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A times series of laboratory experiments were conducted to investigate the effect of land deposition of contaminated sediments on the bioavailability and mobility of metals. Four sandy sediments were sampled at sites expected to have elevated levels of cadmium and zinc. The physical and chemical characteristics and ecotoxicity of sediments, pore waters, and leachates were evaluated after periods ranging from 1 to 45 days of land deposition. Cd and Zn retardation and leaching potential were calculated and this simulation gave good predictions of subsequently observed Cd and Zn mobility. The mobility and leaching of Cd and Zn in the sediments increased with decreasing pH and with decreasing content of organic matter. During the deposition an increase in sediment toxicity to plants and an increase in eluate toxicity to invertebrates were observed. A high rate of water flow through the sediment resulted in a lower toxicity enhancement of the sediments and a higher toxicity enhancement of the eluates. This result suggests that water flow through the sediment reduces the actual toxicity of the upper layer of deposited sediment but at the same time intensifies the risk of groundwater contamination., (Copyright 2002 Elsevier Science (USA))

Currently, soil quality criteria or soil risk assessments of metals are based on laboratory toxicity tests which are carried out in soils freshly spiked with metal salts. With these data, species sensitivity distributions are fitted, from which hazardous concentrations and predicted no effect concentrations are derived. However, due to long-term processes, called ageing, soil metal availability decreases with time. Here we show that pH is the most important parameter determining the effect of ageing on zinc partitioning in soils, with the effect of ageing becoming more important with increasing pH. Furthermore, zinc bioavailability, expressed as the internal zinc concentrations in red clover (Trifolium pratense) is closely related to pore water zinc concentration. In addition, there is a clear dose-response relationship between the survival of the earthworm Eisenia fetida and the calcium chloride-extracted zinc fraction. These results indicate that zinc partitioning can be used to predict zinc bioavailability to terrestrial organisms. However, the use of spiked soils in toxicity assays can result in an over-estimation of the effects of zinc, especially at a high pH.

Application of 5% (w:w) novel metal immobilizing agent reduced the water soluble, the calcium chloride extracted as well as the pore water concentration of zinc in soils from Maatheide, a metal contaminated site in the northeast of Belgium. Addition of the metal immobilizing agents also eliminated acute toxicity to the potworm Enchytraeus albidus and the earthworm Eisenia fetida and chronic toxicity to the springtail Folsomia candida. Cocoon production by E. fetida, however, was still adversely affected. These differences may be explained by the species dependent routes of metal uptake: F. candida is probably mainly exposed via pore water while in E. fetida dietary exposure is probably also important. From these results it is clear that organisms with different exposure routes should be used simultaneously to assess the environmental risk of metal contaminated soils.

The characterisation of the risk of (new) chemicals to species/communities, when both the exposure/environmental concentration and effects (species sensitivity) are variable and uncertain, is the central issue in Probabilistic Environmental Risk Assessment. The spatial variability is one of the largest components of the total variability. This paper tries to explicitly account for this spatial variability by geo-referencing the exposure, effect and finally probabilistic risk. Geo-referencing makes the risk assessment more refined and realistic. In addition, it is also highlighted that geo-referencing the effects of chemicals (species sensitivity distribution) is still a large unexplored area but has large potential to improve probabilistic ecological risk assessments.

Zinc accumulation in Daphnia magna was investigated, and the results were linked to the previously established optimal concentration range for zinc and D. magna. It was observed that organisms cultured in this optimal range (300-600 microg Zn/L) contained 212 +/- 57 to 254 +/- 79 microg Zn/g dry weight. Lower and higher zinc contents were obtained after acclimation to previously established culture concentrations inducing deficiency and toxicity, respectively. The calculation of bioconcentration factors indicated that zinc was actively regulated, at least up to a concentration of 600 microg Zn/L. Zinc uptake and elimination are rapid processes; major increases and decreases in body content occurred within 1 day. Zinc concentrations in daphnids exposed to 600 microg Zn/L fluctuated with 2- to 3-day intervals, suggesting a role of molting in the regulation and elimination of zinc.

Biotic ligand models have been developed for various metals (e.g. Cu, Ag, Zn) and different aquatic species. These models incorporate the effect of physico-chemical water characteristics (major cations, pH, dissolved organic carbon) on the bioavailability and toxicity of the metal. In this study, the individual effects of calcium, magnesium, potassium, sodium and pH on zinc toxicity to the green alga Pseudokirchneriella subcapitata (formerly and better known as Selenastrum capricornutum and Raphidocelis subcapitata) were investigated. Stability constants for binding to algal cells (K(BL)) were derived for those cations affecting zinc toxicity, using the mathematical approach proposed by De Schamphelaere and Janssen [Environ. Sci. Technol. 63, (2002) 48-54]. Potassium proved to be the only cation tested that did not alter zinc toxicity to algae significantly. Log (K(BL)) values for Ca, Mg and Na, derived at pH 7.5, were 3.2, 3.9 and 2.8, respectively. Toxicity tests performed at different pH values (5.5-8.0) indicated that competition between H(+) and Zn(2+) reduces zinc toxicity. However, the observed relationship between (H(+)) and the 72h-EbC(50) [expressed as microM (Zn(2+))] is not linear and suggests that pH affects the physiology of the biotic ligand. Although, in general, our findings seem to suggest that zinc toxicity to algae can be modelled as a function of key water characteristics, the results also demonstrate that the part of the conventional BLM-hypothesis-i.e. that the binding characteristics of the biotic ligand are independent of the test medium characteristics-is not valid for algae. The observed pH-dependent change of stability constants should therefore be further investigated and incorporated in future BL-modelling efforts with algae.

