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Boldenone (17-hydroxy-androsta-1,4-diene-3-one, Bol) and boldione (androst-1,4-diene-3,17-dione, ADD), are currently listed as exogenous anabolic steroids by the World Anti-Doping Agency. However, it has been reported that these analytes can be produced endogenously. Interestingly, only for Bol a comment is included in the list on its potential endogenous origin. In this study, the endogenous origin of ADD in human urine was investigated, and the potential influence of phytosterol consumption was evaluated. We carried out a 5-week in vivo trial with both men (n=6) and women (n=6) and measured alpha-boldenone, beta-boldenone, boldione, androstenedione, beta-testosterone and alpha-testosterone in their urine using gas chromatography coupled to multiple mass spectrometry (GC-MS-MS). The results demonstrate that endogenous ADD is sporadically produced at concentrations ranging from 0.751 ng mL(-1) to 1.73 ng mL(-1), whereas endogenous Bol could not be proven. We also tested the effect of the daily consumption of a commercially available phytosterol-enriched yogurt drink on the presence of these analytes in human urine. Results from this study could not indicate a relation of ADD-excretion with the consumption of phytosterols at the recommended dose. The correlations between ADD and other steroids were consistently stronger for volunteers consuming phytosterols (test) than for those refraining from phytosterol consumption (control). Excretion of AED, bT and aT did not appear to be dependent on the consumption of phytosterols. This preliminary in vivo trial indicates the endogenous origin of boldione or ADD in human urine, independent on the presence of any structural related analytes such as phytosterols.

Little research on the epigenetic phenomenon of DNA methylation has been performed with invertebrates. However, a few studies have shown that effects of transient chemical exposure can be transferred to non-exposed generations through epigenetic inheritance. If this is a wide-spread phenomenon it may have major consequences for current ecological risk assessments. The presence of DNA methylation in Daphnia magna has only very recently been demonstrated, although not quantified. In the present study, the overall content of 5-methyl-2'-deoxycytidine (5 mdC) in the DNA of D. magna was determined. Additionally, the effect of transgenerational Zn exposure on the 5 mdC content was assessed. An optimized LC-MS-MS method proved to be very suitable for measuring overall levels of 5 mdC in relatively small samples of D. magna DNA. The average [5 mdC]/[dG] ratio ranged from 0.13% to 0.81%. A slight but significant decrease in DNA methylation was found in the offspring (F1) of Zn exposed daphnids in the parental generation. However, this effect did not propagate into the next generation (F2). The presence of DNA methylation in D. magna and the finding that different exposure histories can entail different levels of methylation, open the way for further investigations on the implications of epigenetics in aquatic toxicology.

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.

Total concentrations of metals in soil are poor predictors of toxicity. In the last decade, considerable effort has been made to demonstrate how metal toxicity is affected by the abiotic properties of soil. Here this information is collated and shows how these data have been used in the European Union for defining predicted-no-effect concentrations (PNECs) of Cd, Cu, Co, Ni, Pb, and Zn in soil. Bioavailability models have been calibrated using data from more than 500 new chronic toxicity tests in soils amended with soluble metal salts, in experimentally aged soils, and in field-contaminated soils. In general, soil pH was a good predictor of metal solubility but a poor predictor of metal toxicity across soils. Toxicity thresholds based on the free metal ion activity were generally more variable than those expressed on total soil metal, which can be explained, but not predicted, using the concept of the biotic ligand model. The toxicity thresholds based on total soil metal concentrations rise almost proportionally to the effective cation exchange capacity of soil. Total soil metal concentrations yielding 10% inhibition in freshly amended soils were up to 100-fold smaller (median 3.4-fold, n = 110 comparative tests) than those in corresponding aged soils or field-contaminated soils. The change in isotopically exchangeable metal in soil proved to be a conservative estimate of the change in toxicity upon aging. The PNEC values for specific soil types were calculated using this information. The corrections for aging and for modifying effects of soil properties in metal-salt-amended soils are shown to be the main factors by which PNEC values rise above the natural background range.

Molting in insects is regulated by ecdysteroids and juvenile hormones. Several synthetic non-steroidal ecdysone agonists are on the market as insecticides. These ecdysone agonists are dibenzoylhydrazine (DBH) analogue compounds that manifest their toxicity via interaction with the ecdysone receptor (EcR). Of the four commercial available ecdysone agonists, three (tebufenozide, methoxyfenozide and chromafenozide) are highly lepidopteran specific, one (halofenozide) is used to control coleopteran and lepidopteran insects in turf and ornamentals. However, compared to the very high binding affinity of these DBH analogues to lepidopteran EcRs, halofenozide has a low binding affinity for coleopteran EcRs. For the discovery of ecdysone agonists that target non-lepidopteran insect groups, efficient screening systems that are based on the activation of the EcR are needed. We report here the development and evaluation of two coleopteran-specific reporter-based screening systems to discover and evaluate ecdysone agonists. The screening systems are based on the cell lines BRL-AG-3A and BRL-AG-3C that are derived from the weevil Anthonomus grandis, which can be efficiently transduced with an EcR reporter cassette for evaluation of induction of reporter activity by ecdysone agonists. We also cloned the almost full length coding sequence of EcR expressed in the cell line BRL-AG-3C and used it to make an initial in silico 3D-model of its ligand-binding pocket docked with ponasterone A and tebufenozide.

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.

Cerium dioxide nanoparticles (CeO2 NPs) are increasingly being used as a catalyst in the automotive industry. Consequently, increasing amounts of CeO2 NPs are expected to enter the environment where their fate in and potential impacts are unknown. In this paper we describe the fate and effects of CeO2 NPs of three different sizes (14, 20, and 29 nm) in aquatic toxicity tests. In each standard test medium (pH 7.4) the CeO2 nanoparticles aggregated (mean aggregate size approximately 400 nm). Four test organisms covering three different trophic levels were investigated, i.e., the unicellular green alga Pseudokirchneriella subcapitata, two crustaceans: Daphnia magna and Thamnocephalus platyurus, and embryos of Danio rerio. No acute toxicity was observed for the two crustaceans and D. rerio embryos, up to test concentrations of 1000, 5000, and 200 mg/L, respectively. In contrast, significant chronic toxicity to P. subcapitata with 10% effect concentrations (EC10s) between 2.6 and 5.4 mg/L was observed. Food shortage resulted in chronic toxicity to D. magna, for wich EC10s of > or = 8.8 and < or = 20.0 mg/L were established. Chronic toxicity was found to increase with decreasing nominal particle diameter and the difference in toxicity could be explained by the difference in surface area. Using the data set, PNEC(aquatic)S > or = 0.052 and < or = 0.108 mg/L were derived. Further experiments were performed to explain the observed toxicity to the most sensitive organism, i.e., P. subcapitata. Toxicity could not be related to a direct effect of dissolved Ce or CeO2 NP uptake or adsorption, nor to an indirect effect of nutrient depletion (by sorption to NPs) or physical light restriction (through shading by the NPs). However, observed clustering of NPs around algal cells may locally cause a direct or indirect effect.

