The Physiological Basis for Altered Na\u3csup\u3e+\u3c/sup\u3e and Cl\u3csup\u3e-\u3c/sup\u3e Movement Across the Gills of Rainbow Trout (\u3cem\u3eOncorhynchus mykiss\u3c/em\u3e) in Alkaline (pH=9.5) Water

To test the hypothesis that internal ion imbalances at high pH are caused by altered branchial ion transporting capacity and permeability, radiotracers (24Na+ and 36Cl-) were used to measure ion movements across the gills of intact rainbow trout (Oncorhynchus mykiss) during 3 d exposure to pH 9.5. At control pH (pH 8.0), the trout were in net ion balance, but by 8 h at high pH, 60%–70% reductions in Cl- influx (Cl) and Na+ influx (JNa/in) led to net Cl- and Na+ losses of -200 µmol kg-1 h-1. Outflux (diffusive efflux plus renal ion losses) was not initially altered. By 72 h, net Cl- balance was reestablished because of a restoration of JCl/in. Although JNa/in remained 50% lower at this time, counterbalancing reductions in Na+ outflux restored net Na+ balance. One-substrate ion-uptake kinetics analyses indicated that reduced ion influx after 8 h at pH 9.5 was caused by 50% decreases in Cl- and Na+ maximal transport rates (JCl/max, JNa/max), likely reflecting decreased numbers of functional transport sites. Two-substrate kinetic analyses indicated that reduced internal HCO3- and H+ supply for respective branchial Cl-/base and Na+/acid transport systems also contributed to lower JCl/in and, to a lesser extent, lower JNa/in at pH 9.5. Recovery in in of JCl/in after 3 d accounted for restoration of Cl- balance and max likely reflected increased numbers of transport sites. In contrast, JNa/in remained 33% lower after 3 d, but a lower affinity of the max gills for Na+ (fourfold greater KNa/m) accounted for the chronic m reduction in Na+ influx at pH 9.5. Thus, reestablishment of Cl- uptake capacity and counterbalancing reductions in Na+ outflux allows rainbow trout to reestablish net ion balance in alkaline waters

Effects of Chronic Waterborne Nickle Exposure on Two Successive Generations of \u3cem\u3eDaphnia Magna\u3c/em\u3e

In a 21-d chronic toxicity test in which an F0 generation of Daphnia magna were exposed to waterborne Ni, the noobservable-effect concentration (for survival, reproduction, and growth) was 42 μg Ni L-1, or 58% of the measured 21-d median lethal concentration (LC50) of 71.9 μg Ni L-1 (95% confidence interval, 56.5–95.0). Chronic exposure to 85 μg 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 μg 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 μg Ni L-1 (decreased glycogen and adenosine triphosphate [ATP]) and 21 μg 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 μg 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 μg 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 μg 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

Characterization of Freshwater Natural Dissolved Organic Matter (DOM): Mechanistic Explanations for Protective Effects Against Metaltoxicity and Direct Effects on Organisms

Dissolved organic matter (DOM) exerts direct and indirect influences on aquatic organisms. In order to better understand how DOM causes these effects, potentiometric titration was carried out for a wide range of autochthonous and terrigenous freshwater DOM isolates. The isolates were previously characterized by absorbance and fluorescence spectroscopy. Proton binding constants (pKa) were grouped into three classes:acidic (pKa ≤ 5), intermediate (5 \u3c pKa ≤ 8.5) and basic (pKa \u3e 8.5). Generally, the proton site densities (LT) showed maximum peaks at the acidic and basic ends around pKa values of 3.5 and 10, respectively. More variably positioned peaks occurred in the intermediate pKa range. The acid–base titrations revealed the dominance of carboxylic and phenolic ligands with a trend for more autochthonous sources to have higher total LT. A summary parameter, referred to as the Proton Binding Index (PBI), was introduced to summarize chemical reactivity of DOMs based on the data of pKa and LT. Then, the already published spectroscopic data were explored and the specific absorbance coefficient at 340 nm (i.e. SAC340), an index of DOM aromaticity,was found to exhibit a strong correlation with PBI. Thus, the tendencies observed in the literature that darker organic matter is more protective against metal toxicity and more effective in altering physiological processes in aquatic organisms can now be rationalized on a basis of chemical reactivity to protons

