Gene expression profiling of three different stressors in the water flea Daphnia magna.

Microarrays are an ideal tool to screen for differences in gene expression of thousands of genes simultaneously. However, often commercial arrays are not available. In this study, we performed microarray analyses to evaluate patterns of gene transcription following exposure to two natural and one anthropogenic stressor. cDNA microarrays compiled of three life stage specific and three stressor-specific EST libraries, yielding 1734 different EST sequences, were used. We exposed juveniles of the water flea Daphnia magna for 48, 96 and 144 h to three stressors known to exert strong selection in natural populations of this species i.e. a sublethal concentration of the pesticide carbaryl, infective spores of the endoparasite Pasteuria ramosa, and fish predation risk mimicked by exposure to fish kairomones. A total of 148 gene fragments were differentially expressed compared to the control. Based on a PCA, the exposure treatments were separated into two main groups based on the extent of the transcriptional response: a low and a high (144 h of fish or carbaryl exposure and 96 h of parasite exposure) stress group. Firstly, we observed a general stress-related transcriptional expression profile independent of the treatment characterized by repression of transcripts involved in transcription, translation, signal transduction and energy metabolism. Secondly, we observed treatment-specific responses including signs of migration to deeper water layers in response to fish predation, structural challenge of the cuticle in response to carbaryl exposure, and disturbance of the ATP production in parasite exposure. A third important conclusion is that transcription expression patterns exhibit stress-specific changes over time. Parasite exposure shows the most differentially expressed gene fragments after 96 h. The peak of differentially expressed transcripts came only after 144 h of fish exposure, while carbaryl exposure induced a more stable number of differently expressed gene fragments over time.

Baseline toxic mixtures of non-toxic chemicals: "solubility addition" increases exposure for solid hydrophobic chemicals.

This study addresses the question whether hydrophobic organic chemicals exerting no toxicity at their solubility limit (saturation) can form a toxic mixture. Spiking methods generally do not allow testing exactly at saturation without introducing microcrystals. Passive dosing was thus applied to test the acute toxicity of several high melting point PAHs and their mixtures at the respective saturation levels to aquatic and terrestrial invertebrates. With the aquatic Daphnia magna, anthracene, chrysene, and benzo(a)pyrene resulted in no or limited acute toxicity (0-20%), whereas binary and tertiary mixtures of these resulted in significant acute toxicity (70-88%). Toxicity of PAHs and their mixtures could be fitted with one (sum) chemical activity-response curve in accordance with a similar mode of toxic action (i.e., concentration addition). The effective chemical activity (Ea-50) of 0.029 and the effective concentration on a lipid basis (EC(lipid, eq.)-50) of 95.7 mM were well within the range for baseline toxicity. Similar mixtures showed less toxicity to the terrestrial Folsomia candida due to steady-state body-burdens being below equilibrium partitioning levels. The results of the present study raise questions about the focus of risk assessment schemes and toxicity testing guidelines on individual substances, since apparently non-toxic chemicals might become toxic in a mixture.

Discrepancies in the acute versus chronic toxicity of compounds with a designated narcotic mechanism.

In this study, it was illustrated that even for certain simple organic compounds with a designated mode of action (MOA) (i.e. narcotic toxicity) unexpected differences in acute and chronic toxicity can be observed. In a first part of the study, species sensitivity distributions (SSDs) based on either acute or chronic toxicity data of three narcotic test compounds (methanol, ethanol and 2-propanol) were constructed. The results of the acute SSDs were as expected for narcotic compounds: rather similar sensitivity and small differences in toxicity were observed among different species. On the contrary, the chronic SSDs of methanol and ethanol indicated larger interspecies variation in sensitivity. Furthermore, the chronic toxicity trend (ethanol>methanol>2-propanol) was unexpectedly different from the acute toxicity trend (2-propanol>ethanol>methanol) and acute versus chronic extrapolation could not be successfully described for methanol and ethanol using an ACR of 10 (as suggested for narcotic compounds). In contrast to the interspecies approach in the first part of this study, the second part of the study was focused on the assessment of acute and chronic toxicity of the three test compounds in Daphnia magna, which was identified as one of the most sensitive organisms to methanol and ethanol. Here, the differences in acute and chronic toxicity trend were in accordance to the results of the SSDs. The enhancement of membrane penetration due to the small molecular size of methanol and ethanol, in combination with the higher toxicity of their respective biotransformation products were suggested as potential causes of the increased chronic toxicity. Furthermore, it was stressed that larger awareness of these irregularities in acute to chronic extrapolations of narcotic compounds is required and should receive additional attention in further environmental risk assessment procedure.

