More than inorganic copper is bioavailable to aquatic mosses at environmentally relevant concentrations

The present study investigates how dissolved organic matter (DOM) alters copper bioavailability at environmentally relevant concentrations (1-5 microg/L of dissolved copper, 1-4 mg/L of dissolved organic copper). A methodology combining two biological endpoints (short-term and steady-state bioaccumulation of copper by the aquatic moss Fontinalis antipyretica) and a sampling of labile copper with diffusion gradient in thin films (DGT) is proposed for batch experiments conducted with mineral water and various DOM, ethylenediaminetetra-acetic acid (EDTA), humic acid, and natural Seine River (France) extracts (hydrophobic and transphilic fractions). All types of DOM reduce the bioavailability of copper to aquatic mosses, and this reduction was more pronounced for the short-term biological endpoint, which was taken as being representative for environmental exposure. Labile copper sampled with DGT made it possible to estimate short-term bioaccumulation in the case of EDTA and natural Seine River extracts. With humic acid solutions, however, labile copper was lower than bioavailable copper. This result suggests that at realistic metal concentrations and with certain types of natural DOM, bioavailable copper might comprise not only inorganic copper but also some weak organic complexes. Hence, labile copper, in situ sampled with DGT, might not systematically overestimate bioavailable copper, as suggested previously on the basis of in vitro toxicity studies.     

Development and field validation of a predictive copper toxicity model for the green alga Pseudokirchneriella subcapitata

In this study, the combined effects of pH, water hardness, and dissolved organic carbon (DOC) concentration and type on the chronic (72-h) effect of copper on growth inhibition of the green alga Pseudokirchneriella subcapitata were investigated. Natural dissolved organic matter (DOM) was collected at three sites in Belgium and The Netherlands using reverse osmosis. A full central composite test design was used for one DOM and a subset of the full design for the two other DOMs. For a total number of 35 toxicity tests performed, 72-h effect concentration resulting in 10% growth inhibition (EbC10s) ranged from 14.2 to 175.9 micrograms Cu/L (factor 12) and 72-h EbC50s from 26.9 to 506.8 micrograms Cu/L (factor 20). Statistical analysis demonstrated that DOC concentration, DOM type, and pH had a significant effect on copper toxicity; hardness did not affect toxicity at the levels tested. In general, an increase in pH resulted in increased toxicity, whereas an increase of the DOC concentration resulted in decreased copper toxicity. When expressed as dissolved copper, significant differences of toxicity reduction capacity were noted across the three DOM types tested (up to factor 2.5). When expressed as Cu2+ activity, effect levels were only significantly affected by pH; linear relationships were observed between pH and the logarithm of the effect concentrations expressed as free copper ion activity, that is, log(EbC50Cu2+) and log(EbC10Cu2+): (1) log(EbC50Cu2+)= - 1.431 pH + 2.050 (r2 = 0.95), and (2) log(EbC10cu2+) = -1.140 pH -0.812 (r2 = 0.91). A copper toxicity model was developed by linking these equations to the WHAM V geochemical speciation model. This model predicted 97% of the EbC50dissolved and EbC10dissolved values within a factor of two of the observed values. Further validation using toxicity test results that were obtained previously with copper-spiked European surface waters demonstrated that for 81% of tested waters, effect concentrations were predicted within a factor of two of the observed. The developed model is considered to be an important step forward in accounting for copper bioavailability in natural systems.     

Measuring the bioavailability of two hydrophobic organic compounds in the presence of dissolved organic matter

Bioavailability of benzo[a]pyrene (BaP) and 3,3',4,4'-tetrachlorobiphenyl (TCB) was studied in natural lake water containing dissolved organic matter (DOM). Lake water was diluted to give a dissolved organic carbon (DOC) range of 1 to 20 mg/L. Partition coefficients for the model compounds were assessed at different DOM concentrations and over time with three different methods, namely equilibrium dialysis and reverse-phase and liquid-liquid extraction. In addition, biological partition coefficients were estimated from the difference in the bioconcentration of the model compounds in Daphnia magna in the presence and absence of DOM. Results showed that bioavailability of the model compounds was reduced by the presence of DOM. The equilibrium dialysis method gave the best estimates for bioavailability of the model compounds when compared with biologically determined values. Both the reverse-phase and the liquid-liquid extraction overestimated the bioavailable fraction. The more pronounced overestimation of bioavailable fraction of TCB suggested that the sorption of TCB was not only lower but the interaction was also weaker than that of BaP. Increasing DOM concentration produced lower partition coefficients and the effect seemed to be more pronounced when measured by the reverse-phase and the extraction methods.     

