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

Speract induces calcium oscillations in the sperm tail

Sea urchin sperm motility is modulated by sperm-activating peptides. One such peptide, speract, induces changes in intracellular free calcium concentration ([Ca2+]i). High resolution imaging of single sperm reveals that speract-induced changes in [Ca2+]i have a complex spatiotemporal structure. [Ca2+]i increases arise in the tail as periodic oscillations; [Ca2+]i increases in the sperm head lag those in the tail and appear to result from the summation of the tail signal transduction events. The period depends on speract concentration. Infrequent spontaneous [Ca2+]i transients were also seen in the tail of unstimulated sperm, again with the head lagging the tail. Speract-induced fluctuations were sensitive to membrane potential and calcium channel blockers, and were potentiated by niflumic acid, an anion channel blocker. 3-isobutyl-1-methylxanthine, which potentiates the cGMP/cAMP-signaling pathways, abolished the [Ca2+]i fluctuations in the tail, leading to a very delayed and sustained [Ca2+]i increase in the head. These data point to a model in which a messenger generated periodically in the tail diffuses to the head. Sperm are highly polarized cells. Our results indicate that a clear understanding of the link between [Ca2+]i and sperm motility will only be gained by analysis of [Ca2+]i signals at the level of the single sperm

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