Assessment of aquatic experimental versus predicted and extrapolated chronic toxicity data of four structural analogues.

The present study was developed to assess the chronic toxicity predictions and extrapolations for a set of chlorinated anilines (aniline (AN), 4-chloroaniline (CA), 3,5-dichloroaniline (DCA) and 2,3,4-trichloroaniline (TCA)). Daphnia magna 21 d chronic experimental data was compared to the chronic toxicity predictions made by the US EPA ECOSAR QSAR tools and to acute-to-chronic extrapolations. Additionally, Species Sensitivity Distributions (SSDs) were constructed to assess the chronic toxicity variability among different species and to investigate the acute versus chronic toxicity in a multi-species context. Since chlorinated anilines are structural analogues with a designated polar narcotic mode of action, similar toxicity responses were assumed. However, rather large interchemical and interspecies differences in toxicity were observed. Compared to the other three test compounds, TCA exposure had a significantly larger impact on growth and reproduction of D. magna. Furthermore, this study illustrated that QSARs or a fixed ACR are not able to account for these interchemical and interspecies differences. Consequently, ECOSAR was found to be inadequate to predict the chronic toxicity of the anilines and the use of a fixed ACR (of 10) led to under of certain species. The experimental ACRs determined in D. magna were substantially different among the four aromatic amines (ACR of 32 for AN, 16.9 for CA, 5.7 for DCA and 60.8 for TCA). Furthermore, the SSDs illustrated that Danio rerio was rather insensitive to AN in comparison to another fish species, Phimphales promelas. It was therefore suggested that available toxicity data should be used in an integrative multi-species way, rather than using individual-based toxicity extrapolations. In this way, a relevant overview of the differences in species sensitivity is given, which in turn can serve as the basis for acute to chronic extrapolations.

Physiological and molecular effect assessment versus physico-chemistry based mode of action schemes: Daphnia magna exposed to narcotics and polar narcotics.

Structural analogues are assumed to elicit toxicity via similar predominant modes of action (MOAs). Currently, MOA categorization of chemicals in environmental risk assessment is mainly based on the physicochemical properties of potential toxicants. It is often not known whether such classification schemes are also supported by mechanistic biological data. In this study, the toxic effects of two groups of structural analogues (alcohols and anilines) with predefined MOA (narcotics and polar narcotics) were investigated at different levels of biological organization (gene transcription, energy reserves, and growth). Chemical similarity was not indicative of a comparable degree of toxicity and a similar biological response. Categorization of the test chemicals based on the different biological responses (growth, energy use, and gene transcription) did not result in a classification of the predefined narcotics versus the predefined polar narcotics. Moreover, gene transcription based clustering profiles were indicative of the observed effects at higher level of biological organization. Furthermore, a small set of classifier genes could be identified that was discriminative for the clustering pattern. These classifier genes covaried with the organismal and physiological responses. Compared to the physico-chemistry based MOA classification, integrated biological multilevel effect assessment can provide the necessary MOA information that is crucial in high-quality environmental risk assessment. Our findings support the view that transcriptomics tools hold considerable promise to be used in biological response based mechanistic profiling of potential (eco)toxicants.

Nickel response in function of temperature differences: effects at different levels of biological organization in Daphnia magna.