Most of the research on the epigenetic phenomenon of DNA methylation has been performed with vertebrates and plants. Knowledge on DNA methylation in Daphnia magna, a key test organism in aquatic toxicology, is completely lacking. Through epigenetic inheritance, effects of transient chemical exposure could be transferred to non-exposed generations, which could have a major impact on ecological risk assessment procedures. In this study, we determined if CpG methylation occurs in D. magna and if this can be influenced by exposure to toxic substances. Homologs of human DNA methyltransferases DNMT1, DNMT2 and DNMT3A were found in the partially available D. magna genome. Using an optimized "Amplification of Intermethylated Sites (AIMS)" technique, two methylated fragments were discovered in D. magna DNA. No homology was found for these sequences. The methylation and the D. magna origin of the fragments were confirmed with Southern analysis. This optimized AIMS technique was then applied to DNA of D. magna which were exposed to 180 microg/L Cd for two generations. Exposure resulted in a significant decrease in reproduction. The same methylated fragments with the same band intensity were observed in DNA of both non-exposed and exposed daphnids. As such, it could not be demonstrated that Cd exposure altered DNA methylation. However, the presence of DNA methylation in D. magna shows that potentially epigenetic effects may occur in this species.

The major research questions addressed in this study were (i) whether green microalgae living in soft water (operationally defined water hardness<10mg CaCO(3)/L) are intrinsically more sensitive to Ni than green microalgae living in hard water (operationally defined water hardness >25mg CaCO(3)/L), and (ii) whether a single bioavailability model can be used to predict the effect of water hardness on the toxicity of Ni to green microalgae in both soft and hard water. Algal growth inhibition tests were conducted with clones of 10 different species collected in soft and hard water lakes in Sweden. Soft water algae were tested in a 'soft' and a 'moderately hard' test medium (nominal water hardness=6.25 and 16.3mg CaCO(3)/L, respectively), whereas hard water algae were tested in a 'moderately hard' and a 'hard' test medium (nominal water hardness=16.3 and 43.4 mg CaCO(3)/L, respectively). The results from the growth inhibition tests in the 'moderately hard' test medium revealed no significant sensitivity differences between the soft and the hard water algae used in this study. Increasing water hardness significantly reduced Ni toxicity to both soft and hard water algae. Because it has previously been demonstrated that Ca does not significantly protect the unicellular green alga Pseudokirchneriella subcapitata against Ni toxicity, it was assumed that the protective effect of water hardness can be ascribed to Mg alone. The logK(MgBL) (=5.5) was calculated to be identical for the soft and the hard water algae used in this study. A single bioavailability model can therefore be used to predict Ni toxicity to green microalgae in soft and hard surface waters as a function of water hardness.

In this study, increasing Mg concentrations and decreasing pH were observed to decrease Ni toxicity to the green alga Pseudokirchneriella subcapitata. To investigate to what extent the original biotic ligand model (BLM) concept could explain Ni toxicity as a function of water chemistry, the protective effects of Mg(2+) and H(+) were modeled as BLM-type single-site competition effects. The model parameters representing these effects were log K(MgBL)=3.3 and log K(HBL)=6.5. The BLM was capable of predicting Ni toxicity by an error of less than a factor of 2 in most synthetic and natural waters used in this study. However, since the relationship between 72-h E(r)C50(Ni(2+)) (i.e. the 72-h E(r)C50 expressed as Ni(2+) activity) and H(+) activity was not linear over the entire tested pH range, only the 'linear part' between pH 6.45 and 7.92 was used for derivation of log K(HBL). This nonlinearity indicates that the effect of pH can probably not be attributed to H(+) competition with Ni(2+) for a single site alone. When modeling the effect of pH as a linear relation between 72-h E(r)C50(pNi(2+)) (=-log (72-h E(r)C50(Ni(2+)) corrected for the presence of Mg)) and pH, the applicability of the model was successfully extended to pH levels as low as 6.01. This type of empirical model has also been used in our previous studies on the development of a chronic Ni bioavailability model for Daphnia magna and a long-term Ni bioavailability model for rainbow trout. Finally, we could not detect a statistically significant interactive effect of pH and Mg on the toxicity of Ni(2+) to P. subcapitata and this is in line with the formulation of our empirical model.

Most ecological effect assessment methodologies use effect concentrations derived from single-species testing (ECx,single-species-test) as the basis to estimate 'safe' environmental concentrations (such as environmental quality criteria). Here, we examined to what extent such ECx,single-species-test are representative for population-level effect concentrations in a community setting (ECx,multi-species-test). Data from USEPA's ECOTOX database revealed the existence of considerable scatter around the relationship between ECx,single-species-test (endpoint: mortality) and ECx,multi-species-test (endpoint: population abundance). However, we demonstrate that this scatter is reduced when ECx,single-species-test and ECx,multi-species-test are determined simultaneously and by the same research group. Indeed, if these conditions are fulfilled, the quotient of both ECx values for invertebrates approaches 1 for chemicals that directly target invertebrates. Unfortunately, comparable data for other classes of chemicals and/or taxonomic groups were not found. However, theoretical ecosystem model simulations, which confirmed the results based on the above-mentioned analysis of the ECOTOX database, indicated that for phytoplankton, EC10,single-species-test>EC10,multi-species-test, for chemicals that directly target invertebrates. For chemicals that directly target phytoplankton, the ecosystem model simulations suggest that ECx,single-species-test>ECx,multi-species-test for both phytoplankton and invertebrates. Hence, our observation based on the analysis of existing experimental data that the ECx,single-species-test is similar to the ECx,multi-species-test may be biased by the fact that only data were available for invertebrates and for chemicals targeting invertebrates. Experimental research is required to test the predictions made by the model simulations for phytoplankton as well as for chemicals directly targeting phytoplankton.

Calcium is involved in a wide variety of biological processes and has an important structural role in crustaceans. The present study aimed at exploring the possible link between Ca body concentrations and the ingestion rate and the role of soft tissue vs. total tissue Ca accumulation in Daphnia magna. D. magna was cultured for 21 days at different water Ca concentrations ranging from 3.4 to 32.5 mg/L. Every week Ca body concentrations (soft and total tissues), ingestion rate, growth, survival and reproduction were measured. Daily, algal food that was not deficient in Ca was supplied. Ca in the soft tissues represented 8 to 26% of the total Ca body concentrations. The ratio Ca in soft tissue/Ca in total tissue was generally not influenced by the Ca exposure concentration but decreased with time, i.e., age (from an average of 0.24 at day 7 to 0.09 at day 21). During week 1, a 54% decrease in Ca body concentrations was observed in daphnids exposed in medium with 3.4 mg/L Ca compared to those exposed to 32.5 mg/L. The concurrent decrease in ingestion rate was 14%. No significant differences among Ca treatments were observed during week 2 for ingestion rate and week 3 for calcium body concentrations. Also, no effects on growth and reproduction were observed, although these were expected at the lowest Ca concentration tested. It is hypothesised that Ca absorption from food in combination with an increased ingestion rate is used to maintain Ca homeostasis under Ca limiting conditions.

We exposed Daphnia magna for 21 days to dietary Zn, incorporated in a diet of the green alga Pseudokirchneriella subcapitata at 720 microg Zn/g dry wt and compared its response to D. magna fed with a control diet (116 microg Zn/g dry wt). Exposure to dietary Zn resulted in an increased body burden of D. magna (93.7 microg/g dry wt vs. 61.3 microg/g dry wt in the control) but did not affect survival, growth, or feeding rate. Only reproduction was significantly reduced from the 2nd brood onwards. Gene expression analysis, using microarray analysis and RT-PCR, showed that dietary Zn exposure resulted in the differential expression of several genes involved in molting-associated processes (i.e., chitin binding, cuticle metabolism), especially after 6 days of exposures (but not after 13 or 21 days of exposure). Monitoring of time to molt and intermolt-period confirmed this molting effect at the organism level in the first week of exposure. The data suggest a possible link between Zn-induced effects on molting-related processes and reproductive inhibition, but this link is only obvious for effects on the 2nd brood size and not for later broods. Reproductive inhibition in later broods may also be explained by a disturbed mitochondrial function, but more research is clearly needed to give a more definitive integrated explanation of the observed effects at the molecular and organism level.

