The biotic ligand model as a promising tool to predict Cu toxicity in amazon blackwaters.

The Rio Negro basin of Amazonia (Brazil) is a hotspot of fish biodiversity that is under threat from copper (Cu) pollution. The very ion-poor blackwaters have a high dissolved organic carbon (DOC) concentration. We investigated the Cu sensitivity of nine Amazonian fish species in their natural blackwaters (Rio Negro). The acute lethal concentration of Cu (96 h LC50) was determined at different dilutions of Rio Negro water (RNW) in ion-poor well water (IPW), ranging from 0 to 100%. The IPW was similar to RNW in pH and ionic composition but deficient in DOC, allowing this parameter to vary 20-fold from 0.4 to 8.3 mg/L in tests. The Biotic Ligand Model (BLM; Windward version 3.41.2.45) was used to model Cu speciation and toxicity over the range of tested water compositions, and to estimate lethal Cu accumulations on the gills (LA50). The modeling predicted a high relative abundance of Cu complexes with DOC in test waters. As these complexes became more abundant with increasing RNW content, a concomitant decrease in free Cu2+ was observed. In agreement with this modeling, acute Cu toxicity decreased (i.e. 96 h LC50 values increase) with increasing RNW content. The three most sensitive species (Hemigrammus rhodostomus, Carnegiella strigatta and Hyphessobrycon socolofi) were Characiformes, whereas Corydoras schwartzi (Siluriformes) and Apistogramma agassizii (Cichliformes) were the most tolerant. These sensitivity differences were reflected in the BLM-predicted lethal gill copper accumulation (LA50), which were generally lower in Characiformes than in Cichliformes. Using these newly estimated LA50 values in the BLM allowed for accurate prediction of acute Cu toxicity in the nine Amazonian fish. Our data emphasize that the BLM approach is a promising tool for assessing Cu risk to Amazonian fish species in blackwater conditions characterized by very low concentrations of major ions but high concentrations of DOC.

The effects of temperature on nickel bioaccumulation and toxicity in the freshwater snail Lymnaea stagnalis.

It is well known that temperature can have important effects on the toxicity of metals (and other contaminants) to aquatic organisms. To date, research has mostly focused on thermal effects on acute metal toxicity, and there is a data gap on thermal effects on chronic metal toxicity to sensitive organisms that are particularly relevant to environmental risk assessment. This latter research is especially needed in the context of increased global temperature and heat waves frequency associated with climate change. We investigated temperature effects on chronic nickel (Ni) bioaccumulation and toxicity to the metal-sensitive freshwater snail Lymnaea stagnalis. In the laboratory, we conducted a series of experiments with juvenile snails that were pre-acclimated to different temperatures since their embryonic stage. We found that temperature and nickel separately had strong effects on juvenile growth rate and survival. Rising temperature from 18 to 26 °C had no noticeable effect on Ni-induced growth inhibition and Ni bioaccumulation in juvenile L. stagnalis exposed over 40 days to 0, 30 and 60 µg L-1 of dissolved Ni. These results agreed with estimates of Ni uptake and elimination rates (ku and ke), which were either unaffected by temperature or increased by similar factors from 18 to 26 °C. On the other hand, a temperature increase from 18 to 26 °C appeared to exacerbate Ni lethality to juvenile snails in the 40-day toxicity test. This exacerbation might have been due to a combination of factors, including detrimental changes in metabolically available Ni pools and/or to sensitization of the organism under sub-optimal temperatures. Overall, our study shows that thermal effects on metal chronic toxicity are complex, with effects that can be response-specific and not directly related to metal toxicokinetic.

The effects of winter cold acclimation on acute and chronic cadmium bioaccumulation and toxicity in the banded killifish (Fundulus diaphanus).

