Rapid ecotoxicological bioassay using delayed fluorescence in the green alga Pseudokirchneriella subcapitata

A rapid and simple ecotoxicological bioassay allows quick estimation of the effects of Simazine (CAT) or 3,5-dichlorophenol (3,5-DCP) on the growth of the green alga Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum). The effects of a 15-min exposure to CAT or 3,5-DCP on delayed fluorescence (DF) in P. subcapitata were compared to the results of standard growth inhibition tests involving 72h of exposure to these chemicals at the same concentrations. Integrated DF intensity in the time period from 0.6 to 50s was found to correlate with algal growth inhibition as measured by the standard tests. In addition, the behaviour of DF in this time period exhibited characteristic kinetics indicative of the chemical being tested.     

Effect of Pseudokirchneriella subcapitata (Chlorophyceae) exudates on metal toxicity and colloid aggregation

The effects of Pseudokirchneriella subcapitata exudates on the acute toxicity of metals (Cd, Cu, Pb and Zn) were studied using (1h) algal photosynthesis inhibition tests. The metal concentrations tested were 30, 60, 120, 250 and 500microgL(-1) for Cd and Zn; and 500, 1000, 2000, 3000, 4000microgL(-1) for Cu and Pb. It was established that P. subcapitata exudates markedly decreased metal toxicity. This effect was ascribed to: (i) the presence of organic ligands that bind metals and reduce the concentration of free ionic metal, and/or (ii) interaction of exudates with the cell surface with a subsequent decrease in metal uptake. The effects of the exudates on colloid aggregation were also examined using two different types of single particle counters (SPC). Exudates facilitated the colloid removal, likely via acceleration of aggregation (bridging). The results clearly demonstrate that algal exudates play an important role in the biogeochemical cycling of metals in natural surface water: (1) by reducing free metal concentrations and toxicity to living organisms and (2) by favoring colloid aggregation leading to the removal of colloid-bound metals (colloidal pumping). Such results highlight one potential application of the algae in the remediation of metal-contaminated waters. The results also suggest that current algal toxicity testing protocols, in particular long-term and static tests, may underestimate metal toxicity because of the presence of algal exudates.     

Toxicity and transformation of fenamiphos and its metabolites by two micro algae Pseudokirchneriella subcapitata and Chlorococcum sp

The acute toxicity of an organophosphorous pesticide, fenamiphos and its metabolites, fenamiphos sulfoxide (FSO), fenamiphos sulfone (FSO(2)), fenamiphos phenol (FP), fenamiphos sulfoxide phenol (FSOP) and fenamiphos sulfone phenol (FSO(2)P), to the aquatic alga Pseudokirchneriella subcapitata and the terrestrial alga Chlorococcum sp. was studied. The toxicity followed the order: fenamiphos phenol>fenamiphos sulfone phenol>fenamiphos sulfoxide phenol>fenamiphos. The oxidation products of fenamiphos, FSO and FSO(2) were not toxic to the algal species up to 100 mg L(-1). Both algae were able to transform fenamiphos, FSO and FSO(2), while the phenols were found to be stable in the incubation media. Bioaccumulation of both fenamiphos and its metabolites was observed in the case of Chlorococcum sp. while only metabolites were accumulated in P. subcapitata. This study demonstrates that (i) the hydrolysis products of fenamiphos, FSOP and FSO(2)P are more toxic to both fresh water and soil algae than their parent chemicals, (ii) further fenamiphos can be transformed and bioconcentrated by these algae. Therefore, contamination of natural environments such as waterbodies with fenamiphos or its metabolites can have adverse impacts on the food chain and associated biota (especially to the primary consumers such as Daphnia) since algae are the primary producers located at the base of the food chain. Further, the finding that the fenamiphos phenols are more toxic to algae highlights the need to consider the transformation products in ecological risk assessment of fenamiphos.     

