James C. McGeer

The biotic ligand model: a historical overview

During recent years, the biotic ligand model (BLM) has been proposed as a tool to evaluate quantitatively the manner in which water chemistry affects the speciation and biological availability of metals in aquatic systems. This is an important consideration because it is the bioavailability and bioreactivity of metals that control their potential to cause adverse effects. The BLM approach has gained widespread interest amongst the scientific, regulated and regulatory communities because of its potential for use in developing water quality criteria (WQC) and in performing aquatic risk assessments for metals. Specifically, the BLM does this in a way that considers the important influences of site-specific water quality. This journal issue includes papers that describe recent advances with regard to the development of the BLM approach. Here, the current status of the BLM development effort is described in the context of the longer-term history of advances in the understanding of metal interactions in the environment upon which the BLM is based. Early developments in the aquatic chemistry of metals, the physiology of aquatic organisms and aquatic toxicology are reviewed first, and the degree to which each of these disciplines influenced the development of water quality regulations is discussed. The early scientific advances that took place in each of these fields were not well coordinated, making it difficult for regulatory authorities to take full advantage of the potential utility of what had been learned. However, this has now changed, with the BLM serving as a useful interface amongst these scientific disciplines, and within the regulatory arena as well. The more recent events that have led to the present situation are reviewed, and consideration is given to some of the future needs and developments related to the BLM that are envisioned. The research results that are described in the papers found in this journal issue represent a distinct milestone in the ongoing evolution of the BLM approach and, more generally, of approaches to performing ecological assessments for metals in aquatic systems. These papers also establish a benchmark to which future scientific and regulatory developments can be compared. Finally, they demonstrate the importance and usefulness of the concept of bioavailability and of evaluative tools such as the BLM.

Effects of chronic sublethal exposure to waterborne Cu, Cd or Zn in rainbow trout. 1: Iono-regulatory disturbance and metabolic costs.

The relationships among growth, feeding behaviour, ion regulation, swimming performance and oxygen consumption in rainbow trout (Oncorhynchus mykiss) were compared during chronic exposure (up to 100 days) to sublethal levels of waterborne Cd (3 µg.l(-1)), Cu (75 µg.l(-1)) or Zn (250 µg.l(-1)) in moderately hard water (hardness of 140 mg.l(-1), pH 8). A pattern of disturbance, recovery and stabilization was evident for all three metal exposures, although the degree of disturbance, specific response and time course of events varied. Growth was unaffected by any of the metals under a regime of satiation feeding but appetite was increased and decreased in Cu- and Cd-exposed trout respectively. Critical swimming speed was significantly lowered in fish chronically exposed to Cu, an effect associated with elevated O(2) consumption rate at higher swimming speeds. Branchial Na(+)/K(+) ATPase activity was elevated in Cu-exposed fish but not in Cd-exposed trout. Disruption of carcass Na(+) and Ca(2+) balance was evident within 2 days of exposure to either Cd, Cu or Zn, with subsequent recovery to control levels. The loss of Ca(2+) in trout exposed to waterborne Cd persisted longest, and recovery took approximately a month. The physiological response of trout to chronic Cu exposure involves mechanisms that result in an associated metabolic cost. In comparison, Cd is neither a loading nor a limiting stress and acclimation to chronic Cd-exposure does not appear to involve a long term metabolic cost.

Effects of chronic sublethal exposure to waterborne Cu, Cd or Zn in rainbow trout 2: tissue specific metal accumulation

Tissue specific metal accumulations (gills, liver, kidney and whole body) in rainbow trout (Oncorhynchus mykiss) were compared during chronic exposure (up to 100 days) to sublethal levels of waterborne Cd (3 µg.l(-1)), Cu (75 µg.l(-1)) or Zn (250 µg.l(-1)) in moderately hard water (hardness of 140 mg.l(-1), pH 8.0). A general pattern of tissue metal increase and stabilization was evident for all three metals, although the degree and time course of accumulation varied. The exception to this general pattern was a lack of Zn accumulation in the liver and kidney although small amounts did accumulate in the gills and whole body. Accumulation of Cu occurred primarily in the liver while for Cd the kidney was the major organ of accumulation. Exponential modeling was employed to compare and contrast the saturation concentration and time to half saturation of various tissues. Accumulation of essential metals (Cu and Zn), if it occurred, was rapid and increases were relatively low. For example the time to half saturation during Cu exposures was always less than 2 weeks and the maximum level of accumulation was less than four times background levels. For non-essential Cd, time to half saturation for the liver and kidney was always longer than 5 weeks and modeled saturation concentrations were up to 80-fold higher than background. The response to Cu and Zn suggested an active regulation of tissue burdens while that of Cd appears to be more passive, resulting in continuous metal accumulation over an extended time course. While the initial patterns of accumulation for each metal were generally consistent with the damage, repair and acclimation pattern from concurrent physiological measurements it was clear that tissue metal accumulation was not a good indicator of either exposure of physiological impact.