Temperate freshwater fishes can experience large seasonal temperature fluctuations that could affect their exposure and sensitivity to trace metals. Yet, temperature effects are overlooked in ecotoxicology studies, especially for cold temperatures typical of the winter. In the present study, the effects of long-term cold acclimation on Cd bioaccumulation and toxicity were investigated in a freshwater fish, the banded killifish (Fundulus diaphanus). Killifish were acclimated to 14 °C or gradually cooled (2 °C/week) to 4 °C and cold acclimated for 6 weeks. Then, both acclimation groups were exposed to environmentally realistic waterborne Cd concentrations (0, 0.5 or 5 µg Cd L-1) for a further 28 d at their respective acclimation temperatures. Tissue metal bioaccumulation, fish survival, condition, and markers of oxidative and ionoregulation stress, were measured after 0, 2, 5 and 28 days of Cd exposure. Cadmium tissue accumulation increased over the exposure duration and was typically lower in cold-acclimated fish. In agreement with this lower bioaccumulation, fewer Cd toxic effects were observed in cold-acclimated fish. There was little evidence of a difference in intrinsic Cd sensitivity between 4 °C- and 14 °C-acclimated fish, as Cd toxicity appeared to closely follow Cd bioaccumulation. Our study suggests that current environmental water quality guidelines would be protective in the winter for the abundant and ecologically-important banded killifish.

State of polycyclic aromatic hydrocarbon (PAH) contamination in the Saint John Harbour, New Brunswick, Canada.

This study examined the concentrations and compositions of polycyclic aromatic hydrocarbon (PAH) contaminants in the surficial sediments of the Saint John Harbour (SJH) and assessed PAH exposure to local aquatic biota. Our findings suggest that sedimentary PAH contamination is heterogeneous and widespread in the SJH, with several sites exceeding the Canadian and NOAA recommended guidelines for the protection of aquatic life. Despite high concentrations of PAHs at some sites, there was no indication that local nekton was affected. Lack of a biological response may be due in part to a low bioavailability of sedimentary PAHs, presence of confounding factors (e.g., trace metals), and/or adaptation of local wildlife to the historic PAH contamination in this region. Overall, although no indication of effects to wildlife was observed with the data collected in the present study, continued efforts should be made to remediate highly contaminated areas and reduce the prevalence of these compounds.

Investigating the mechanisms of dissolved organic matter protection against copper toxicity in fish of Amazon's black waters.

We investigated how natural dissolved organic matter (DOM) of the Rio Negro (Amazon) affects acute copper (Cu) toxicity to local fish: the cardinal tetra (Paracheirodon axelrodi) and the dwarf cichlid (Apistogramma agassizii). It is established that Cu2+ complexation with DOM decreases Cu bioavailability (and thus toxicity) to aquatic organisms, as conceptualized by the Biotic Ligand Model (BLM). However, we also know that Rio Negro's DOM can interact with fish gills and have a beneficial effect on Na+ homeostasis, the main target of acute Cu toxicity in freshwater animals. We aimed to tease apart these potential protective effects of DOM against Cu-induced Na+ imbalances in fish. In the laboratory, we acclimated fish to Rio Negro water (10 mg L-1 DOC) and to a low-DOM water (1.4 mg L-1 DOC) with similar ion composition and pH (5.9). We measured 3-h Cu uptake in gills and unidirectional and net Na+ physiological fluxes across a range of Cu concentrations in both waters. Various DOM pre-acclimation times (0, 1 and 5 days) were evaluated in experiments with P. axelrodi. Copper exposure led to similar levels of net Na+ loss in the two fish, but with distinct effects on Na+ influx and efflux rates reflecting their different ionoregulation strategies. Rio Negro DOM protected against Cu uptake and toxicity in the two fish species. Both Cu uptake in fish gills and Na+ regulation disturbances were relatively well predicted by the modelled aqueous free Cu2+ ion concentration. These findings suggest that protection by DOM occurs mainly from Cu complexation under the tested conditions. The prevalence of this geochemical-type protection over a physiological-type protection agrees with the BLM conceptual framework, supporting the use of the BLM to assess the risk of Cu in these Amazonian waters.