In this study, gene transcription profiling in combination with the assessment of systemic parameters at individual and population levels were applied to study the (toxic) effects induced through temperature stress in the presence or the absence of an additional chemical stressor (nickel) in Daphnia magna. It was illustrated that lower temperatures were mainly characterized by a reduction of growth and lipid content, while higher temperatures caused an increase of both endpoints. Many of the differentially regulated transcripts could be correlated with processes affected at higher hierarchical levels of biological organization. Gene clusters with probable roles in producing offspring (peak expression at 22°C), enhancing the metabolic rate (temperature related expression) and translational processes (increased expression at 14°C) were identified. However, it was not possible to pinpoint a specific subset of genes, exclusively responding to temperature or nickel and allowing a retrospective identification of the particular stressor. Overall, extreme temperatures caused a higher level of stress in the organisms in comparison to nickel exposure. Moreover, organisms subjected to the natural stressor appeared to be less capable of dealing with the additional chemical stressor and as a result activate or repress more gene pathways.

Bacterial gene profiling assay applied as an alternative method for mode of action classification: pilot study using chlorinated anilines.

Polar narcotic structural analogues (e.g., chlorinated anilines with a differing degree of chlorosubstitution, such as aniline, 4-chloroaniline, 3,5-dichloroaniline, and 2,3,4-trichloroaniline) are assumed to induce their toxic effects via the same predominant mode of action (MOA; membrane damage) at equitoxic exposure concentrations. In this study, a bacterial gene profiling assay consisting of 14 general stress genes was used to test this hypothesis for these four compounds. Although we found a consistent induction of membrane damage, the response cascade and the extent of the response differed among the different chemical treatments. The higher chlorosubstituted anilines also triggered significantly more genes involved in other general stress MOA classes (oxidative stress and protein perturbation). These findings illustrate that, along with the commonly used physicochemistry-based MOA categorization methods, alternative tests such as the bacterial gene profiling assay can yield valuable biological information on the MOA of a certain chemical or group of chemicals that is crucial in high-quality environmental risk assessment. In a second phase, the experimental gene profiling data sets of the chlorinated anilines were analyzed and weighed against existing data on other polar and non polar narcotic compounds to obtain a broader comparison in which the predefined chemical MOAs (narcosis and polar narcosis) were contrasted with the biological MOAs (gene expression profiles). Although additional optimization of the assay is needed, our results show that the bacterial gene profiling assay opens new perspectives for biology-based chemical grouping, thereby further enabling targeted MOA-based risk assessment.

Aquatic multi-species acute toxicity of (chlorinated) anilines: experimental versus predicted data.

Aquatic toxicity information is essential in environmental risk assessment to determine the potential hazards and risks of new and existing chemicals. Prediction and modelling techniques, such as quantitative structure activity relationships (QSAR) and species sensitivity distributions (SSDs), are applied to fill data gaps and to predict, assess and extrapolate the toxicity of chemicals. In this study, both techniques (i.e. the ECOSAR programme as QSAR tool and SSDs) were assessed for a set of polar narcotic structural analogues that differ in their degree of chloro-substitution (aniline, 4-chloroaniline, 3,5-dichloroaniline and 2,3,4-chloroaniline). The acute toxicity of these compounds was tested in one prokaryote species (Escherichia coli) and three eukaryote aquatic species (Pseudokirchneriella subcapitata, Daphnia magna and Danio rerio). Consequently, the experimental acute toxicity data were compared to the QSAR predictions made by the ECOSAR programme and compared to the species sensitivity modelling results. Large interspecies differences in sensitivity were observed (D. magna>P. subcapitata>D. rerio>E. coli). 4-Chloroaniline acted as an outlier in P. subcapitata toxicity. Whereas in D. magna, toxicity decreased rather than increased with increasing logK(ow) of the test compounds. In general, large interchemical and interspecies differences in toxicity of these relatively simple chemical structures were observed. Moreover, this species variation could not entirely be characterized by the ECOSAR tool. SSD modelling is particularly focussed on species variations and emphasis is put on protecting those species that are most affected by chemical exposure. Compared to QSARs, SSDs offer broader perspectives regarding species sensitivity ranking, however, in this study they could only be applied for aniline and 4-chloroaniline.

