Chris M. Wood

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.

A new paradigm for ammonia excretion in aquatic animals: Role of rhesus (RH) glycoproteins

SUMMARY Ammonia excretion at the gills of fish has been studied for 80 years, but the mechanism(s) involved remain controversial. The relatively recent discovery of the ammonia-transporting function of the Rhesus (Rh) proteins, a family related to the Mep/Amt family of methyl ammonia and ammonia transporters in bacteria, yeast and plants, and the occurrence of these genes and glycosylated proteins in fish gills has opened a new paradigm. We provide background on the evolution and function of the Rh proteins, and review recent studies employing molecular physiology which demonstrate their important contribution to branchial ammonia efflux. Rhag occurs in red blood cells, whereas several isoforms of both Rhbg and Rhcg occur in many tissues. In the branchial epithelium, Rhcg appears to be localized in apical membranes and Rhbg in basolateral membranes. Their gene expression is upregulated during exposure to high environmental ammonia or internal ammonia infusion, and may be sensitive to synergistic stimulation by ammonia and cortisol. Rhcg in particular appears to be coupled to H+ excretion and Na+ uptake mechanisms. We propose a new model for ammonia excretion in freshwater fish and its variable linkage to Na+ uptake and acid excretion. In this model, Rhag facilitates NH3 flux out of the erythrocyte, Rhbg moves it across the basolateral membrane of the branchial ionocyte, and an apical “Na+/NH +4 exchange complex” consisting of several membrane transporters (Rhcg, V-type H+-ATPase, Na+/H+ exchanger NHE-2 and/or NHE-3, Na+ channel) working together as a metabolon provides an acid trapping mechanism for apical excretion. Intracellular carbonic anhydrase (CA-2) and basolateral Na+/HCO –3 cotransporter (NBC-1) and Na+/K+-ATPase play indirect roles. These mechanisms are normally superimposed on a substantial outward movement of NH3 by simple diffusion, which is probably dependent on acid trapping in boundary layer water by H+ ions created by the catalysed or non-catalysed hydration of expired metabolic CO2. Profitable areas for future investigation of Rh proteins in fish are highlighted: their involvement in the mechanism of ammonia excretion across the gills in seawater fish, their possible importance in ammonia excretion across the skin, their potential dual role as CO2 transporters, their responses to feeding, and their roles in early life stages prior to the full development of gills.

The Effects of Chronic Plasma Cortisol Elevation on the Feeding Behaviour, Growth, Competitive Ability, and Swimming Performance of Juvenile Rainbow Trout

Plasma cortisol elevation, a common consequence of stress, occurs in salmonids of subordinate rank; these fish acquire a smaller share of available food and grow more slowly. This study examined the role of cortisol itself in these phenomena. Cortisol implants, with parallel sham and control treatments, were used to create a chronic threefold elevation in plasma cortisol levels in juvenile rainbow trout, and the individual feeding patterns of the fish were evaluated using X‐ray radiography. The three treatment groups were (1) held alone and fed to satiation, thereby providing a measure of voluntary appetite, or mixed together in equal proportions and fed to either (2) satiation or (3) half‐satiation, thereby allowing assessment of the additional effects of competitive interaction and food limitation. Chronic plasma cortisol elevation had significant negative effects on individual appetite, growth rate, condition factor, and food conversion efficiency, independent of whether the fish were held under unmixed or mixed conditions. Under the latter, mean share of meal was reduced and fin damage increased in cortisol‐treated fish; negative growth effects were more severe with food limitation, but the response patterns were otherwise unchanged. Even in the absence of other groups, cortisol‐treated fish showed more variable feeding patterns. When compared at the same individual ration levels, cortisol‐treated fish had lower growth rates, reflecting a higher “cost of living.” Cortisol treatment had no effect on aerobic swimming performance. These results suggest that the structure of the feeding hierarchy may not be determined solely by competitive ability but may also be greatly influenced by differences in the feeding behaviour of unstressed fish versus stressed fish caused by cortisol elevation in the latter.

The physiology of waterborne silver toxicity in freshwater rainbow trout (Oncorhynchus mykiss) 1. The effects of ionic Ag

Abstract Adult rainbow trout, fitted with arterial catheters, were exposed to AgNO3 for 6 days at a concentration (10 μg Ag l−1, flow-through) close to the 7 day LC50 in moderately hard freshwater. Approximately 35% of total Ag occurred as free ionic Ag+, and the remainder as silver chlorides. Ag accumulated on the gills and increased about 4-fold above control levels in blood plasma, stabilizing by 48 h. Much greater concentrations of Ag accumulated in the liver, but not the kidney, at 6 days. Metallothionein induction did not occur. Plasma [Na+] and [Cl−] declined steadily to 70% of control levels by day 6, accompanied by a progressive metabolic acidosis, a 5-fold increase in blood [glucose], a 40% decrease in relative plasma volume, contraction of the spleen, and marked hemoconcentration. Plasma [Ca2+] and [K+] were largely unaffected. Respiratory suffocation did not occur: plasma [lactate] remained constant, arterial PO2 increased and PCO2 decreased, the latter compensating the metabolic acidosis so arterial pH fell only moderately. Comparably sampled control fish exhibited negligible disturbance. Unidirectional Na+ influx from the water, measured in juvenile trout, was inhibited by 42% immediately, and abolished by 48 h of AgNO3 exposure. These symptoms suggested a similar toxic mechanism of action to that of low environmental pH. We speculate that Ag+ interferes with net Na+ and Cl− uptake at the gills, and causes death by secondary fluid volume disturbance, hemoconcentration, and eventual cardiovascular collapse.