Speciation of aqueous methylmercury influences uptake by a freshwater alga (Selenastrum capricornutum)

Uptake of methylmercury (MeHg) by the alga Selenastrum capricornutum was measured in freshwater batch culture bioassays. The concentration of MeHg in the alga increased rapidly (within 15 min), reached a maximum by 6 h, and then declined because of growth dilution. The alga's rapid growth rate (doubling time, approximately 10 h) contributed to the importance of growth dilution. Conditional first-order rate constants were calculated for uptake (k1 = 6.95 x 10(-9) L/cell/h) and growth (kG = 0.07/h). A competitive synthetic ligand, disodium ethylenediaminetetra-acetate, formed strong complexes with MeHg and reduced MeHg uptake, consistent with the biotic ligand model. A conditional equilibrium formation constant (K) for the MeHg-algae complex was estimated to be approximately 10(16) and was used to model the influence of natural ligands on MeHg bioavailability. Model results suggested MeHg would be most bioavailable at concentrations of dissolved organic matter (DOM) less than 10 mg/L and increasingly unavailable at higher DOM concentrations for the specific humic acid modeled. Similarly, at molar concentrations of sulfide (and, possibly, metal-sulfide clusters) equal to approximately half the MeHg concentration, MeHg was predicted to be unavailable to algae because of the formation of strong 2:1 MeHg-sulfide complexes.     

Influence of dissolved organic matter on the complexation of mercury under sulfidic conditions

The complexation of Hg under sulfidic conditions influences its bioavailability for microbial methylation. Neutral dissolved Hg-sulfide complexes are readily available to Hg-methylating bacteria in culture, and thermodynamic models predict that inorganic Hg-sulfide complexes dominate dissolved Hg speciation under natural sulfidic conditions. However, these models have not been validated in the field. To examine the complexation of Hg in natural sulfidic waters, octanol/water partitioning methods were modified for use under environmentally relevant conditions, and a centrifuge ultrafiltration technique was developed. These techniques demonstrated much lower concentrations of dissolved Hg-sulfide complexes than predicted. Furthermore, the study revealed an interaction between Hg, dissolved organic matter (DOM), and sulfide that is not captured by current thermodynamic models. Whereas Hg forms strong complexes with DOM under oxic conditions, these complexes had not been expected to form in the presence of sulfide because of the stronger affinity of Hg for sulfide relative to its affinity for DOM. The observed interaction between Hg and DOM in the presence of sulfide likely involves the formation of a DOM-Hg-sulfide complex or results from the hydrophobic partitioning of neutral Hg-sulfide complexes into the higher-molecular-weight DOM. An understanding of the mechanism of this interaction and determination of complexation coefficients for the Hg-sulfide-DOM complex are needed to adequately assess how our new finding affects Hg bioavailability, sorption, and flux.     

Differential bioavailability of copper complexes to bioluminescent Pseudomonas fluorescens reporter strains

Increased levels of Cu in agricultural soils are of concern, because Cu toxicity may adversely affect important soil microorganisms, including pseudomonads. Because total metal concentrations correlate poorly with bioavailability and toxicity, a need exists for more information linking Cu speciation, bioavailability, and toxicity. The objective of the present study was to determine the bioavailability of different Cu complexes to Pseudomonas spp. A Cu-specific bioluminescent Pseudomonas fluorescens reporter strain was used to determine bioavailable Cu, which was operationally defined as those Cu species that induced expression of bioluminescence. Another strain of P. fluorescens, which continuously expressed bioluminescence, was used as a toxicity reporter. Experiments were performed in a defined aqueous medium containing 0.04 microM Cu, which was amended with ethylenediaminetetraacetic acid (EDTA), citrate, or a well-characterized pool of dissolved organic matter (DOM). Bioluminescence emitted by the biosensors was related to data for Cu speciation obtained by geochemical modeling. Changes in Cu bioavailability in the presence of EDTA coincided with modeled changes in Cu2+ activity, indicating that Cu-EDTA complexes were not bioavailable to the Cu-specific reporter. In contrast, changes of Cu bioavailability in the presence of citrate did not correspond to changes in Cu2+, indicating that Cu-citrate complexes were fully bioavailable to the reporter strain. Finally, the response of the Cu-reporter strain to Cu in the presence of DOM indicated that Cu formed bioavailable as well as unavailable complexes with DOM. We conclude that free Cu2+ activity is a poor predictor of Cu bioavailability to Pseudomonas spp. in samples containing organic ligands.     