Utility of Tissue Residues for Predicting Effects of Metals on Aquatic Organisms

As part of a SETAC Pellston Workshop, we evaluated the potential use of metal tissue residues for predicting effects in aquatic organisms. This evaluation included consideration of different conceptual models and then development of several case studies on how tissue residues might be applied for metals, assessing the strengths and weaknesses of these different approaches. We further developed a new conceptual model in which metal tissue concentrations from metal-accumulating organisms (principally invertebrates) that are relatively insensitive to metal toxicity could be used as predictors of effects in metal-sensitive taxa that typically do not accumulate metals to a significant degree. Overall, we conclude that the use of tissue residue assessment for metals other than organometals has not led to the development of a generalized approach as in the case of organic substances. Species-specific and site-specific approaches have been developed for one or more metals (e.g., Ni). The use of gill tissue residues within the biotic ligand model is another successful application. Aquatic organisms contain a diverse array of homeostatic mechanisms that are both metal- and species-specific. As a result, use of whole-body measurements (and often specific organs) for metals does not lead to a defensible position regarding risk to the organism. Rather, we suggest that in the short term, with sufficient validation, species- and site-specific approaches for metals can be developed. In the longer term it may be possible to use metal-accumulating species to predict toxicity to metal-sensitive species with appropriate field validation.

The role of dissolved organic carbon in moderating the bioavailability and toxicity of Cu to rainbow trout during chronic waterborne exposure

We examined the influence of dissolved organic carbon (DOC) on the bioavailability of waterborne Cu to rainbow trout (Oncorhynchus mykiss) during chronic sublethal exposure. Juvenile rainbow trout were exposed to Cu (as CuSO(4)) and DOC as humic acid (HA, as sodium salt) for one month in synthetic soft water to give treatments with varying combinations of free ionic and HA complexed Cu. The total Cu concentration was 7 microg/l for all treatments (except controls) and HA was added at levels of 0, 2.5 and 7.5 mg/l which corresponded to DOC levels of 1.2, 2.2 and 4.0 mg/l. Fish grew well in all treatments and no mortalities occurred. Cu was highly bioavailable in the treatment with no added HA; gill and liver Cu accumulation occurred as well as a disruption of Na(+) regulation. In Cu treatments with additions of both 2.5 and 7.5 mg/l HA, there was no significant tissue accumulation of Cu. The addition of HA alleviated and delayed the disruption of iono-regulatory mechanisms. A recovery of plasma Na(+) losses was observed and this was associated with an increase in gill Na(+)/K(+) ATPase activity by the end of the exposure. Following the month of chronic exposure the uptake and turnover rates of Cu at the gills and into various tissue compartments were measured through radioisotopic techniques ((64)Cu). While chronic Cu exposure did not result in acclimation (i.e. increased LC50), the uptake rate and extent of Cu uptake into the gills and liver was increased. This study demonstrates that growth and tissue accumulation of Cu are poor predictors of the chronic effects of Cu, and illustrates that HA moderates chronic Cu bioavailability. The lack of a link between Cu bioaccumulation and Cu impact and the role of organic matter in reducing the bioavailability of Cu are important considerations in the context of ecological risk assessment.

The effect of water chemistry on the acute toxicity of nickel to the cladoceran Daphnia pulex and the development of a biotic ligand model

The goal of this study was to evaluate the influence of water chemistry parameters on the acute toxicity of waterborne Ni to Daphnia pulex in soft waters and using this information to develop a biotic ligand model. The effects of Ca, Mg, Na, K, Cl, pH (two differently buffered sets) and natural organic matter (NOM) from two sources were evaluated in standardized 48h acute toxicity tests. Increases in Ca2+ had a protective effect on Ni toxicity, suggesting that this ion competes with Ni at the site of biological uptake. Increased waterborne Mg2+ also reduced Ni toxicity, but to a lesser degree compared with Ca2+. EC50 values increased at higher pH when the organic buffer 3-morpholinepropanesulfonic acid was used to adjust test pH, however in tests series where pH was varied using HCO(3)(-) the results were equivocal. Other testing showed that Na, K and Cl did not influence the toxicity response of D. pulex to Ni. Complexation of Ni by NOM reduced toxicity but Nordic Reservoir NOM was much more protective compared to Suwannee River NOM. Geochemical modeling of organic matter complexation of Ni was done using the HydroQual Biotic Ligand Model (BLM ver. 2.3.3; research mode) and the Windermere Humic Aqueous Model (WHAM ver 6.0). Results showed dramatic differences between the two models in dissolved organic matter complexation. Modelling of Ni geochemistry for test solutions other than those containing NOM showed consistent and minor differences between the WHAM and the BLM. The latter model was used to develop a comprehensive prediction model of Ni toxicity. logK values developed for competitive cationic effects showed that Ca and Mg have a much higher protective effect in soft water compared to models developed for Daphnia magna in hard water. The BLM developed for this species in soft water provided good predictions of toxicity across a wide range of Ni concentrations but also highlighted the need for an improved understanding of the effects of NOM and pH on Ni toxicity in soft waters.