The mechanism of acute silver nitrate toxicity in freshwater rainbow trout (Oncorhynchus mykiss) is inhibition of gill Na+ and Cl−1 transport

Rainbow trout (Oncorhynchus mykiss) were exposed to 2 and 10 μg l−1 silver (as AgNO3) for up to 75 h in moderately hard freshwater. At 10 μg l−1 total Ag, branchial Na+ and Cl− influxes were inhibited by over 50% immediately and by almost 100% at 8 h, and showed no signs of recovery over the duration of the experiment. Na+ and Cl− effluxes were much less affected. These changes in unidirectional fluxes resulted in a large net loss of both Na+ and Cl− across the gills and a significant decrease in plasma [Na+] and [Cl−1]. The effects of exposure to 2 μg l−1 Ag on Na+ and Cl−1 transport were generally similar to those at 10 μg l−1, but were of a lesser magnitude. Unidirectional Na+ fluxes recovered immediately following removal of silver, after 48 h exposure to 2 μg l−1. Michaelis-Menten kinetic analysis demonstrated that the maximal rate of Na+ influx (Jmax) was significantly reduced after 48 h exposure to 2 μg l−1 Ag, whereas the affinity of the transport sites for Na+ (1/Km) was unaffected, indicating that the inhibition of Na+ influx by silver was of a non-competitive nature. Fish exposed to 10 μg l−1 Ag for 48 h also had significantly lower activities of the branchial enzymes Na+K+ ATPase (85% inhibition) and carbonic anhydrase (28% inhibition). The results of this study suggest that a disturbance of branchial ionoregulation, as a result of inhibition of branchial enzymes involved in ion transport, is the principal mechanism of the physiological toxicity of silver nitrate to freshwater fish.

Cadmium disrupts behavioural and physiological responses to alarm substance in juvenile rainbow trout (Oncorhynchus mykiss)

SUMMARY Alarm substance is a chemical signal released from fish skin epithelial cells after a predator causes skin damage. When other prey fish detect alarm substance by olfaction, they perform stereotypical predator-avoidance behaviours to decrease predation risk. The objective of this study was to explore the effect of sublethal cadmium (Cd) exposure on the behavioural and physiological responses of juvenile rainbow trout (Oncorhynchus mykiss) to alarm substance. Waterborne exposure to 2 μg Cd l–1 for 7 days eliminated normal antipredator behaviours exhibited in response to alarm substance, whereas exposures of shorter duration or lower concentration had no effect on normal behaviour. Furthermore, dietary exposure to 3 μg Cd g–1 in the food for 7 days, which produced the same whole-body Cd accumulation as waterborne exposure to 2 μg l–1, did not alter normal behaviour, indicating that an effect specific to waterborne exposure alone (i.e. Cd accumulation in the olfactory system) results in behavioural alteration. Whole-body phosphor screen autoradiography of fish exposed to 109Cd demonstrated that Cd deposition in the olfactory system (rosette, nerve and bulb) during waterborne exposure was greater than in all other organs of accumulation except the gill. However, Cd could not be detected in the brain. A short-term elevation in plasma cortisol occurred in response to alarm substance under control conditions. Cd exposures of 2 μg l–1 waterborne and 3 μg g–1 dietary for 7 days both inhibited this plasma cortisol elevation but did not alter baseline cortisol levels. Our results suggest that exposure to waterborne Cd at environmentally realistic levels (2 μg l–1) can disrupt the normal behavioural and physiological responses of fish to alarm substance and can thereby alter predator-avoidance strategies, with potential impacts on aquatic fish communities.

Ion balance, acid-base regulation, and chloride cell function in the common killifish, Fundulus heteroclitus-a euryhaline estuarine teleost

