D. Scott Smith

The two faces of DOC.

Dissolved organic carbon (DOC), through its ability to complex metals and thereby reduce their bioavailability, plays a major role in ameliorating metal toxicity in natural waters. Indeed DOC is a key variable in the Biotic Ligand Model (BLM) for predicting metal toxicity on a site-specific basis. However, recent evidence indicates that all DOCs are not alike, but rather heterogeneous in their ability to protect organisms against metal toxicity, at least in fresh water. The degree of protection appears to correlate with optical properties, such that dark, aromatic-rich compounds of allochthonous origin, with greater humic acid content, are more effective in this regard, particularly against Cu, Ag, and Pb toxicity. The specific absorption coefficient of the DOC in the 300-350nm range (SAC(300-350)) has proven to be a simple and effective index of this protective ability. PARAFAC, a multivariate statistical technique for analysis of excitation-emission fluorescence spectroscopy data, also holds promise for quantifying the humic-like and fulvic-like fluorophores, which tend to be positively and negatively correlated with protective ability, respectively. However, what has been largely missing in the toxicological realm is any appreciation that DOC may also affect the physiology of target organisms, such that part of the protection may occur by a mechanism other than metal complexation. Recently published evidence demonstrates that DOC has effects on Na(+) transport, diffusive permeability, and electrical properties of the gills in fish and crustaceans in a manner which will promote Na(+) homeostasis. These actions could thereby protect against metal toxicity by physiological mechanisms. Future research should investigate potential direct interactions of DOC molecules with the branchial epithelium. Incorporation of optical properties of DOC could be used to improve the predictive capabilities of the BLM.

Physicochemical and spectroscopic properties of natural organic matter (NOM) from various sources and implications for ameliorative effects on metal toxicity to aquatic biota

Natural organic matter (NOM), expressed as dissolved organic carbon (DOC in mgCL(-1)), is an ubiquitous complexing agent in natural waters, and is now recognized as an important factor mitigating waterborne metal toxicity. However, the magnitude of the protective effect, judged by toxicity measures (e.g. LC50), varies substantially among different NOM sources even for similar DOC concentrations, implying a potential role of NOM physicochemical properties or quality of NOM. This review summarizes some key quality parameters for NOM samples, obtained by reverse osmosis, and by using correlation analyses, investigates their contribution to ameliorating metal toxicity towards aquatic biota. At comparable and environmentally realistic DOC levels, molecular spectroscopic characteristics (specific absorbance coefficient, SAC, and fluorescence index, FI) as well as concentrations of fluorescent fractions obtained from mathematical mixture resolution techniques (PARAFAC), explain considerable variability in the protective effects. NOM quality clearly influences the toxicity of copper (Cu) and lead (Pb). NOM quality may also influence the toxicity of silver (Ag), cadmium (Cd) and inorganic mercury (Hg), but as yet insufficient data are available to unequivocally support the latter correlations between toxicity reduction and NOM quality predictors. Cu binding capacities, protein-to-carbohydrate ratio, and lipophilicity, show insignificant correlation to the amelioration offered by NOMs, but these conclusions are based on data for Norwegian NOMs with very narrow ranges for the latter two parameters. Certainly, various NOMs alleviate metal toxicity differentially and therefore their quality measures should be considered in addition to their quantity.

Effects of dissolved organic matter and reduced sulphur on copper bioavailability in coastal marine environments

