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.     

Development of multispecies algal bioassays using flow cytometry

Multispecies algal bioassays, suitable for assessing copper toxicity, were developed with three marine (Micromonas pusilla, Phaeodactylum tricornutum, and Heterocapsa niei) and three freshwater (Microcystis aeruginosa, Pseudokirchneriella subcapitata, and Trachelomonas sp.) microalgae. Flow cytometry was used to separate and count algal signals based on pigment fluorescence and cell size. Species were mixed together on the basis of equivalent surface areas to avoid the confounding effect on toxicity of increased biomass for metal binding. Under control conditions (no added copper), M. pusilla growth was inhibited in the presence of the other marine microalgae compared to single-species tests, while the opposite was true (i.e., growth stimulation) for M. aeruginosa and P. subcapitata in freshwater mixtures. Competition for nutrients, including CO2, and algal exudate production may account for these effects. Interactions between microalgal species also had a significant effect on copper toxicity to some species. In freshwater multispecies bioassays, the toxicity of copper to Trachelomonas sp. was greater in the presence of other species, with copper concentrations required to inhibit growth (cell division) rate by 50% (72-h [IC50]) decreasing from 9.8 to 2.8 microg Cu/L in single- and multispecies bioassays, respectively. In contrast, in marine multispecies bioassays, copper toxicity to the marine diatom P. tricornutum was reduced compared to single-species bioassays, with an increase in the 72-h IC50 value from 13 to 24 microg Cu/L. This reduction in copper toxicity was not explained by differences in the copper complexing capacity in solution (as a result of exudate production) because labile copper, measured by anodic stripping voltammetry, was similar for P. tricornutum alone and in the mixture. These results demonstrate that single-species bioassays may over- or underestimate metal toxicity in natural waters.     

Toxicity of Euphorbia milii latex and niclosamide to snails and nontarget aquatic species

The toxicity of Euphorbia milii molluscicidal latex and niclosamide (NCL) to target snails (Biomphalaria glabrata and Biomphalaria tenagophila) and nontarget aquatic organisms is evaluated. Planorbidae snails were killed by very low concentrations of lyophilized latex (48-h LC(50), mg/L: B. glabrata, 0.12; B. tenagophila, 0.09; Helisoma duryi, 0.10). Latex was less toxic (48-h LC(50) or EC(50), mg/L) to oligochaeta (Tubifex tubifex, 0.31), planktonic crustacea (Daphnia similis, 0.38; C. dubia, 1.07; Artemia sp., 0.93), and fishes (Danio rerio, 0.96; Poecilia reticulata, 1. 39), and considerably less toxic to Ampullariidae snails (Pomacea sp. , 10.55) and frog tadpoles (Rana catesbeiana, 7.50). Latex (up to 100 mg/L) was not toxic to bacteria (P. putida and V. fischeri), algae (Selenastrum capricornutum and Chlorella vulgaris), and mosquito larvae (Anopheles albitarsis, Aedes aegypti, Aedes fluviatilis). NCL was very toxic (48-h LC(50) or EC(50), mg/L) to Planorbidae snails (B. glabrata, 0.15, B. tenagophila, 0.13; H. duryi, 0.10), T. tubifex (0.11), crustacea (D. similis, 0.19; Ceriodaphnia dubia, 0.47; Artemia sp. 0.18), fishes (D. rerio, 0.25; P. reticulata, 0.29), R. catesbeiana (0.16), and Pomacea sp. (0.76). NCL was toxic to bacteria, algae (96-h IC(50), mg/L: S. capricornutum, 0.34; C. vulgaris, 1.23) and slightly toxic to mosquito larvae. In conclusion, E. milii latex, as compared with the reference molluscicide niclosamide, presents a higher degree of selectivity toward snails which are intermediate hosts of Schistosoma trematodes.     