In the present study, the individual effects of Ca, Mg, and pH on the chronic toxicity of Ni to Daphnia magna were examined in a series of 21-d reproduction tests in synthetic test solutions. Based on the linear increase of 21-d median effective concentrations expressed as Ni2+ activity (21-d EC50Ni2+) with increasing activities of Ca2+ and Mg2+, the effects of Ca and Mg were modeled according to single-site competition with log KCaBL = 3.53 and log KMgBL = 3.57 (BL = biotic ligand). Because the increase of 21-d EC50Ni2+ with increasing H+ activity was nonlinear, the effect of pH could not be described appropriately by single-site competition between Ni2+ and H+. Instead, the effect of pH was modeled based on an empirical linear relationship between pH and 21-d EC50pNi2+* (equal to -log [21-d EC50Ni2+ corrected for the presence of Ca and Mg]) and was superimposed on the effects of Ca and Mg. For all test solutions used for model development, the developed model predicted the observed 21-d EC50 expressed as dissolved Ni concentration with an error of less than a factor of two. The importance of dissolved organic carbon in protecting D. magna against chronic Ni toxicity was demonstrated by conducting 21-d reproduction tests in a series of Ni-spiked natural waters. Because the model tended to systematically overestimate chronic Ni toxicity in these natural waters, it was further optimized to yield more accurate predictions in natural waters. Although some room still exists for improvement, the developed model is, to our knowledge, the first to present a useful tool for assessing the risk of Ni to aquatic invertebrates.

To date, (eco)toxicological information on industrial nanoparticles is very limited. In the present study, the hypothesis that the ecotoxicity of nanoparticles (NPs) is related to their surface area and not to their mass was tested using a freshwater green algal species. Particle diameter and morphology were assessed using light scattering and electron microscopy techniques. To assess the toxicity of silica (SiO2) nanoparticles, the growth inhibition of the alga Pseudokirchneriella subcapitata when exposed to stable silica suspensions was monitored. Commercial LUDOX suspensions of nanoparticles with 12.5 and 27.0 nm diameter were found to be toxic, with 72-h 20% effect concentrations for growth rate (E(r)C20) values +/- standard deviation (n = 5) of 20.0 +/- 5.0 and 28.8 +/- 3.2 mg/L, respectively. The toxicity was attributable to the solid nanospheres, because no aggregation was observed and dissolution of the nanoparticles was negligible. When expressing the concentration as a surface area, the difference in toxicity was not significant. In the latter case, 72-h E(r)C20 values +/- standard deviation (n = 5) were 4.7 +/- 1.2 and 3.9 +/- 0.4 m2/L. Silica bulk material was found to be nontoxic up to 1 g/L. In an additional experiment with 100 mg/L of 12.5 and 27.0 nm SiO2 NPs, the interaction between the nanoparticles and algal cells was studied using transmission electron microscopy. Although the particles clearly adhered to the outer cell surface, no evidence was found for particle uptake.

The ecological risk assessment and the development of water-quality criteria for Co are currently still hampered by insufficient knowledge about the toxicity of Co to freshwater organisms. A relevant group of organisms, for which no toxicity data with Co are available, is the class of the herbivorous pulmonate freshwater snails, which fulfil a pivotal role in the consumption and decomposition of aquatic plants and epihyton. We measured the growth rate of the pond snail Lymnaea stagnalis chronically exposed for 28 days to a series of Co concentrations. The no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) for growth rate were 26 and 79 microg Co/L, respectively. Growth rate of snails exposed to 79 microg Co/L and higher concentrations was more impaired in the final 2 weeks of exposure than in the first 2 weeks of exposure. The reduced growth rate at 79 microg Co/L was accompanied by a reduced concentration of Ca in the haemolymph at the end of the exposure. Possible mechanisms of toxicity of Co to snail growth were suggested to be an impairment of Ca uptake and homeostasis and/or feeding inhibition. Although additional research is needed to investigate the relative importance of these mechanisms, as well as the interrelatedness between them, the toxicity data currently presented can assist in risk assessment and water-quality criteria development.

Although a large body of evidence indicates that metal toxicity to soil organisms is affected by physicochemical soil properties, use of this knowledge in ecological risk assessments is limited because of the lack of a model applicable to a wide range of soils. To study the effect of soil characteristics on the toxicity of copper to terrestrial invertebrates, chronic toxicity tests with Eisenia fetida and Folsomia candida were performed in 19 European field soils. These soils were carefully selected to cover the range of toxicity-influencing parameters encountered in the European Union. Toxicity values varied greatly among soils, with 28-d median effect concentrations ranging from 72.0 to 781 mg Cu/kg dry weight for E. fetida and from 45.4 to 2,270 mg Cu/kg dry weight for F. candida. For both species, variation in copper toxicity values was best explained by differences in the actual cation-exchange capacity (CEC) at soil pH. Using the obtained regression algorithms, the observed toxicity could, in most cases, be predicted within a factor of two for E. fetida and within a factor of three for F. candida. The developed models were validated in three additional European field soils, a standard artificial soil and a standard field soil. The presented regression equations, based on the actual CEC, offer an easy-to-apply method for taking the influence of soil properties on metal toxicity into account.

In the present study the induction of metallothioneins (MTs) and its relation to cytosolic metal concentrations (Zn, Cu and Cd) in the euryhaline crustacean Neomysis integer exposed to Cd at different salinities was studied. N. integer was exposed to the same free cadmium ion activity of 5.74 x 10(-9) mol l(-1) (i.e. 1/5 of the 96 h LC(50) value expressed as cadmium activity) in hypo-osmotic (5 psu), isosmotic (16 psu) and hyper-osmotic media (25 psu) for 7 days. In this way, the effect of salinity on cadmium speciation was eliminated and therefore the physiological effect of salinity on Cd accumulation and MT induction could be studied. The accumulation of cytosolic Cd in N. integer changed with salinity from 1.11+/-0.05 micromol l(-1) at 5 psu up to 1.43+/-0.17 micromol l(-1) at 25 psu. This could indicate that the physiological response of euryhaline estuarine invertebrates like N. integer to salinity changes can influence the rate of trace metal uptake from solution. While the salinity changes did not cause significant differences in cytosolic Zn concentrations (mean value of all tested salinities: 34.4+/-2.8 micromol l(-1)), an inverse relationship between salinity and cytosolic Cu concentration was observed. The highest concentration of 15.7+/-2.3 micromol Cul(-1) was determined at 5 psu and the lowest 10.9+/-1.4 micromol Cul(-1) at 25 psu. This could point to a possible relationship between the copper concentration and the hemocyanin metabolism in N. integer. This is the first time that differential pulse voltammetry method was applied to MT assays with N. integer. Although the exposure to Cd resulted in a higher Cd cytosolic concentration, no subsequent MT increase was detected. The significant positive correlation between MT levels and cytosolic Cu concentrations (Spearman correlation coefficient r(s)=0.356, p=0.009) implies a strong relationship between MT and Cu in N. integer.