During recent years, the biotic ligand model (BLM) has been proposed as a tool to evaluate quantitatively the manner in which water chemistry affects the speciation and biological availability of metals in aquatic systems. This is an important consideration because it is the bioavailability and bioreactivity of metals that control their potential to cause adverse effects. The BLM approach has gained widespread interest amongst the scientific, regulated and regulatory communities because of its potential for use in developing water quality criteria (WQC) and in performing aquatic risk assessments for metals. Specifically, the BLM does this in a way that considers the important influences of site-specific water quality. This journal issue includes papers that describe recent advances with regard to the development of the BLM approach. Here, the current status of the BLM development effort is described in the context of the longer-term history of advances in the understanding of metal interactions in the environment upon which the BLM is based. Early developments in the aquatic chemistry of metals, the physiology of aquatic organisms and aquatic toxicology are reviewed first, and the degree to which each of these disciplines influenced the development of water quality regulations is discussed. The early scientific advances that took place in each of these fields were not well coordinated, making it difficult for regulatory authorities to take full advantage of the potential utility of what had been learned. However, this has now changed, with the BLM serving as a useful interface amongst these scientific disciplines, and within the regulatory arena as well. The more recent events that have led to the present situation are reviewed, and consideration is given to some of the future needs and developments related to the BLM that are envisioned. The research results that are described in the papers found in this journal issue represent a distinct milestone in the ongoing evolution of the BLM approach and, more generally, of approaches to performing ecological assessments for metals in aquatic systems. These papers also establish a benchmark to which future scientific and regulatory developments can be compared. Finally, they demonstrate the importance and usefulness of the concept of bioavailability and of evaluative tools such as the BLM.

A previously developed biotic ligand model (BLM) was validated for its capacity to predict acute 48-h EC(50) values of copper to Daphnia magna in 25 reconstituted media with different pH values and concentrations of artificial dissolved organic carbon, Ca, Mg and Na. Before the BLM validation, fitting of measured (with a copper ion-selective electrode) and calculated (with the BLM) Cu(2+)-activity was performed by adjusting the WHAM model V (i.e. the metal-organic speciation part of the BLM) copper-proton exchange constant to pK(MHA)=1.9. Using this value, the 48-h EC(50) values observed agreed very well with BLM-predicted EC(50) values for tests performed at pH<8, but not at all for tests performed at pH>8. Additional experiments demonstrated that this was due to toxicity of the CuCO(3) complex, which is the most abundant inorganic copper species at pH>8. This was incorporated into the initial BLM by allowing the binding of CuCO(3) (next to Cu(2+) and CuOH(+)) to the biotic ligand of D. magna. The affinity of CuOH(+) and CuCO(3) for the biotic ligand was approximately five- and 10-fold lower than that of Cu(2+), respectively. With the refined BLM, 48-h EC(50) values could be accurately predicted within a factor of two not only in all 25 reconstituted media, but also in 19 natural waters. This validated and refined BLM could support efforts to improve the ecological relevance of risk assessment procedures applied at present.

Juvenile rainbow trout, Oncorhynchus mykiss, were exposed to the synthetic estrogen 17alpha-ethinylestradiol (EE(2)) through injection (1, 10, 25 and 50 microg EE(2)/g fish/week) and via water exposure (1, 10 and 100 ng EE(2)/l). After seven (injection and water exposure) and 14 days (only for water exposure), blood and plasma vitellogenin concentrations were quantified using indirect endpoints, i.e. plasma alkaline-labile phosphorus (ALP), plasma protein and plasma calcium. In addition, the relative gonad (GSI) and liver weight (HSI) were recorded. Actual plasma vitellogenin concentrations were measured with an enzyme immunoassay. Only fish injected with 50 microg EE(2)/g fish had a significantly higher gonad weight. No concentration-dependent changes in the HSI were detected in fish exposed via the water, but a significant dose-dependent increase of the HSI was observed in fish injected with EE(2). Exposure of rainbow trout to EE(2) had a significant effect on all tested plasma parameters. Plasma protein, phosphoprotein and calcium concentrations were significantly higher after two weeks exposure to 100 ng EE(2)/l. Fish injected with 10, 25 and 50 microg EE(2)/g fish exhibited increased plasma protein concentrations after 1 week. Compared to the controls, plasma ALP and calcium levels were significantly higher in all injected fish. A significant and positive correlation was observed between all three plasma parameters and between these indirect parameters and the actual plasma vitellogenin concentrations. These findings indicate that both the plasma ALP and the plasma calcium assay have a similar sensitivity as that of available antibody-based assays (EIA), at least in EE(2) exposure studies, and thus these assays can provide a rapid, simple and cost-effective alternative to available immunoassays.

Corrosion and runoff from zinc-coated materials and outdoor structures is an important source for the dispersion of zinc in the environment. Being part of a large inter-disciplinary research project, this study presents the bioavailability of zinc in runoff water immediately after release from the surface of 15 different commercially available zinc-based materials exposed to the urban environment of Stockholm, Sweden. Runoff water was analysed chemically and evaluated for its possible environmental impact, using both a biosensor test with the bacteria Alcaligenes eutrophus (Biomet) and the conventional 72 h growth inhibition test with the green alga Raphidocelis subcapitata. Chemical speciation modelling revealed that most zinc (94.3-99.9%) was present as the free Zn ion, the most bioavailable speciation form. These findings were confirmed by the results of the biosensor test (Biomet) which indicated that all zinc was indeed bioavailable. Analysis of the ecotoxicity data also suggested that the observed toxic effects were due to the presence of Zn2+ ions. Finally, regression analysis showed that, for this type of runoff samples, the rapid screening biosensor was capable of predicting (a) the total amount of zinc present in the runoff samples (R2 of 0.93-0.98; p < 0.05) and (b) the observed 72 h-EbC50s (R2 of 0.69-0.97; p < 0.05).