Testing for the toxicity of chemicals with Tetrahymena pyriformis

A test was developed using Tetrahymena pyriformis in order to determine the toxicity of various chemicals. Pre-cultured T. pyriformis was exposed for 24 h at 30 degrees C to various concentrations of chemicals, and the number of T. pyriformis surviving were then counted. The concentration of the chemical, at which the proliferation of T. pyriformis was restricted to one-half of the blank test (EC50), was determined. The method, applied to 57 chemicals, demonstrated that it could be used to detect the chemicals at low concentrations rapidly and with ease. The EC50 values showed a good relationship with 48 h LC50 values for Himedaka (Orizias latipes), and could be explained on the basis of the partition coefficient between water and n-octanol.     

A novel algal toxicity testing technique for assessing the toxicity of both metallic and organic toxicants

This study presents a closed-system algal toxicity test technique that is capable of detecting the effects of both organic and metallic toxicants. Toxicity testing was conducted by transferring adequate amounts of algal suspension, dilution water (with culture growth medium), and toxicants into 300-mL BOD bottles. The BOD bottles were completely filled up with no head-space left. The initial cell density and the exposure time were 15,000 cells/mL and 48 h, respectively. The performance of the above test method was evaluated using three heavy metals and six organic toxicants based on three different test endpoints, i.e., dissolved oxygen production, algal growth rate, and cell density. The proposed test revealed excellent test sensitivity and reproducibility. Currently, none of the existing algal toxicity test protocols is adequate for assessing the toxicity of organic chemicals. The closed-system algal toxicity tests developed by previous researchers also may not be ideal because the enlarged headspace and/or enriched bicarbonate buffer may result in either underestimations of the exposure concentrations or insensitive responses to both heavy metals and organic toxicants. Compared to the aforementioned algal toxicity test methods, the proposed technique in the present study has a more general applicability under conditions such as effluent samples containing both metals and organic toxicants or samples with unknown compositions.     

Evaluation of toxic and interactive toxic effects of three agrochemicals and copper using a battery of microbiotests

Three commonly used test organisms of different trophic levels (Vibrio fischeri, Pseudokirchneriella subcapitata and Daphnia magna) were exposed to selected agrochemicals (fosthiazate, metalaxyl-M, imidacloprid) and copper, in single doses or in binary mixtures. The toxicity of each single compound varied up to two orders of magnitude, depending on the test species examined. V. fischeri was the most sensitive test organism regarding fosthiazate and metalaxyl-M, indicating an IC₅₀ value of 0.20 mg/L (0.17-0.25 mg/L) and 0.88 mg/L (0.35-1.57 mg/L), respectively. Imidacloprid was the least toxic compound, indicating an EC₅₀ value on D. magna of 64.6 mg/L (43.3-122.5 mg/L) and an IC₅₀ value on V. fischeri of 226 mg/L (159-322 mg/L), while for imidacloprid at a concentration of 1000 mg/L the effect on P. subcapitata was lower than 50%. Copper was the most toxic compound towards all test organisms exhibiting the highest toxic effect on P. subcapitata, with an IC₅₀ value of 0.05 mg/L (0.003-0.008 mg/L). The toxic effects of the binary mixtures have been compared to the theoretically expected effect, resulting from a simple mathematical model based on the theory of probabilities. The independent action model was used in order to predict the theoretically expected effect. The interactive effects were mostly antagonistic or additive, while in few cases (interactive effects of metalaxyl-M and copper on V. fischeri) a synergistic mode of action was observed for some concentration combinations. Experiments showed that interactive effects of chemicals may vary depending on the test species used as well as on the chemicals and their respective concentrations. Although most of the concentrations of chemicals tested in this study are higher than the ones usually found in natural environment, the evaluation of their interactive toxic effects using a battery of bioassays may comprise a useful tool for the estimation of the environmental hazard of chemicals.     