The effects of Ca, Mg, Na and pH on the acute toxicity of Ni to Daphnia magna were investigated in a series of 48-h immobilization assays in synthetic test solutions. Both Ca and Mg reduced Ni toxicity, while Na did not. Ni toxicity was not affected in the pH range of 5.7-7.5, but a further increase of pH up to 8.1 resulted in an increase of toxicity of the free Ni2+ ion. Based on the results of these experiments, a biotic ligand model (BLM) was developed in which the effects of Ca and Mg were modeled as single-site competition effects. Stability constants representing the binding strength between Ca2+ and Mg2+ and the biotic ligand (BL) were logK(CaBL)=3.10 and logK(MgBL)=2.47, respectively. The effect of pH could not be appropriately described by single-site competition between Ni2+a nd H+. Since the overall variation of toxicity within the tested pH range was relatively small, we decided not to incorporate the effect of pH in the current model. The model was able to predict 48-h EC50s in all synthetic test solutions by an error less than factor 2. The model's predictive capacity was also evaluated using results of toxicity tests in Ni-spiked natural surface waters. For 15 out of 16 tested waters, 48-h EC50s were predicted by an error less than factor 2. Additionally, after calibration to account for interclonal or interspecies sensitivity differences, the model was able to accurately predict earlier published 48-h EC50s for another D. magna clone as well as for Ceriodaphnia dubia. Finally, the predictive capacity of the model was demonstrated to be better than that of previously proposed models that include a logK(NaBL), a logK(HBL) and a logK(CaBL), but did not incorporate a logK(MgBL). An in-depth comparison of these models learned that (i) there is no need to incorporate a logK(NaBL), (ii) it is important to recognize the protective effect of Mg, and (iii) the incorporation of a logK(HBL) does not adequately describe the effect of pH. Although our model seems very promising, further research, especially into the effects of elevated pH and alkalinity levels, is needed to allow further refinement.

The species sensitivity distribution, a technique currently used to derive water-quality standards of chemicals, is associated with a set of inadequately tested assumptions. One of these assumptions is that ecosystem structure is as or more sensitive than ecosystem function, i.e., that structure is the target of concern. In this paper, we tested this assumption for a simple freshwater ecosystem exposed to different toxicants. Using an ecosystem model, we calculated no observed effect concentrations (NOECs) for ecosystem structure (ecosystem structure-NOECs) and function (ecosystem function-NOECs) for each of 1000 hypothetical toxicants. For 979 of these toxicants, the ecosystem structure-NOEC was lower than or equal to the ecosystem function-NOEC, indicating that the tested assumption can be considered valid. For 239 of these 979 toxicants, both NOECs were equal. For half of the 1000 toxicants, the structure of lower trophic levels (i.e., phytoplankton) appears to be more sensitive than the structure of higher trophic levels (i.e., fish). As such, ecosystem structure-NOECs are primarily determined by the sensitivity of the structure of lower trophic levels. In contrast, ecosystem functions associated with higher trophic levels (e.g., total ingestion by fish) are more sensitive than functions associated with lower trophic levels (e.g., total photosynthesis by phytoplankton) for 749 toxicants.

Species sensitivity distributions (SSDs) are statistical distributions which extrapolate single-species toxicity test results to ecosystem effects. This SSD approach assumes that ecological interactions between populations, such as grazing and competition, do not influence the sensitivity of ecosystems. The validity of this assumption in a simple freshwater pelagic ecosystem was tested using ecosystem modelling. For each of a 1000 hypothetical toxicants, a lognormal SSD was fitted to chronic single-species EC10s of the species present. As such, these distributions did not account for ecological interactions and were therefore termed 'conventional SSDs' (cSSDs). Next, sensitivity distributions that did take into account ecological interactions were constructed (eco-SSD) for the same 1000 toxicants, using an ecosystem model. For 254 of the 1000 hypothetical toxicants, mean and/or variance of the cSSD were significantly higher than mean and/or variance of the eco-SSD, as such rejecting the general validity of the tested assumption. A classification tree approach indicated that especially toxicants which directly affect phytoplankton (i.e. herbicides) may have a higher mean for cSSD than for eco-SSD. Conversely, means of eco-SSD and cSSD tend to be equal for toxicants directly affecting zooplankton and fish, e.g. insecticides. For the 254 hypothetical toxicants for which the tested assumption was false, a predicted no effect concentration (PNEC) calculated as the lowest single-species EC10 divided by an application factor of 10 was on average a factor 10 lower than the corresponding ecosystem-NOEC calculated by the ecosystem model.

In ecotoxicology, derivation of a "safe" environmental concentration is usually achieved by the use of extrapolation factors or by statistical extrapolation from a set of single species toxicity data. These approaches ignore ecological interactions between species in the field. An ecology-based alternative to this pragmatic approach can be ecosystem modelling, which can account for ecological interactions. However, it is largely unexplored how well the predictions of these models quantitatively agree with large-scale experimental studies. Therefore, we evaluated the capacity of a flexible ecosystem model to predict population and ecosystem-level no observed effect concentrations (NOECs) of 7 organic toxicants. These NOECs were compared with population and ecosystem-NOECs observed in 11 micro- and mesocosm studies. For each of the latter studies, the model was customized to account for the specific ecological interactions within these systems and combined with appropriate single-species toxicity data from literature. Population-NOEC predictions were accurate, or at least protective, for 60, and 85% of all considered model populations, respectively. For all 11 studies, a protective ecosystem-NOEC could be derived, being accurate in 7 cases, and conservative in 4 cases. In general, it can be stated that this type of models can serve as an ecology-based alternative to current extrapolation techniques in EEAs and water quality standard setting.

Ecosystem models, combining a food web model with a toxic effect sub-model, have been proposed to incorporate ecological interactions in ecological effect assessments. Toxic effect sub-models in different studies tend to differ in (1) the used single-species toxicity data, (2) the effects they consider, (3) the concentration-effect function used. In this paper, we constructed four ecosystem models, each with a different toxic effect sub-model, and tested their capacity to predict biomass changes, and no observed effect concentrations (NOECs) established in an experimental microcosm. For most populations, these predictions depended heavily on the type of ecosystem model. The ecosystem model with a toxic effect sub-model incorporating mortality effects using a logistic concentration-effect function made accurate predictions for most populations. Additional incorporation of sub-lethal effects did not result in better predictions. Ecosystem models using linear concentration-effect functions predict biomass decreases at concentrations that are four times lower than the observed NOECs.

