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.     

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.     

Chronic toxicity of copper and ammonia to juvenile freshwater mussels (Unionidae)

The objectives of the present study were to develop methods for conducting chronic toxicity tests with juvenile mussels under flow-through conditions and to determine the chronic toxicity of copper and ammonia to juvenile mussels using these methods. In two feeding tests, two-month-old fatmucket (Lampsilis siliquoidea) and rainbow mussel (Villosa iris) were fed various live algae or nonviable algal mixture for 28 d. The algal mixture was the best food resulting in high survival (>or=90%) and growth. Multiple copper and ammonia toxicity tests were conducted for 28 d starting with two-month-old mussels. Six toxicity tests using the algal mixture were successfully completed with a control survival of 88 to 100%. Among copper tests with rainbow mussel, fatmucket, and oyster mussel (Epioblasma capsaeformis), chronic value ([ChV], geometric mean of the no-observed-effect concentration and the lowest-observed-effect concentration) ranged from 8.5 to 9.8 microg Cu/L for survival and from 4.6 to 8.5 microg Cu/L for growth. Among ammonia tests with rainbow mussel, fatmucket, and wavy-rayed lampmussel (L. fasciola), the ChV ranged from 0.37 to 1.2 mg total ammonia N/L for survival and from 0.37 to 0.67 mg N/L for growth. These ChVs were below the U.S. Environmental Protection Agency 1996 chronic water quality criterion (WQC) for copper (15 microg/L; hardness 170 mg/L) and 1999 WQC for total ammonia (1.26 mg N/L; pH 8.2 and 20 degrees C). Results indicate that toxicity tests with two-month-old mussels can be conducted for 28 d with >80% control survival; growth was frequently a more sensitive endpoint compared to survival; and the 1996 chronic WQC for copper and the 1999 chronic WQC for total ammonia might not be adequately protective of the mussel species tested. However, a recently revised 2007 chronic WQC for copper based on the biotic ligand model may be more protective in the water tested.     

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.     

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.     

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.     

Effects of nutrient enrichment, depuration substrate, and body size on the trophic transfer of cadmium associated with microalgae to the benthic amphipod Leptocheirus plumulosus

Bioavailability and nutrient effects on the trophic transfer of Cd associated with microalgae to the marine benthic amphipod Leptocheirus plumulosus were investigated. Cadmium assimilation efficiency (AE) of suspension-feeding L. plumulosus significantly varied among three algal species tested (Nitzschia punctata, Thalassiosira weissflogii, and Isochrysis galbana). Depuration substrate greatly influenced Cd AE for L. plumulosus (AE was much higher for nonburrowed amphipods), probably because sediment burrowing allowed L. plumulosus to feed as a surface deposit feeder. The L. plumulosus body size, ranging from 0.5 to 2.0 mm, did not affect Cd AE. Nitrate enrichment from 0 to 180 microM in algal culture significantly increased Cd AE from 9.4 to 18.8% for T. weissflogii, from 10.0 to 27.3% for N. punctata, and from 10.0 to 16.2% for I. galbana; nitrate enrichment from 0 to 60 microM did not influence Cd AE in any algal species tested. Physiological turnover rate constants of Cd in L. plumulosus ranged from 0.016 to 0.025/h for the three species and were independent of nitrate addition. Nitrate enrichment strongly enhanced Cd distribution in algal cytoplasm. Phosphate enrichment (0-7.5 miroM) did not significantly affect Cd AEs in L. plumulosus. Overall, a significant linear relationship was observed between the Cd AE of L. plumulosus and the fraction of Cd available in algal cytoplasm. Our work suggests that eutrophication by nitrate enrichment has the potential to enhance the trophic transfer of Cd from microalgae to suspension-feeding benthic invertebrates.     

Copper toxicity in relation to surface water-dissolved organic matter: biological effects to Daphnia magna