Water- and sediment-quality effects on Pimephales promelas spawning vary along an agriculture-to-urban land-use gradient

Many streams in the U.S. are “impaired” due to anthropogenic influence. For watershed managers to achieve practical understanding of these impairments, a multitude of factors must be considered, including point and nonpoint-source influence on water quality. A spawning assay was developed in this study to evaluate water- and sediment-quality effects that influenced Pimephales promelas (fathead minnow) egg production over a gradient of urban and agricultural land use in 27 small watersheds in Eastern Wisconsin. Six pairs of reproducing fathead minnows were contained in separate mesh cartridges within one larger flow-through chamber. Water- and sediment quality were sampled for an array of parameters. Egg production was monitored for each pair providing an assessment of spawning success throughout the 21-day test periods. Incidences of low dissolved oxygen (DO) in many of these streams negatively impacted spawning success. Nine of 27 streams experienced DO less than 3.1 mg/L and 15 streams experienced DO less than 4.8 mg/L. Low DO was observed in urban and agricultural watersheds, but the upper threshold of minimum DO decreased with increasing urban development. An increase in specific conductance was related to a decrease in spawning success. In previous studies for streams in this region, specific conductance had a linear relation with chloride, suggesting the possibility that chloride could be a factor in egg production. Egg production was lower at sites with substantial urban development, but sites with low egg production were not limited to urban sites. Degradation of water- and sediment-quality parameters with increasing urban development is indicated for multiple parameters while patterns were not detected for others. Results from this study indicate that DO must be a high priority watershed management consideration for this region, specific conductance should be investigated further to determine the mechanism of the relation with egg production, and water- and sediment-quality degrade in relation to urban influence.     

Assessment of ammonia toxicity in tests with the microalga,Nephroselmis pyriformis,Chlorophyta

Previous studies of an industrial effluent have indicated toxic effects by the ammonium/water system in tests with the unicellular green alga, Nephroselmis pyriformis. This investigation was undertaken to determine the toxicity of ammonia to this alga, and to identify the dominant toxicant in an industrial process effluent. The algal standard test was modified to improve the pH control, i.e. by testing at shorter exposure time and in the presence of a buffering agent, 3 mM HEPES. Ammonia was found to be the predominant toxic form in the dissociating ammonium/water system and the specific toxicity of ammonia (EC50, 24 h exposure) was 2.34 microM (32.8 microg ammonia nitrogen/L). Due to the dissociation, the toxicity is strongly pH dependent. Joint toxicity with additive effects of ammonia and ammonium ions was indicated, but the toxicity of ammonium ions was almost a factor 100 less (EC50, 24 h exposure, 224 microM or 3140 microg ammonium-nitrogen/L). According to the results the EC50 for effect on growth rate of N. pyriformis in seawater of pH 8.0 is 71 microM total ammonium. Hence, this alga appears to be more sensitive to ammonia than other studied marine plankton algae. Ammonia was found to be the dominant toxicant in the industrial effluent, using N. pyriformis as test organism.     

Toxicity of benzotriazole and benzotriazole derivatives to three aquatic species

Benzotriazole and its derivatives comprise an important class of corrosion inhibitors, typically used as trace additives in industrial chemical mixtures such as coolants, deicers, surface coatings, cutting fluids, and hydraulic fluids. Recent studies have shown that benzotriazole derivatives are a major component of aircraft deicing fluids (ADFs) responsible for toxicity to bacteria (Microtox). Our current research compared the toxicity of benzotriazole (BT), two methylbenzotriazole (MeBT) isomers, and butylbenzotriazole (BBT). Acute toxicity assays were used to model the response of three common test organisms: Microtox bacteria (Vibrio fischeri), fathead minnow (Pimephales promelas) and water flea (Ceriodaphnia dubia). The response of all the three organisms varied over two orders of magnitude among all compounds. Vibrio fischeri was more sensitive than either C. dubia or P. promelas to all the test materials, while C. dubia was less sensitive than P. promelas. The response of test organisms to unmethylated benzotriazole and 4-methylbenzotriazole was similar, whereas 5-methylbenzotriazole was more toxic than either of these two compounds. BBT was the most toxic benzotriazole derivative tested, inducing acute toxicity at a concentration of < or = 3.3 mg/l to all organisms.     