Copper-induced toxicity in aqueous systems depends on its speciation and bioavailability. Natural organic matter (NOM) and reduced sulphur species can complex copper, influencing speciation and decreasing bioavailability. NOM composition in estuaries can vary, depending on inputs of terrigenous, autochthonous, or wastewater source material. At a molecular level, variability in NOM quality potentially results in different extents of copper binding. The aims of this study were to measure acute copper EC(50) values in coastal marine and estuarine waters, and identify the relationships between total dissolved copper EC(50) values and measured water chemistry parameters proportional to NOM and reduced sulphur composition. This has implications on the development of marine-specific toxicity prediction models. NOM was characterised using dissolved organic carbon (DOC) concentration and fluorescence measurements, combined with spectral resolution techniques, to quantify humic-, fulvic-, tryptophan-, and tyrosine-like fractions. Reduced sulphur was measured by the chromium-reducible sulphide (CRS) technique. Acute copper toxicity tests were performed on samples expressing extreme DOC, fluorescent terrigenous, autochthonous, and CRS concentrations. The results show significant differences in NOM quality, independent of DOC concentration. CRS is variable among the samples; concentrations ranging from 4 to 40 nM. The toxicity results suggest DOC as a very good predictive measure of copper EC(50) in estuaries (r(2)=0.87) independent of NOM quality. Furthermore, for filtered samples, CRS exists at concentrations that would be saturated with copper at measured EC(50), suggesting that while CRS might bind Cu and decrease bioavailability, it does not control copper speciation at toxicologically relevant concentrations and therefore is not a good predictive measure of copper toxicity in filtered samples.

Characterization of freshwater natural dissolved organic matter (DOM): Mechanistic explanations for protective effects against metal toxicity 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 (58.5). 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 340nm (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.

A comparison of the copper sensitivity of six invertebrate species in ambient salt water of varying dissolved organic matter concentrations

The copper sensitivity of four saltwater invertebrates (the mussel Mytilus galloprovincialis, the oyster Crassostrea virginica, the sand dollar Dendraster excentricus, and the sea urchin Strongylocentrotus purpuratus) was determined experimentally using chronic-estimator embryo-larval test procedures. The effect of sample dissolved organic matter (DOM) content on Cu bioavailability was determined for these species using commonly prescribed test procedures. Comparisons were made among these test results and test results reported previously for two other invertebrate species: the mussel Mytilus edulis and the copepod Eurytemora affinis. All six species exhibited a direct and significant relationship between the sample dissolved organic carbon (DOC; a surrogate measure of DOM) and either the dissolved Cu median lethal concentration (LC50) values or median effect concentration (EC50) values. This relationship is significant even when the DOM has different quality as evidenced by molecular fluorescence spectroscopy. Once normalized for the effects of DOM, the Cu sensitivity of these species from least to most sensitive were E. affinis < D. excitricus < C. virginica approximately S. purpuratus approximately M. edulis approximately M. galloprovincialis. This ranking of species sensitivity differs from the saltwater species sensitivity distribution proposed in 2003 by the U.S. Environmental Protection Agency. These results support the need to account for factors that modify Cu bioavailability in future saltwater Cu criteria development efforts. More specifically, Cu saltwater species sensitivity distribution data will need to be normalized by factors affecting Cu bioavailability to assure that accurate and protective criteria are subsequently developed for saltwater species and their uses.

Specific surface chemical interactions between hydrous ferric oxide and iron-reducing bacteria determined using pKa spectra

A modified regularized least squares pK(a) spectrum approach is applied to determine disassociation constants and proton binding site concentrations on bacteria, hydrous ferric oxide (HFO), and bacteria/HFO composite surfaces. This involves fitting experimental acid-base titration data to a continuous binding site model for a chemically heterogeneous surface with a variety of reactive groups yielding a pK(a) spectrum. The modified parameter fitting method optimizes simultaneously for both smoothness of the pK(a) spectrum and goodness of fit, whereas other methods optimize for goodness of fit given a fixed smoothness factor. Uncertainty estimates in pK(a) spectra were made by taking the mean and standard deviation of the spectra from replicate titration data. Titration of Shewanella putrefaciens strain CN32, a facultative iron-reducing bacterial species, demonstrate five types of binding sites consistent with known cell surface groups on bacteria, with mean pK(a) values of 3.62, 4.97, 6.92, 8.22, and 9.97. Composite surfaces formed by precipitation of HFO onto bacteria surfaces were also titrated. These surfaces no longer yielded low pK(a) sites in pK(a) spectra, indicating that ferric iron interacts with the bacteria via carboxylic (low pK(a)) sites during precipitation. In addition, mechanically mixed HFO bacterial samples also showed removal of carboxylic binding sites, suggesting that solid phase HFO interacts directly with carboxylic sites on bacterial cells. Moreover, the pK(a) spectra for HFO bacterial composites were not dependent on how the composite was formed; the mechanically mixed or surface-precipitated samples exhibited very similar binding site distributions. The determined pK(a) spectra imply that the overall binding mechanism for bacteria interactions with HFO involve carboxylic groups on the bacteria binding to the most basic sites on the HFO surface in approximately 1:1 stoichiometry.