Effects of chronic waterborne nickel exposure on two successive generations of Daphnia magna

In a 21-d chronic toxicity test in which an F0 generation of Daphnia magna were exposed to waterborne Ni, the no-observable-effect concentration (for survival, reproduction, and growth) was 42 microg Ni L(-1), or 58% of the measured 21-d median lethal concentration (LC50) of 71.9 microg Ni L(-1) (95% confidence interval, 56.5-95.0). Chronic exposure to 85 microg Ni L(-1) caused marked decreases in survival, reproduction, and growth in F0 animals. In the F1 generation (daphnids born of mothers from the chronically exposed F0 generation), animals chronically exposed to 42 microg Ni L(-1) for 11 d weighed significantly less (20%) than controls, indicating increased sensitivity of F1 animals. Additionally, in this successive generation, significant decreases in whole-body levels of metabolites occurred following exposure to both 42 microg Ni L(-1) (decreased glycogen and adenosine triphosphate [ATP]) and 21 microg Ni L(-1) (decreased ATP). No significant changes were observed in whole-body total lipid, total protein, and lactate levels at any concentration. Whereas F1 neonates with mothers that were exposed to 21 microg Ni L(-1) showed increased resistance to acute Ni challenge, as measured by a significant (83%) increase in the acute (48-h) LC50, F1 neonates with mothers that were exposed to 42 microg Ni L(-1) were no more tolerant of acute Ni challenge than control animals were. Nickel accumulations in F1 animals chronically exposed to 21 and 42 microg Ni L(-1) were 11- and 18-fold, respectively, above control counterparts. The data presented suggest that chronic Ni exposure to two successive generations of D. magna lowered the overall energy state in the second generation. Whereas the quantity of neonates produced was not affected, the quality was; thus, environmentally meaningful criteria for regulating waterborne Ni concentrations in freshwater require consideration of possible multigenerational effects.

Modes of metal toxicity and impaired branchial ionoregulation in rainbow trout exposed to mixtures of Pb and Cd in soft water.

Models such as the Biotic Ligand Model (BLM) predict how natural organic matter (NOM) and competing ions (e.g., Ca(2+), H(+) and Na(+)) affect metal bioavailability and toxicity in aquatic organisms. However, such models focus upon individual metals, not metal mixtures. This study determined whether Pb and Cd interact at the gill of rainbow trout (Oncorhynchus mykiss) when trout were exposed to environmentally relevant concentrations of these metals (Cd<100 nmol L(-1); Pb<500 nmol L(-1)) in soft (<100 micromol Ca(2+)L(-1)), moderately acidic (pH 6.0) water. The 96-h LC50 for Pb was 482 nmol L(-1), indicating that Pb was one-order of magnitude more toxic in soft, acidic water than in harder, circumneutral pH waters. The LC50 for Cd alone was also low, 6.7 nmol L(-1). Surprisingly, fish acclimated to soft water had multiple populations of Pb-gill and Cd-gill binding sites. A low capacity, high affinity population of Pb-gill binding sites had a B(max) of 18.2 nmol g(-1) wet weight (ww) and apparent K(Pb-gill)=7.05, but a second low affinity population could not be saturated up to free Pb concentrations approaching 4000 nmol L(-1). Two populations of Cd-gill binding sites were characterized: a high affinity, low capacity population with an apparent K(Cd-gill)=7.33 and B(max)=1.73 nmol g(-1) ww, and a low affinity, high capacity population with an apparent K(Cd-gill)=5.86, and B(max)=13.7 nmol g(-1) ww. At low concentrations, Cd plus Pb accumulation was less than additive because Cd out-competed Pb for gill binding sites, which were likely apical Ca(2+)-channels. While disturbances to Ca(2+) influx were caused by Cd alone, Pb alone had no effect. However, Pb exacerbated Cd-induced disturbances to Ca(2+) influx demonstrating that, although Pb- plus Cd-gill binding was less than additive due to competition, the effects (ionic disturbances) were more than additive (synergistic). Pb was also likely binding to intracellular targets, such as branchial carbonic anhydrase, which led to inhibited Na(+) influx. This ionic disturbance was exacerbated by Cd. We conclude that exposure to environmentally relevant concentrations of Pb plus Cd results in less than additive metal-gill binding in soft, moderately acidic waters. However, ionic disturbances caused by Cd plus Pb are greater than additive, and this may ultimately increase the toxicity of Cd-Pb mixtures to fishes. Our findings suggest that it may be necessary to re-evaluate water quality criteria and assumptions of the BLM for fish exposed to mixtures of Pb and Cd in the acidic, soft waters found in the Canadian Shield, Scandinavia and other sensitive regions.