The common killifish,Fundulus heteroclitus, is a euryhaline teleost common throughout estuaries of eastern North America. This symposium paper reviews the important contributions of the killifish to our present understanding of ionoregulation in seawater (SW) fish and their mechanisms of euryhalinity, and presents new data developing the killifish as a freshwater (FW) model system. Experiments on killifish have characterized (i) drinking in SW and its reduction in FW; (ii) the adaptive roles of the kidney to SW and FW conditions; (iii) the instantaneous (Phase I) and delayed (Phase II) reductions in Na+ outflux that occur upon transfer from SW to FW; (iv) the importance of prolactin secretion in the Phase II effect; (v) the cortisol-stimulated induction of branchial Na+, K+-ATPase that occurs upon transfer from FW to SW; (vi) the accompanying changes in morphology of the mitochondria-rich (MR) or “chloride cells” on the gills; (vii) the localization of this Na+, K+-ATPase activity to the basolateral membrane of chloride cells; and (viii) the NaCl-secretory function of these cells in SW. The opercular epithelium, which is rich in chloride cells, has been used as an in vitro model to characterize the mechanisms and control of NaCl secretion in SW fish. Much less is known about gill function in fresh water (inward NaCl transport), primarily due to the absence of a comparable freshwater model. Here we show that killifish acclimated to dilute FW ([NaCl] = 1 mmol I−1) possess large numbers of MR cells on the opercular epithelium. When mounted in vitro with FW on the outside, the preparation develops a large inside negative transepithelial potential (TEP) that is a Na+ diffusion potential. By the Ussing flux ratio criterion, Na+ fluxes are passive, but a small active influx of Cl− occurs, an observation that supports the involvement of MR cells in active Cl− uptake. This FW opercular epithelium if bathed with isotonic saline on both sides does not secrete Cl−, indicating that the MR cells indeed are of the FW type. In vivo, the fish exhibits a high rate of Na+ influx and outflux; Cl− outflux is much lower, and there is no detectable Cl− influx. Experimental variation of FW [NaCl] reveals a saturable, low affinity Na+ uptake mechanism, a Cl− influx mechanism that is activated only at much higher concentrations, and no evidence of exchange diffusion. Acid-base disturbance appears to be corrected by differential regulation of the outflux components only. Hence, the FW killifish ionoregulates somewhat differently from the few other FW teleosts that have been examined, and its opercular epithelium will serve as a very useful model system.

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.

Mechanism of branchial apical silver uptake by rainbow trout is via the proton-coupled Na+ channel

The branchial uptake mechanism of the nonessential heavy metal silver from very dilute media by the gills of freshwater rainbow trout was investigated. At concentrations >36 nM AgNO(3), silver rapidly entered the gills, reaching a peak at 1 h, after which time there was a steady decline in gill silver concentration and a resulting increase in body silver accumulation. Below 36 nM AgNO(3), there was only a very gradual increase in gill and body silver concentration over the 48-h exposure period. Increasing water sodium concentration ([Na(+)]; 0.05 to 21 mM) significantly reduced silver uptake, although, in contrast, increasing ambient [Ca(2+)] or [K(+)] up to 10 mM did not reduce silver uptake. Kinetic analysis of silver uptake at varying [Na(+)] showed a significant decrease in maximal silver transport capacity (173 +/- 34 pmol. g(-1). h(-1) at 0.1 mM [Na(+)] compared with 35 +/- 9 at 13 mM [Na(+)]) and only a slight decrease in the affinity for silver transport (K(m); 55 +/- 27 nM at 0.1 mM [Na(+)] compared with 91 +/- 47 nM at 13 mM [Na(+)]). Phenamil (a specific blocker of Na(+) channels), at a concentration of 100 microM, blocked Na(+) uptake by 78% of control values (58% after washout), and bafilomycin A(1) (a specific blocker of V-type ATPase), at a concentration of 2 microM, inhibited Na(+) uptake by 57% of control values, demonstrating the presence of a proton-coupled Na(+) channel in the apical membrane of the gills. Phenamil (after washout) and bafilomycin A(1) also blocked silver uptake by 62 and 79% of control values, respectively, indicating that Ag(+) is able to enter the apical membrane via the proton-coupled Na(+) channel.The branchial uptake mechanism of the nonessential heavy metal silver from very dilute media by the gills of freshwater rainbow trout was investigated. At concentrations >36 nM AgNO3, silver rapidly entered the gills, reaching a peak at 1 h, after which time there was a steady decline in gill silver concentration and a resulting increase in body silver accumulation. Below 36 nM AgNO3, there was only a very gradual increase in gill and body silver concentration over the 48-h exposure period. Increasing water sodium concentration ([Na+]; 0.05 to 21 mM) significantly reduced silver uptake, although, in contrast, increasing ambient [Ca2+] or [K+] up to 10 mM did not reduce silver uptake. Kinetic analysis of silver uptake at varying [Na+] showed a significant decrease in maximal silver transport capacity (173 ± 34 pmol ⋅ g-1 ⋅ h-1 at 0.1 mM [Na+] compared with 35 ± 9 at 13 mM [Na+]) and only a slight decrease in the affinity for silver transport ( K m; 55 ± 27 nM at 0.1 mM [Na+] compared with 91 ± 47 nM at 13 mM [Na+]). Phenamil (a specific blocker of Na+ channels), at a concentration of 100 μM, blocked Na+ uptake by 78% of control values (58% after washout), and bafilomycin A1 (a specific blocker of V-type ATPase), at a concentration of 2 μM, inhibited Na+ uptake by 57% of control values, demonstrating the presence of a proton-coupled Na+ channel in the apical membrane of the gills. Phenamil (after washout) and bafilomycin A1 also blocked silver uptake by 62 and 79% of control values, respectively, indicating that Ag+ is able to enter the apical membrane via the proton-coupled Na+ channel.