The geometry of the toxicity of silver nanoparticles to freshwater mussels.

The question about the influence of the geometry of silver nanoparticle (nAg) towards toxicity in aquatic organisms is largely unanswered. The purpose of this study was to examine if different geometries of nAg could initiate biophysical stress in the soft tissues of mussels. Freshwater Dreissenna bugensis mussels were exposed for 48 h at 15 °C to 10 and 50 µg/L of ionic Ag and to 3 forms of polyvinylpyrrolidone (PVP)-coated nAg of similar size: sphere, cube and prism. At the end of the exposure period, mussels were allowed to depurate overnight and the post-mitochondrial fraction of the soft tissues were analyzed for the levels of liquid crystals (LCs), changes in the activity and fractal dimensions of pyruvate kinase-lactate dehydrogenase (PK-LDH), F-actin and protein-ubiquitin (UB) levels. The data revealed that exposure to nAg forms lead to increased formation of LCs in increasing order of intensity: prismatic > cubic > spherical nAg. The activity in PK-LDH was decreased by all forms of nAg but not by ionic Ag+ (as with the following effects). Fractal kinetics of the PK-LDH system revealed that the nAg forms increased the spectral dimension (sD) in increasing order: spherical > cubic > prismatic nAg. A decrease in the fractal diffusion rate (fDR) with small changes in the fractal dimension (fD) was also obtained. The levels of F-actin and protein-UB were significantly affected for most forms of nAg and followed a pattern similar to LCs levels. In conclusion, the geometry of nAg could influence the formation of LCs, alter the fractal kinetics of the PK-LDH system, F-actin levels and protein damage in the soft tissues of freshwater mussels.

The influence of surface coatings on the toxicity of silver nanoparticle in rainbow trout.

Silver nanoparticles (nAg) are often produced with different coatings that could influence bioavailability and toxicity in aquatic organisms. The purpose of this study was to examine the influence of 4 surface coatings of nAg of the same core size towards bioavailability and toxicity in juvenile rainbow trout (Oncorhynchus mykiss). Juveniles were exposed to 50 µg/L of 50 nm diameter nAg for 96 h at 15 °C with the following coatings: branched polyethylenimine (bPEI), citrate, polyvinylpyrrolidone (PVP) and silicate (Si). The data revealed that the coatings influenced hepatic Ag loadings in the following trend PVP > citrate > bPEI and Si with estimated bioavailability factors of 28, 18, 6 and 2 L/kg respectively. Hepatic Ag levels were significantly associated with DNA damage and inflammation as determined by arachidonate cyclooxygenase activity. The bPEI and citrate-coated nAg consistently produced the observed effects above in addition to increased mitochondrial electron transport activity and glutathione S-transferase activity. The absence of metallothionein and lipid peroxidation suggests that mechanisms other than the liberation of Ag+ were at play. In conclusion, surface coatings were shown to significantly influence bioavailability and toxic properties of nAg to rainbow trout juveniles.

Effects of inhaled nano-TiO2 aerosols showing two distinct agglomeration states on rat lungs.

Nano-aerosols composed of large agglomerates (LA) (>100nm) are more likely to promote pulmonary clearance via macrophages phagocytosis. Small agglomerates (SA) (<100nm) seem to escape this first defense mechanism and are more likely to interact directly with biological material. These different mechanisms can influence pulmonary toxicity. This hypothesis was evaluated by comparing the relative pulmonary toxicity induced by aerosolized nano-TiO(2) showing two different agglomeration states: SA (<100nm) and LA (>100nm) at mass concentrations of 2 or 7mg/m(3). Groups of Fisher 344 male rats were nose-only exposed for 6h. The median number aerodynamic diameters were 30 and 185nm at 2mg/m(3), and 31 and 194nm at 7mg/m(3). We found in rat's bronchoalveolar lavage fluids (BALF) a significant 2.1-fold increase in the number of neutrophils (p<0.05) in the group exposed to the 7mg/m(3) LA nano-aerosol suggesting a mild inflammatory response. Rats exposed to the 7mg/m(3) SA nano-aerosol showed a 1.8-fold increase in LDH activity and 8-isoprostane concentration in BALF, providing evidence for cytotoxic and oxidative stress effects. Our results indicate that biological responses to nanoparticles (NP) might depend on the dimension and concentration of NP agglomerates.

Transcriptomic signatures in Chlamydomonas reinhardtii as Cd biomarkers in metal mixtures.