Toxicity of metal mixtures to a tropical freshwater alga (Chlorella sp): the effect of interactions between copper,cadmium, and zinc on metal cell binding and uptake

The individual and combined effects of copper, cadmium, and zinc on the cell division rate of the tropical freshwater alga Chlorella sp. were determined over 48 to 72 h. Metal mixtures were prepared based on multiples of their single-metal median effective concentration (EC50) values, i.e., toxic units (TU) using a triangular mixture design with five toxicant levels (0, 0.75, 1.0, 1.25, and 1.5 TU). Single-metal EC50 values after a 72-h exposure were 0.11, 0.85, and 1.4 microM for copper, cadmium, and zinc, respectively. Significant interactions were observed for all metal combinations after 48 and 72 h. An equitoxic mixture of Cu + Cd was more than concentration additive (synergistic) to the growth of Chlorella sp., while combinations of Cu + Zn, Cd + Zn, and Cu + Cd + Zn were all less than concentration additive or were antagonistic. To determine the effect of each metal on the uptake of the other, extracellular (membrane-bound) and intracellular metal concentrations, both alone and in mixtures, were compared. The increased growth inhibition observed for mixtures of Cu + Cd was due to higher concentrations of cell-bound and intracellular copper in the presence of cadmium compared with copper alone (i.e., cadmium-enhanced copper uptake). In contrast, both extra- and intracellular cadmium concentrations were reduced in the presence of copper. In mixtures of Cu + Zn, copper also inhibited the binding and cellular uptake of zinc, which resulted in decreased toxicity. Zinc had no appreciable effect on the uptake of copper by Chlorella sp. Our results suggest that all three metals share some common uptake and transport sites on Chlorella cells and that copper out competes both cadmium and zinc for cell binding. Determination of metal cell distribution coefficients (K(d)) confirmed that K(d) values for cadmium and zinc in single-metal exposures decreased in the presence of copper.     

Influence of cellular density on determination of EC(50) in microalgal growth inhibition tests

Growth inhibition tests for copper were carried out on four marine microalgal species: Chlorella autotrophyca, Nannochloris atomus (Chlorophyceae), Phaeodactylum tricornutum (Bacillariophyceae), and Isochrysis aff. galbana (Primnesiophyceae). The test initial cellular densities were reduced to 50 and 10% from the recommended initial cellular density in most of standardized assays. OECD test protocol (originally described for freshwater) was adapted for seawater. The EC(50) values were reduced when initial cellular density decreased. The green algae used in this study exhibited lower sensitivity than P. tricornutum and quite lower than I. aff. galbana. The latter species was found to be very sensitive to copper. The concept of cellular toxic quote (amount of toxic per cell) is defined in order to improve the results of toxicity tests.     

Acute toxicity of copper, ammonia, and chlorine to glochidia and juveniles of freshwater mussels (Unionidae)

The objective of the present study was to determine acute toxicity of copper, ammonia, or chlorine to larval (glochidia) and juvenile mussels using the recently published American Society for Testing and Materials (ASTM) Standard guide for conducting laboratory toxicity tests with freshwater mussels. Toxicity tests were conducted with glochidia (24- to 48-h exposures) and juveniles (96-h exposures) of up to 11 mussel species in reconstituted ASTM hard water using copper, ammonia, or chlorine as a toxicant. Copper and ammonia tests also were conducted with five commonly tested species, including cladocerans (Daphnia magna and Ceriodaphnia dubia; 48-h exposures), amphipod (Hyalella azteca; 48-h exposures), rainbow trout (Oncorhynchus mykiss; 96-h exposures), and fathead minnow (Pimephales promelas; 96-h exposures). Median effective concentrations (EC50s) for commonly tested species were >58 microg Cu/L (except 15 microg Cu/L for C. dubia) and >13 mg total ammonia N/L, whereas the EC50s for mussels in most cases were <45 microg Cu/L or <12 mg N/L and were often at or below the final acute values (FAVs) used to derive the U.S. Environmental Protection Agency 1996 acute water quality criterion (WQC) for copper and 1999 acute WQC for ammonia. However, the chlorine EC50s for mussels generally were >40 microg/L and above the FAV in the WQC for chlorine. The results indicate that the early life stages of mussels generally were more sensitive to copper and ammonia than other organisms and that, including mussel toxicity data in a revision to the WQC, would lower the WQC for copper or ammonia. Furthermore, including additional mussel data in 2007 WQC for copper based on biotic ligand model would further lower the WQC.     