The Influence of Dissolved Organic Matter (DOM) on Sodium Regulation and Nitrogenous Waste Excretion in the Zebrafish (Danio rerio)

Dissolved organic matter (DOM) is both ubiquitous and diverse in composition in natural waters, but its effects on the branchial physiology of aquatic organisms have received little attention relative to other variables (e.g. pH, hardness, salinity, alkalinity). Here, we investigated the effects of four chemically distinct DOM isolates (three natural, one commercial, ranging from autochthonous to highly allochthonous, all at ∼6 mg C l−1) on the physiology of gill ionoregulation and nitrogenous waste excretion in zebrafish acclimated to either circumneutral (7.0–8.0) or acidic pH (5.0). Overall, lower pH tended to increase net branchial ammonia excretion, net K+ loss and [3H]PEG-4000 clearance rates (indicators of transcellular and paracellular permeability, respectively). However, unidirectional Na+ efflux, urea excretion and drinking rates were unaffected. DOM sources tended to stimulate unidirectional Na+ influx rate and exerted subtle effects on the concentration-dependent kinetics of Na+ uptake, increasing maximum transport capacity. All DOM sources reduced passive Na+ efflux rates regardless of pH, but exerted negligible effects on nitrogenous waste excretion, drinking rate, net K+ loss or [3H]PEG4000 clearance, so the mechanism of Na+ loss reduction remains unclear. Overall, these actions appear beneficial to ionoregulatory homeostasis in zebrafish, and some may be related to physicochemical properties of the DOM sources. They are very different from those seen in a recent parallel study on Daphnia magna using the same DOM isolates, indicating that DOM actions may be both species and DOM specific

Effects of Chronic Cd Exposure via the Diet or Water on Internal Organ-Specific Distribution and Subsequent Gill Cd Uptake Kinetics in Juvenile Rainbow Trout (\u3cem\u3eOncorhynchus mykiss\u3c/em\u3e)

New regulatory approaches to metal toxicity (e.g., biotic ligand model [BLM]) focus on gill metal binding and tissue specific accumulation of waterborne metals; the dietary route of exposure and dietary/waterborne interactions are not considered, nor are the consequences of chronic exposure by either route. Therefore, we studied the effect of the same gill Cd load (∼μ2.5 mg/g), achieved by a chronic, 30-d exposure to Cd either via the diet (1,500 mg/kg) or the water (2 μg/L), on tissue-specific Cd distribution and subsequent uptake of waterborne Cd in juvenile rainbow trout (Oncorhynchus mykiss). These two exposure regimes resulted in a branchial Cd load that had been taken up across either apical gill membranes (waterborne Cd) or basolateral gill membranes (through the bloodstream for dietary Cd). The BLM characteristics of the gills (i.e., short-term Cd uptake kinetics) were altered: affinity (log KCd-Gill [95% confidence level]) decreased from 7.05 (6.75–8.76) for control to 6.54 (6.32–7.03) for waterborne Cd and 5.92 (5.83–6.51) for dietary Cd, whereas binding capacity (Bmax) increased from 3.12 (2.14–4.09) to 4.80 (3.16–6.43) and 5.50 (2.86–8.17) nmol·g-1 for control, waterborne, and dietary Cd, respectively. Fish exposed to dietary Cd accumulated a much greater overall chronic Cd body burden relative to fish exposed to waterborne Cd or control fish. The carcass accumulated the greatest percentage of total body Cd in control and waterborne-exposed fish, whereas the intestinal tissue accumulated the greatest percentage in dietary-exposed fish. Tissue-specific Cd burdens were highest in the kidney in both dietary and waterborne treatments. We conclude that chronic Cd exposure alters Cd uptake dynamics, and that the route of Cd exposure, whether waterborne or dietary, results in differences of internal Cd accumulation and branchial Cd uptake characteristics. These factors should be considered in future BLM development

Protective Effects of Calcium Against Chronic Waterborne Cadmium Exposure to Juvenile Rainbow Trout