The individual effect of different major cations (Ca2+, Mg2+, Na+, K+, and H+) on the acute toxicity of zinc to the waterflea Daphnia magna was investigated. The 48-h median effective concentration (EC50) in the baseline test medium (i.e., a standard medium with very low ion concentrations) was about 6 microM (Zn2+). An increase of Ca2+ (from 0.25 mM to 3 mM), Mg2+ (from 0.25 mM to 2 mM), and Na+ activity (from 0.077 mM to 13 mM) reduced zinc toxicity by a factor of 6.3, 2.1, and 3.1, respectively. No further toxicity reduction was observed when Ca2+ and Mg2+ activities exceeded 3.0 and 2.0 mM, respectively. Both K+ and H+ did not significantly alter zinc toxicity (expressed as Zn2+ activity). From these data, conditional stability constants for Ca2+ (log K = 3.24), Mg2+ (log K = 2.97), Na+ (log K = 2.16), and Zn2+ (log K = 5.31) were derived and incorporated into a biotic ligand model (BLM) predicting acute zinc toxicity to D. magna in surface waters with different water quality characteristics. Validation of the developed BLM using 17 media with different pH, hardness, and dissolved organic carbon (DOC) content resulted in a significant correlation coefficient (R2 = 0.76) between predicted and observed 48-h EC50. Eighty-eight percent of the predictions were within a factor of 1.3 of the observed 48-h EC50.

Central composite designs were used to develop surface response relationships for predicting the chronic toxicity of mixtures of zinc, cadmium, copper, and lead to the potworm Enchytraeus albidus. Surface response relationships were obtained for all binary mixtures of zinc, cadmium, copper, and lead as well as for mixtures with all four metals. For the binary mixture of zinc and cadmium, this approach and the toxit unit approach gave similar results. However, with the toxit unit approach no such surface response models can be developed. The experimental results indicated that effect predictions based on the concentration addition model were always higher than those obtained with the independent action model. The observed effect was lower than the effect predicted by the concentration addition model for all metal mixtures. Therefore it can be concluded that the concentration addition model represents a reasonable worst-case scenario for the risk assessment of metal mixtures in terrestrial ecosystems.

Testosterone metabolism by Neomysis integer (Crustacea; Mysidacea) was assessed to obtain initial data on its metabolic capacity. N. integer were exposed to both testosterone and [(14)C]testosterone. Identification of testosterone metabolites and endogenous steroids was performed using thin-layer chromatography and liquid chromatography with multiple mass spectrometry. Endogenous production of testosterone in mysids was detected for the first time. N. integer were exposed to testosterone and metabolized administered testosterone extensively. At least 11 polar testosterone metabolites (R(f,metabolite) < R(f,testosterone)), androstenedione, dihydrotestosterone, and testosterone were produced in vivo by N. integer. A sex-specific testosterone metabolism was also observed, although this observation requires further confirmation. The anabolic steroid beta-boldenone was also identified for the first time in invertebrates. The metabolic pathway leading to the formation of beta-boldenone remains unknown, since the steroidal precursor androstadienedione could not be detected. These results reveal interesting similarities in enzyme systems in invertebrate and vertebrate species. Alterations in steroid hormone metabolism may be used as a new biomarker for the effects of endocrine disruptors in invertebrates., ((c) 2002 Elsevier Science (USA))

Despite growing concerns about the potential adverse effects of elevated chromium concentrations in the environment, only a few terrestrial toxicity data are available. Therefore, chronic toxicity of chromium(III) was assessed for Eisenia fetida, Enchytraeus albidus, and Folsomia candida, the three invertebrates for which standard test protocols are available. The 21-day EC(50) for the cocoon production of E. fetida was 892 (679-1110) mg Cr/kg dry weight. Based on the reproduction, a 42-days EC(50) of 637 (355-791) mg Cr/kg dry weight was observed for E. albidus, while for F. candida a 28-day EC(50) of 604 (254-3380) mg Cr/kg dry weight was noted. Although the presented data can be considered a step forward in the assessment of the potential risks of chromium(III) in terrestrial environments, further research is needed to evaluate the influence of soil parameters on the toxicity of chromium and to quantify the effect of aging on bioavailability., ((c) 2002 Elsevier Science (USA).)

Acute and chronic ecotoxicity tests with lindane were carried out using the soil invertebrates Eisenia fetida, Enchytraeus albidus, and Folsomia candida. To assess the influence of soil type on the bioavailability, these tests were carried out in a standard artificial OECD soil and in sandy and loamy field soil. For each species, differences in lindane toxicity were observed for the three soil types. These differences were, however, not related to the organic matter content. The relative differences in lindane toxicity between the soils was species-specific. These results therefore indicate that the pore-water hypothesis, i.e., the pore-water contaminant fraction being the toxicological bioavailable fraction, is not always applicable for organic substances. NOEC, NEC, as well as EC10 data were subsequently used to calculate hazardous concentrations for 5% of the species; this methodology, aimed at setting environmental quality criteria, is discussed.

Embryo-larval toxicity tests with the African catfish (Clarias gariepinus) were performed to assess the comparative sensitivity of different endpoints. Measured test responses included embryo and larval survival, hatching, morphological development, and larval growth. Chromium, cadmium, copper, sodium pentachlorphenol (NaPCP), and malathion were used as model toxicants. Hatching was not affected by any of the chemicals tested, and embryo survival was only affected by chromium at > or = 36 mg/L. The growth of larvae was significantly reduced at > or = 11 mg/L Cr, > or = 0.63 mg/L Cu, > or = 0.03 mg/L NaPCP, and > or = 1.25 mg/L malathion. Morphological development of C. gariepinus was affected by all of the toxicants tested. Different types of morphological aberrations were observed, i.e., reduction of pigmentation in fish exposed to cadmium and copper, yolk sac edema in fish exposed to NaPCP and malathion, and deformation of the notochord in fish exposed to chromium and malathion. The sensitivity of the endpoints measured can be summarized as follows: growth > abnormality > larval survival > embryo survival > hatching.