Evaluating the Potential of Effluents and Wood Feedstocks from Pulp and Paper Mills in Brazil, Canada, and New Zealand to Affect Fish Reproduction: Chemical Profiling and In Vitro Assessments

This study investigates factors affecting reproduction in fish exposed to pulp and paper mill effluents by comparing effluents from countries with varying levels of documented effects. To explore the hypothesis of wood as a common source of endocrine disrupting compounds, feedstocks from each country were analyzed. Analyses included in vitro assays for androgenic activity (binding to goldfish testis androgen receptors), estrogenic activity (yeast estrogen screen), and neurotransmitter enzyme inhibition (monoamine oxidase and glutamic acid decarboxylase). Chemical analyses included conventional extractives, known androgens, and gas chromatograph index (GCI) profiles. All effluents and wood contained androgenic activity, particularly in nonpolar fractions, although known androgens were undetected. Effluents with low suspended solids, having undergone conventional biotreatment had lower androgenic activities. Estrogenic activity was only associated with Brazilian effluents and undetected in wood. All effluents and wood inhibited neurotransmitter enzymes, predominantly in polar fractions. Kraft elemental chlorine free mills were associated with the greatest neurotransmitter inhibition. Effluent and wood GCI profiles were correlated with androgenic activity and neurotransmitter enzyme inhibition. Differences in feedstock bioactivities were not reflected in effluents, implying mill factors mitigate bioactive wood components. No differences in bioactivities could be discerned on the basis of country of origin, thus we predict effluents in regions lacking monitoring would affect fish reproduction and therefore recommend implementing such programs.

Surface chemistry and relative Ni sorptive capacities of synthetic hydrous Mn oxyhydroxides under variable wetting and drying regimes

Abstract The surface binding site characteristics and Ni sorptive capacities of synthesized hydrous Mn oxyhydroxides experimentally conditioned to represent three hydrological conditions—MnOXW, freshly precipitated; MnOXD, dried at 37°C for 8 d; and MnOXC, cyclically hydrated and dehydrated (at 37°C) over a 24-h cycle for 7 d—were examined through particle size analysis, surface acid-base titrations and subsequent modelling of the pKa spectrum, and batch Ni sorption experiments at two pH values (2 and 5). Mineralogical bulk analyses by XRD indicate that all three treatments resulted in amorphous Mn oxyhydroxides; i.e., no substantial bulk crystalline phases were produced through drying. However, drying and repeated wetting and drying resulted in a non-reversible decrease in particle size. In contrast, total proton binding capacities determined by acid-base titrations were reversibly altered with drying and cyclically re-wetting and drying from 82 ± 5 μmol/m2 for the MnOXW to 21 ± 1 μmol/m2 for the MnOXD and 37 ± 5 μmol/m2 for the MnOXC. Total proton binding sites measured decreased by ≈75% with drying from the MnOXW and then increased to ≈50% of the MnOXW value in the MnOXC. Thus, despite a trend of higher surface area for the MnOXD, a lower total number of sites was observed, suggesting a coordinational change in the hydroxyl sites. Surface site characterization identified that changes also occurred in the types and densities of surface sites for each hydrologically conditioned Mn oxyhydroxide treatment (pH titration range of 2–10). Drying decreased the total number of sites as well as shifted the remaining sites to more acidic pKa values. Experimentally determined apparent pHzpc values decreased with drying, from 6.82 ± 0.06 for the MnOXW to 3.2 ± 0.3 for the MnOXD and increased again with rewetting to 5.05 ± 0.05 for the MnOXC. Higher Ni sorption was observed at pH 5 for all three Mn oxyhydroxide treatments compared to pH 2. However, changes in relative sorptive capacities among the three treatments were observed for pH 2 that are not explainable simply as a function of total binding site density or apparent pHzpc values. These results are the first to our knowledge, to quantitatively link the changes induced by hydrologic variability for surface acid base characteristics and metal sorption patterns. Further, these results likely extend to other amorphous minerals, such as Fe oxyhydroxides, which are commonly important geochemical solids for metal scavenging in natural environments.