In the natural environment, toxicant effects can be monitored by the signature mRNA expression patterns of genes that they generate in test organisms. The specificity and sensitivity of these transcriptome-based bioassays to a given toxicant can be confounded by temporal changes in biomarker mRNA expression, effects of other toxicants and hardness ions, and non-linear mRNA expression responses of genes. This study provides the foundation for the development of a transcriptomic-based bioassay for bioavailable Cd in the freshwater alga, Chlamydomonas reinhardtii. It characterizes: (1) the Cd regulation of nine genes with respect to their mRNA induction kinetics; (2) the effects of two additional metals common to freshwaters, Cu2+ and Pb2+, and (3) the relationships between metal bioaccumulation and the transcriptomic responses. Quantitative real time PCR was used to monitor mRNA levels of nine Cd-induced genes following an exposure to 0.01, 0.11 and 1.16 µM Cd2+. Several distinct mRNA expression patterns were observed with time. While the presence of Cu2+ and Pb2+ decreased Cd biouptake, mRNA levels increased for six genes, showing lack of Cd2+ specificity. Nonetheless, the transcriptomic effects of binary metal exposures rarely adhered to a simple additive model based on single metal exposures; rather most exhibited synergistic or antagonistic interactions. While none of these genes could be used as a specific Cd biomarker, the signature mRNA expression profile obtained from a select subset of Cd sensitive genes was a useful biomarker of sublethal effects.

Diffusion coefficients of several rhodamine derivatives as determined by pulsed field gradient-nuclear magnetic resonance and fluorescence correlation spectroscopy.

Rhodamine derivatives are popular, photostable fluorophores that are used in a number of fluorescent based techniques, including fluorescence correlation spectroscopy (FCS). Indeed, in FCS, both rhodamine 6G (R6G) and rhodamine 110 (R110) are used as calibration standards to determine the dimensions of the instrument confocal volume. In spite of a requirement for precise values of the diffusion coefficients, literature values are scarce and vary over an order of magnitude. In this paper, the diffusion coefficients of four rhodamine fluorophores (rhodamine 6G (R6G), rhodamine B (RB), rhodamine 123 (R123), rhodamine 110 (R110)) were determined by pulsed field gradient nuclear magnetic resonance (PFG-NMR) spectrometry and then validated by comparison with fluorescence correlation spectroscopy. With the objective of validating the FCS calibration, diffusion coefficients of several dextrans and a polystyrene nanoparticle were also determined and compared with literature values or theoretical values that were based upon the Stoke-Einstein equation. The work presented here lead us to conclude that the diffusion coefficients for R6G and R110 have generally been underestimated in the literature. We propose revised values of 4.4x10(-10) m2 s(-1) for R110 and 4.0x10(-10) m2 s(-1) for R6G. Using the revised D value for R110 to calibrate the FCS instrument, diffusion coefficients have then been systematically determined for different conditions of pH, ionic strength and concentration. To correct for differences due to solvent effects (D2O vs. H2O), an isotopic correction factor, DD2O/DH2O of 1.23, was determined from both FCS and from the solvent auto-diffusion coefficients obtained by NMR.

Bioavailability of trace metals to aquatic microorganisms: importance of chemical, biological and physical processes on biouptake.

An important challenge in environmental biogeochemistry is the determination of the bioavailability of toxic and essential trace compounds in natural media. For trace metals, it is now clear that chemical speciation must be taken into account when predicting bioavailability. Over the past 20 years, equilibrium models (free ion activity model (FIAM), biotic ligand model (BLM)) have been increasingly developed to describe metal bioavailability in environmental systems, despite the fact that environmental systems are always dynamic and rarely at equilibrium. In these simple (relatively successful) models, any reduction in the available, reactive species of the metal due to competition, complexation or other reactions will reduce metal bioaccumulation and thus biological effects. Recently, it has become clear that biological, physical and chemical reactions occurring in the immediate proximity of the biological surface also play an important role in controlling trace metal bioavailability through shifts in the limiting biouptake fluxes. Indeed, for microorganisms, examples of biological (transport across membrane), chemical (dissociation kinetics of metal complexes) and physical (diffusion) limitation can be demonstrated. Furthermore, the organism can employ a number of biological internalization strategies to get around limitations that are imposed on it by the physicochemistry of the medium. The use of a single transport site by several metals or the use of several transport sites by a single metal further complicates the prediction of uptake or effects using the simple chemical models. Finally, once inside the microorganism the cell is able to employ a large number of strategies including complexation, compartmentalization, efflux or the production of extracellular ligands to minimize or optimize the reactivity of the metal. The prediction of trace metal bioavailability will thus require multidisciplinary advances in our understanding of the reactions occurring at and near the biological interface. By taking into account medium constraints and biological adaptability, future bioavailability modeling will certainly become more robust.

Cd bioaccumulation by a freshwater bacterium, Rhodospirillum rubrum.

Cd bioaccumulation by Rhodospirillum rubrum, a Gram-negative freshwater bacterium, was studied in a synthetic medium. The free ion (Cd2+) was the best predictor of the Cd internalization fluxes. Representation of the short-term uptake fluxes as a function of [Cd2+] in the medium demonstrated a linear relationship, as would be expected for a rate-limiting, first-order internalization with a single transporter. Nonetheless, several different accumulation profiles were observed, depending on the Cd concentration. Cd uptake was regulated differently for concentrations above and below 10(-6) M (or was regulated only above [Cd2+] = 10(-6) M). Short-and long-term studies revealed that regulation was rapidly initiated for the highest Cd concentrations examined, effectively decreasing both adsorbed and internalized Cd. Anodic stripping voltammetry demonstrated that a Cd complexing ligand was produced within minutes upon exposure to 5 x 10(-6) M Cd2+ and that an extracellular sequestration of Cd was one mechanism regulating Cd uptake. Competition studies with other cations revealed a competitive inhibition of Cd uptake by Zn and an uptake enhancement in the presence of Mn and Cu.