Despite growing concern about the potential adverse effects of elevated nickel concentrations in the environment, only a few toxicity data are available for terrestrial invertebrates. Therefore, chronic toxicity of nickel was assessed for Eisenia fetida, Enchytraeus albidus and Folsomia candida, the three invertebrates for which standard test protocols are available. The 21 d EC50 for the cocoon production of E. fetida was 362 (241-508) mg Ni/kg dry wt. For the reproduction of E. albidus, a 42 d EC50 of 275 (217-346) mg Ni/kg dry wt was observed. The 28 d EC50 for the reproduction of F. candida was 476 (347-671) mg Ni/kg dry wt. The obtained toxicity data were very similar to those of related species reported in literature. Although the presented data can be considered as a step forward in the assessment of the potential risks of nickel in terrestrial environments, further research is needed to evaluate the influence of soil parameters on the toxicity of nickel and to quantify the effect of ageing on bioavailability.

The extent to which Ca2+, Mg2+, Na+, K+ ions and pH independently mitigate acute copper toxicity for the cladoceran Daphnia magna was examined. Higher activities of Ca2+, Mg2+, and Na+ (but not K+) linearly increased the 48-h EC50 (as Cu2+ activity), supporting the concept of competitive binding of these ions and copper ions to toxic action or transport sites at the organism-water interface (e.g. fish gill, the biotic ligand). The increase of the EC50 (as Cu2+ activity) with increasing H+, however, seemed to suggest cotoxicity of CuOH+ rather than proton competition. Based on the biotic ligand model (BLM) concept, we developed a methodology to estimate stability constants for the binding of Cu2+, CuOH+, Ca2+, Mg2+, Na+, and H+ to the biotic ligand, solely based on toxicity data. Following values were obtained: log K(CuBL) = 8.02, log K(CuOHBL)= 7.45, log K(CaBL) = 3.47, log K(MgBL) = 3.58, log K(NaBL) = 3.19, and log K(HBL) approximately 5.4. Further, we calculated that on average 39% of the biotic ligand sites need to be occupied by copper to induce a 50% acute effect for D. magna after 48 h of exposure. Using the estimated constants, a BLM was developed that can predict acute copper toxicity for D. magna as a function of water characteristics. The presented methodology can easily be applied for BLM development for other organisms and metals. After validation with laboratory and natural waters (including DOC), the developed model will support efforts to improve the ecological relevance of presently applied risk assessment procedures.

In standard chronic terrestrial toxicity tests with invertebrates, adult organisms are exposed to the contaminants and the number of offspring is quantified. These procedures do not allow the assessment of possible effects on all life stages of the organism, which may lead to an underestimation of the toxicity of the test substance. To evaluate the importance of this issue, the potworm Enchytraeus albidus was exposed to zinc, cadmium, copper and lead for two subsequent generations. Juvenile production was assessed for both generations. Considering the variability of metal toxicity data reported in the literature, it is concluded that the two generation assay did not markedly increase the sensitivity of the standard E. albidus test for the tested metals. Therefore, toxicity data obtained with the proposed test guideline with E. albidus are protective for all life stages.

Zinc is an essential metal for all living organisms. However, so far, little or no attention has been paid to the consequences of zinc deficiency or acclimation to this metal during culturing and testing on toxicity test results. In this study, the cladoceran Ceriodaphnia dubia was acclimated for 10 generations to four zinc concentrations ranging from 0 to 100 microg Zn/l and changes in zinc tolerance were monitored using acute (48 h) and chronic (9 days) assays. C. dubia deprived of zinc and acclimated to 13 microg Zn/l had a lower fitness in comparison with organisms acclimated to 50 and 100 microg Zn/l. In the two lowest versus the two highest acclimation concentrations the 9dEC50 values (on immobility) were 358-387 microg Zn/l versus 486-489 microg Zn/l; the mean number of young per female was 11-18 versus 25-32; and the time to first brood was 4.7-5.0 days versus 4.0-4.3 days. Moreover, the coefficient of variation of all parameters tested was highest in the two lowest acclimation concentrations. The results indicate that culturing test animals in media lacking trace metals such as zinc could give rise to animals that are unnaturally sensitive to those same metals daring toxicity tests.

The zinc tolerance of two Daphnia magna populations collected at a zinc contaminated site was studied. One clone was isolated from each population in order to determine interclonal variation in zinc tolerance. 48hEC50-values, life table parameters, carapace lengths and cellular energy allocation (CEA) were used as test endpoints and compared with the results obtained with a standard laboratory clone. The natural clones were more tolerant to acute zinc toxicity (up to a factor of 4) and exhibited a higher reproduction rate (factor 2) and carapace length (factor 1.2). The optimal zinc concentrations for the natural clones ranged from 80 to 200 microg Zn/l. When cultured without zinc, the natural clones gradually lost their zinc tolerance. Therefore, the environmental relevance of using toxicity data obtained with organisms (natural, as well as laboratory clones) acclimated to culture media containing no or very small amounts of zinc can be questioned.