Characterization,DBPs formation,and mutagenicity of different organic matter fractions in two source waters

Dissolved organic matters (DOM) are critical in the formation of the mutagenic disinfection byproducts (DBPs). In this study, five DOM fractions were isolated and investigated from a contaminated river and a clean reservoir source waters using resin adsorption. The DOM fractions were characterized with excitation–emission matrix, and several typical DBPs formation potentials and the mutagenicity of each DOM fraction were measured. Among these fractions, hydrophobic neutrals (HON), hydrophilic acids (HIA) and hydrophilic bases (HIB) generated the highest carbon-containing DBPs in the river source water, as did HIB and hydrophobic bases (HOB) in reservoir water. Hydrophobic acids (HOA), HON, and HIA were the three most important fractions forming nitrogen-containing DBPs. Following chlorination, the mutagenicity of HON, HIA, HOA and HIB was 1503, 626, 422 and 116 ng 4-NQO/mg DOC in river water, respectively. Only HIA and HOA were mutagenic with 85 and 10 ng 4-NQO/mg DOC in reservoir water, respectively. The soluble microbial products like substances and aromatic proteins contributed significantly to the mutagenicity of river water; whereas the humic acid-like and fulvic acid-like substances were the primary contributors to the mutagenicity of reservoir water.     

Effect of dissolved organic matter of various origins and biodegradabilities on the bioaccumulation of polycyclic aromatic hydrocarbons in Daphnia magna

As a preliminary study of the influence of urban organic matter on the bioavailability of polycyclic aromatic hydrocarbons (PAHs), the effect of different types of dissolved organic matter (DOM) on the bioaccumulation of fluoranthene, pyrene, or benzo[a]pyrene in Daphnia magna was studied. Commercial humic substances, DOM from the aeration basin of a wastewater treatment plant, and highly biodegradable DOM (algae or animal extracts) were tested. The bioaccumulation of benzo[a]pyrene was reduced by each DOM (up to 80% reduction with humic substances). Pyrene bioaccumulation was also decreased by each DOM to a lesser extent. Fluoranthene bioaccumulation was affected by the presence of humic acids only. In each experiment, the solution containing humic DOM led to the lowest bioaccumulation. Supposing that only dissolved PAHs were bioavailable, the reduction of bioaccumulation allowed a biological estimate of the partition coefficients of DOM and PAH, K(DOC). The estimated coefficients were positively related to the aromaticity of DOM and negatively related to its biodegradability.     

UV-B-Induced acute toxicity of pyrene to the waterflea Daphnia magna in natural freshwaters

The effects of various water characteristics in natural freshwaters on the acute toxicity of one polycyclic aromatic hydrocarbon (PAH), pyrene, to a pelagic invertebrate Daphnia magna was studied under ultraviolet B (UV-B) radiation and in the dark. Pyrene was photoactivated and was more toxic to D. magna in the presence of UV-B radiation. Dissolved organic material (DOM), measured as dissolved organic carbon (DOC), significantly reduced the photoenhanced toxicity of pyrene. Under UV-B radiation the EC(50) values were lower and in relation to the amount of DOM, ranging from 3.0 to 30.0 microg/L pyrene, whereas in the dark they were between 29.2 and 54.8 microg/L and not related to the amount of DOM in the waters. Although the condition and mortality of the daphnids in the control groups were not affected by UV-B irradiation, the increased toxicity was considered to be either an additive or a synergistic effect of both the photomodified pyrene and the stressing light conditions of UV-B. The measured binding of pyrene to DOM was low, although it was related to the amount of DOC. Despite the relatively high intensity of UV-B used, humic substances in the waters remained undegraded. It was thus concluded that with their brownish-yellowish color, waters rich in humic substances decreased the photomodification of the freely dissolved parent compound simply by diminishing the light penetration in these waters and, by implication, contact with the intact compound. These results suggest that DOM in surface waters plays an important role in protecting against the photoinduced toxicity of PAHs.     