Environmental risk of nickel in aquatic Arctic ecosystems.

The Arctic faces many environmental challenges, including the continued exploitation of its mineral resources such as nickel (Ni). The responsible development of Ni mining in the Arctic requires establishing a risk assessment framework that accounts for the specificities of this unique region. We set out to conduct preliminary assessments of Ni exposure and effects in aquatic Arctic ecosystems. Our analysis of Ni source and transport processes in the Arctic suggests that fresh, estuarine, coastal, and marine waters are potential Ni-receiving environments, with both pelagic and benthic communities being at risk of exposure. Environmental concentrations of Ni show that sites with elevated Ni concentrations are located near Ni mining operations in freshwater environments, but there is a lack of data for coastal and estuarine environments near such operations. Nickel bioavailability in Arctic freshwaters seems to be mainly driven by dissolved organic carbon (DOC) concentrations with bioavailability being the highest in the High Arctic, where DOC levels are the lowest. However, this assessment is based on bioavailability models developed from non-Arctic species. At present, the lack of chronic Ni toxicity data on Arctic species constitutes the greatest hurdle toward the development of Ni quality standards in this region. Although there are some indications that polar organisms may not be more sensitive to contaminants than non-Arctic species, biological adaptations necessary for life in polar environments may have led to differences in species sensitivities, and this must be addressed in risk assessment frameworks. Finally, Ni polar risk assessment is further complicated by climate change, which affects the Arctic at a faster rate than the rest of the world. Herein we discuss the source, fate, and toxicity of Ni in Arctic aquatic environments, and discuss how climate change effects (e.g., permafrost thawing, increased precipitation, and warming) will influence risk assessments of Ni in the Arctic.

Effects of Non-native Fish on Lacustrine Food Web Structure and Mercury Biomagnification along a Dissolved Organic Carbon Gradient.

Although the introduction of non-native fish species has been shown to alter trophic ecology in aquatic ecosystems, there has been limited research on how invasive species alter methylmercury (MeHg) biomagnification in lacustrine food webs. We sampled surface water and biota from 8 lakes in Quebec, Canada, spanning a range of dissolved organic carbon (DOC) concentrations (2.9-8.4 mg/L); 4 lakes were inhabited by native brook trout (Salvelinus fontinalis), and the remaining lakes contained brook trout and a non-native fish, Allegheny pearl dace (Margariscus margarita). Periphyton, zooplankton, macroinvertebrates, and fish were analyzed for: 1) stable carbon (δ13 C) and nitrogen (δ15 N) isotope ratios to delineate food webs, and 2) total Hg (THg) or MeHg. Compared with the brook trout from reference lakes, fish from invaded lakes had higher length-standardized THg concentrations as well as a narrower dietary range and elevated trophic level, inferred from unadjusted δ13 C and δ15 N values, respectively. The rate of Hg biomagnification was similar across invaded and reference lakes, implying little effect of the invasive fish on the trophic transfer of MeHg. Despite differences in food web structure due to pearl dace invasion, DOC was the strongest predictor of brook trout THg levels for all lakes, suggesting that underlying environmental factors exerted a stronger influence on brook trout THg concentrations than the presence of a non-native forage fish. Environ Toxicol Chem 2020;39:2196-2207. © 2020 SETAC.

A Mystery Tale: Nickel Is Fickle When Snails Fail-Investigating the Variability in Ni Toxicity to the Great Pond Snail.