Different extractable zinc fractions and the ecotoxicity of zinc in Enchytraeus albidus were assessed using freshly spiked artificial soils and spiked soils which had been aged for 8 weeks. Standard artificial Organization for Economic Cooperation and Development (OECD)-soils were aged in four different ways: (1) storing at 20 degrees C; (2) percolation followed by storing at 20 degrees C; (3) alternately heating at 60 degrees C and storing at 20 degrees C; and (4) alternately freezing at -20 degrees C and storing at 20 degrees C. Ageing had no clear influence on the pore water concentration, the water soluble and the calcium chloride extractable fraction of zinc in the artificial soils. Similarly, the 21d LC50 and the 42d EC50(reproduction) for E. albidus were not influenced by the different treatments. This absence of zinc fixation in the artificial soil during ageing was probably due to the use of kaolinite clay in OECD-soil.

Cadmium tolerance in Enchytraeus albidus was assessed for non-acclimatised organisms, as well as populations acclimatised for 18 months to 32, 100 and 320 mg Cd/kg dry wt. of standard artificial soil. Despite the increased concentration of metallothionein-like proteins in the acclimatised potworms, no changes in sensitivity to acute and chronic (juvenile production) cadmium toxicity could be observed. The 21-day LC50s ranged from 627 for the non-acclimatised enchytraeids to 748 mg Cd/kg dry wt. for the organisms acclimatised for 18 months to a cadmium concentration of 320 mg/kg dry wt. The 42-day EC50s varied from 96.2 (73.6-115) for the organisms acclimatised to 32 mg Cd/kg dry wt. to 234 (186-290) mg Cd/kg dry wt. for those acclimatised to 100 mg Cd/kg dry wt., while the value was intermediate for the non-acclimatised organisms and those acclimatised to the highest cadmium concentration. The elevated content of metallothionein-like proteins apparently only served for the detoxification of the increased cadmium body burdens.

The ecotoxicological and chemical response of five metals (Cd, Cr, Cu, Ni, and Zn) to selective fractionation techniques was assessed using the organism Daphnia magna. The metals Cu and Zn revealed similar toxicity patterns, showing an increased toxicity when lowering the pH, a marked reduction in toxicity after addition of ethylenediaminetetraacetic acid (EDTA). Similar toxicity reductions were obtained by passing the metal solutions over ion exchangers, activated charcoal, and filtration/solid-phase extraction units at neutral and basic pHs. The discrimination between Cu- and Zn-toxicity occurred in the oxidant reduction test, i.e., Zn toxicity was not affected by the addition of Na2S2O3, while Cu toxicity was strongly reduced. A second cluster was formed by Cd and Ni. The toxicity of both metals markedly decreased in the EDTA, ion exchangers, activated charcoal, filtration, and solid-phase extraction tests at high pHs. Discrimination between the toxicity of both metals was accomplished with the graduated pH test, in which a pH-independent toxicity response was found for Ni while the toxicity of Cd increased at lower pHs. Compared with the other metals, a very distinct toxicity pattern for Cr was observed. From the applied fractionation techniques, only the graduated pH and the anion resin tests resulted in a marked change in Cr toxicity. The produced toxicity patterns for each individual metal could therefore be used as fingerprints (i.e., toxicity identification fingerprints) in order to identify the toxicity caused by individual metals for D. magna.

Metal bioavailability is influenced by soil characteristics and aging period. In the present study, both factors were modeled by comparing metal bioavailability in spiked artificial soil and historically contaminated field soils. The chronic toxicity of zinc to Folsomia candida in spiked artificial soils could be predicted with a model based on pH, cation exchange capacity, and total zinc concentration. However, this model could not adequately predict chronic zinc toxicity in contaminated field soils. Porewater concentration and water- and calcium chloride-extracted zinc fractions of the contaminated field soils were lower than those predicted using models developed for spiked artificial soils, indicating that the effect of aging on metal bioavailability should be taken into account. The reproduction of F. candida in contaminated field soils was lower than predicted with models developed using zinc concentration in the porewater and the water- and calcium chloride-extracted fractions in spiked artificial soils. This suggests that these fractions are not the only bioavailable zinc fractions and that dietary metal exposure might also be an important route of uptake under environmentally relevant conditions. Aging and dietary uptake should be studied urgently in order to be able to perform effect-based risk assessments of metal contaminated soils.

The effect of zinc acclimation of Raphidocelis subcapitata (syn. Selenastrum capricornutum) and Chlorella vulgaris on their sensitivity towards this metal was examined in a series of laboratory experiments. These two commonly used algal species were acclimated to 65 microg Zn/l and changes in zinc tolerance were monitored using standard growth inhibition tests. The chemically defined ISO medium was used as a control culture medium. Both species demonstrated a maximum increase in zinc tolerance of a factor of 3 after 100 days of acclimation. Shifts in the shape of the concentration-response curve due to acclimation were observed for R. subcapitata. Compared to non-acclimated algae, acclimated R. subcapitata exhibited higher growth rates in all zinc treatments as well as in the controls. This suggests that the use of ISO-medium results in sub-optimal growth due to zinc deficiency. These effects could not be demonstrated for C. vulgaris. The zinc tolerance of both species decreased significantly one week after returning the acclimated algae to control (ISO) medium. 72hEC50 values based on growth rate were two to four times higher than those calculated using biomass measurements. Algal toxicity test results, particularly if used for metal risk assessments, must not be conducted using nutrient deficient media.

Acute and chronic ecotoxicity tests with cadmium were conducted with the earthworm Eisenia fetida, the potworm Enchytraeus albidus and the springtail Folsomia candida. To assess the influence of the soil type on cadmium bioavailability, these tests were carried out in a standard artificial soil, in a sandy and a loamy field soil. It was not possible to evaluate the influence of soil parameters on the bioavailability on the basis of the experiments that were conducted in only three different soil types, therefore, literature data were also included. However, even in the same standard artificial soils, toxicity data in the literature for Eisenia fetida and Folsomia candida varied considerably. Consequently, no models could be developed that allow a normalization of the ecotoxicity of cadmium to parameters controlling bioavailability. In contrast to zinc, effect concentrations of cadmium for terrestrial invertebrates were always much higher than background concentrations. As the effect of aging on the bioavailability of cadmium was never taken into account, because toxicity experiments were always carried out in freshly spiked soilds, these effect concentrations may even be regarded as conservative. Furthermore, the zinc-cadmium ratio in soils is usually so high that the risk of zinc ecotoxicity for terrestrial invertebrates will usually be much greater in comparison to cadmium ecotoxicity.