Mixtures of similarly acting compounds in Daphnia magna: from gene to metabolite and beyond.

Daphnia are an important and widely studied model species in ecological and toxicological studies throughout the world and an official (OECD) recommended test organism. Their small size, wide distribution and easy growth conditions make this organism ideal for functional genomics based studies, including metabolic profiling and transcriptomics. In this study we used an integrated systems approach in which transcriptomic, metabolomic and energetic responses of juvenile (4days old) daphnids were evaluated in response to exposure to two poly aromatic hydrocarbons (pyrene and fluoranthene) and binary mixtures thereof. In addition, these responses were linked to responses measured during chronic experiments (21days) assessing survival, growth and reproductive traits. Custom Daphnia magna microarrays were used to assess transcriptomic changes. Hierarchical cluster analysis did not result in a clear distinction between the single compounds suggesting similar molecular modes of action. Cluster analysis with both the single compounds and the binary mixture treatments resulted in a separation of treatments based on differences in toxic ratios rather than component differences. Changes in the metabolic profiles of the organisms were investigated using Nuclear Magnetic Resonance Spectroscopy and Gas and Liquid Chromatography Mass Spectrometry. These multivariate metabolomic datasets were analyzed with Principal Components Analysis and Partial Least Squares Discriminant Analysis. The major metabolite changes responsible for the differences observed indicated a disturbance in aminosugar metabolism in all cases. The study demonstrates the potential of 'omics' to provide screening tools for monitoring of the freshwater environment--in invertebrate species--which is reasonably rapid, cost-effective and has the potential to greatly increase the amount of information obtained from aquatic toxicology testing.

Controlling and maintaining exposure of hydrophobic organic compounds in aquatic toxicity tests by passive dosing.

The risk assessment of hydrophobic organic compounds (HOCs) in aquatic toxicity or bioconcentration tests is a challenge due to their low aqueous solubilities, sorption and losses leading to poorly defined exposure and reduced test sensitivity. Passive dosing overcomes these problems via the continual partitioning of HOCs from a dominating reservoir loaded in a biocompatible polymer such as silicone, providing defined and constant freely dissolved concentrations and eliminating spiking with co-solvents. This study characterised the performance of a passive dosing format for aquatic tests with small organism such as invertebrates and algae, consisting of PDMS silicone cast into the base of the glass test vessel. The PDMS silicone was loaded by partitioning from a methanol solution containing PAHs (logK(OW) 3.56-6.63) as model compounds, followed by removal of the methanol with water. This resulted in highly reproducible PDMS silicone HOC concentrations. When shaking, release of PAHs into aqueous solution was rapid and reproducible, and equilibrium partitioning was reached within 5h for all compounds. The buffering capacity was sufficient to maintain stable concentrations over more than 10 weeks. This format was applied in a 48h Daphnia magna immobilisation assay to test the toxicity of a range of PAHs at their aqueous solubility. D. magna immobilisation did not show a trend with aqueous solubility or hydophobicity (K(OW)) of the PAHs. However, the immobilisation data for all compounds could be fitted with one maximum chemical activity response curve. Those PAHs with the lowest maximum chemical activities resulted in no immobilisation. Naphthalene and phenanthrene showed full toxicity at aqueous solubility, and passive dosing was also used for the concentration-response testing of these compounds. The freely dissolved aqueous concentrations causing 50% immobilisation (EC-50) were 1.96 mg L(-1) for naphthalene and 0.48 mg L(-1) for phenanthrene. Therefore, passive dosing is a practical and economical means of improving the exposure of HOCs in aquatic toxicity or bioconcentration tests.