Influence of Dissolved Organic Matter on Acute Toxicity of Zinc to Larval Fathead Minnows (Pimephales promelas)

We conducted laboratory toxicity tests in support of the development of a biotic ligand model (BLM) to predict acute toxicity of zinc (Zn) to fathead minnows (Pimephales promelas). To test the effect of dissolved organic matter (DOM) on Zn toxicity, we exposed larval fathead minnows to Zn in water containing elevated concentrations of dissolved organic carbon (DOC) in 96-h static-renewal toxicity tests. We tested DOM isolated from four surface waters: Cypress Swamp, Delaware; Edisto River, South Carolina; Suwannee River, Georgia; and Wilmington, Delaware, wastewater treatment effluent. The DOM isolates from the Edisto River and Wilmington wastewater treatment effluent contained elevated concentrations of NaCl (20–110× control NaCl) due to the use of a Na⁺-exchange resin to remove Ca²⁺ and Mg²⁺ during the DOM isolation process. Therefore, we also performed Zn toxicity tests in which we added up to 20 mM NaCl to exposure solutions containing Cypress Swamp and Suwannee River DOM. A threshold concentration of 11 mg DOC/L was needed to decrease Zn toxicity, after which the 96 h Zn LC50 was positively correlated with DOC concentration. Elevated NaCl concentrations did not alter Zn toxicity in the presence of DOM. In conjunction with data from other studies with fish and invertebrates, results of this study were used to calibrate Version 2.1.1 of the Zn BLM. BLM-predicted LC50s for our exposure waters containing elevated DOM concentrations were within the range of acceptable deviation relative to the observed LC50s (i.e., 0.5–2× observed LC50s); however, BLM-predicted LC50s for our exposure waters containing < 1 mg DOC/L were 2–3× lower than the observed LC50s (i.e., the BLM over-predicted the toxicity). Therefore, the current composite-species BLM for Zn could be improved for fathead minnows if that species were modeled separately from the other species used to calibrate Version 2.1.1.     

Modification of the equilibrium partitioning approach for volatile organic compounds in sediment

Although weakly hydrophobic chemicals such as volatile organic compounds (VOCs) tend not to persist in sediments, they may nevertheless be present in some sediments because of recent or ongoing releases. Standard methods are not available for assessing risks to benthic invertebrates due to VOCs in sediment. More strongly hydrophobic organic chemicals are frequently assessed by using the equilibrium partitioning (EqP) approach, which predicts the bioavailable fraction of chemical (the concentration dissolved in pore water) from the sediment-sorbed fraction, assuming that partitioning is at equilibrium. As typically applied, the EqP approach is ineffective for assessing VOCs in sediment, because the standard EqP equation fails to account for the contribution of dissolved chemical to the total chemical concentration in sediment. For chemicals with low organic carbon-water partition coefficients (Kocs), this results in nonsensical sediment-quality benchmarks that are more conservative (i.e., lower) than benchmarks calculated by assuming 100% bioavailability. A modified EqP equation is presented that accounts for the dissolved fraction of total chemical concentrations in sediment. Results of the standard and modified EqP equations converge with increasing Koc and are essentially identical at log Koc values exceeding approximately 3.5.     

