Using biofilms for monitoring metal contamination in lotic ecosystems: The protective effects of hardness and pH on metal bioaccumulation.

Biofilms can make good bioindicators and biomarkers, offering a convenient tool to monitor metal contamination in streams that results from mine tailing sites. Biofilm metal content (Cu, Zn, Cd, Pb) as well as diatom diversity and the presence of teratologies (diatom abnormalities) were determined for biofilms from rivers with a variety of physicochemical properties across a metal contamination gradient. The results of metal accumulation were highly consistent from year to year, with significant relationships between calculated free metal ion concentrations and biofilm metal contents for samples from different rivers. This indicates the "universal nature" of the metal accumulation process in biofilms. The authors observed that protons and major cations protected against metal accumulation. A very low number of diatom taxa were found at the most contaminated sites, and the highest proportions of deformities were observed at these sites. However, it was difficult to distinguish the effect of metal contamination from the effect of other parameters, especially pH. The results suggest that the development of biofilm-based proxies for metal bioavailability is useful and that incorporation of the effects of hardness and pH in this metal contamination monitoring tool is important. Environ Toxicol Chem 2016;35:1489-1501. © 2015 SETAC.

Uptake and subcellular partitioning of trivalent metals in a green alga: comparison between Al and Sc.

Despite 40+ years of research on aluminum (Al) toxicity in aquatic organisms, Al transport mechanisms through biological membranes, and the intracellular fate of Al once assimilated, remain poorly understood. The trivalent metal scandium shares chemical similarities with Al and, unlike Al, it has a convenient radioactive tracer (Sc-46) allowing for relatively simple measurements at environmentally relevant concentrations. Thus, we investigated the potential of Sc to substitute for Al in uptake and intracellular fate studies with the green alga Chlamydomonas reinhardtii. Short-term (<60 min) competitive uptake experiments indicated that Al does not inhibit Sc influx, implying that these metals do not share a common transport mechanism. Also, internalized Al concentrations were ~4 times higher than Sc concentrations after long-term (72 h) exposures under similar conditions (4.5 µM AlT or ScT, 380 µM FT, pH 7.0, 3.8 pM Al calc (3+) and 1.0 pM Sc calc (3+) ). However, interesting similarities were observed in their relative subcellular distributions, suggesting possible common toxicity/tolerance mechanisms. Both metals mostly distributed to the organelles fraction and almost no association was found with the cytosolic proteins. The greatest difference was observed in the cellular debris fraction (membranes and nucleus) where Al was much more concentrated than Sc. However, it is not clear whether or not this fraction contained extracellular metal associated with the algal surface. To summarize, Sc does not seem to be an adequate substitute of Al for transport/uptake studies, but could be for investigations of toxicity/tolerance mechanisms in C. reinhardtii. Further work is needed to verify this latter suggestion.

Assessment of trace metal binding kinetics in the resin phase of diffusive gradients in thin films.

The dynamic technique of diffusive gradients in thin films (DGT), that measures metal speciation in situ, has found wide environmental application. Simple interpretation of the metal accumulation in terms of a solution concentration has assumed that trace metals do not penetrate beyond the surface of the binding layer, but penetration, although theoretically discussed has not yet been directly measured. Multiple binding layers were used to enable analysis of different depths of a DGT binding phase (Chelex-100 or iminodiacetate resins). In simple metal solution (no ligand) at pH 7, metal penetration to the back layer was low and similar for all metals. However, at lower pH up to 42% of an individual metal accumulated in the back resin layer. This was most noticeable for Mn at pH 4 and 5, but Cd and Co were also affected at pH 4. These results were consistent with rate limited binding, particularly for Mn. A kinetic model successfully fitted the data and allowed derivation of a binding rate constant and the mean distance that metals penetrate into a resin gel (λ(M)). Only for Mn, Co and Cd were experimentally derived λ(M) values greater than the diameter of a Chelex-100 resin bead. For most situations, then, the penetration into the binding layer is negligible and binding of trace metal ions can be regarded as instantaneous, validating the simple use and interpretation of DGT. For weakly binding metals at low pH the slower binding allows penetration, which may affect the DGT measurement.

Kinetic signatures of metals in the presence of Suwannee River fulvic acid.

This work provides new information on the dissociation kinetics of metal-fulvic acid (FA) complexes. Diffusive gradients in thin-film (DGT) devices deployed in solutions containing metals and 30 mg L(-1) Suwannee River FA at pH 5 and 7, at two different metal-to-ligand ratios, were used to estimate an apparent diffusive boundary layer (ADBL) thickness at the gel-solution interface. The discrepancy between the ADBL thickness measured for metals that are known to dissociate from complexes quickly (e.g., Cd) and that of other trace metals was exploited to calculate the rate of complex dissociation. When the ADBL thickness is plotted for a suite of metals, a "kinetic signature" is created. There was a clear kinetic signature at pH 7, with substantial kinetic limitation for Cu, Pb, and Ni and none for Cd, Co, and Mn (i.e., Cu-, Pb-, and Ni-FA complexes dissociated more slowly). At pH 5, the kinetic signature was less distinct, due in part to slow association kinetics of Mn, and possibly Cd and Co, with the resin. The good sensitivity of the method to small changes in dissociation kinetics was able to show that the dissociation of most metal-FA complexes is sufficiently fast to not limit the DGT measurement.

The effect of bacteria on the sensitivity of microalgae to copper in laboratory bioassays.

