registration, Evaluation, Authorization and Restriction of Chemical substances (REACH) (EC, 2006) requires the characterization of the chronic toxicity of many chemicals in the aquatic environment, including molybdate (MoO(4)(2-)). Our literature review on the ecotoxicity of molybdate revealed that a limited amount of reliable chronic no observed effect concentrations (NOECs) for the derivation of a predicted no-effect concentration (PNEC) existed. This paper presents the results of additional ecotoxicity experiments that were conducted in order to fulfill the requirements for the derivation of a PNEC by means of the scientifically most robust species sensitivity distribution (SSD) approach (also called the statistical extrapolation approach). Ten test species were chronically exposed to molybdate (added as sodium molybdate dihydrate, Na(2)MoO(4)·2H(2)O) according to internationally accepted standard testing guidelines or equivalent. The 10% effective concentrations (EC10, expressed as measured dissolved molybdenum) for the most sensitive endpoint per species were 62.8-105.6 (mg Mo)/L for Daphnia magna (21day-reproduction), 78.2 (mg Mo)/L for Ceriodaphnia dubia (7day-reproduction), 61.2-366.2 (mg Mo)/L for the green alga Pseudokirchneriella subcapitata (72h-growth rate), 193.6 (mg Mo)/L for the rotifer Brachionus calyciflorus (48h-population growth rate), 121.4 (mg Mo)/L for the midge Chironomus riparius (14day-growth), 211.3 (mg Mo)/L for the snail Lymnaea stagnalis (28day-growth rate), 115.9 (mg Mo)/L for the frog Xenopus laevis (4day-larval development), 241.5 (mg Mo)/L for the higher plant Lemna minor (7day-growth rate), 39.3 (mg Mo)/L for the fathead minnow Pimephales promelas (34day-dry weight/biomass), and 43.2 (mg Mo)/L for the rainbow trout Oncorhynchus mykiss (78day-biomass). These effect concentrations are in line with the few reliable data currently available in the open literature. The data presented in this study can serve as a basis for the derivation of a PNEC(aquatic) that can be used for national and international regulatory purposes and for setting water quality criteria. Using all reliable data that are currently available, a HC(5,50%) (median hazardous concentration affecting 5% of the species) of 38.2 (mg Mo)/L was derived with the statistical extrapolation approach.

Non-simultaneous ecotoxicity testing of single chemicals and their mixture results in erroneous conclusions about the joint action of the mixture.

The ecotoxicity of binary chemical mixtures with a common mode of action is often predicted with the concentration addition model. The assumption of concentration addition is commonly tested statistically based on results of toxicity experiments with the two single chemicals and their binary mixture. The present simulation study shows that if not all these experiments are performed simultaneously, one has a 20-80% chance of concluding synergism or antagonism while the mixture is actually additive (false positive rate). Truly synergistic or antagonistic mixtures have a 10-50% chance of being falsely categorized as additive (false negative rate). Additionally, false positive rates decrease with increasing experimental error, while false negative rates increase with increasing experimental error. Based on these results, we put forward a number of recommendations for future mixture ecotoxicity evaluation.