Assessing the ecotoxicity of vinyl chloride using green alga P. subcapitata, nematode C. elegans, and the SOS chromotest in a closed system without headspace

The ecotoxicity of vinyl chloride (VC) was evaluated using green alga, nematode, and the SOS chromotest. The green alga and nematode tested were Pseudokirchneriella subcapitata, and Caenorhabditis elegans, respectively. Because of the tendency of VC to escape from an aqueous exposure medium to the air phase, all tests in the present study were performed in a closed system without headspace to minimize the losses of VC. Previous studies on VC toxicity were performed in an open system or closed system with headspace. VC inhibits the growth of P. subcapitata. The 48-h IC50 value for P. subcapitata exposed to VC was calculated to be 5.15 mg/L. The survival of C. elegans was not influenced at concentrations of up to 60 mg/L; however, VC has an adverse effect on the reproduction of C. elegans. In a stress-related gene expression test using C. elegans, a significant and concentration-dependent expression of heat shock protein 16.2 was observed, indicating that VC induces the stress of C. elegans at the genetic level. The results of the SOS chromotest using Escherichia coli PQ37 showed an IF_max value of 1.11, indicating that VC is not genotoxic. The present study demonstrated that VC has an adverse effect on the algal growth and reproductive and genetic levels of C. elegans. A closed system without headspace is an effective method of testing the aquatic toxicity of volatile organic compounds such as VC.     

Assessment of copper bioavailability and toxicity in vineyard runoff waters by DPASV and algal bioassay

The aim of this study was to assess the toxicity of runoff waters in an agricultural multipollution context through an in-depth assessment of copper bioavailability and toxicity. Runoff waters were screened for major ions, metals and diuron. The potential environmental impact of these runoff waters was evaluated using the conventional 72-h growth inhibition test with the green alga Pseudokirchneriella subcapitata. The results suggested that the toxicity detected in the calcareous vineyard field was due to the presence of diuron, whereas the non-calcareous runoff waters were non-toxic. Chemical speciation modelling by MINEQL revealed that most of the copper present in the non-toxic natural runoff waters was complexed by organic matter. These samples were spiked with copper, and then the toxicity and the electrochemically bioavailable copper fraction were measured. Differential pulse anodic stripping voltammetry (DPASV) was used to detect labile complexes and free copper. This combined approach highlighted the presence of some labile copper complexes in samples reaching the EC10-these could have contributed to the copper toxicity.     

Use of multidimensional scaling in the selection of wastewater toxicity test battery components

In aquatic toxicity testing, no single test species is sensitive to all toxicants. Therefore, test batteries consisting of several individual assays are becoming more common. The organisms in a test battery should be representative of the entire system of interest. The results of the assays should be complementary to other components in the test battery to avoid redundancy. With the aid of multidimensional scaling (MDS), a multivariate statistical method, we examined the toxicity data of five bioassays (the continuous Shk1, Polytox, activated sludge respiration inhibition, Nitrosomonas, and Tetrahymena assays) that could serve as test battery components for the assessment of wastewater toxicity to activated sludge. MDS mapped the five assays into a two-dimensional space and showed that the Nitrosomonas assay should be included in test batteries plus one of the remaining four assays for assessing wastewater toxicity to activated sludge.     