Phase distribution of synthetic pyrethroids in runoff and stream water

Synthetic pyrethroids (SPs) are a group of hydrophobic compounds with significant aquatic toxicity. Their strong affinity to suspended solids and humic materials suggests that SPs in natural surface water are distributed in solid-adsorbed, dissolved organic matter (DOM)-adsorbed, and freely dissolved phases. The freely dissolved phase is of particular importance because of its mobility and bioavailability. In the present study, we used solid-phase microextraction to detect the freely dissolved phase, and we evaluated the phase distribution of bifenthrin and permethrin in stream and runoff waters. In stream water, most SPs were associated with the suspended solids and, to a lesser extent, with DOM. The freely dissolved phase contributed only 0.4% to 1.0%. In runoff effluents, the freely dissolved concentration was 10% to 27% of the overall concentration. The predominant partitioning into the adsorbed phases implies that the toxicity of SPs in surface water is reduced because of decreased bioavailability. This also suggests that monitoring protocols that do not selectively define the freely dissolved phase can lead to significant overestimation of toxicity or water-quality impacts by SPs.     

Dissolved organic matter modulating the uptake, biotransformation, and elimination rates of pyrene in Daphnia magna

In the current study, the toxicokinetics of pyrene in Daphnia magna was determined in detail, and the role of dissolved organic matter (DOM) in these processes was investigated. Uptake, biotransformation, parent compound and metabolite elimination rates (k(u), k(m), k(ep), k(em)), depuration rates, bioconcentration factors (BCFs), and biological half-lives were studied in one natural humic water, in two waters made from DOM isolates, and in DOM-free artificial fresh water. The daphnids were exposed to pyrene for 24 h and depurated for another 24 h. To analyze the data, the two-compartment model was used to obtain toxicokinetic parameters, and the depuration data were analyzed by linear regression. Extensive biotransformation of pyrene resulted in high metabolite concentration in the organisms (83% of total radioactivity) after 24 h exposure. Depuration of the parent compound seemed to be biphasic: at first, depuration was rapid, but after 4 to 10 h in uncontaminated water, the disappearance rate came close to zero. After a 24-h depuration time, 7.8% of the pyrene accumulated remained in the organism. Overall, the elimination of parent compound was faster than the elimination of metabolites, which might imply inefficient excretion of water-soluble biotransformation products from daphnids. The effect of DOM on accumulation of pyrene was seen in a decreased uptake rate, indicating lower bioavailability of the compound in waters that contain high-aromaticity DOM. In the same waters, DOM significantly accelerated pyrene biotransformation and overall elimination. Altogether, DOM appears to be able to affect toxicokinetics through mechanisms that are connected to interaction with the contaminant but also directly through interaction with biotransformation systems.     

The evaluation for alterations of DOM components from upstream to downstream flow of rivers in Toyama (Japan) using three-dimensional excitation-emission matrix fluorescence spectroscopy

The dissolved organic matter (DOM) is one of the important factors for controlling water quality. The behavior and constitutions of DOM is related to the risk of human health because it is able to directly or indirectly affect the behavior, speciation and toxicity of various environmental pollutants. However, it is not easy to know the contents of DOM components without using various complicated and time consuming analytical methods because DOM is a complex mixture and usually exists at low concentration. Here, we describe the fluorescence properties of DOM components in water samples collected from four rivers in Toyama, Japan by means of the three-dimensional excitation-emission matrix (3DEEM) fluorescence spectroscopy. In order to evaluate the alterations of DOM components in each of the river during the flow from upstream to downstream, the patterns of relative fluorescence intensity (RFI) at six peaks which are originated from fluorophores including humic-like and protein-like components were investigated. The changes in the patterns of RFI values at each of the peak and the concentration of dissolved organic carbon (DOC) for each river water sample were discussed in connection with the differences of land use managements and basic water quality parameters, such as pH, EC, turbidity, Fe(3+), T-N, NO(3)-N, T-P, PO(4)-P, chlorophyll a, DOC and N/P ratio. The DOC concentrations in the water samples collected from these rivers were relatively low (0.63-1.16 mg/L). Two main peaks which have a strong RFI value expressed a positive correlation with the DOC concentration (r = 0.557, 0.535). However, the correlations between the RFI values for other four peaks and the DOC concentration were below 0.287. The alterations of DOM components during the flow of a river from upstream to downstream were investigated from the changes in the patterns of RFI values for six fluorescent peaks. It was clarified that the great increase of RFI values in peak A and peak T from river water located in urban area showed high concentration of PO(4)-P and Fe(3+), and low N/P ratio due to the high biological activities. The values of fluorescence index (FIX) and biological index (BIX) were as high as 1.60 and 0.72, respectively.     