Dissolved Ni concentrations inhibiting the growth of juvenile great pond snails (Lymnaea stagnalis) have been documented to vary from about 1 to 200 µg L-1 Ni. This variability makes L. stagnalis either a moderately sensitive or the most sensitive freshwater species to chronic Ni exposure tested to date. Given the role of sensitive species in environmental risk assessment frameworks, it is particularly important to understand this variability, i.e., to characterize the factors that modulate Ni toxicity and that may confound toxicity test outcomes when uncontrolled. In the present study, we tested if this variability was due to analytical (growth calculation: biomass versus growth rate), environmental (water quality), lab-specific practices, and/or snail population differences among earlier studies. Specifically, we reanalyzed previously published Ni toxicity data and conducted additional measurements of Ni aqueous speciation, short-term Ni uptake, and chronic Ni toxicity with test waters and snail cultures used in previous studies. Corrections for Ni bioavailability and growth calculations explained a large degree of variability in the published literature. However, a residual 16-fold difference remained puzzling between 2 studies: Niyogi et al. (2014) (low ECxs) and Crémazy et al. (2018) (high ECxs). Indeed, differences in metal bioavailability due to water chemistry, lab-specific practices, and snail population sensitivity could not explain the large variation in Ni toxicity in these 2 very similar studies. Other potentially important toxicity-modifying factors were not directly evaluated in the present work: test duration, diet, snail holding conditions, and snail age at onset of testing. The present analysis highlights the need for further studies to elucidate 1) the mechanisms of growth inhibition in Ni-exposed L. stagnalis and 2) the important abiotic and biotic factors affecting this biological response. Until these processes are understood, substantial uncertainties will remain about inclusion of this species in Ni environmental risk assessment. Integr Environ Assess Manag 2020;16:983-997. © 2020 SETAC.

Unravelling Metal Speciation in the Microenvironment Surrounding Phytoplankton Cells to Improve Predictions of Metal Bioavailability.

A lack of knowledge on metal speciation in the microenvironment surrounding phytoplankton cells (i.e., the phycosphere) represents an impediment to accurately predicting metal bioavailability. Phycosphere pH and O2 concentrations from a diversity of algae species were compiled. For marine algae in the light, the average increases were 0.32 pH units and 0.17 mM O2 in the phycosphere, whereas in the dark the average decreases were 0.10 pH units and 0.03 mM O2, in comparison to bulk seawater. In freshwater algae, the phycosphere pH increased by 1.28 units, whereas O2 increased by 0.38 mM in the light. Equilibrium modeling showed that the pH alteration influenced the chemical species distribution (i.e., free ion, inorganic complexes, and organic complexes) of Al, Cd, Co, Cu, Fe, Hg, Mn, Ni, Pb, Sc, Sm, and Zn in the phycosphere, and the O2 fluctuation increased oxidation rates of Cu(I), Fe(II) and Mn(II) from 2 to 938-fold. The pH/O2-induced changes in phycosphere metal chemistry were larger for freshwater algae than for marine species. Reanalyses of algal metal uptake data in the literature showed that uptake of the trivalent metals (Sc, Sm and Fe), in addition to divalent metals, can be better predicted after considering the phycosphere chemistry.

Effects of natural light and depth on rates of photo-oxidation of dissolved organic carbon in a major black-water river, the Rio Negro, Brazil.

Systems rich in terrigenous dissolved organic carbon (DOC), like the Rio Negro, can contribute significant amounts of carbon dioxide back to the atmosphere and support important microbial communities. We investigated photo-oxidation in the Rio Negro: (1) the depth to which light causes complete photo-oxidation to CO2 and changes in DOC structure, (2) the daily rate of change of absorbance indices, (3) the relationship between sub-surface rates of photo-oxidation to CO2 and light exposure, (4) the areal rates of photo-oxidation, and (5) the stability of fluorophore signals. Experiments were run in an outdoor pool of Rio Negro water, under natural sunlight during the dry seasons of 2015 and 2018. In 2018, rates of complete photo-oxidation and changes in absorbance indices decayed exponentially, approaching their asymptotes between 9 and 15 cm depth. In 2015, direct absorbance indices ceased changing at 14 cm depth. Fluorescence of humic acid-like moieties continued to decrease, sometimes to 35-43 cm depth. This indicates that partial photo-oxidation of DOC, and thus interaction with the microbial community, occurs to greater depths than previously expected. Areal rates of CO2 production were 28.8 and 39.3 mg C m-2 d-1 (two experiments, October 2018). Sub-surface (1.1 cm) rates were strongly related to light levels, reaching a maximum of 0.68 mg C l-1 d-1 in September. Complete photo-oxidation ceased below 29.6 mW cm-2 d-1 UV radiation, providing a daily baseline for observable production of CO2. Absorbance indices changed by 9 to 14% d-1 at high light levels, except for R254/365 (4.4% d-1). Fluorophore emission ranges were stable between 2014 and 2018, indicating that emissions can be compared across time and space. This study contributes to better estimates and understanding of photo-oxidation in tropical, black-water rivers, which will be useful for carbon modelling.