Short-term algal toxicity test based on phosphate uptake

In order to develop a short-term algal toxicity test, the growth of and the phosphate uptake by the green alga Selenastrum capricornutum during batch culture were observed. In the control medium, S. capricornutum took up phosphate earlier than it grew. It was also observed that the phosphate uptake was inhibited by the presence of a toxicant. From these results, phosphate uptake was considered as one of the useful effect parameters for a short-term algal toxicity test. As the removal rate of phosphate from the medium is a function of the amount of algal cell initially inoculated, the test period is variable. The relationship between the amount of inoculation and phosphate uptake was examined and the test conditions suitable for a 3-h toxicity test were established as one example. According to this test procedure, the inhibitory effect of some toxicants on the phosphate uptake was determined. For comparison, a conventional algal assay based on algal growth was also performed. The EC50s for both tests were close. This indicated that the algal toxicity test method proposed in this paper would be useful for the uses where rapidity is required.     

Use of highly sensitive sublethal stress responses in the social amoeba Dictyostelium discoideum for an assessment of freshwater quality

In this work, the sensitivity of a battery of tests on the social amoeba Dictyostelium discoideum has been assessed within a freshwater toxicity study. The results obtained from the evaluation of survival and replication rate of D. discoideum were compared to those derived with a series of widely used tests for freshwater toxicity assessment, i. e. bioassays using Vibrio fischeri, Daphnia magna and Pseudokirchneriella subcapitata. The effects on sublethal endpoints, i.e. lysosomal membrane stability (LMS) and endocytotic rate, were analysed in conjunction with high-level endpoints to verify the potential to make a typical bioassay more sensitive. The field ecotoxicological investigation employing D. discoideum is part of a monitoring study assessing environmental quality of the Bormida River (Italy), subjected until recently to a chronic industrial pollution. The survey was carried out at several stations (upstream and downstream of a chemical factory outlet) in two different periods. In 2002, the results of chemical analyses performed on river water indicated no contamination. The ecotoxicological data obtained in this period showed that no evidence of biological effects was observed using V. fischeri and D. magna bioassays. In spite of the previous classical acute toxicity tests, significant differences in cell viability of D. discoideum were found. By analysing the effects measured on LMS and endocytotic rate, more relevant changes were observed for these sublethal stress biomarkers compared to survival. The chronic toxicity data showed significant changes in cell growth both of P. subcapitata and D. discoideum. Nevertheless, more sensitive and rapid responses were obtained when assessing the effects of exposure on D. discoideum. The chemical and ecotoxicological data obtained in 2006 indicated a full recovery of the quality of the river water (neither contamination nor toxicity found). Altogether, the results reported in this study underline that the use of a battery of biomarkers in conjunction with high-level endpoints may help follow the pollutant-induced stress syndrome in the organisms from early sublethal effects to starting mortality.     

Cross-species extrapolation of chronic nickel Biotic Ligand Models

The use of Biotic Ligand Models (BLMs) to normalize metal ecotoxicity data and predict effects in non-BLM organisms should be supported by quantitative evidence. This study determined the ability of chronic nickel BLMs developed for the cladocera Daphnia magna and Ceriodaphnia dubia to predict chronic nickel toxicity to three invertebrates for which no specific BLMs were developed. Those invertebrates were the snail Lymnaea stagnalis, the insect Chironomus tentans, and the rotifer Brachionus calyciflorus. Similarly, we also determined the ability of chronic nickel BLMs developed for the alga Pseudokirchneriella subcapitata and the terrestrial vascular plant Hordeum vulgare to predict chronic nickel toxicity to the aquatic vascular plant Lemna minor. Chronic nickel toxicity to the three invertebrates and the aquatic plant were measured in five natural waters that varied in pH, Ca, Mg, and dissolved organic carbon (DOC), which are known to affect chronic nickel toxicity and are the important input variables for the chronic nickel BLMs. Nickel toxicity to the three invertebrates varied considerably among the test waters, i.e., a 14-fold variation of EC50s in L. stagnalis, a 3-fold variation in EC20s in C. tentans, and a 10-fold variation in EC20s in B. calyciflorus, but the cladoceran BLMs were able to predict nickel effect concentrations within a factor of two. Nickel toxicity (EC50s) to L. minor varied by 6-fold among the test waters. Although the P. subcapitata and H. vulgare BLMs offered reasonable predictions of nickel EC50s to L. minor, the D. magna and C. dubia BLM showed better predictions. Our results confirm the influence of site-specific pH, hardness, and DOC on chronic nickel toxicity to aquatic organisms, and support the use of chronic nickel BLMs to manage this influence through normalizations of ecotoxicity data.     