Water quality standards for copper are usually stated in total element concentrations. It is known, however, that a major part of the copper can be bound in complexes that are biologically not available. Natural organic matter, such as humic and fulvic acids, are strong complexing agents that may affect the bioavailable copper (Cu2+) concentration. The aim of this study was to quantify the relation between the concentration of dissolved natural organic matter and free Cu2+ in surface waters, and the biological effect, as measured in a standardized ecotoxicological test (48 h-median effective concentration [EC50] Daphnia magna, mobility). Six typical Dutch surface waters and an artificial water, ranging from 0.1 to 22 mg/L dissolved organic carbon (DOC), were collected and analyzed quarterly. Chemical speciation modeling was used as supporting evidence to assess bioavailability. The results show clear evidence of a linear relation between the concentration of dissolved organic carbon (in milligrams DOC/L) and the ecotoxicological effect (as effect concentration, EC50, expressed as micrograms Cu/L): 48-h EC50 (Daphnia, mobility) = 17.2 x DOC + 30.2 (r2 = 0.80, n = 22). Except for a brook with atypical water quality characteristics, no differences were observed among water type or season. When ultraviolet (UV)-absorption (380 nm) was used to characterize the dissolved organic carbon, a linear correlation was found as well. The importance of the free copper concentration was demonstrated by speciation calculations: In humic-rich waters the free Cu2+ concentration was estimated at approximately 10(-11) M, whereas in medium to low dissolved organic carbon waters the [Cu2+] was approximately 10(-10) M. Speciation calculations performed for copper concentrations at the effective concentration level (where the biological effect is considered the same) resulted in very similar free copper concentrations (approximately 10(-8) M Cu) in these surface waters with different characteristics. These observations consistently show that the presence of organic matter decreases the bioavailability, uptake, and ecotoxicity of copper in the aquatic environment. It demonstrates that the DOC content must be included in site-specific environmental risk assessment for trace metals (at least for copper). It is the quantification of the effects described that allows policy makers to review the criteria for copper in surface waters.     

Influences of metal concentration in phytoplankton and seawater on metal assimilation and elimination in marine copepods

Radiotracer experiments were conducted to examine the influence of the concentration of Cd, Se, and Zn in ingested phytoplankton (dinoflagellate Prorocentrum minimum and diatom Thalassiosira weissflogii) and in ambient seawater on metal assimilation and elimination efficiencies of three marine copepods, Acartia spinicauda, Paracalanus aculeatus, and Calanus sinicus. The assimilation efficiencies (AEs) decreased by 1.7 to 2.0 times, 1.4 to 4.1 times, and 1.3 to 2.2 times in the copepods with an increase in metal concentration in ingested algae by 16 to 84 times, 14 times, and 45 to 153 times, for Cd, Se, and Zn, respectively. However, the physiologic turnover rate constant was relatively independent of the metal concentration in copepods. No evidence was found of any interaction between Cd and Zn in their assimilation by copepods. Assimilation efficiencies of Cd were higher in copepods feeding on the dinoflagellate P. minimum, whereas the AEs of Zn were higher in copepods feeding on the diatom T. weissflogii. Differences in metal distribution in algal cytoplasm at different ambient metal concentrations may be partially responsible for the observed influence of metal concentration in algal cells on metal assimilation in copepods. However, metal desorption within the gut of the copepod may have little influence on metal assimilation, as a result of the short gut residence time of food particles and the neutral gut pH. Our study also indicated that the ingestion rate of copepods was reduced by a higher concentration of Cd and Se, but was not affected by Zn concentration in the food particles. Consequently, partial regulation of metal trophic transfer in response to increasing metal contamination may be achieved by a change in metal assimilation efficiency and the ingestion activity of the copepod, but not by changes in metal turnover rates from the animals.     

Use of ethylenediaminetetraacetic acid in pulp mills and effects on metal mobility and primary production

Over the last decade, the use of ethylenediaminetetraacetic acid (EDTA) has increased by a factor of three within the Swedish pulp industry. The effects of pulp mill wastewater in combination with EDTA on metal mobility and the consequences for ecologically relevant organisms have been studied. Ethylenediaminetetraacetic acid was added to pulp mill effluent water in the laboratory and compared to wastewater without EDTA. Degradation of the water was followed over a experimental period up to eight weeks and samples were taken five times for chemical analysis and at the beginning and the end for biological tests. Half a year later, the experiment was repeated. The results from both experiments showed that EDTA markedly increased the solubility of zinc, cadmium, lead, iron, nickel, and copper, whereas the solubility of vanadium, molybdenum, thallium, arsenic, and chromium was not changed by the presence of EDTA. No measurable degradation of EDTA occurred even after 5.5 months. The toxicity of the pulp mill effluent water was low, and only a slight toxic effect was observed on the reproduction of Ceramium strictum. The most pronounced effect was a stimulation in the growth of three algal species in 5 to 20% (v/v) total effluent wastewater with and without EDTA. The presence of EDTA in the dissolved fraction of the pulp mill effluent water increased the growth stimulation in 20 to 40% (v/v), possibly because of the increased availability of essential metals like iron, zinc, and copper. The capacity of the pulp mill wastewater to support algal growth was nearly as good as a rich nutrient medium for algae. The combination of poorly degradable EDTA and the increasing availability of metals might further enhance eutrophication in the recipient areas of pulp mills.     