Juvenile rainbow trout (Oncorhynchus mykiss [Walbaum]) on 1% daily ration were exposed to 0 (control) or 2 μg of cadmium as Cd(NO3)2·4H2O per liter added to four different calcium (Ca) concentrations: 260 (background), 470 (low), 770 (medium), or 1200 (high) μM of Ca added as Cd(NO3)2·4H2O in synthetic soft water for 30 d. Mortality was highest (;80%) in the background 1 Cd treatment. Approximately 40% mortality was observed in the low 1 Cd exposure; mortality was 10% or less for all other treatments. No growth effects were seen for any of the exposures. Kidneys accumulated the greatest concentration of Cd during the 30 d, followed by gills and livers. Accumulation of Cd in gills, kidney, and liver decreased at higher water Ca concentrations. No differences in whole-body or plasma Ca concentrations were found. Swimming performance was impaired in the low + Cd-exposed fish. Influx of Ca2+ into whole bodies decreased as water Ca concentrations increased; influx of Ca2+ into background + Cd–treated fish was significantly reduced compared to that in control fish. Experiments that measured uptake of new Cd into gills showed that the affinity of gills for Cd (KCd-gill) and the number of binding sites for Cd decreased as water Ca concentrations increased. Acute accumulation of new Cd into gills and number of gill Cd-binding sites increased with chronic Cd exposure, whereas the affinity of gills for Cd decreased with chronic Cd exposure. Longer-term gill binding (72 h) showed reduced uptake of new Cd at higher water Ca levels and increased uptake with chronic Cd exposure. Complications were found in applying the biotic ligand model to fish that were chronically exposed to Cd because of discrepancies in the maximum number of gill Cd-binding sites among different studies

Physiological Effects of Chronic Copper Exposure to Rainbow Trout (\u3cem\u3eOncorhynchus Mykiss\u3c/em\u3e) in Hard and Soft Water: Evaluation of Chronic Indicators

Effects of chronic copper exposure on a suite of indicators were examined: acute toxicity, acclimation, growth, sprint performance, whole-body electrolytes, tissue residues, and gill copper binding characteristics. Juvenile rainbow trout were exposed for 30 d to waterborne copper in hard water (hardness = 120 μg/L as CaCO3, pH = 8.0, Cu = 20 and 60 μg/L) and soft water (hardness = 20 μg/L as CaCO3, pH = 7.2, Cu = 1 and 2 μg/L). Significant acclimation to the metal occurred only in fish exposed to 60 mg/L, as seen by an approx. twofold increase in 96-h LC50 (153 vs 91 μg Cu/L). Chronic copper exposure had little or no effect on survival, growth, or swimming performance in either water hardness, nor was there any initial whole-body electrolyte loss (Na+ and Cl-). The present data suggest that the availability of food (3% wet body weight/day, distributed as three 1% meals) prevented growth inhibition and initial ion losses that usually result from Cu exposure. Elevated metal burdens in the gills and livers of exposed fish were measures of chronic copper exposure but not of effect. Initial gill binding experiments revealed the necessity of using radiolabeled Cu (64Cu) to detect newly accumulated Cu against gill background levels. Using this method, we verified the presence of saturable Cu-binding sites in the gills of juvenile rainbow trout and were able to make estimates of copperbinding affinity (log Kgill=Cu) and capacity (Bmax). Furthermore, we showed that both chronic exposure to Cu and to low water calcium had important effects on the Cu-binding characteristics of the gills

Costs of Chronic Waterborne Zinc Exposure and the Consequences of Zinc Acclimation on the Gill/Zinc Interactions of Rainbow Trout in Hard and Soft Water

Juvenile rainbow trout were exposed to zinc in both moderately hard water (hardness 5 120 mg CaCO3/L, pH = 8.0, Zn = 150 μg/L or 450 μg/L) and soft water (hardness = 20 mg CaCO3/L, pH = 7.2, Zn = 50 μg/L or 120 μg/L) for 30 d. Only the 450 mg/L zinc–exposed fish experienced significant mortality (24% in the first 2 d). Zinc exposure caused no effect on growth rate, but growth affected tissue zinc levels. Whole body zinc levels were elevated, but gills and liver showed no consistent increases relative to controls over the 30-d. Therefore, tissue zinc residues were not a good indicator of chronic zinc exposure. After the 30-d exposure, physiological function tests were performed. Zinc was 5.4 times more toxic in soft water (control 96 h LC50s in hard and soft water were 869 μg/L and 162 μg/L, respectively). All zinc-exposed trout had acclimated to the metal, as seen by an increase in the LC50 of 2.2 to 3.9 times over that seen in control fish. Physiological costs related to acclimation appeared to be few. Zinc exposure had no effect on whole body Ca2+ or Na+ levels, on resting or routine metabolic rates, or on fixed velocity sprint performance. However, critical swimming speed (UCrit) was significantly reduced in zinc-exposed fish, an effect that persisted in zinc-free water. Using radioisotopic techniques to distinguish new zinc incorporation, the gills were found to possess two zinc pools: a fast turnover pool (T1/2 = 3–4 h) and a slow turnover pool (T1/2 = days to months). The fast pool was much larger in soft water than in hard water, but at most it accounted for \u3c3.5% of the zinc content of the gills. The size of the slow pool was unknown, but its loading rate was faster in soft water. Chronic zinc exposure was found to increase the size of the fast pool and to increase the loading rate of the slow pool