The Effects of Natural Suspended Solids on Copper Toxicity to the Cardinal Tetra in Amazonian River Waters.

The aim of the present study was to characterize the effects of particles on metal aquatic toxicity in a tropical system. To this end, we investigated the effects of natural suspended solids on copper (Cu) geochemistry and acute toxicity to the cardinal tetra (Paracheirodon axelrodi), in 2 Amazonian rivers with different total suspended solids (TSS) levels: the Rio Negro (low TSS ~ 8 mg L-1 ) and the Rio Solimões (high TSS ~ 70 mg L-1 ). The effect of particles on Cu aqueous geochemistry was assessed by measuring total, dissolved, and free ionic Cu concentrations in filtered (<0.45 µm) and unfiltered waters. Furthermore, acute Cu toxicity to fish was assessed in both filtered and unfiltered waters, by measuring physiological net fluxes of Na+ , Cl- , K+ , and total ammonia (in both river waters) and 96-h fish mortality (in Rio Solimões only). The particles in the Rio Negro were not abundant enough to play a significant role in our study. On the other hand, the Rio Solimões particles bound approximately 70% of total aqueous Cu in our tests. In agreement with bioavailability-based models, this decrease in dissolved (and free ionic) Cu concentration decreased Cu lethality in the 96-h toxicity tests. In the physiological measurements, the best evidence of particle protection was the total alleviation of Cu-induced Cl- losses. These flux tests also suggested that the particles themselves may negatively affect Na+ balance in the fish. Overall, the present study supports the use of bioavailability concepts to account for the role of natural suspended solids on metal biological effects in the Amazon River basin. Environ Toxicol Chem 2019;38:2708-2718. © 2019 SETAC.

Using the Biotic Ligand Model framework to investigate binary metal interactions on the uptake of Ag, Cd, Cu, Ni, Pb and Zn in the freshwater snail Lymnaea stagnalis.

There is growing interest in the development of mechanistically-based models, such as the Biotic Ligand Model (BLM), for assessing the environmental risk of metal mixtures. However, the derivation of such models requires insights into the mechanisms of multimetal interactions that are often lacking for aquatic organisms. In the present study, we investigated how binary mixtures of six metals (Ag, Cd, Cu, Ni, Pb and Zn) interact for uptake in the great pond snail Lymnaea stagnalis, a freshwater species particularly sensitive to metals in chronic exposure. For each metal, short-term (2-3 h) uptake experiments on juvenile snails were performed with the metal alone and in combination with a second metal, at concentrations encompassing the chronic toxicity concentration range. These experiments showed significant binary metal interactions for 7 out of 15 mixtures. Most interactions were inhibitory in nature, not reciprocal and caused by either Ag or Cu. They led to relative changes of uptake that did not exceed 50% within the range of metal chronic toxicity. The BLM proved to be successful at explaining most of the interactions, via competitive inhibition. This study is in support of using bioavailability-based models, such as the BLM, to model metal mixture interactions in L. stagnalis.

Chronic Toxicity of Binary Mixtures of Six Metals (Ag, Cd, Cu, Ni, Pb, and Zn) to the Great Pond Snail Lymnaea stagnalis.