Relationships between surface-bound and internalized copper and cadmium and toxicity in Chlamydomonas reinhardtii

In the present study, the adsorption and uptake of copper (Cu) and cadmium (Cd) in Chlamydomonas reinhardtii were examined to establish fundamental toxicity relationships to glutathione and cell-growth endpoints. Establishing these fundamental relationships of metal accumulation and toxicity metrics is necessary to subsequently implement an algal biotic ligand model. The glutathione response was similar to the response measured from growth endpoints for both internal and adsorbed Cu, indicating that glutathione may be a useful biomarker of toxicity. The glutathione response with Cd contrasted markedly with that observed with Cu and was therefore observed to be a metal-specific biomarker. The density of sites binding metals and the related stability constants for the algal cell surface were also determined. Short exposures to metals (2 h) were conducted, and we determined 6.0 × 10(-6) mol/g sites binding Cu and 2.0 × 10(-6) mol/g sites binding Cd and conditional stability constants as log K' = 7.2 and log K' = 6.7 for Cu and Cd, respectively. Experiments were also conducted to determine the effect on toxicity endpoints of varying nitrate concentrations and different humic acids (HA) in the exposure media. Varying nitrate concentrations did not have an effect on cell growth over 24 h. The surface-adsorbed Cu measurements from the experiments with HA depended on the type and concentration of HA.     

The influence of mucus on copper speciation in the gill microenvironment of carp (Cyprinus carpio)

pH and mucus (as total organic carbon) in the gill microenvironment of carp, Cyprinus carpio, exposed to various concentrations of total copper were measured in an exposure experiment using Playle's apparatus. The activities of free copper ions in both inspired and expired waters were determined using a copper ion selective electrode. A trial-and-error procedure of coupling species distribution calculations (MINEQA2) with single-site complexation modeling (mucus-copper) was proposed for computation of both copper speciation and complexation capacity of gill mucus for copper in an interactive loop. The method was demonstrated to be valid in calculating the metal speciation in this study. It was found that pH and alkalinity in the gill microenvironment were slightly higher than in the surrounding water of pH 6.7. The rates of excretion of CO(2), HCO(-)(3), and mucus at the gills increased, either linearly or nonlinearly with increases in the total copper concentration in the surrounding water. The conditional complexation stability constant of the mucus was calculated as log k=5.32, while the complexation stability equivalent concentration of the mucus was derived as 0.95 mmolCu/mgC. As a consequence of changes in pH and alkalinity, in conjunction with occurrence of mucus, copper speciation in the gill microenvironment is significantly different from that in the surrounding water, with 3-20% of the copper complexed by the gill mucus. The sequence of species domination also shifted from CuCO(0)(3), Cu(2+), CuHCO(+)(3), and Cu(OH)(0)(2) in the surrounding water to CuCO(0)(3), Cu(2+), Cu(OH)(0)(2), CuHCO(+)(3), and mucus-Cu in the gill microenvironment.     

Use of anodic stripping voltammetry in predicting toxicity of copper in river water

The labile concentration and toxicity of Cu as influenced by alkalinity and different concentrations of ethylenediaminetetraacetic acid (EDTA) and naturally derived fulvic acid (FA) were determined by bioassays carried out in the culture media for Daphnia magna (D. magna). The labile concentration of Cu was obtained by differential pulse anodic stripping voltammetry with a double-acidification method (DAM-DPASV). Changes in water alkalinity did not affect the labile concentration of Cu, but increase in alkalinity did reduce the mortality of D. magna. In the presence of EDTA and FA, both labile concentration of Cu and mortality were reduced. By excluding Cu-carbonate complexes from the labile concentration, a bioavailable concentration of Cu ([Cu*]) was obtained and was used to predict the acute toxicity of Cu on D. magna. For natural waters, the labile concentration of Cu was measured by DAM-DPASV, and [Cu*] was calculated using MINTEQ A2 software (developed by the U.S. Environmental Protection Agency) based on the anion composition of waters. This procedure was tested for waters and sediment elutriates sampled from the Le An River (Jiangxi Province, China) that were severely polluted by the discharges from a copper mine. The results showed that [Cu*] was a good indicator for Cu toxicity and could be used under field conditions.     