Physiological Responses to Acute Silver Exposure in the Freshwater Crayfish (\u3cem\u3eCambarus diogenes diogenes\u3c/em\u3e)—A Model Invertebrate?

Adult crayfish (Cambarus diogenes diogenes) exposed to 8.41 ± 0.17 μg silver/L (19.4% as Ag+) in moderately hard freshwater under flow-through conditions for 96 h exhibited ionoregulatory disturbance, elevated metabolic ammonia (Tamm) production and substantial silver accumulation in the gills, hemolymph, and hepatopancreas. The ionoregulatory disturbance included both a generally reduced unidirectional Na1 influx and an increased unidirectional Na+ efflux, leading to a substantial net loss of Na+ from the silver-exposed crayfish. The Na+ uptake in silver-exposed crayfish differed overall from controls, while the increased Na+ efflux recovered to control values 48 h into the 96 h of exposure. The general inhibition of Na+ uptake could be explained by a reduced sodium/potassium-adenosine triphosphatase (Na/K-ATPase) activity in terminally obtained gill samples from the silver exposed crayfish. The silver-induced effect on Na+ uptake and loss translated to reduced hemolymph Na+ concentrations but not significantly reduced hemolymph Cl- concentrations. Hemolymph Tamm and Tamm efflux both increased in silver-exposed crayfish, indicating an increased metabolic Tamm production. The present study demonstrates that the toxic mechanism of waterborne silver exposure in freshwater crayfish resembles that of freshwater teleost fish. The crayfish might therefore be a useful model system for extending current environmental regulatory strategies, currently based on teleost fish, to invertebrates

Physiological Effects of Five Different Marine Natural Organic Matters (NOMs) and Three Different Metals (Cu, Pb, Zn) on Early Life Stages of the Blue Mussel (Mytilus galloprovincialis)

Metals are present in aquatic environments as a result of natural and anthropogenic inputs, and may induce toxicity to organisms. One of the main factors that influence this toxicity in fresh water is natural organic matter (NOM) but all NOMs are not the same in this regard. In sea water, possible protection by marine NOMs is not well understood. Thus, our study isolated marine NOMs by solid-phase extraction from five different sites and characterized them by excitation-emission fluorescence analysis—one inshore (terrigenous origin), two offshore (autochthonous origin), and two intermediate in composition (indicative of a mixed origin). The physiological effects of these five NOMS alone (at 8 mg/L), of three metals alone (copper, lead and zinc at 6 µg Cu/L, 20 µg Pb/L, and 25 µg Zn/L respectively), and of each metal in combination with each NOM, were evaluated in 48-h exposures of mussel larvae. Endpoints were whole body Ca2++Mg2+-ATPase activity, carbonic anhydrase activity and lipid peroxidation. By themselves, NOMs increased lipid peroxidation, Ca2++Mg2+-ATPase, and/or carbonic anhydrase activities (significant in seven of 15 NOM-endpoint combinations), whereas metals by themselves did not affect the first two endpoints, but Cu and Pb increased carbonic anhydrase activities. In combination, the effects of NOMs predominated, with the metal exerting no additional effect in 33 out of 45 combinations. While NOM effects varied amongst different isolates, there was no clear pattern with respect to optical or chemical properties. When NOMs were treated as a single source by data averaging, NOM had no effect on Ca2++Mg2+-ATPase activity but markedly stimulated carbonic anhydrase activity and lipid peroxidation, and there were no additional effects of any metal. Our results indicate that marine NOMs may have direct effects on this model marine organism, as well as protective effects against metal toxicity, and the quality of marine NOMs may be an important factor in these actions