Trace metal distribution in soluble organic matter from municipal solid waste compost determined by size-exclusion chromatography

Municipal solid waste (MSW) composts carry high amounts of trace metals and organic complexing agents that may influence metal bioavailability and mobility after application to soils. In order to assess the degree of organic complexation of trace metals in the solution phase of MSW compost and the relevance of organic ligand type, size exclusion chromatography (SEC) was applied to compost-extracted organic ligands. Adjustment of the elution conditions minimized the interaction with the gel matrix for compost humic substances and dissolved organic matter (DOM) fractions. The SEC was then used to separate the aqueous compost extract into samples with distinct differences in chemical constituents. The highest quantities of Cu, Zn, Ni, Mn, and Cd were found to coelute with the main peak of the SEC elution curve, which, as observed by Fourier-transformed infrared (FTIR) spectroscopy, also had the highest density of carboxyl groups. The ratio of aromatic to aliphatic structures was higher for eluates with low retention times, and cations such as Al, Cr, and Fe were preferably associated with these larger organic molecules. All trace metals in the compost solution phase were bound mostly to DOM rather than forming inorganic complexes.     

Bioavailability models for predicting acute and chronic toxicity of zinc to algae, daphnids, and fish in natural surface waters

Bioavailability models predicting acute and/or chronic zinc toxicity to a green alga (Pseudokirchneriella subcapitata), a crustacean (Daphnia magna), and a fish (Oncorhynchus mykiss) were evaluated in a series of experiments with spiked natural surface waters. The eight selected freshwater samples had varying levels of bioavailability modifying parameters: pH (5.7-8.4), dissolved organic carbon (DOC, 2.48-22.9 mg/L), Ca (1.5-80 mg/L), Mg (0.79-18 mg/L), and Na (3.8-120 mg/L). In those waters, chronic zinc toxicity (expressed as 10% effective concentrations [EC10]) varied up to 20-fold for the alga (72-h EC10 from 27.3 to 563 microg Zn/L), and approximately sixfold for the crustacean (21-d EC10 from 59.2 to 387 microg Zn/L), and fivefold for the fish (30-d LC10, lethal concentration for 10% of the organisms, from 185 to 902 microg Zn/L). For P. subcapitata a refined bioavailability model was developed by linking an empirical equation, which predicts toxicity expressed as free Zn2+ activity as a function of pH, to the geochemical speciation model WHAM/Model V. This model and previously developed acute and/or chronic biotic ligand models for D. magna and 0. mykiss generally predicted most effect concentrations by an error of less than a factor of two. In waters with pH > 8, however, chronic toxicity to D. magna was underestimated by a factor 3 to 4. Based on the results of this validation exercise and earlier research, we determined applicability ranges for pH (6-8) and Ca (5-160 mg/L) in which all three developed models are valid. Within these ranges, all three models may be considered useful tools for taking into account bioavailability in regulatory assessments of zinc.     

Effect of suspended solids on bioavailability of pyrethroid insecticides

Runoff and surface-water effluents commonly contain suspended solids. Adsorption to suspended particles and the associated dissolved organic matter (DOM) may significantly decrease the freely dissolved concentration of a hydrophobic compound and, hence, its availability to aquatic organisms. In the present study, we evaluated phase distribution and bioaccumulation of two synthetic pyrethroids, bifenthrin and permethrin, in water samples containing suspended solids from different source sediments. Uptake of [14C]bifenthrin or [14C] permethrin by Daphnia magna after 24 h consistently decreased with increasing levels of suspended solids in the range of 0 to 200 mg/L. The trend of decrease was closely mimicked by pesticide accumulation on polydimethylsiloxane (PDMS) fibers exposed under the same conditions, and the ratio of body residues in D. magna to the concentration detected in the PDMS fiber was consistently around 2.4. Regression analysis showed that the pesticide adsorbed on particles or DOM was completely unavailable to D. magna for uptake during the 24-h exposure. The relative contribution of particles and DOM to the reduced bioavailability depended on the organic matter content and the texture of the source sediment. The influence from particles was predominant for sandy sediments, but contribution from DOM became comparable to or even greater than particles when the organic matter content of the source sediment was 1% or greater. The inhibitory effects of suspended solids on bioavailability should be considered when monitoring runoff and surface-water effluents for synthetic pyrethroids. The proposed PDMS method is simple and inexpensive, and it may serve as an effective option for obtaining ecotoxicologically relevant concentrations.     