Effect of combined physico-chemical processes on the phytotoxicity of olive mill wastewaters

A pool of laboratory experiments is planned with the aim of evaluating the possibility to reduce the phytotoxicity of olive mill wastewater (OMW) with combined physico-chemical processes (centrifugation-ozonation, centrifugation-solar photolysis, centrifugation-solar modified photoFenton, centrifugation-solar modified photoFenton-ozonation). A moderate COD removal of an OMW is reached by using ozonation or solar modified photoFenton separately or solar modified photoFenton/O(3) combined process even for prolonged treatment times. The O(3)-treated OMWs are still toxic towards algal growth (Pseudokirchneriella subcapitata) and only for dilutions equal to or higher than 1:160 a stimulation of algal growth is observed. The sole ozonation does not reduce significantly the phytotoxicity of tested OMW measured through the GI calculation of Raphanus sativus L., Cucumis sativus L. and Lactuca sativa L. A marked reduction of OMW inhibition, higher than 50%, is evidenced for 1:8 dilution OMW samples ozonated for 2h. The long-term storage of OMW associated with solar irradiation without or with Fe(III) ions under continuous aeration is less efficient than ozonation, and the combined action of the two former treatments does not significantly contribute to enhance both COD removal and germination index. Better results are obtained on seed germination and root elongation of plantlets of the three selected species, which germinated on OMW-free solidified medium and were then transferred on a solidified culture medium containing O(3)-treated OMW diluted 1:2 and 1:4. The operating costs are estimated for the solar modified photoFenton-ozonation process.     

Selecting a battery of bioassays for ecotoxicological characterization of wastes

This study was conducted in France within the context of waste classification (Hazardous Waste Council Directive 91/689/EEC), and focused on "ecotoxic" property (H14). In 1998, an experimental test strategy was developed to assess ecotoxicological properties of wastes using a battery of six standardized bioassays. This combined direct and indirect approaches integrating two solid-phase tests: emergence and growth inhibition of Lactuca sativa (14 days), mortality of Eisenia fetida (14 days) and four standardized tests performed on water extracts from wastes: growth inhibition of Pseudokirchneriella subcapitata (3 days), inhibition of mobility of Daphnia magna (48 h), inhibition of reproduction of Ceriodaphnia dubia (7 days), inhibition of light emission of Vibrio fischeri (30 min). This study aimed to set up preliminary conclusions on relevancy of this experimental test strategy, based on data obtained since 1998. Results were analyzed from the combined use of Hierarchical Cluster Analysis, Principal Component Analysis and Nonlinear Mapping. These multivariate analyses clearly showed that it was possible to reduce this number of tests without changing the typology of the wastes. A battery of bioassays including one solid phase test and two tests performed on water extracts (L. sativa, V. fischeri and C. dubia) was found as an optimal solution for characterizing the toxicity of the studied wastes. This optimal battery represents a good basis for determining the H14 property.     