Although single-species laboratory toxicity tests with microalgae are sensitive and highly reproducible, they lack environmental realism. Interactions between algae and their associated bacteria, either in the plankton or in biofilms, may alter algal sensitivity to contaminants, which are not mimicked in laboratory toxicity tests. This study investigated the effects of simple algal-bacterial relationships on the sensitivity of laboratory-cultured algae to copper using 72-h algal growth-rate inhibition bioassays. Four species of microalgae were used, two isolates of each; a strain of algae with no microscopically visible and no culturable bacteria present (operationally defined as axenic) and a non-axenic strain. The four algae used were the marine diatom Nitzschia closterium, the freshwater green alga Pseudokirchneriella subcapitata and two tropical Chlorella spp. Under control conditions (no copper), N. closterium and P. subcapitata grew better in the presence of the bacterial community. Sensitivity to copper (assessed as the concentration to inhibit the growth rate by 50% after 72-h (IC50)) was not significantly different for the axenic and non-axenic strains of N. closterium, P. subcapitata or for Chlorella sp. (PNG isolate). At pH 5.7, the axenic Chlorella sp. (NT isolate) had a 72-h IC50 of 46mugCuL(-1), while in the presence of bacteria the IC50 increased (i.e., sensitivity decreased) to 208mugCuL(-1). However, when the bacterial status of both the operationally defined axenic and non-axenic cultures of N. closterium and Chlorella sp. (NT isolate) was investigated using polymerase chain reaction (PCR) amplification of 16S rRNA followed by DNA fingerprinting using denaturing gradient gel electrophoresis (DGGE), it was found that bacteria were actually present in all the algal cultures, i.e. the axenic cultures were not truly bacteria-free. Based on sequence information, the bacteria present were nearly all identified as alphaproteobacteria, and a number of isolates had high similarity to bacteria previously identified as symbionts or species endophytically associated with marine organisms. The "axenic" cultures contained less bacterial phylotypes than the non-axenic cultures, and based on band-intensity, also contained less bacterial DNA. This supported the findings of few differences in copper sensitivity between strains, and suggests that standard microalgal toxicity tests probably inadvertently use non-axenic cultures in metal assessment.

Uptake and internalisation of copper by three marine microalgae: comparison of copper-sensitive and copper-tolerant species.

Although it has been well established that different species of marine algae have different sensitivities to metals, our understanding of the physiological and biochemical basis for these differences is limited. This study investigated copper adsorption and internalisation in three algal species with differing sensitivities to copper. The diatom Phaeodactylum tricornutum was particularly sensitive to copper, with a 72-h IC50 (concentration of copper to inhibit growth rate by 50%) of 8.0 microg Cu L(-1), compared to the green algae Tetraselmis sp. (72-h IC50 47 microg Cu L(-1)) and Dunaliella tertiolecta (72-h IC50 530 microg Cu L(-1)). At these IC50 concentrations, Tetraselmis sp. had much higher intracellular copper (1.97+/-0.01 x 10(-13)g Cu cell(-1)) than P. tricornutum (0.23+/-0.19 x 10(-13)g Cu cell(-1)) and D. tertiolecta (0.59+/-0.05 x 10(-13)g Cu cell(-1)), suggesting that Tetraselmis sp. effectively detoxifies copper within the cell. By contrast, at the same external copper concentration (50 microg L(-1)), D. tertiolecta appears to better exclude copper than Tetraselmis sp. by having a slower copper internalisation rate and lower internal copper concentrations at equivalent extracellular concentrations. The results suggest that the use of internal copper concentrations and net uptake rates alone cannot explain differences in species-sensitivity for different algal species. Model prediction of copper toxicity to marine biota and understanding fundamental differences in species-sensitivity will require, not just an understanding of water quality parameters and copper-cell binding, but also further knowledge of cellular detoxification mechanisms.

Sensitivity of marine microalgae to copper: the effect of biotic factors on copper adsorption and toxicity.

Microalgae are sensitive indicators of environmental change and, as the basis of most freshwater and marine ecosystems, are widely used in the assessment of risk and development of environmental regulations for metals. However, interspecies differences in sensitivity to metals are not well understood. The relationship between metal-algal cell binding and copper sensitivity of marine microalgae was investigated using a series of 72-h growth-rate inhibition bioassays and short-term (1-h) uptake studies. A range of marine algae from different taxonomic groups were screened to determine whether copper adsorption to the cell membrane was influenced by biotic factors, such as the ultrastructure of cell walls and cell size. Minutocellus polymorphus was the most sensitive species to copper and Dunaliella tertiolecta the least sensitive, with 72-h IC50 values (concentration to inhibit growth-rate by 50%) of 0.6 and 530 microg Cu/L, respectively. Copper solution-cell partition coefficients at equilibrium (K(d)) were calculated for six species of algae on a per cell and surface area basis. The largest and smallest cells had the lowest and highest K(d) values, respectively (on a surface area basis), with a general (non-linear) trend of decreasing K(d) with increasing cell surface area (p=0.026), however, no relationship was found between K(d) and copper sensitivity, nor cell size and copper sensitivity. Interspecies differences in copper sensitivity were not related to cell size, cell wall type, taxonomic group or K(d) values. The differences in sensitivity may be due to differences in uptake rates across the plasma membrane, in internal binding mechanisms and/or detoxification mechanisms between the different microalgal species.

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.