Initiated by regulatory restrictions on the use of zinc for various building and construction applications, together with a lack of knowledge related to the release of zinc induced by atmospheric corrosion, a major interdisciplinary research project was implemented to generate data to be used in future risk assessment. Runoff rates from a large number of commercially available zinc-based materials have been determined on panels inclined 45 degrees from the horizon, facing south, during a 1-year atmospheric exposure in an urban environment in Sweden. Possible environmental effects of runoff water immediately after leaving the surface of the various materials have been evaluated during two different sampling periods of varying season and zinc concentration, using the standard growth inhibition test with algae. Raphidocelis subcapitata (formerly Selenastrum capricornutum). Zinc-specific biosensors with the bacterial strain of Alcaligenes eutrophus, and computer modeling using the water-ligand model MINTEQA2 and the humic aquatic model WHAM, have been used to assess the bioavailability and chemical speciation of zinc in the runoff water. An excellent consistency between the different methods was observed. The results show considerably lower runoff rates of zinc (0.07-3.5 g m(-2) year(-1)) than previously being used for regulatory restrictions, and the concentration of zinc to be predominantly responsible for the observed toxicity of the runoff water towards the green algae. The majority of the released zinc quantity was found to be present as free hydrated zinc ions and, hence, bioavailable. The data do not consider changes in bioavailability and chemical speciation or dilution effects during entry into the environment, and should therefore only be used as an initial assessment of the potential environmental effect of zinc runoff from building applications. This interdisciplinary approach has the potential for studies on the environmental fate of zinc in soil or aquatic systems.

Acute and chronic ecotoxicity tests with zinc were performed with the earthworm Eisenia fetida, the potworm Enchytraeus albidus, and the springtail Folsomia candida. To assess the influence of the soil type on zinc toxicity for these soil invertebrates, these tests were carried out in a standard artificial soil, a sandy and a loamy field soil. Based on the results of this experimental work and data taken from literature, models were developed relating the ecotoxicity of zinc to the most important parameters controlling bioavailability: pH and cation exchange capacity. Models were developed for E. fetida and F. candida using the regression technique partial least squares projection to latent structures (PLS). Acute as well as chronic toxicity data of both organisms could be normalized on the basis of the pH and the cation exchange capacity of the test soils. For other terrestrial invertebrates, not enough data were available to develop reliable models.

Clay and organic matter are considered as important parameters influencing bioavailability and ecotoxicity of metals in soils. As there exists a large variation in the type of clay and organic matter in field soils, the quantity of these variables alone may not be good indicators of metal bioavailability. To test this hypothesis, toxicity experiments with the potworm Enchytraeus albidus were conducted in artificial soils with three types of clay and seven types of organic matter, while the soil pH was kept constant. The 14d LC50 of zinc and cadmium varied from 83.0 to 1,140 mg Zn/kg D.W. and from 55.2 to 704 mg Cd/kg D.W., respectively, depending on the type of clay and organic matter that were used. Simultaneous measurements of the cation exchange capacity showed that this soil parameter is a better indicator of the bioavailability as it takes into account the type of clay and organic matter as well as other adsorption phases such as metal oxyhydroxides.

Uptake and elimination kinetics of zinc and cadmium were studied in the oligochaetes Enchytraeus albidus and Eisenia fetida. Even at the highest exposure concentrations where E. fetida survived, the internal zinc concentration was still regulated to a constant level. Enchytraeus albidus could not regulate the internal zinc concentration as well, and the body burden increased after exposure to high zinc concentrations. After transfer to clean soil, the internal zinc concentration dropped back to the control level within a few weeks. For both species, internal cadmium concentrations did not reach equilibrium during the uptake experiments. The internal concentrations causing 50% reduction in cocoon production for E. fetida exposed to cadmium varied between different soil types, indicating that no fixed critical body burdens exist. For both zinc and cadmium, bioaccumulation factors decreased with increasing soil metal concentrations. Bioaccumulation factors may therefore be poor indicators of environmental risk. Their dependence on the total soil concentration makes bioaccumulation factors also unsuitable for assessing the influence of soil characteristics on the bioavailability of metals in contaminated field soils. For the same reason, uptake rate constants are probably not suited for this purpose.

Development of zinc tolerance is described for the cladoceran Daphnia magna Straus. Zinc tolerance (i.e., toxicity and deficiency) was monitored during successive generations of D. magna acclimated to different zinc concentrations. Survival, reproduction, carapax length measurements, and cellular energy allocation assessments were used as test endpoints. Special attention was paid to the consequences of zinc deficiency. The zinc acclimation concentration clearly influenced the overall fitness of the organism. After several generations of acclimation, an optimal concentration curve was observed, with an optimum zinc concentration between 300 and 450 microg/L. Zinc deficiency resulted in a lower zinc tolerance, a higher coefficient of variation for brood size, and an increased pH sensitivity. These results clearly indicate that (background) zinc concentrations present in test and culture media have to be considered in the evaluation of toxicity test results, especially when the toxicity data are used for water-quality guideline derivation and/or ecological risk assessment. Culture and test media containing very little or no zinc do not provide a basis for useful ecotoxicological data.