Aquatic toxicity evaluation of para-methylstyrene

The aquatic toxicity of para-methylstyrene was evaluated in acute toxicity studies using fathead minnows (Pimephales promelas), daphnids (Daphnia magna), and freshwater green algae (Selenastrum capricornutum). Static tests were performed in sealed containers with no headspace to minimize loss of this volatile compound to the atmosphere. Concentrations of para-methylstyrene in test solutions were analyzed by gas chromatography equipped with a purge and trap module and flame ionization detection. Test results are based on mean, measured concentrations. para-Methylstyrene was moderately toxic to fathead minnows, daphnids, and green algae. The 96-h LC(50) and NOEC for fathead minnows were 5.2 and 2.6 mg/L, respectively. The 48-h EC(50) and NOEC for daphnids were 1.3 and 0.81 mg/L, respectively. The 72-h EC(50) and NOEC for green algae were 2.3 and 0.53 mg/L, respectively; these effects were algistatic rather than algicidal. para-Methylstyrene's potential impact on aquatic ecosystems is significantly mitigated by its volatility, an important fate process.     

The Effect of pH on the Uptake and Toxicity of Copper and Zinc in a Tropical Freshwater Alga (Chlorella sp.)

Copper and zinc toxicity to the freshwater alga Chlorella sp. was determined at a range of pH values (5.5–8.0) in a synthetic softwater (hardness 40–48 mg CaCO₃/L). The effects of the metals on algal growth (cell division) rate were determined after 48-h exposure at pH 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0. The toxicity of both metals was pH dependent. As pH decreased from 8.0 to 5.5, the copper concentration required to inhibit the algal growth rate by 50% (IC50) increased from 1.0 to 19 μg/L. For zinc, the IC50 increased from 52 to 2700 μg/L over the same pH range. Changes in solution speciation alone did not explain the increased toxicity observed as the pH increased. Modelled Cu²⁺ and Zn²⁺ concentrations decreased with increasing pH, whereas toxicity was observed to increase. Measurements of extracellular (cell-bound) metal concentrations support the biotic ligand model (BLM) theory of competition between protons (H⁺) and metals for binding sites at the algal cell surface. Higher extracellular metal concentrations were observed at high pH, indicating reduced competition. Independent of pH, both extracellular and intracellular copper were directly related to growth inhibition in Chlorella sp., whereas zinc toxicity was related to cell-bound zinc only. These findings suggest that the algal cell surface may be considered as the biotic ligand in further development of a chronic BLM with microalgae. Conditional binding constants (log K) were determined experimentally (using measured intracellular metal concentrations) and theoretically (using concentration-response curves) for copper and zinc for Chlorella sp. at selected pH values. Excellent agreement was found indicating the possibility of using concentration-response data to estimate conditional metal-cell binding constants.     

Reproductive toxicity of dietary copper to a saltwater cladoceran, Moina monogolica Daday

In the present study, the chronic toxicity of dietary copper to Moina monogolica Daday was investigated. Microalgal growth inhibition tests were conducted for 96 h with the green algae Chlorella pyrenoidosa exposed to copper. The 96-h median effective concentration (95% confidence interval) was 509.12 (388.68-629.56) microg/L. Then, C. pyrenoidosa was exposed for 96 h to a control and to seven dissolved copper concentrations. Cellular copper concentration accumulated in a dose-dependent manner and was plotted against cell density. These algae were used as food in a 21-d bioassay with M. monogolica in seawater to which no dissolved copper was added. Brood size was not reduced in the first brood, but significant reductions at all algal-exposure copper concentrations (44.78-817.17 microg/L) were observed in all subsequent broods, with increasing magnitude in each brood. Neither longevity nor number of broods per female was significantly affected, even at the highest copper exposure, though both endpoints did show a consistent downward trend with increasing copper exposure. Total reproduction, brood size, and net reproductive rate were decreased significantly in all dietary copper exposures (algae exposed to 44.78-817.17 microg/L). In contrast, the intrinsic rate of natural increase was reduced significantly only with algae exposed to greater than 619.27 microg/L, most likely because of the heavy influence of early reproduction on this metric. Because cell density in algal cultures decreased with increasing copper concentrations, it is possible that changes in the nutritional content of the algal diet could have played a role in causing the observed changes in reproduction of M. monogolica.     