Aquatic toxicity of manufactured nanomaterials: challenges and recommendations for future toxicity testing

Environmental context The increased use of nanomaterials in industrial and consumer products requires robust strategies to identify risks when they are released into the environment. Aquatic toxicologists are beginning to possess a clearer understanding of the chemical and physical properties of nanomaterials in solution, and which of the properties potentially affect the health of aquatic organisms. This review highlights the main challenges encountered in aquatic nanotoxicity testing, provides recommendations for overcoming these challenges, and discusses recent studies that have advanced our understanding of the toxicity of three important OECD nanomaterials, titanium dioxide, zinc oxide and silver nanomaterials. Abstract Aquatic nanotoxicologists and ecotoxicologists have begun to identify the unique properties of the nanomaterials (NMs) that potentially affect the health of wildlife. In this review the scientific aims are to discuss the main challenges nanotoxicologists currently face in aquatic toxicity testing, including the transformations of NMs in aquatic test media (dissolution, aggregation and small molecule interactions), and modes of NM interference (optical interference, adsorption to assay components and generation of reactive oxygen species) on common toxicity assays. Three of the major OECD (Organisation for Economic Co-operation and Development) priority materials, titanium dioxide (TiO2), zinc oxide (ZnO) and silver (Ag) NMs, studied recently by the Natural Sciences and Engineering Research Council of Canada (NSERC), National Research Council of Canada (NRC) and the Business Development Bank of Canada (BDC) Nanotechnology Initiative (NNBNI), a Canadian consortium, have been identified to cause both bulk effect, dissolution-based (i.e. free metal), or NM-specific toxicity in aquatic organisms. TiO2 NMs are most toxic to algae, with toxicity being NM size-dependent and principally associated with binding of the materials to the organism. Conversely, dissolution of Zn and Ag NMs and the subsequent release of their ionic metal counterparts appear to represent the primary mode of toxicity to aquatic organisms for these NMs. In recent years, our understanding of the toxicological properties of these specific OECD relevant materials has increased significantly. Specifically, researchers have begun to alter their experimental design to identify the different behaviour of these materials as colloids and, by introducing appropriate controls and NM characterisation, aquatic nanotoxicologists are now beginning to possess a clearer understanding of the chemical and physical properties of these materials in solution, and how these materials may interact with organisms. Arming nanotoxicologists with this understanding, combined with knowledge of the physics, chemistry and biology of these materials is essential for maintaining the accuracy of all future toxicological assessments.

Development of a biotic ligand model for the acute toxicity of zinc to Daphnia pulex in soft waters.

The goal of this study was to develop a biotic ligand model (BLM) for the acute toxicity of zinc to the Daphnia pulex in soft water. In different tests Ca (as CaSO(4)), Na (as NaCl), Mg (as MgSO(4)), K (as KCl), pH (using the buffer 3-(N morpholino)-propanesulphonic acid (MOPS)) and dissolved organic carbon (DOC) were altered to determine possible effects on the 48h EC50 for Zn. Increases in waterborne Ca(2+) had a protective effect on Zn toxicity, suggesting that this ion competes with Zn and that they share a common site of biological uptake. Increased waterborne Mg(2+) also reduced Zn toxicity, but to a lesser degree compared with Ca(2+). No significant effects of other cations on EC50 for Zn were observed, indicating that the toxicity of Zn in D. pulex is not linked to Na(+) and K(+). Increasing DOC concentrations resulted in higher EC50 values for Zn due to the complexation of Zn by organic matter in solution and the resulting reduction of free Zn(2+) ion concentrations. Tests to characterize the effect of pH on Zn toxicity showed a small rise in EC50 values between pH 6.3 and 7.1 and no further change as pH was increased to 8.0. Two existing BLM implementations for acute Zn effects on D. magna were tested to determine their applicability to D. pulex in soft water. The existing models underestimated the protective effect of Ca(2+) and Mg(2+) but accurately predict the effects of DOC. A modified BLM, with revised equilibrium constants for competitive cation effects was developed. This study shows that the acute effects of Zn on D. pulex in soft water can be characterized and incorporated into a predictive BLM.