In order to study the effect of anthropogenic substances on freshwater and marine ecosystems and to develop methods to derive water-quality criteria, ecotoxicological testing is required. While toxicity assessments are traditionally based on dissolved metal concentrations, assuming that toxicity is caused by waterborne metal only, it was recently pointed out that also the dietary exposure route should be carefully considered and interpreted in regulatory assessments of zinc. In this context, the aim of this experimental study was to develop a method which allows the uptake of waterborne and dietary zinc by Daphnia magna and the interaction between both exposure routes to be studied. Therefore, the setup of a dual isotopic tracer study was required. During several days, daphnids were exposed to 67Zn and 68Zn via the dietary and the waterborne routes, respectively, and after several time intervals the daphnids were sampled and subjected to isotopic analysis by means of inductively coupled plasma mass spectrometry (ICP-MS). In order to obtain reliable and accurate results for zinc, special care was taken to prevent contamination and to deal with the spectral interferences traditionally hindering the determination of zinc. The figures of merit of both a quadrupole-based ICP-MS instrument equipped with a dynamic reaction cell, and a sector field ICP-MS unit were studied, and it was concluded that by using a sector field mass spectrometer operated at medium mass resolution all interferences could be overcome adequately. Although the set-up of the exposure experiments seems to be rather simple at first sight, it was shown in this work that several (dynamic) variables can have an influence on the results obtained and on the subsequent data interpretation. The importance of these confounding factors was examined, and on the basis of preliminary calculations it became clear that not only the isotopic composition of the daphnids has to be studied--adequate monitoring of the isotopic composition of the dissolved phase and the algae during the exposure of the daphnids is also required to accurately discriminate between uptake from water and from food.

Five benthic organisms commonly used for sediment toxicity testing were chronically (28 to 35 days) exposed to copper in standard laboratory-formulated sediment (following Organization for Economic Cooperation and Development guidelines) and lethal and sub-lethal toxicities were evaluated. Sub-lethal endpoints considered were reproduction and biomass production for Lumbriculus variegatus, growth and reproduction for Tubifex tubifex, growth and emergence for Chironomus riparius, and growth for Gammarus pulex and Hyalella azteca. Expressed on whole-sediment basis the observed lethal sensitivity ranking (from most to least sensitive) was: G. pulex>L. variegatus>H. azteca=C. riparius=T. tubifex, with median chronic lethal concentrations (LC50) between 151 and 327 mg/kg dry wt. The sub-lethal sensitivity ranking (from most to least sensitive, with the most sensitive endpoint between parentheses): C. riparius (emergence)>T. tubifex (reproduction)=L. variegatus (reproduction)>G. pulex (growth)>H. azteca (growth), with median effective concentrations (EC50) between 59.2 and 194 mg/kg dry wt. No observed effect concentrations (NOEC) or 10% effective concentrations (EC10) for the five benthic invertebrates were used to perform a preliminary risk assessment for copper in freshwater sediment by means of (a) the "assessment factor approach" or (b) the statistical extrapolation approach (species sensitivity distribution). Depending on the data (NOEC or EC10) and the methodology used, we calculated a Predicted No Effect Concentration (PNEC) for sediment between 3.3 and 47.1 mg Cu/dry wt. This range is similar to the range of natural (geochemical) background concentrations of copper in sediments in Europe, i.e. 90% of sediments have a concentration between 5 and 49 mg Cu/kg dry wt. A detailed analysis of the outcome of this preliminary exercise highlighted that multiple issues need to be explored for achieving a scientifically more sound risk assessment and for the development of robust sediment quality criteria for copper, including (i) the use of the assessment factor approach vs. the statistical extrapolation approach, (ii) the importance of bioavailability modifying factors (e.g., organic carbon, acid volatile sulfide), and (iii) the influence of prevailing geochemical (bioavailable) background concentrations on the copper sensitivity of local benthic biota.

Standardized toxicity tests are generally performed with juvenile test organisms, e.g., in Daphnia magna assays neonates<24 h old are used. The purpose of this research was to investigate the influence of a delayed exposure to Cu and Zn on population parameters and toxicity values derived from these endpoints. Juveniles (<24 h; T0) and 7-d old daphnids (T7) were exposed for 21 and 14 d, respectively. For Cu, juveniles were significantly more sensitive than 7d old organisms following acute (48 h) as well as chronic (14 d) exposure. After 14 d of exposure to 130 microg/L Cu, mortality was 80% and 10% in T0 and T7, respectively. Juveniles per surviving female at this concentration decreased by 78% and 14% compared to the control. 14 d-NOEC and LOEC values (based on juveniles per surviving female) were 75 and 90 microg/L Cu for T0 and both>130 microg/L for T7. For Zn, survival in T0 and T7 was similar. Although T7 organisms produced significantly more offspring, 14 d-NOEC and LOEC values were equal to those of T0, i.e., 80 and 115 microg/L, respectively. For Cu as well as for Zn effect concentrations based on 14 and 21 d exposure were similar (results from T0). It can be concluded that acute and chronic toxicity data obtained from juvenile D. magna are more sensitive or equally sensitive than obtained from 7d old organisms. As differences are observed between the two metals extrapolations of these conclusions to other toxicants and other aquatic species cannot be made without further investigation.

The extent to which Ca(2+), Mg(2+), Na(+), K(+) ions and pH independently influence copper toxicity to barley (Hordeum vulgare) was assessed by measuring root growth in nutrient solutions. Increased Ca(2+) activity resulted in a sixfold decrease in [EC50(cu2+)] values, while a positive relationship between the cation activity and the EC50 was expected. Increased Mg(2+) activity resulted in a twofold increase in [EC50(cu2+)] values. Na(+), K(+) and H(+) activities did not significantly affect Cu(2+) toxicity. The obtained results indicated that competition for binding sites between Cu(2+) and cations such as Ca(2+), Mg(2+), Na(+), K(+) and H(+) is not an important factor in determining Cu(2+) toxicity to H. vulgare. However, the EC50s could, with one exception, be predicted within a factor three based on the free Cu(2+) activity, indicating that the free Cu(2+) activity cannot only be used to predict metal toxicity to aquatic, but also to terrestrial organisms.

The aims of the present study were to investigate (1) whether cladocerans living in soft water (operationally defined hardness < 10 mg CaCO(3)/L) are intrinsically more sensitive to Ni than cladocerans living in hard water (operationally defined hardness > 25 mg CaCO(3)/L) and (2) whether a single bioavailability model can be used to predict the protective effect of water hardness on the toxicity of Ni to cladocerans in both soft and hard water. To address these research questions, acute and chronic bioassays were conducted with 10 different cladoceran species collected in soft and hard water lakes in Sweden. Soft water organisms were tested in a 'soft' and a 'moderately hard' test water (nominal hardness = 6.25 and 16.3 mg CaCO(3)/L, respectively). Hard water organisms were tested in a 'moderately hard' and a 'hard' test water (nominal hardness = 16.3 and 43.4 mg CaCO(3)/L, respectively). The results of the toxicity tests in the 'moderately hard' test water revealed no significant differences between the intrinsic sensitivity of soft versus hard water organisms. Modeling exercises indicated that water hardness significantly reduced Ni toxicity to both the soft and the hard water organisms tested. Although predictions of chronic toxicity were sufficiently accurate using the same logK(CaBL) and logK(MgBL) (i.e. the model parameters describing the effect of hardness) for all organisms under consideration, predictions of acute toxicity were significantly more accurate when separate logK(CaBL) and logK(MgBL) values were derived for the soft and the hard water organisms tested. This is due to the fact that the relative decrease of acute Ni toxicity to soft water organisms in 'moderately hard' compared to 'soft' test water was significantly higher than for hard water organisms in 'hard' compared to 'moderately hard' test water.

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.