Effects of dissolved organic matter and reduced sulphur on copper bioavailability in coastal marine environments

Copper-induced toxicity in aqueous systems depends on its speciation and bioavailability. Natural organic matter (NOM) and reduced sulphur species can complex copper, influencing speciation and decreasing bioavailability. NOM composition in estuaries can vary, depending on inputs of terrigenous, autochthonous, or wastewater source material. At a molecular level, variability in NOM quality potentially results in different extents of copper binding. The aims of this study were to measure acute copper EC₅₀ values in coastal marine and estuarine waters, and identify the relationships between total dissolved copper EC₅₀ values and measured water chemistry parameters proportional to NOM and reduced sulphur composition. This has implications on the development of marine-specific toxicity prediction models. NOM was characterised using dissolved organic carbon (DOC) concentration and fluorescence measurements, combined with spectral resolution techniques, to quantify humic-, fulvic-, tryptophan-, and tyrosine-like fractions. Reduced sulphur was measured by the chromium-reducible sulphide (CRS) technique. Acute copper toxicity tests were performed on samples expressing extreme DOC, fluorescent terrigenous, autochthonous, and CRS concentrations. The results show significant differences in NOM quality, independent of DOC concentration. CRS is variable among the samples; concentrations ranging from 4 to 40 nM. The toxicity results suggest DOC as a very good predictive measure of copper EC₅₀ in estuaries (r²=0.87) independent of NOM quality. Furthermore, for filtered samples, CRS exists at concentrations that would be saturated with copper at measured EC₅₀, suggesting that while CRS might bind Cu and decrease bioavailability, it does not control copper speciation at toxicologically relevant concentrations and therefore is not a good predictive measure of copper toxicity in filtered samples.     

A phytoplankton growth assay for routine in situ environmental assessments

This study proposes an ecologically relevant and cost-effective phytoplankton growth assay for routine in situ toxicity assessments. Assay procedures were developed applying, to the extent possible, the rationale behind the design of standard algal assays. Chlorella vulgaris was selected as test species because it grows well immobilized in alginate beads and has a wide geographic distribution. The performance of the assay in a freshwater system impacted by acid mine drainage demonstrated the suitability of assay chambers and procedures. The test system, made of inexpensive materials, allowed the rapid and easy deployment of the assay. The deployment of extra chambers at reference sites provided the ability to periodically check whether algal growth had already reached recommended growth criteria (time at which the assay should end). By deploying chambers filled with control medium at all sites, temperature was identified to explain 95% of the variation in growth. By using an artificial nutrient source shown capable of promoting algal growth according to recommended standards, toxicity from the mine effluent was distinguish from in situ nutrient limitation effects. The very good agreement (r2 = 90%) between mean in situ growth rates estimated by microscopy and by spectrophotometry and their similar coefficient of variation showed the latter to be a suitable straightforward methodology for assay endpoint estimation.     

Challenges for the development of a biotic ligand model predicting copper toxicity in estuaries and seas

An effort is ongoing to develop a biotic ligand model (BLM) that predicts copper (Cu) toxicity in estuarine and marine environments. At present, the BLM accounts for the effects of water chemistry on Cu speciation, but it does not consider the influence of water chemistry on the physiology of the organisms. We discuss how chemistry affects Cu toxicity not only by controlling its speciation, but also by affecting the osmoregulatory physiology of the organism, which varies according to salinity. In an attempt to understand the mechanisms of Cu toxicity and predict its impacts, we explore the hypothesis that the common factor linking the main toxic effects of Cu is the enzyme carbonic anhydrase (CA), because it is a Cu target with multiple functions and salinity-dependent expression and activity. According to this hypothesis, the site of action of Cu in marine fish may be not only the gill, but also the intestine, because in this tissue CA plays an important role in ion transport and water adsorption. Therefore, the BLM of Cu toxicity to marine fish should also consider the intestine as a biotic ligand. Finally, we underline the need to incorporate the osmotic gradient into the BLM calculations to account for the influence of physiology on Cu toxicity.     

Development of photosynthetic biofilms affected by dissolved and sorbed copper in a eutrophic river

Photosynthetic biofilms are capable of immobilizing important concentrations of metals, therefore reducing bioavailability to organisms. But also metal pollution is believed to produce changes in the microalgal species composition of biofilms. We investigated the changes undergone by natural photosynthetic biofilms from the River Meuse, The Netherlands, under chronic copper (Cu) exposure. The suspended particles in the river water had only a minor effect on reduction of sorption and toxicity of Cu to algae. Biofilms accumulated Cu proportionally to the added concentration, also at the highest concentration used (9 microM Cu). The physiognomy of the biofilms was affected through the growth of the chain-forming diatom Melosira varians, changing from long filaments to short tufts, although species composition was not affected by the Cu exposure. The Cu decreased phosphate uptake and algal biomass measured as chl a, which degraded exponentially in time. Photosynthetic activity was always less sensitive than algal biomass; the photon yield decreased linearly in time. The protective and insulating role of the biofilm, supported by ongoing autotrophic activity, was indicated as essential in resisting metal toxicity. We discuss the hypothesis that the toxic effects of Cu progress almost independently of the species composition, counteracting ongoing growth, and conclude that autotrophic biofilms act as vertical heterogeneous units. Effective feedback mechanisms and density dependence explain several discrepancies observed earlier.     