Influence of multigeneration acclimation to copper on tolerance, energy reserves, and homeostasis of Daphnia magna straus

A multigeneration acclimation experiment was performed with Daphnia magna exposed to copper to assess possible changes in tolerance and to establish the optimal concentration range (OCEE) of this species. The hypothesis was tested that as the bioavailable background concentration of an essential metal increases (within realistic limits), the natural tolerance (to the metal) of the acclimated/adapted organisms and communities will increase. During 18 months the daphnids were exposed to six different, environmentally relevant, copper background concentrations ranging between 0.5 and 100 microg Cu L(-1) (7 x 10(-15) and 3.7 x 10(-9) M Cu2+). An increase in acute (effect concentration resulting in 50% immobility: 48-h EC50) and chronic copper (effect concentration resulting in 50% or 10% reproduction reduction: 21-d EC50, 21-d EC10) tolerance was observed with increasing exposure concentration. The 48-h EC50 increased significantly from 204 +/- 24 microg Cu L(-1) to 320 +/- 43 microg Cu L(-1). A nonsignificant change from 48.0 (47.9-48.0) microg Cu L(-1) to 78.8 (66.3-93.6) microg Cu L(-1) was noted in the chronic toxicity assays. The optimal concentration range was assessed using different biological parameters (i.e., net reproduction [R0]), energy reserves (Ea), body length measurements, filtration rates, and body burdens. After three generations of acclimation the OCEE ranged between 1 and 35 microg Cu L(-1) (2 X 10(-14) to 80 x 10(-12) M Cu2+). Body burden measurements revealed an active copper regulation up to 35 microg Cu L(-1) (80 pM Cu2+). It can be concluded that acclimation of D. magna to copper does occur in laboratory experiments, even at realistic copper background concentrations (10(-11) - 10(-9) M Cu2+). However, it is suggested that this phenomenon is of less importance in the context of regulatory risk assessments. An optimal copper concentration range for D. magna was observed between 1 and 35 microg Cu L(-1) (10(-14) - 10(-11) M Cu2+), indicating that copper deficiency can occur in routine laboratory cultures.     

Development of a whole-sediment toxicity test using a benthic marine microalga

An acute whole-sediment toxicity test with a benthic marine microalga was developed and optimized using flow cytometry to distinguish algae (based on their chlorophyll a autofluorescence) from sediment particles. Of seven benthic marine algae screened, the diatom Entomoneis cf punctulata was most suitable because of its tolerance of a wide range of water and sediment physicochemical parameters, including salinity, pH, ammonia, and sulfide. A whole-sediment and water-only toxicity test based on inhibition of esterase activity in this species was developed. Enzyme activity rather than growth was used as the test endpoint, as nutrient release from sediments has previously been found to stimulate algal growth, potentially masking contaminant toxicity. The sensitivity of the bioassay to a range of metals (copper, zinc, cadmium, lead, arsenic, manganese) and phenol in water-only exposures was compared to the standard 72-h growth rate inhibition test. The esterase enzyme inhibition test was sensitive to copper, with a 3-h inhibitory concentration to cause a 50% (IC50) reduction in a fluorescein diacetate fluorescence value of 97 +/- 39 microg Cu/L. A concentration-dependent response was also observed in the presence of sediment particles (copper tailings), with and without dilution, using a control clean sediment. The primary route of exposure to copper was via pore water rather than by direct contact with tailings particles. This is the first whole-sediment bioassay developed with a benthic alga suitable for sediment quality assessment in marine/estuarine systems, and its advantages and limitations are discussed.     