Controlled flooding of lowlands is considered as a potential water management strategy to minimize the risk of flooding of inhabited areas during high water periods. However, due to industrial activities, river water, sediments and soils are often contaminated with metals which may have adverse effects on the ecosystem's structure and functioning. Additionally, salinity may greatly affect the bioavailability and toxicity of metals present or imported into these systems. The effect of contaminated soils under different flooding and salinity exposure scenarios on the growth, reproduction and metal accumulation in the oligochaete Tubifex tubifex (Müller, 1774) were examined. In these bioassays metal contaminated soils were flooded with water of different salinities (0 and 3 psu), and tested after 0, 6 and 12 months of permanent inundation. We indeed found that inundation time had significant decreasing effects on Cu and Zn accumulation; although initial accumulation of Cu and Zn was higher in the previously unflooded soil at the start of the flooding treatment, these differences seem to disappear after 6 months of permanent inundation. Moreover, the complex interaction between substrate type and salinity suggests that redox potential is probably of major importance.

As part of the Endis-Risks project, the current study describes the occurrence of the chlorotriazine pesticides atrazine, simazine and terbutylazine in water, sediment and suspended matter in the Scheldt estuary (B-Nl) from 2002 to 2005 (3 samplings a year, 8 sampling points). Atrazine was found at the highest concentrations, varying from 10 to 736 ng/l in water and from 5 up to 10 ng/g in suspended matter. Simazine and terbutylazine were detected at lower concentrations. Traces of the targeted pesticides were also detected in sediments, but these were below the limit of quantification. As part of an ecotoxicological assessment, we studied the potential effect of atrazine on molting of Neomysis integer (Crustacea:Mysidacea), a resident invertebrate of the Scheldt Estuary and a proposed test organism for the evaluation of endocrine disruption. Following chronic exposure ( approximately 3 weeks), atrazine did not significantly affect mysid molting at environmentally relevant concentrations (up to 1 microg/l).

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.

Choline saccharinate and choline acesulfamate are two examples of hydrophilic ionic liquids, which can be prepared from easily available starting materials (choline chloride and a non-nutritive sweetener). The (eco)toxicity of these ionic liquids in aqueous solution is very low in comparison to other types of ionic liquids. A general method for the synthesis and purification of hydrophilic ionic liquids is presented. The method consists of a silver-free metathesis reaction, followed by purification of the ionic liquid by ion-exchange chromatography. The crystal structures show a marked difference in hydrogen bonding between the two ionic liquids, although the saccharinate and the acesulfamate anions show structural similarities. The optimized structures, the energetics, and the charge distribution of cation-anion pairs in the ionic liquids were studied by density functional theory (DFT) and second-order (Møller-Plesset) perturbation theory calculations. The occupation of the non-Lewis orbitals was considered to obtain a qualitative picture of the Lewis structures. The calculated interaction energies and the dipole moments for the ion pairs in the gas phase were discussed.

The effects of Ca, Mg and pH on the toxicity of Ni to juvenile rainbow trout (Oncorhynchus mykiss) were examined during 17-26-day exposures to Ni in 15 synthetic test solutions. Higher chemical activities of Ca2+, Mg2+ and H+ reduced Ni toxicity, as demonstrated by increased 17-day median lethal concentrations expressed as Ni2+ activity (17-d LC50(Ni2+)). A non-linear increase of the 17-d LC50(Ni2+) with increasing H+ suggested that the effect of pH could not be appropriately described by single-site competition between Ni(2+) and H+ for sensitive sites on the fish gill. Instead, a linear increase of pNi2+ (=-log 17-d LC50(Ni2+)) with increasing pH was observed with a slope of 0.32. This slope was used as the basis for modelling the effect of pH. The effects of Ca and Mg were modelled according to single-site competition with logK(CaBL)=logK(MgBL)=3.6, both assumed to be independent of pH. The effect of pH was superimposed on this competition effect and was also assumed to be independent of Ca and Mg concentrations. The model was able to predict 17-d LC50s (expressed as dissolved Ni) in most synthetic test waters within a factor 2 deviation from observed toxicity. The model's predictive capacity was also evaluated using results of similar laboratory toxicity tests with juvenile rainbow trout in Ni-spiked European natural surface waters. For most of these waters, predicted 17-d LC50s did not deviate more than a factor 2 from observed toxicity. The same model, calibrated to account for sensitivity differences between species, life stages and/or exposure durations, was able to accurately predict 96-h LC50s for larval and juvenile fathead minnow (Pimephales promelas) and juvenile rainbow trout, based on data taken from literature. Although the developed model seems very promising, the uncertainty around the role of alkalinity and the exact mechanisms by which Ca, Mg and pH modify Ni toxicity need to be further explored.

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.

Current evidence suggests that neo formation of the anabolic steroid boldenone (androsta-1,4-diene-17-ol-3-one) occurs in calves' faecal material, making it difficult to distinguish between illegally administered boldenone and its potential endogenous presence. This strengthens the urgent need to elucidate the pathway leading to boldenone formation. In our laboratory, the invertebrate Neomysis integer (Crustacea, Mysidacea) was used since 2004 as an alternative model for the partial replacement of vertebrate animals in metabolisation studies with illegal growth promotors and veterinary drugs, e.g. boldenone. The present study evaluates the metabolic capacity of other invertebrates, the brine shrimp Artemia franciscana and maggots of the greenbottle fly Lucilia sericata. The first results indicate that maggots of L. sericata are able to convert phytosterols and -stanols, nowadays in substantial amounts added to animal feed, into androsta-1,4-diene-3,17-dione (ADD), the precursor of boldenone, at a yield of 0.10-0.14% (p<0.001, significance compared to endogenous excretion of maggots) but not to boldenone itself. Furthermore, beta-testosterone, an endogenous hormone, was transformed into androst-4-ene-3,17-dione (AED), ADD and beta-boldenone at a significant (p<0.001, significance compared to endogenous excretion of maggots) yield of circa 13%, 0.80% and 2.2%, respectively. In future studies these results are of value to further evaluate the use of maggots of L. sericata as an invertebrate model in metabolisation studies.

The acute biotic ligand model (BLM) is proposed by the U.S. Environmental Protection Agency (U.S. EPA) to incorporate bioavailability in calculating aquatic life criteria for Cu in freshwater. This approach currently assumes that the effects of water chemistry on acute Cu toxicity can be described with one single set of identical BLM parameters for all organisms. An important water characteristic is the concentration of Na, which protects aquatic organisms against Cu toxicity. Based on physiological considerations related to the mechanism of Cu toxicity and the possible role of Na therein, we hypothesized that an interspecies variability of the protective effect of sodium on Cu toxicity might exist among freshwater organisms. To test this hypothesis, acute 48-h toxicity assays with Cu were conducted with 16 field-collected cladoceran populations and a laboratory-reared clone of Ceriodaphnia dubia at Na concentrations of between 0.077 and 10 mM. Increased Na protected all but one population. Contrary to what the BLM predicts, however, an upper limit to this protective effect was observed for some populations at Na concentrations of greater than 4 mM. This may suggest that processes other than just Cu-Na competition at a single unidentate biotic ligand site may be involved in Cu toxicity. Between populations, conditional stability constants for binding of Na to the biotic ligand (log K*(NaBL)), which quantify the protective effect of Na, varied between 2.2 and 4.4. Higher log K values generally were associated with more sensitive populations. Although a full mechanistic explanation for our observations is lacking, our data may potentially be used to refine the U.S. EPA procedure to establish site-specific water-quality criteria for Cu.

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.