Speciation and Size Distribution of Copper and Zinc in Urban Road Runoff

The impact of pollutants from road runoff on receiving water bodies becomes increasingly serious. However, less is known about the study on the distribution of metal speciation in particles with different sizes, and the interaction among metal speciation. Our research is aimed at investigating speciation distribution of copper (Cu) and zinc (Zn) in particles and the interaction among metal speciation. Stainless steel sieves in different sizes were used to accomplish filtration scheme. Sequential extraction procedures contained five steps for the chemical fractionation of metals. Flame atomic absorption spectrometry (Shimadzu, AA-6800) was used to determine the concentration of metal speciation. Speciation distribution of Cu was similar to that of Zn. Size distribution implied that small particles (<75 μm) determined the distribution for both Cu and Zn, as well as their migration. Correlation analysis indicated that the interaction among speciation of Cu was different from that of Zn.     

A review of total dissolved copper and its chemical speciation in San Francisco Bay, California

Following basin-wide contamination from industrial emissions and urban development, total dissolved copper concentrations in some regions of San Francisco Bay have exceeded national and state guidelines for water quality. In the face of dramatic improvements in wastewater treatment and point source control, persisting elevated dissolved copper concentrations in the Bay have prompted multiple studies and extensive monitoring of this estuary since 1989. Statistical analyses of monitoring data show that total dissolved copper concentrations have declined in the North (by 17%) and South (29%) San Francisco Bay as well as in the Southern Sloughs (44%) from 1993 to 2001. Concentrations remain elevated in the farthest reaches of the Bay (Delta and Estuary Interface), and in the Central Bay. Dissolved copper concentrations throughout the Bay have also been positively correlated (r = 0.632, P < 0.0005, n = 598) with dissolved organic matter, supporting results from complimentary chemical speciation studies which indicate that high-affinity copper-binding organic ligands dominate the chemical speciation of dissolved copper in the Bay. These organic ligands typically bind > 99.9% of the dissolved copper, effectively buffering the system against small changes in dissolved copper concentrations, and maintaining free Cu(2+) concentrations well below the toxicity threshold of ambient aquatic microorganisms. In response to these findings, site-specific water quality criteria for dissolved copper concentrations are now being developed by the Regional Water Quality Board to provide a more appropriate standard for copper toxicity in the Bay-one based on its chemical speciation and bioavailability.     

Development of flow cytometry-based algal bioassays for assessing toxicity of copper in natural waters

Copper toxicity to the freshwater algae Selenastrum capricornutum and Chlorella sp. and the marine algae Phaeodactylum tricornutum and Dunaliella tertiolecta was investigated using different parameters measured by flow cytometry: cell division rate inhibition, chlorophyll a fluorescence, cell size (i.e., light-scattering), and enzyme activity. These parameters were assessed regarding their usefulness as alternative endpoints for acute (1-24 h) and chronic (48-72 h) toxicity tests. At copper concentrations of 10 micrograms/L or less, significant inhibition (50%) of the cell division rate was observed after 48- and 72-h exposures for Chlorella sp., S. capricornutum, and P. tricornutum. Bioassays based on increases in algal cell size were also sensitive for Chlorella sp. and P. tricornutum. Copper caused both chlorophyll a fluorescence stimulation (48-h EC50 of 10 +/- 1 micrograms Cu/L for P. tricornutum) and inhibition (48-h EC50 of 14 +/- 6 micrograms Cu/L for S. capricornutum). For acute toxicity over short exposure periods, esterase activity in S. capricornutum using fluorescein diacetate offered a rapid alternative (3-h EC50 of 90 +/- 40 micrograms Cu/L) to growth inhibition tests for monitoring copper toxicity in mine-impacted waters. For all the effect parameters measured, D. tertiolecta was tolerant to copper at concentrations up to its solubility limit in seawater. These results demonstrate that flow cytometry is a useful technique for toxicity testing with microalgae and provide additional information regarding the general mode of action of copper (II) to algal species.