Toxicological evaluation of nitrofurazone and furaltadone on Selenastrum capricornutum, Daphnia magna, and Musca domestica

Short-term toxicity of nitrofurans, nitrofurazone, furaltadone tartrate, and furaltadone chlorohydrate, was tested in the laboratory on two freshwater organisms, Selenastrum capricornutum (algae) and Daphnia magna (crustaceans). Toxicity studies with nitrofurazone were also carried out on larval development of the house fly Musca domestica L. Nitrofurazone was invariably the most toxic compound (the 96-hr EC50 of algal species was 1.45 mg/liters; the EC50 values for D. magna were 40.04 and 28.67 mg/liter after 24 and 48 hr, respectively) followed by furaltadone tartrate and furaltadone chlorohydrate. This study provides some evidence of the potential ecotoxicity of nitrofurans, indicating the need for further investigations.     

Comparison of Cd, Cu, and Zn toxic effects on four marine phytoplankton by pulse-amplitude-modulated fluorometry

The toxic effects of Cd, Cu, and Zn on four different marine phytoplankton, Dunaliella tertiolecta, Prorocentrum minimum, Synechococcus sp., and Thalassiosira weissflogii, were examined by comparing the cell-specific growth rate, pulse-amplitude-modulated (PAM) parameters (maximum photosystem II quantum yield phiM and operational quantum yield phi'M, chlorophyll a content, and cellular metal concentration, over a 96-h period. The calculated no-observed-effect concentration (NOEC) based on both cell-specific growth rate and two PAM parameters (phiM and phi'M) were mostly identical. Thus, these PAM parameters and cell-specific growth rate were comparable in their sensitivities as the biomarkers for trace metal toxicity to marine phytoplankton. The cyanobacteria Synechococcus sp. was the most sensitive species among the four algal species tested because of its higher cell surface to volume ratio. The toxicity of the three tested metals followed the order of Cd > Cu > Zn based on the cellular metal concentration of the four algae at the NOEC. The cellular metal bioaccumulation followed the same Freundlich isotherm for each metal regardless of the algal species, indicating that the metal accumulation was a nonmetabolic process under high ambient metal concentrations and that the cell surface metal binding was comparable among the different species. For all the algae examined in our study, the bioaccumulation potentials of Cu and Zn were similar to each other, while the Cd bioaccumulation was much lower under environmentally realistic metal concentration.     

Determination of short-term copper toxicity in a multispecies microalgal population using flow cytometry

This study was conducted to determine the role of algal-algal interactions in a multispecies microalgal population on their sensitivities to copper based on an enzyme inhibition assay using flow cytometric measures. Autofluorescence (chlorophyll a and phycocyanin) was used to identify species and count algal signals. The effect of multispecies population on copper toxicity of Microcystis aeruginousa was detected (1) at the same initial cell density, (2) at the same surface area, and (3) in the presence and absence of Chlorella pyrenoidosa and Scenedesmus obliquus. As copper concentrations increased, esterase activity of M. aeruginosa changed in a concentration-dependent manner. The 24 h EC(50) value of M. aeruginosa in the multispecies population was significantly (P < 0.05) higher than those in the single-species population. Compared with S. obliquus, the effect of C. pyrenoidosa on M. aeruginosa was more marked (the 24 h EC(50) value of copper on fluorescin diacetate fluorescence of M. aeruginosa was 11 microg/L). At 48 h copper exposure (6 microg/L) analysis of intracellular reactive oxygen species levels also showed similar algal-algal interactions in multispecies microalgal populations. The pigment assay suggested that these algal-algal interactions occurred only at low concentrations (< 13 microg/L, 24 and 48 h copper exposure). This study demonstrates the importance of using multispecies populations to estimate metal toxicity in natural waters.     

Toxicity, biotransformation, and mode of action of arsenic in two freshwater microalgae (Chlorella sp. and Monoraphidium arcuatum)