Statistical test designs were used to identify and quantify the soil parameters important for determining the ecotoxicological effects of copper and lead to the potworm Enchytraeus albidus. The application of a Fractional Factorial Design revealed that the acute toxicity of copper and lead to E. albidus can vary over more than two orders of magnitude depending on the physico-chemical characteristics of the (artificial) soils. The differences in metal ecotoxicity were mainly determined by pH and organic matter content or cation exchange capacity (CEC). Using a Central Composite Design, models were developed describing the ecotoxicity of copper and lead to E. albidus as a function of these parameters. To validate the developed response surface models, two field soils and the standard artificial soil prescribed by OECD were spiked with copper and lead and the acute toxicity to E. albidus was assessed. These validation experiments confirmed that the toxicity of copper and lead could be predicted using information on the total metal concentration, the pH and the CEC of the soil with toxicity decreasing with increasing pH and CEC.

Aspects of the carbohydrate metabolism of Daphnia magna exposed for 48 and 96 h to sublethal concentrations of mercury and lindane were investigated. General as well as toxicant-specific perturbations in the intermediary metabolism were observed. Both model toxicants caused an increase in glycolytic and hexose-monophosphate shunt activity. Mercury exposure increased lactate dehydrogenase and isocitrate activity (only after 96 h), while lindane exposure, on the contrary, inhibited the cellular lactate formation and increased the Krebs' cycle activity (only after 48 h). Daphnids exposed to sublethal mercury concentrations clearly exhibited increased glycogenolytic activity, while in lindane-exposed organisms mainly glycogen phosphorylase inhibition was detected. The short-term enzyme-based effect levels (48--96 h LOEC and EC(10) values) were compared with the effects on the population dynamics. This evaluation for both model toxicants suggests that threshold levels (LOEC or EC(10) values) based on pyruvate kinase activity after 48 and 96 h of exposure could be potential early warning signals for long-term effects. A set of enzymatic endpoints, based on the intermediary metabolism, is suggested to characterize the metabolic state of the daphnids., (Copyright 2001 Academic Press.)

Embryo-larval toxicity tests with the African catfish (Clarias gariepinus) were conducted with five chemicals (Cr, Cd, Zn, NaPCP and malathion) and three environmental samples. The sensitivity of the 5-day assay was compared to that of the 12-day embryo-larval toxicity tests with the zebra fish (Danio rerio). The ratios of the C. gariepinus and D. rerio LC50 values ranged from 0.4 for Cr to 8.9 for Zn. The ratios of subchronic values ranged from 0.25 for NaPCP to 3.1 for Cd indicating a more comparable sensitivity of the two species. For the three sediment pore waters, the ratios were 0.6, 1.1, and 2.4 and the subchronic values were identical for the two species. The results suggest that, considering the short-test duration and its sensitivity, the 5-day embryo-larval tests with C. gariepinus may be a potential alternative for short-term embryo-larval toxicity testing with fish.

Environmental risk assessment is a key feature of regulations controlling the placing of new, and the maintenance of existing, chemicals products in the market place. For example, European Commission Directive 93/67/EC on Risk Assessment for New Notified Substances and Commission Regulation (EC) No. 1488/94 on Risk Assessment for Existing Substances requires that risk assessments be carried out for new and existing substances in the European Community. The process of environmental risk assessment seeks to determine the balance of probability of species and communities being damaged by chemical releases. The process relies upon a valid estimation of a predicted environmental concentration (PEC) in relevant environmental compartments and a predicted no effect concentration (PNEC) below which the organisms present in that compartment are unlikely to be significantly affected. If the PEC exceeds the PNEC there is a potential for damaging effects to occur. This article focuses on the determination of PNECs for risk assessment. Methods for determining a PNEC described in OECD Monograph 26 (1989, Report of the OECD Workshop on Ecological Effect Assessment, Paris, France, have been applied to data derived for the four chemicals lindane, 3,4-dichloroaniline, atrazine, and copper in a series of collaborative research projects funded by the European Commission., (Copyright 2000 Academic Press.)

Intravascular elastography is a new technique to obtain mechanical properties of the vessel wall and plaque. Mechanical information of vascular tissue is important for characterisation of different plaque components, detection of plaque vulnerability and thus choosing the proper interventional technique. The feasibility of the technique is investigated using phantoms and diseased human arteries. These studies demonstrated that elastography reveals information that is unavailable or inconclusive from the echogram alone. The technique is based on the principle that tissue strain is directly related to its mechanical properties. In intravascular elastography, the tissue is compressed using different intravascular pressures. The strain is determined using cross-correlation techniques of the radio frequency (r.f.) signals. Reliable strain estimates are only obtained when signals of corresponding tissue are correlated. Owing to catheter motion, off-centre position and non-uniform rotation of the intravascular transducer, the r.f. traces at low and at high pressure may be misaligned. Four algorithms are tested to track the corresponding ultrasound signals. Three methods (l1norm, l2norm and cross-correlation) are applied on the r.f. signal and one (l1norm) on the envelope (speckle tracking). Simulations are performed to obtain a data set with a priori knowledge of the scattering particles positions in the tissue at high and low pressure. Different positions of the catheter in the lumen, compression levels of the material and signal-to-noise ratios (SNRs) are investigated. Finally, these findings are corroborated with a phantom experiment in a water tank. From the simulations, it can be concluded that the speckle tracking algorithm has the best performance, under all circumstances. The performance decreases with larger eccentricity of the catheter and larger compression of the material. The SNR is only of minor influence. The speckle tracking algorithm has also the best performance in the phantom experiment. The performance of the speckle tracking algorithm is better than the three r.f.-based algorithms. For intravascular elastography, implementation of this method may improve the quality of the elastogram.