Although metal-mixture toxicity has recently received increasing attention, there is still insufficient knowledge on joint effects occurring in chronic exposures to relatively low metal concentrations. We characterized the chronic toxicity of binary mixtures of six metals (Ag, Cd, Cu, Ni, Pb, and Zn) in 14 day growth tests with juveniles of the metal-sensitive freshwater snail Lymnaea stagnalis. Observations were compared with predictions from individual metals and from the two most frequently used mixture models: concentration addition (CA) and independent action (IA). Predictions based on measured total dissolved concentrations and on calculated free-ion activities did not differ greatly because multimetal geochemical interactions in the tests were limited. In around half of the tests, mixture toxicity was higher than the greatest effect caused by the individual metals, arguing in favor of considering joint effects. When the additive models were used, the great majority of interactions were either additive or less than additive (i.e., antagonism). In general, the IA model was the most accurate, while the CA model was the most conservative. Along with other studies, these findings suggest that, at least for binary combinations, the simple CA model may provide satisfactory protection from the chronic metal toxicity of metal mixtures to aquatic organisms.

Characterization of the effects of binary metal mixtures on short-term uptake of Cd, Pb, and Zn by rainbow trout (Oncorhynchus mykiss).

Biotic Ligand Models (BLMs) for individual metals improve our ability to regulate metals in the aquatic environment by considering the effects of water quality parameters (ionic composition, pH, DOC) on metal bioavailability. However, in natural aquatic systems, organisms are often simultaneously exposed to multiple metals and these interactions are not currently considered in BLMs or most environmental regulations. Recently, several different mixture BLMs (mBLMs) have been developed to begin assessing this issue. Some of these models assume competitive interactions between all metals, while others assume only metals with similar modes of action (e.g., Na+ or Ca2+ antagonists) will competitively interact. In this study, we used standard in vivo 3-h gill metal binding assays to characterize the uptake of Cd, Pb, and Zn individually and in binary mixtures with Ag, Cd, Cu, Pb, Ni, and Zn across a range of concentrations that encompassed the 96-h LC50 for each metal. Inhibition of Cd, Pb, and Zn uptake at the gill by introduction of a second metal was consistent with mode of action in some cases, but not others. Further, contrary to expectations, inhibition was always either non-competitive or could not be defined statistically. We also observed one example of stimulated metal uptake (Ni stimulated Zn uptake). Consistent with our previous experiments on Ag, Cu, and Ni, these studies suggest that current mBLM frameworks will need revision to better reflect the mechanisms underlying metal mixture interactions.

Experimentally derived acute and chronic copper Biotic Ligand Models for rainbow trout.

We evaluated the effects of varying water chemistry ([Ca2+]=0.2-3mM, [Mg2+]=0.05-3mM, dissolved organic matter (DOM, natural, from maple leaves)=0.3-10mg of CL-1, pH=5.0-8.5) on the acute (96-h, unfed fish) and chronic (30-d, fed fish) toxicity of waterborne Cu to juvenile rainbow trout (Oncorhynchus mykiss) exposed in flow-through conditions. Acute and chronic Biotic Ligand Models (BLMs) were developed from the obtained toxicity data-sets, using the Visual MINTEQ software. Our results indicate that Cu is predominantly an acute toxicant to rainbow trout, as there were no observable growth effects and the 96-h and 30-d LC50 values were similar, with mortality mostly occurring within the first few days of exposure. Calcium and DOM were greatly protective against both acute and chronic Cu toxicity, but Mg seemed to only protect against chronic toxicity. Additional protection by pH 5.0 in acute exposure and by pH 8.5 in chronic exposure occurred. In the range of conditions tested, the observed 96-h LC50 and 30-d LC20 values varied by a factor of 39 and 27 respectively. The newly developed acute and chronic BLMs explained these variations reasonably well (i.e. within a 2-fold error), except at pH≥8 where the high observed acute toxicity could not be explained, even by considering an equal contribution of CuOH+ and Cu2+ to the overall Cu toxicity. The 96-h LC50 values of 59% of 90 toxicity tests from 19 independent studies in the literature were reasonably well predicted by the new acute BLM. The LC20 predictions from the new chronic BLM were reasonable for 7 out of 14 toxicity tests from 6 independent chronic studies (with variable exposure durations). The observed deviations from BLM predictions may be due to uncertainties in the water chemistry in these literature studies and/or to differences in fish sensitivity. A residual pH effect was also observed for both the acute and the chronic data-sets, as the ratio of predicted vs. observed LC values generally increased with the pH. Additional mechanistic studies are required to understand the influence of pH, Na, and Mg on Cu toxicity to trout. The present study presents the first experimentally developed chronic Cu BLM for the rainbow trout. To the best of our knowledge, it also presents the first acute Cu BLM that is based on a published data-set for trout. These newly developed BLMs should contribute to improving the risk assessment of Cu to fish in freshwater.