Diffusion coefficients of humic substances in agarose gel and in water.

Measurements of the diffusion coefficients of five different humic substances (HS) have been performed in water and in agarose hydrogels at several pH values (in the range of 3-10) and gel concentrations (in the range of 0.7-3% w/w). Fluorescence correlation spectroscopy (FCS) and classical diffusion cells were used in parallel to probe diffusion over both microscopic and mesoscopic distance scales. In general, agreement between the techniques was reasonable, which indicated that local nonhomogenities in the gel did not play an important role. Diffusion coefficients (D) in the gel were generally in the range of 0.9-2.5 x 10(-10) m2 s(-1) but were generally only 10-20% lower than in solution. At low pH values, one of the studied humic substances (a peat humic acid, PPHA) formed large aggregates that could not penetrate into the gel and therefore could not be defined by a single D value. The observed decreases of D in the gel for other HS were too large to be explained by the tortuousity and obstructive effects of the gel alone. D decreased slightly with increasing gel concentration and increased slightly with pH. Because modifications of D due to pH were similar in both the gel and the free solution, it is unlikely that complexation with the gel was greatly influenced by the pH. Rather, the main effect that appeared to decrease the diffusive flux in gels was likely small increases in the hydrodynamic radii of the humic macromolecules. An anomalous diffusion model was used to describe the FCS data in the gel. The characteristic exponent determined by fitting the autocorrelation functions with this model decreased only slightly (from 0.96 to 0.90) with increasing gel concentration providing support that HS complexation with the gel fibers was not very important. The results have important implications for our understanding of the fate and behavior of the HS and their associated pollutants and for interpreting metal speciation data obtained using gel-covered analytical sensors.

Determination of electrophoretic mobilities and hydrodynamic radii of three humic substances as a function of pH and ionic strength.

Capillary electrophoresis (CE) and fluorescence correlation spectroscopy (FCS) were employed to determine electrophoretic mobilities and hydrodynamic sizes of three humic substances (IHSS aquatic fulvic acid (FA), IHSS aquatic humic acid (HA), and IHSS peat humic acid (PHA)) as a function of pH and ionic strength. A slight aggregation corresponding to the formation of dimers and trimers was observed at low pH using fluorescence correlation spectroscopy (FCS). For example, for the peat humic acid, diffusion coefficients decreased from 2.1 x 10(-10) m2 s(-1) at pH 4 to 2.4 x 10(-10) m2 s(-1) at pH 11. For all three humic substances, electrophoretic mobilities were also shown to decrease significantly below pH 6. Calculated zeta potentials observed at high pH of -69 mV (FA), -62 mV (HA), and -63 mV (PHA) decreased to -39, -50, and -47 mV, respectively, under slightly acidic pH (4.5-4.8) conditions. No evidence of ionic strength induced aggregation was found using fluorescence correlation spectroscopy (FCS); diffusion coefficients increased slightly (<25%) with increasing ionic strength (up to 1 M). Negative electrophoretic mobilities decreased to a maximum measured ionic strength of 0.18 M. Above this ionic strength, no peaks were observed due to an increased HS adsorption to the capillary wall and an important decrease in electroosmotic flow. Interpretation of electrophoretic mobilities determined by CE is complicated by the fact that under certain conditions, HS appeared to be complexed by CE buffer systems, including MES, BES, and AMPSO.

Single molecule study of xanthan conformation using atomic force microscopy.

Conformations of individual macromolecules of the biopolymer xanthan were investigated using atomic force microscopy (AFM). Xanthan from very dilute solutions (1 ppm) was allowed to adsorb onto freshly cleaved mica and examined using tapping mode AFM under ambient conditions. The secondary structure of xanthan was probed by heating the polymer and gradually cooling, which denatured and renatured the polymer. When salt was present, renatured xanthan formed a double helical structure, consistent with the structure of native xanthan. In pure water, renaturation was not complete as what appeared to be single helical structures were observed. The number-average contour length (L(n)) of the polymer in its single helical state was 1651 nm. In the double helical state, induced by the addition of salt, L(n) decreased to 450 nm (in 0.5 M KCl). The chains also became less rigid as salt was added. The persistence length decreased from 417 nm in pure water to approximately 150 nm in 0.1 or 0.5 M KCl. This indicated a trend toward more flexible molecules when salt was present. Calculations of end-to-end distances based on equilibrium and projected conformations confirmed that the xanthan chain conformation on the mica surface was at equilibrium and was therefore representative of the conformation of xanthan in solution. The single-molecule AFM technique eliminates one common bias of solution techniques, which is the determination of an average signal between aggregates and dissolved molecules. It is thus a useful complement to solution-based methods for determining physical-chemical properties of biopolymers.