Effect of initial cell density on the bioavailability and toxicity of copper in microalgal bioassays

Algal toxicity tests based on growth inhibition over 72 h have been extensively used to assess the toxicity of contaminants in natural waters. However, these laboratory tests use high cell densities compared to those found in aquatic systems in order to obtain a measurable algal response. The high cell densities and test duration can result in changes in chemical speciation, bioavailability, and toxicity of contaminants throughout the test. With the recent application of flow cytometry to ecotoxicology, it is now possible to use lower initial cell densities to minimize chemical speciation changes. The speciation and toxicity of copper in static bioassays with the tropical freshwater alga Chlorella sp. and the temperate species Selenastrum capricornutum (Pseudokirchneriella subcapitata) were investigated at a range of initial cell densities (10(2)-10(5) cells/ml). Copper toxicity decreased with increasing initial cell density. Copper concentrations required to inhibit growth (cell division) rate by 50% (72-h median effective concentration [EC50]) increased from 4.6 to 16 microg/L for Chlorella sp. and from 6.6 to 17 microg/L for S. capricornutum as the initial cell density increased from 10(2) to 10(5) cells/ml. Measurements of anodic stripping voltammetry-labile, extracellular, and intracellular copper confirmed that at higher initial cell densities, less copper was bound to the cells, resulting in less copper uptake and lower toxicity. Chemical measurements indicated that reduced copper toxicity was due primarily to depletion of dissolved copper in solution, with solution speciation changes due to algal exudates and pH playing a minor role. These findings suggest that standard static laboratory bioassays using 10(4) to 10(5) algal cells/ml may seriously underestimate metal toxicity in natural waters.     

Reduction in acute toxicity of soils to terrestrial oligochaetes following the removal of bioavailable polycyclic aromatic hydrocarbons with mild supercritical carbon dioxide extraction

Three soil samples contaminated with polycyclic aromatic hydrocarbons (PAHs) that caused 100% mortality to terrestrial oligochaetes were extracted with supercritical carbon dioxide to remove the bioavailable fraction of PAHs. Although the remaining PAH concentrations were high after supercritical fluid extraction (SFE), 650 to 8,000 mg/kg, acute toxicity to Eisenia fetida and Enchytraeus albidus essentially was eliminated. These results demonstrate that mild SFE with pure carbon dioxide preferentially extracts PAH molecules that are bioavailable toxicologically to the oligochaetes, although biologically unavailable PAHs are not extracted, suggesting that SFE could be used for the removal of toxicity due to hydrophobic organic chemicals in soils during toxicity identification evaluations.     

Effects of water hardness and dissolved organic material on bioavailability of selected organic chemicals

The influence of water hardness and dissolved organic matter (DOM) on bioavailability of organic chemicals to Daphnia magna was studied by using benzo[a]pyrene (BaP), pyrene, atrazine, and 3,3',4,4'-tetrachlorobiphenyl (TCB) as model compounds. Two types of DOM were used, namely Lake Kontiolampi, Joensuu, Finland water (KL) and Nordic reference fulvic acid (NoFA) dissolved in artificial freshwater. Binding of the four contaminants by KL DOM decreased with increasing water hardness. Furthermore, increasing hardness reduced the binding of BaP and pyrene to NoFA. The binding of atrazine and TCB by NoFA was low and was not significantly affected by water hardness. In the DOM-free samples, the bioconcentration of the four contaminants in D. magna usually was not affected by water hardness. In the presence of DOM, the bioconcentration factors (BCFs) were lower (except for atrazine) than in the DOM-free controls. In the presence of both types of DOM, increasing water hardness resulted in higher BCFs for BaP. The bioconcentration of pyrene and TCB increased with increasing water hardness in the presence of KL DOM. In conclusion, the effects of DOM and water hardness on bioavailability of hydrophobic chemicals depend on the type of chemical and on the properties of DOM.