Investigating copper toxicity in the tropical fish cardinal tetra (Paracheirodon axelrodi) in natural Amazonian waters: Measurements, modeling, and reality.

Copper at high concentrations is an ionoregulatory toxicant in fish and its toxicity is known to be strongly modulated by the water chemistry. The toxicity of Cu to the tropical fish cardinal tetra (Paracheirodon axelrodi) was investigated in waters from two major rivers of the Amazon watershed: the Rio Negro (filtered <0.45µm, pH 5.6, DOC=8.4 mgL-1, Na=33µM, Ca=8µM) and the Rio Solimões (filtered <0.45µm, pH 6.7, DOC=2.8 mgL-1, Na=185µM, Ca=340µM), as well as in a natural "reference water" (groundwater) which was almost DOC-free (pH 6.0, DOC=0.34 mgL-1, Na=53µM, Ca=5µM). Acute 96-h mortality, Cu bioaccumulation and net flux rates of Na+, Cl-, K+ and total ammonia were determined in P. axelrodi exposed in each water. Copper speciation in each water was determined by two thermodynamic models and by potentiometry, and its toxicity was predicted based on the biotic ligand model (BLM) framework. Our results indicate that high Na+ loss is the main mode of toxic action of Cu in P. axelrodi, in accordance with general theory. Cardinal tetra showed a particularly high ability to tolerate Cu and to maintain Na+ balance, similar to the ability of this and other endemic Rio Negro species to tolerate low pH and ion-poor conditions. Cu toxicity was lower in Rio Negro than in the other two waters tested, and the free [Cu2+] at the LC50, as determined by any of the three speciation methods tested, was approximately 10-fold higher. This variation could not be captured by a realistic set of BLM parameters. At least in part, this observation may be due to gill physiological alterations induced by the abundant dissolved organic matter of the Rio Negro. The implication of this observation is that, for metals risk assessment in tropical waters, similar to the Rio Negro, care must be used in applying BLM models developed using temperate DOC and temperate species.

Characterization of the effects of binary metal mixtures on short-term uptake of Ag, Cu, and Ni by rainbow trout (Oncorhynchus mykiss).

Single metal Biotic Ligand Models (BLMs) have been developed for a number of metals and model organisms. While these BLMs improve our ability to regulate metals in the aquatic environment, in reality, organisms are often simultaneously exposed to metal mixtures. Recently, several attempts have been made to develop mixture BLMs (mBLMs). Some of these models assume competitive interactions between all metals, while others assume only metals with a similar mode of action (e.g., Na+ or Ca2+ antagonists) will competitively interact. To begin testing these assumptions in the mBLM framework, standard 3-h gill metal binding assays with Ag, Cu, and Ni (primary metals), were performed in vivo on freshwater rainbow trout. Fish were exposed across a range of concentrations encompassing the 96-h LC50 for that metal to characterize uptake kinetics for each of these three primary metals (radiolabelled) in the presence and absence of a secondary metal (Ag, Cd, Cu, Ni, Pb, or Zn; not radiolabelled). We observed a complex series of interactions in binary mixtures that frequently contradicted theoretical expectations. Metals with similar modes of action did competitively interact in some instances, but not others, and when they did compete the competition was not necessarily reciprocal (e.g., Cu inhibited Ag uptake but Ag did not inhibit Cu uptake). We also observed examples of interactions between metals with dissimilar modes of action and several examples of metals stimulating the uptake of other metals. The underlying mechanisms for these unexpected interactions are unclear, but suggest that many of the current assumptions in mBLMs regarding the number and types of metal uptake sites and corresponding metal interactions are not correct. Careful characterization of metal mixture interactions is clearly needed before a reliable mBLM can be developed.