Sediment acid volatile sulfide (AVS) concentrations were measured in wadeable streams of a wide variety of ecoregions of western Europe (84 sites in 10 countries and nine ecoregions) to better understand spatial distribution and ecoregion relationships. Acid volatile sulfide has been shown to be a major factor controlling the bioavailability and toxicity of many common trace metals, such as Cd, Cu, Ni, Pb, and Zn. Sediment characteristics varied widely. The ratio of the sum of the simultaneously extracted metals (SEM) to AVS ranged from 0.03 to 486.59. The sigmaSEM-AVS ranged from -40.02 to 17.71 micromol/g. On a regional scale, sediment characteristics such as dominant parent soil material showed significant trends in AVS distribution and variation by ecoregion. Total Fe and Mn were correlated weakly with SEM concentrations. Three AVS model approaches (i.e., the SEM:AVS ratio, SEM-AVS difference, and carbon normalization) were compared at threshold exceedance levels of SEM/AVS > 9, SEM-AVS > 2, and SEM-AVS/foc > 150 micromol/g organic carbon (OC). Only 4.76% of the sediments exceeded all three AVS thresholds; 22.6% of the sediments exceeded two models; and 13% of the sediments exceeded one model only. Using the SEM:AVS, SEM-AVS, and fraction of organic carbon models, and including site-specific data and regional soil characteristics, ecoregions 1 (Portugal), 3 (Italy), 4 (Switzerland), and 9 (Belgium/Germany) had the highest potential metals toxicity; ecoregions 13 and 8 (Belgium/France) showed the lowest potential toxicity. However, because AVS can vary widely spatially and temporally, these data should not be considered as representative of the sampled ecoregions. The general relationship between AVS levels and sediment characteristics provides some predictive capability for wadeable streams in the European ecoregions.

Embryonic development is a crucial time window within an organism's life history. Relatively few studies have focused on understanding the potential effects of endocrine disruptors on embryogenesis in invertebrates. Mysids (Crustacea: Mysidacea) have been used extensively in regulatory toxicity testing and they are the only invertebrate model currently included in the U.S. EPA's Endocrine Disruptor Screening and Testing Program. We developed a method for studying mysid embryonic development in multiwell plates until the release of free-swimming juveniles. This method was used to evaluate the potential effects of the insecticide methoprene, a juvenile hormone analog, on mysid embryogenesis. Embryos were exposed to nominal concentrations 0.01, 1, and 100 microg methoprene/L. Average percentage survival, hatching success, total development time and duration of each developmental stage were analyzed. Embryos exposed to 1 and 100 microg methoprene/L had a significantly lower hatching success and lower survival rates. Our study indicates that in vitro embryogenesis can be used as a valuable tool to study the impact of endocrine disruptors in mysids.

A biotic ligand model (BLM) was developed to predict nickel toxicity, affecting root growth of barley (Hordeum vulgare), in nutrient solutions. The extent to which Ca(2+), Mg(2+), Na(+), K(+) ions and pH each influenced nickel toxicity was determined. Higher activities of Mg(2+) linearly increased the 4d EC50 Ni (2+) , while Ca(2+), Na(+), K(+) and H(+) activities did not significantly influence Ni(2+) toxicity. Stability constants for the binding of Ni(2+) and Mg(2+) to the biotic ligand were obtained: logK(NiBL)=5.27 and logK(MgBL)=3.47. Further, it was calculated that on average 57% of the biotic ligand sites needed to be occupied by nickel to induce 50% root growth inhibition. Auto-validation of the BLM indicated that predicted EC50s differed from the observed EC50s by a factor of less than 2, indicating that the BLM concept may also be used to predict metal toxicity to terrestrial plants.

Despite the increased research and regulatory interest in numerous bioactive agents, including natural hormones, xeno-hormones and pharmacological agents, little is known about the presence of these compounds in the estuarine and marine environment. In this study, the results of a 2-year survey on the occurrence of the natural female sex hormones, estradiol (E2) and estrone (E1) and the synthetic steroid, ethinylestradiol (EE2) in the Scheldt estuary (Belgium-The Netherlands) are presented. Chemical analysis of the water samples was performed using Speedisk extraction. Suspended matter samples were analyzed with accelerated solvent extraction (ASE) and detection was performed with gas chromatography coupled to multiple ion trap mass spectrometry. Detected concentrations were in the low ng L(-1) range. E1 and betaE2 (beta-isomer of E2) were detected in water and suspended matter, whereas concentrations of EE2 were below the limit of quantification (LOQ). E1 was observed most frequently and at concentrations up to 10 ng L(-1) in water and up to 0.84 ng g(-1) in suspended matter samples.

The Terrestrial Biotic Ligand Model (TBLM) is applied to a number of noncalcareous soils of the European Union for Cu and Ni toxicities using organisms and endpoints representing three levels of terrestrial organisms: higher plants, invertebrates, and microbes. A comparison of the TBLM predictions to soil metal concentration or free metal ion activity in the soil solution shows that the TBLM is able to achieve a better normalization of the wide variation in toxicological endpoints among soils of disparate properties considered in this study. The TBLM predictions of the EC50s were generally within a factor of 2 of the observed values. To our knowledge, this is the first study that incorporates Cu and Ni toxicities to multiple endpoints associated with higher plants, invertebrates, and microbes for up to eleven noncalcareous soils of disparate properties, into a single theoretical framework. The results of this study clearly demonstrate that the TBLM can provide a general framework for modeling metals ecotoxicity in soils.

Because it is unclear if leaching can account for differences in metal bioavailability observed between metal-spiked soils and historically contaminated field soils, we simultaneously assessed Pb toxicity to the springtail Folsomia candida in three transects of Pb-contaminated soils and in leached and unleached soils spiked at similar total Pb concentrations. Total Pb concentrations of 3,877 mg/kg dry weight and higher always caused significant effects on F. candida reproduction in the spiked soils. In the transects, only the soil with the highest Pb concentration of 14,436 mg/kg dry weight significantly affected reproduction. When expressed as pore-water concentrations, reproduction was never significantly affected at Pb concentrations of 0.539 mg/L, whereas reproduction was always significantly affected at Pb concentrations of 0.678 mg/L and higher, independent of the soil treatment. These results indicate that pore-water Pb concentrations can explain, at least in part, the observed differences in the toxicity data expressed as total Pb concentrations. Leaching after the spiking procedure only caused small differences in Pb toxicity and, therefore, cannot account for toxicity differences between laboratory-spiked soils and historically contaminated field soils.

Cytochromes P450 (CYPs) are important enzymes involved in the regulation of hormone synthesis and in the detoxification and/or activation of xenobiotics. CYPs are found in virtually all organisms, from archae, and eubacteria to eukaryota. A number of endocrine disruptors are suspected of exerting their effects through disruption of normal CYP function. Consequently, alterations in steroid hormone metabolism through changes in CYP could provide an important tool to evaluate potential effects of endocrine disruptors. The aim of this study was to investigate the potential effects of the known CYP modulator, benzo(a)pyrene (BaP), on the testosterone metabolism in the invertebrate Neomysis integer (Crustacea; Mysidacea). N. integer were exposed for 96 h to 0.43, 2.39, 28.83, 339.00 and 1,682.86 microg BaP L(-1) and a solvent control, and subsequently their ability to metabolize testosterone was assessed. Identification and quantification of the produced phase I and phase II testosterone metabolites was performed using liquid chromatography coupled with multiple mass spectrometry (LC-MS2). Significant changes were observed in the overall ability of N. integer to metabolize testosterone when exposed to 2.39, 28.83, 339.00 and 1,682.86 microg BaP L(-1) as compared to the control animals.