The toxicity of As(V) and As(III) to two axenic tropical freshwater microalgae, Chlorella sp. and Monoraphidium arcuatum, was determined using 72-h growth rate-inhibition bioassays. Both organisms were tolerant to As(III) (72-h concentration to cause 50% inhibition of growth rate [IC50], of 25 and 15 mg As[III]/L, respectively). Chlorella sp. also was tolerant to As(V) with no effect on growth rate over 72 h at concentrations up to 0.8 mg/L (72-h IC50 of 25 mg As[V]/L). Monoraphidium arcuatum was more sensitive to As(V) (72-h IC50 of 0.25 mg As[V]/L). An increase in phosphate in the growth medium (0.15-1.5 mg PO4(3-)/L) decreased toxicity, i.e., the 72-h IC50 value for M. arcuatum increased from 0.25 mg As(V)/L to 4.5 mg As(V)/L, while extracellular As and intracellular As decreased, indicating competition between arsenate and phosphate for cellular uptake. Both microalgae reduced As(V) to As(III) in the cell, with further biological transformation to methylated species (monomethyl arsonic acid and dimethyl arsinic acid) and phosphate arsenoriboside. Less than 0.01% of added As(V) was incorporated into algal cells, suggesting that bioaccumulation and subsequent methylation was not the primary mode of detoxification. When exposed to As(V), both species reduced As(V) to As(III); however, only M. arcuatum excreted As(III) into solution. Intracellular arsenic reduction may be coupled to thiol oxidation in both species. Arsenic toxicity most likely was due to arsenite accumulation in the cell, when the ability to excrete and/or methylate arsenite was overwhelmed at high arsenic concentrations. Arsenite may bind to intracellular thiols, such as glutathione, potentially disrupting the ratio of reduced to oxidized glutathione and, consequently, inhibiting cell division.     

Responses of marine unicellular algae to brominated organic compounds in six growth media

Marine unicellular algae, Skeletonema costatum, Thalassiosira pseudonana, and Chlorella sp. were exposed to the industrial brominated compounds tetrabromobisphenol A, decabromobiphenyloxide (DBBO), hexabromocyclododecane (HBCD), pentabromomethylbenzene (PBMB), pentabromoethylbenzene (PBEB), and the herbicide bromoxynil (BROM), in six algal growth media. High concentrations of DBBO (1 mg liter-1), PBMB (1 mg liter-1), and PBEB (0.5 mg liter-1) reduced growth by less than 50%. EC50s of the other compounds varied with growth medium, with high EC50/low EC50 ratios between 1.3 and 9.9. Lowest EC50s, 9.3 to 12.0 micrograms liter-1, were obtained with S. costatum and HBCD. It is concluded that responses to toxicants in different media are the results of interactions among algae, growth medium, toxicant, and solvent carrier.     

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.     

Toxicity of a cadmium-contaminated diet to Hyalella azteca

Four- and 10-week chronic toxicity tests were conducted using the freshwater amphipod Hyalella azteca and Cd-contaminated Chlorella sp. as a food source. Chlorella sp. was cultured in various Cd concentrations, filtered from solution, rinsed, dried, and ground into food flakes for the H. azteca. Unlike Cd toxicity from water sources, growth was found to be a more sensitive toxicological endpoint than survival, with calculated 50 and 25% effect concentrations (EC50s and EC25s, respectively) of 5.43 and 2.82 nmol/g, respectively, for Cd measured in food. Based on the regression of Cd in Chlorella sp. against Cd in filtered culture medium, the EC50 and EC25 corresponded to dissolved Cd concentrations of 11.30 and 5.09 nmol/L, respectively. Little or no bioaccumulation of Cd was found in the tissues of H. azteca that were fed contaminated food. These results demonstrate an apparent toxicological effect (either direct or indirect) of Cd-contaminated Chlorella sp. to H. azteca that is not associated with Cd accumulation. Toxicity of Cd-contaminated Chlorella sp. differs from waterborne Cd toxicity both in terms of the most sensitive endpoint (growth vs survival) and the relationship between toxicity and bioaccumulation. Unlike Cd toxicity through water exposure, Cd bioaccumulation by H. azteca cannot, therefore, be used to infer toxicity of Cd in a diet of Chlorella sp. Although the concentration of Cd in the algal culture medium that ultimately reduced growth of H. azteca in the present study was higher than Cd in water, which caused mortality to H. azteca in water-only tests during previous studies, further research regarding the contribution of dietary Cd to overall Cd toxicity is needed to verify that water-quality guidelines and risk assessments based on water-only exposures are fully protective.     