In the presence of fluoride, free Sc³âº is not a good predictor of Sc bioaccumulation by two unicellular algae: possible role of fluoro-complexes.

We investigated the effect of fluoride complexation on scandium accumulation by two unicellular algae, Chlamydomonas reinhardtii and Pseudokirchneriella subcapitata. This trivalent metal was selected for its chemical similarities with aluminum and for its convenient radioisotope (Sc-46), which can be used as a tracer in short-term bioaccumulation studies. Scandium surface-bound concentrations (Sc(ads)) and uptake fluxes (J(int)) were estimated in the two algae over short-term (<1 h) exposures at pH 5 and in the presence of 0 to 40 µM F(-). Although the computed proportion of dissolved Sc(3+) dropped from 20% to 0.01% over this [F(-)] range, Sc(ads) and J(int) values for both algae decreased only slightly, suggesting a participation of Sc fluoro-complexes in both processes. Surface adsorption and uptake of fluoride complexes with aluminum have been reported in the literature. These observations are not taken into account by current models for trace metal bioaccumulation (e.g., the biotic ligand model). Results from a previous study, where the effects of pH on Sc uptake were investigated, suggested that Sc hydroxo-complexes were internalized by C. reinhardtii. There is thus growing evidence that the free ion concentration may not be adequate to predict the accumulation of Sc (and potentially of other trivalent metals) in aquatic organisms.

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.

The biotic ligand model can successfully predict the uptake of a trivalent ion by a unicellular alga below pH 6.50 but not above: possible role of hydroxo-species.

In many reported cases, the biotic ligand model (BLM) has been shown to predict the bioavailability of divalent metals toward aquatic biota successfully. However, studies on the bioavailability of nonessential trivalent metals, including aluminum (Al), are relatively scarce. In the present study, short-term scandium (Sc) internalization fluxes (Jint) were measured in Chlamydomonas reinhardtii in order to explore the applicability of the BLM to trivalent metals. Scandium was selected for its chemical similarities with Al and for its convenient radio-isotope (Sc-46). Apparent affinity constants of Sc(3+) with membrane transport sites (KSc-Rcell app) were surprisingly high, ranging from 10(8.08) M(-1) to 10(13.95) M(-1) over the pH range from 4.50 to 7.90. The competition of H(+) for binding with Sc(3+) transport sites explained this trend within the pH range of 4.50 to 6.00, but not from pH 6.50-7.90. In this latter pH range, predicted fluxes were smaller than observed fluxes and this divergence increased with pH, from a factor of 4 to approximately 1000. Above pH 6.50, the calculated supply of Sc(3+) to the biointerface by physical diffusion of the free Sc(3+) ion and by the dissociation of its hydroxo-complexes (ScOH(2+), Sc(OH)2(+) and Sc(OH)3) was insufficient to support the high observed internalization fluxes. We speculate that this failure of the BLM could be due to the transmembrane transport of undissociated Sc hydroxo-complexes. Scandium uptake could be modeled reasonably well using a simple semiempirical equation considering equal contributions from Sc(3+), ScOH(2+), Sc(OH)2(+), and Sc(OH)3 and no H(+) competition. Our work highlights the importance of studying the possible role of hydroxo-species in trace metal uptake.