We investigated whether an earlier-developed bioavailability model for predicting copper toxicity to growth rate of the freshwater alga Pseudokirchneriella subcapitata could be extrapolated to other species and toxicological effects (endpoints). Hardness and dissolved organic carbon did not significantly affect the toxicity of the free Cu2+ ion to P. subcapitata (earlier study) and Chlorella vulgaris(this study), but a higher pH resulted in an increased toxicity for both species. Regression analysis showed significant linear relationships between ECxpCu (= "effect concentration" that produces x% adverse effect, expressed as pCu = -log of the Cu2+ activity) and pH. By linking these regression models with a geochemical metal speciation model, dissolved copper concentrations that elicit a given adverse effect (EC(X)dissolved) can be predicted. Within the pH range investigated (5.5-8.7), slopes of the linear EC(X)pCu vs pH regression models varied between 1.301 and 1.472 depending on the species and the effect level (10% or 50%) considered. In a statistical sense these slopes were all significantly different from one another (p < 0.05), suggesting that this empirical regression model does not yet capture the full complexity of toxicological copper bioavailability to algae. However, we demonstrated that regression models with an "average" slope of 1.354 had predictive power very similar to those of regression models with species and effect-specific slopes. Additionally, the "average" regression model was further successfully validated for other species (Chlamydomonas reinhardtii and Scenedesmus quadricauda) and for different toxicological effects/endpoints (growth rate, biomass yield, and phosphorus uptake rate). For all these toxicity datasets effect concentrations of copper could be predicted with this "average" model by errors of less than a factor of 2 in 94-100% of the cases. The success of this "average" model suggests the possibility that the pH-based linear regression model may form a sound conceptual basis for modeling the toxicological bioavailability of copper to green algae in regulatory assessments, although a full mechanistic understanding is lacking and should be the focus of future studies.

In order to gain better insights in the integrated response of Daphnia magna following chronic zinc exposure, several physiological parameters were measured in a time-dependent manner. D. magna juveniles were exposed for 21 days to dissolved Zn concentrations up to 340 microg/L. Next to standard endpoints such as mortality, growth and reproduction the following sub-lethal endpoints were measured: filtration and ingestion rate, respiration rate, energy reserves, internal Zn and total Ca concentrations in the organisms. Organisms exposed to 80 microg/L generally performed better than the Zn deprived control organisms. The former were used to elucidate the effects of higher Zn concentrations on the endpoints mentioned above. After 1 week, only 7% of the organisms exposed to 340 microg/L survived. Body Zn contents of these organisms were 281 +/- 76 microg g dry weight and a 37% decrease of the Ca contents was observed. This suggests a competitive effect of Zn on Ca uptake. Filtration rate (-51%), individual weight (-58%) and energy reserves (-35%) also exhibited a decreasing trend as a function of increasing Zn exposure concentrations. During the second and third exposure week an overall repair process was observed. In the surviving organisms mortality and reproduction were only slightly affected. This can be explained by (over)compensation reactions at lower levels of biological organisation: Ca contents (+24%) and filtration rate (+90%) increased as a function of the exposure concentration while respiration rate decreased (-29%) resulting in energy reserves remaining constant as a function of Zn exposure. It is hypothesized that a disturbed Ca balance is probably the first cause for zinc toxicity effects in D. magna.

Short chronic 48-h toxicity tests with the freshwater rotifer Brachionus calyciflorus (Pallas) were conducted to assess the modifying effects of pH and natural dissolved organic carbon (DOC) concentration on reproductive toxicity of Cu. Toxicity tests were carried out in four test waters according to a 2 x 2 design, in which pH (6 and 7.8) and DOC (5 and 15 mg C/L) were the test variables. Concentrations of dissolved Cu with no observed effect at 48 h (NOEC) varied 12-fold between 8.2 and 103 microg/L. Higher DOC and higher pH resulted in a reduction of toxicity, which is in line with the concepts of the biotic ligand model (BLM). A chronic Cu-BLM, originally developed for the cladoceran Daphnia magna, was calibrated to the rotifer dataset and was found to be able to predict all rotifer NOECs with an error factor of less than 1.6. This finding may be of great interest for risk assessment and the establishment of water quality criteria, as it suggests that chronic Cu-BLMs are comparable across phyla (i.e., arthropoda to rotifera).

The present study reports mutagenic and genotoxic activities associated with ambient air collected at 15 sites characteristic for urban, industrial or rural conditions in Flanders. Airborne particulates (PM10) and semi-volatile compounds were collected on quartz filters (QF) and polyurethane foam (PUF) cartridges using a high-volume sampling device. The mutagenic and genotoxic potency of the organic extracts--Soxhlet extraction with acetone--was determined by use of the Salmonella mutagenicity standard plate-incorporation assay and the Vitotox assay, respectively. Concentrations of 16 polycyclic aromatic hydrocarbons (PAHs) in the extracts were determined by reversed-phase high-performance liquid chromatography (HPLC). Ambient air samples contained significant PAH levels and mutagenic activities at all 15 sites: direct mutagenicity of up to 47 revertants per cubic meter was found in the QF extracts and more limited activity of up to 11 rev m(-3) in the PUF extracts. Metabolic activation of PUF extracts resulted in an important increase in mutagenic activity, up to 30 rev m(-3), but no such increase was observed for QF extracts. The highest values were observed outside large cities at industrial sites and at a rural site contaminated by pollution from a chemical plant at a distance of 4 km. Also at the background location near the North Sea a significant mutagenic activity was measured in the QF extracts (+S9: 9 rev m(-3); -S9: 7 rev m(-3)). Apparently, there is in Flanders a significant background exposure level to airborne mutagenicity, even in areas with limited or no nearby pollution sources. Based on the concentrations of 10 mutagenic PAHs and supposing additivity of their specific mutagenicities, only a few percent (mean 3%) of the observed indirect mutagenic activity could be explained. This implies that most mutagenic activity originated from other substances that were not identified or measured in our chemical analysis. This underscores the importance of bio-monitoring measurements.

Acute zinc toxicity was assessed for 10 freshwater cladoceran species collected in six different ecosystems across Europe and for two standard laboratory-reared species (Daphnia magna and Ceriodaphnia dubia). The collected organisms belonged to five different genera: Daphnia (subgenus Daphnia and Ctenodaphnia), Ceriodaphnia, Simocephalus, Acroperus and Chydorus. The 48-h EC50 of the field-collected organisms tested in standard laboratory water ranged from 375+/-141 to 4314+/-1513 microg Znl(-1). The laboratory clone of D. magna was less sensitive than the majority of the field-collected species, while our laboratory Ceriodaphnia dubia was the second most sensitive. Considerable inter-species variation was found within the genus of Ceriodaphnia (factor 6) and within the genus Daphnia (factor 8). Among the different (sub)genera tested, Chydorus and Ctenodaphnia were significantly more tolerant than the others (up to a factor 3 difference). A significant positive relationship (r2=0.67, p<0.05) between the mean cladoceran 48-h EC50 and the ambient zinc concentration of the different aquatic systems was demonstrated, suggesting a role of acclimation and/or adaptation. No significant correlation between the acute zinc tolerance and the length of the organisms was found.