Chronic toxicity of the Sava River (SE Europe) sediments and river water to the algae Pseudokirchneriella subcapitata

The Sava River, the largest and most commercially valuable water body in the riparian countries, receives inputs of organic and inorganic compounds from a variety of domestic and industrial activities that may affect the health of human beings and wildlife. In this work, the chronic toxicity of sediment, sediment porewater and surface water from the Sava River and connecting tributaries to the freshwater algae Pseudokirchneriella subcapitata was assessed to characterise the potential impact on aquatic organisms. Samples of different environmental matrices were either tested directly (porewater) or subjected to pre-concentration (sediments and surface water) prior to testing in a 72 h growth inhibition assay using P. subcapitata. The results show that a combination of pre-concentration and bioassay testing was able to characterise the toxic potential and to identify several compartment-specific "hot spots" along the Sava River. Based on the resulting data, a crude risk assessment identified that some of the locations may represent a risk to algae. Additional testing using multiple species and exposure phases is required to conduct a thorough risk assessment, however.     

The interaction of treated bleached kraft mill effluent and dissolved oxygen concentration on the survival of the developmental stages of the sheepshead minnow (Cyprinodon variegatus)

The interactions of treated bleached kraft mill effluent (BKME) and dissolved oxygen concentration (DO) on the survival of the embryo, fry, juvenile and adult stages of the sheepshead minnow, Cyprinodon variegatus, were studied under continuous-flow 96 h bioassay conditions. Embryo survival was dependent on effluent concentration only and showed no interaction at nominal DO concentrations of 1.0, 3.0 and 5.0 mg l⁻¹. Survival of the fry was related solely to DO concentration with no interaction with BKME concentrations up to 100%. Juvenile sheepshead minnows showed increased sensitivity to BKME at a nominal DO concentration of 1.0 mg l⁻¹. Adult fish were not affected by BKME at any of the DO concentrations tested. This study shows that the acute toxicity of BKME effluent to sheepshead minnows is a function of the developmental stage of the organism and DO concentration in the receiving stream.     

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

The European Union regulation on Registration, Evaluation, Authorization and Restriction of Chemical substances (REACH) (EC, 2006) requires the characterization of the chronic toxicity of many chemicals in the aquatic environment, including molybdate (MoO₄²⁻). Our literature review on the ecotoxicity of molybdate revealed that a limited amount of reliable chronic no observed effect concentrations (NOECs) for the derivation of a predicted no-effect concentration (PNEC) existed. This paper presents the results of additional ecotoxicity experiments that were conducted in order to fulfill the requirements for the derivation of a PNEC by means of the scientifically most robust species sensitivity distribution (SSD) approach (also called the statistical extrapolation approach). Ten test species were chronically exposed to molybdate (added as sodium molybdate dihydrate, Na₂MoO₄·2H₂O) according to internationally accepted standard testing guidelines or equivalent. The 10% effective concentrations (EC10, expressed as measured dissolved molybdenum) for the most sensitive endpoint per species were 62.8-105.6 (mg Mo)/L for Daphnia magna (21 day-reproduction), 78.2 (mg Mo)/L for Ceriodaphnia dubia (7 day-reproduction), 61.2-366.2 (mg Mo)/L for the green alga Pseudokirchneriella subcapitata (72 h-growth rate), 193.6 (mg Mo)/L for the rotifer Brachionus calyciflorus (48 h-population growth rate), 121.4 (mg Mo)/L for the midge Chironomus riparius (14 day-growth), 211.3 (mg Mo)/L for the snail Lymnaea stagnalis (28 day-growth rate), 115.9 (mg Mo)/L for the frog Xenopus laevis (4 day-larval development), 241.5 (mg Mo)/L for the higher plant Lemna minor (7 day-growth rate), 39.3 (mg Mo)/L for the fathead minnow Pimephales promelas (34 day-dry weight/biomass), and 43.2 (mg Mo)/L for the rainbow trout Oncorhynchus mykiss (78 day-biomass). These effect concentrations are in line with the few reliable data currently available in the open literature. The data presented in this study can serve as a basis for the derivation of a PNEC_aquatic that can be used for national and international regulatory purposes and for setting water quality criteria. Using all reliable data that are currently available, a HC_5,50% (median hazardous concentration affecting 5% of the species) of 38.2 (mg Mo)/L was derived with the statistical extrapolation approach.