This article presents a summary of a collaborative research program involving five European research groups, that was partly funded by the European Commission under its Environmental Research Program. The objective of the program was to develop aquatic toxicity tests that could be used to obtain data for inclusion at Level 2 of the Risk Evaluation Scheme for the Notification of Substances as required by the 7th Amendment to EC Directive 79/831/EEC. Currently only a very limited number of test methods have been described that can be used for this purpose and these are based on an even smaller number of test species. Tests based upon algae (Chlamydomonas reinhardi, Scenedesmus subspicatus, and Euglena gracilis), protozoa (Tetrahymena pyriformis), rotifera (Brachionus calyciflorus), crustacea (Gammarus pulex), and diptera (Chironomus riparius) were developed. The tests encompassed a range of end points and were evaluated against four reference chemicals: lindane, 3, 4-dichloroaniline (DCA), atrazine, and copper. The capacity of the tests to identify concentrations that are chronically toxic in the field was addressed by comparing the effects threshold concentrations determined in the laboratory tests with those determined for similar and/or related species and end points in stream and pond mesocosm studies. The lowest no-observed-effect concentrations (NOEC), EC(x), or LC(x) values obtained for lindane, atrazine, and copper were comparable with the lowest values obtained in the mesocosms. The lowest chronic NOEC determined for DCA using the laboratory tests was approximately 200 times higher than the lowest NOEC in the mesocosms., (Copyright 2000 Academic Press.)

Newly hatched larvae of Clarias gariepinus were exposed to malathion concentrations ranging from 0.3 mg/l to 5.0 mg/l. Their development was observed through five days posthatch, i.e., the stage in which the vertebral elements are not yet present and the notochord is the only functional body axis. A significant higher proportion of C. gariepinus larvae with deformed notochord and pericardial edema was detected at 2.5 mg/l malathion and higher. The results suggested that (1) deformed notochord was the result of the uncontrolled contractions of the body musculature due to malathion toxicity and (2) the vertebral deformities observed in the fish which have been exposed to malathion from the notochord-stage onwards were the result of a deformed notochord instead of the deformation of the vertebral elements themselves.

The swimming behavior of the freshwater rotifer Brachionus calyciflorus exposed to copper (Cu), pentachlorophenol (PCP), 3,4-dichloroaniline (DCA), and lindane, for periods ranging from 5 min to 5 hr, was examined. A swimming behavior test is described which is based on the rotifers' movement rates as they swim over a grid. For all four toxicants a clear dose-response was observed, with the swimming activity decreasing with increasing toxicant concentrations. For Cu the EC50's, the concentration that reduced the swimming activity to 50% of that of the control value, sharply decreased from 0.22 mg/liter after an exposure of 5 min to 0.068, 0.038, and 0.014 mg/liter after exposures of 30, 60, and 300 min, respectively. PCP affected the rotifers' swimming behavior more gradually, with EC50's decreasing from 7.0 mg/liter after an exposure of 5 min to 5.9, 5.4, and 1.5 mg/liter after 30, 60, and 300 min, respectively. A similar pattern was found for DCA with EC50's ranging from 193 to 45.5 mg/liter for the 5-min and 3-hr exposures, respectively. Exposed to lindane however, B. calyciflorus swimming activity exhibited a different response, and the EC50's gradually increased from 13.7 mg/liter after an exposure of 5 min to significantly higher values of 16.4 and 18.5 mg/liter after periods of 1 and 5 hr, respectively. The results of the swimming activity assays were compared to those of acute and chronic toxicity tests performed with the same test species. The potential use and relevance of this behavioral test criterion were evaluated and discussed.

The effect of short-term exposure to xenobiotics on the feeding behavior of the freshwater rotifer Brachionus calyciflorus was studied. The filtration and ingestion rates of this rotifer decreased after an exposure of 5 hr to sublethal concentrations of copper, pentachlorophenolate, 3,4-dichloroaniline, and lindane. The effective concentrations at which feeding rate was reduced to 50% of that in controls (EC50) for the respective chemicals are 0.032, 1.85, 41.2, and 8.5 mg/liter. The potential use of feeding behavior as test criterion for toxicity screening tests with aquatic invertebrates is discussed.

Single vegetation plots in forests and bogs in the deciduous and coniferous-hardwood formation in northwestern Minnesota were studied according to the field methods of the Zurich-Montpellier system. Plots and species were arranged so that the greatest coincidence of occurrences was obtained. Species that show approximately similar amplitudes were united in sociological groups. Plant communities typified by these groups were placed in a hierarchy. The plant communities appear to intergrade but are not completely continuous. Definite concentrations of occurrences remain present in the ordination. Fourteen main types of vegetation are recognized. Their relations to the environment are discussed, and their relations to vegetation in other regions are indicated. Thirteen main types of vegetation (alliances) are recognized in the Itasca State Park region. Lowland vegetation has been divided into five alliances. In three of them the pathway of lake filling, starting in a eutrophic sedge mat and proceeding towards a mesotropic Picea mariana bog forest, is depicted. The two remaining lowland types represent eutrophic swamp forest of Fraxinus nigra and Ulmus americana on shallow peat or young alluvial soils. Upland forests havebeen divided into three "rich" deciduous types and five "poor" coniferous types.The rich types are represented by Tilia americana forests, Acer saccharum forests, Quercus rubra forests, and Populus tremuloides-Quercus sp. forests, allshowing representatives of the rich flora of the mesic Tilia-Acer forest. The coniferous forest types are represented by Popuus tremuloides-Quercus sp. forests and closely related Pinus resinosa-Pinus banksiana forests, Pinus banksiana forests on sandy outwash soils, and Abies balsamea forests in a young stage of development. Of these types the Pinus banksiana forest is floristically the best defined., (© 1967 by the Ecological Society of America.)