Effects of using synthetic sea salts when measuring and modeling copper toxicity in saltwater toxicity tests

Synthetic sea salts are often used to adjust the salinity of effluent, ambient, and laboratory water samples to perform toxicity tests with marine and estuarine species. The U.S. Environmental Protection Agency (U.S. EPA) provides guidance on salinity adjustment in its saltwater test guidelines. The U.S. EPA suggests using commercial sea salt brands, such as Forty Fathoms (now named Crystal Sea Marinemix, Bioassay Grade), HW Marinemix, or equivalent salts to adjust sample salinity. Toxicity testing laboratories in Canada and the United States were surveyed to determine synthetic sea salt brand preference. The laboratories (n = 27) reported using four brands: Crystal Sea Marinemix (56%), HW Marinemix (22%), Instant Ocean (11%), and Tropic Marin (11%). Saline solutions (30 g/L) of seven synthetic sea salts were analyzed for dissolved copper and dissolved organic carbon (DOC) content. Brands included those listed above plus modified general-purpose salt (modified GP2), Kent Marine, and Red Sea Salt. The synthetic sea salts added from < 0.1 to 1.2 microg Cu/L to the solution. Solutions of Crystal Sea Marinemix had significantly elevated concentrations of DOC (range = 5.4-6.4 mg C/L, analysis of variance, Tukey, alpha = 0.05, p < 0.001) while other brands generally contained < 1.0 mg C/L. The elevated DOC in Crystal Sea Marinemix was expected to reduce copper toxicity. However, the measured dissolved copper effective concentration 50% (EC50) for Crystal Sea Marinemix was 9.7 microg Cu/L, similar to other tested sea salts. Analysis indicates that the organic matter in Crystal Sea Marinemix differs considerably from that of natural organic matter. On the basis of consistently adding little DOC and little dissolved copper, GP2 and Kent Marine are the best salts to use.

Dissolved organic carbon from the upper Rio Negro protects zebrafish ( Danio rerio ) against ionoregulatory disturbances caused by low pH exposure

The so-called “blackwaters” of the Amazonian Rio Negro are rich in highly coloured dissolved organic carbon (DOC), but ion-poor and very acidic, conditions that would cause fatal ionoregulatory failure in most fish. However these blackwaters support 8% of the world’s ichthyofauna. We tested the hypothesis that native DOC provides protection against ionoregulatory dysfunction in this extreme environment. DOCs were isolated by reverse-osmosis from two Rio Negro sites. Physico-chemical characterization clearly indicated a terrigenous origin, with a high proportion of hydroxyl and phenolic sites, high chemical reactivity to protons, and unusual proteinaceous fluorescence. When tested using zebrafish (a model organism), Rio Negro DOC provided almost perfect protection against ionoregulatory disturbances associated with acute exposure to pH 4.0 in ion-poor water. DOC reduced diffusive losses of Na+ and Cl−, and promoted a remarkable stimulation of Na+ uptake that otherwise would have been completely inhibited. Additionally, prior acclimation to DOC at neutral pH reduced rates of branchial Na+ turnover, and provided similar protection against acid-induced ionoregulatory disturbances, even if the DOC was no longer present. These results reinforce the important roles that DOC molecules can play in the regulation of gill functions in freshwater fish, particularly in ion-poor, acidic blackwaters.