Laboratory evaluation of the toxicity of Perfluorooctane Sulfonate (PFOS) on Selenastrum capricornutum,Chlorella vulgaris,Lemna gibba,Daphnia magna,and Daphnia pulicaria

Perfluorooctane sulfonate (PFOS) is an anthropogenic compound found in trace amounts in many environmental compartments far from areas of production. This, along with the highly persistent nature of PFOS, presents a concern for possible effects in aquatic ecosystems. The objective of this study was to determine the toxicity of PFOS in representative freshwater organisms. Toxicity testing using standard laboratory protocols was performed on the green algae Selenastrum capricornutum and Chlorella vulgaris, the floating macrophyte Lemna gibba, and the invertebrates Daphnia magna and Daphnia pulicaria. No observable effect concentration (NOEC) values were generated from the most sensitive endpoints for all organisms. Autotroph inhibition of growth NOEC values were 5.3, 8.2, and 6.6 mg/L for S. capricornutum, C. vulgaris, and L. gibba, respectively. The 48-h immobility NOEC values for D. magna and D. pulicaria were 0.8 and 13.6 mg/L, respectively. In comparison to immobility, the 21-day lethality NOEC for D. magna was 5.3 mg/L. Based on effect (immobility) values, the most sensitive of all test organisms was D. magna. The most sensitive organism based on 50% inhibition of growth (IC(50)) was L. gibba, with an IC(50) value of 31.1 mg/L determined from wet weight. This is 4.3 times less than the LC(50) for D. pulicaria, which was 134 mg/L. Significant adverse effects (p < or = 0.05) were observed for all organisms in concentrations >134 mg/L. The results indicate that under laboratory conditions PFOS is acutely toxic to freshwater organisms at concentrations at or near 100 mg/L. Based on known environmental concentrations of PFOS, which occur in the low ng/L to low microg/L range, there is no apparent risk to freshwater systems. However, further work is required to investigate long-term effects in these and other freshwater organisms.     

Toxicity and Bioavailability of Copper Herbicides (Clearigate, Cutrine-Plus, and Copper Sulfate) to Freshwater Animals

In designing aquatic herbicides containing copper, an important goal is to maximize efficacy for target species while minimizing risks for nontarget species. To have a margin of safety for nontarget species, the concentration, duration of exposure (i.e., uptake), and form (i.e., species) of copper used for herbicidal properties should not elicit adverse effects on populations of nontarget species. To determine the potential for risk or adverse effects (conversely the margin of safety), data regarding the comparative toxicity of copper-containing herbicides are crucial. A series of comparative toxicity experiments was conducted, including baseline estimates of toxicity (LC50s, LOECs), sensitive species relationships (thresholds and exposure-response slopes), and bioavailability of toxic concentrations and forms of copper 7 days after initial herbicide application. Aqueous 48-h toxicity experiments were performed to contrast responses of Daphnia magna Strauss, Hyalella azteca Saussure, Chironomus tentans Fabricius, and Pimephales promelas Rafinesque to copper herbicides: Clearigate(R), Cutrine(R)-Plus, and copper sulfate. D. magna was the most sensitive aquatic animal tested for all three herbicides; 48-h LC50s for organisms exposed to Clearigate, Cutrine-Plus, and copper sulfate were 29.4, 11.3, and 18. 9 microg Cu/L, respectively. In terms of potency (calculated from the linearized portion of the exposure-response curves, which included 50% mortality), D. magna was the most sensitive animal tested. Organisms exposed to Clearigate, Cutrine-Plus, and copper sulfate had exposure-response slopes of 2.55, 8.61, and 5.07% mortality/microg Cu/L, respectively. Bioavailability of Clearigate and Cutrine-Plus was determined by comparing survival data (LC50s) of test organisms exposed to herbicide concentrations during the first and last 48-h of a 7-day exposure period. Even in these relatively simplified water-only exposures, a transformation of copper to less bioavailable species over time was observed with a 100-200% decrease in toxicity (i.e., an increase in 48-h LC50s) for all four test animals. This series of laboratory experiments provides a worst-case scenario for determining the risk associated with the manufacturer's recommended application rates of Clearigate (100-1,000 microg Cu/L), Cutrine-Plus (200-1,000 microg Cu/L), and copper sulfate (100-500 microg Cu/L) in natural waters for four nontarget freshwater animals.