Does water chemistry affect the dietary uptake and toxicity of silver nanoparticles by the freshwater snail Lymnaea stagnalis?

Silver nanoparticles (AgNPs) are widely used in many applications and likely released into the aquatic environment. There is increasing evidence that Ag is efficiently delivered to aquatic organisms from AgNPs after aqueous and dietary exposures. Accumulation of AgNPs through the diet can damage digestion and adversely affect growth. It is well recognized that aspects of water quality, such as hardness, affect the bioavailability and toxicity of waterborne Ag. However, the influence of water chemistry on the bioavailability and toxicity of dietborne AgNPs to aquatic invertebrates is largely unknown. Here we characterize for the first time the effects of water hardness and humic acids on the bioaccumulation and toxicity of AgNPs coated with polyvinyl pyrrolidone (PVP) to the freshwater snail Lymnaea stagnalis after dietary exposures. Our results indicate that bioaccumulation and toxicity of Ag from PVP-AgNPs ingested with food are not affected by water hardness and by humic acids, although both could affect interactions with the biological membrane and trigger nanoparticle transformations. Snails efficiently assimilated Ag from the PVP-AgNPs mixed with diatoms (Ag assimilation efficiencies ranged from 82 to 93%). Rate constants of Ag uptake from food were similar across the entire range of water hardness and humic acid concentrations. These results suggest that correcting regulations for water quality could be irrelevant and ineffective where dietary exposure is important.

Induction of reactive oxygen species in chlamydomonas reinhardtii in response to contrasting trace metal exposures.

The toxicity of metals to organisms is, in-part, related to the formation of reactive oxygen species (ROS) in cells and subsequent oxidative stress. ROS are by-products of normal respiration and photosynthesis processes in organisms, but environmental factors, like metal exposure, can stimulate excess production. Metals involved in several different mechanisms such as Haber-Weiss cycling and Fenton-type reactions can produce ROS. Some metals, such as Cd, may contribute to oxidative stress indirectly by depleting cellular antioxidants. We investigated the measurement of ROS as a sensitive biomarker of metal toxicity (that could possibly be implemented in a biotic ligand model for algae) and we compared ROS induction in response to several contrasting transition metals (Cu, V, Ni, Zn, and Cd). We also compared the ROS response to glutathione and growth toxicity endpoints measured in a previous study. The cell-permeable dye, 2'7'dichlorodihydrofluorescein diacetate, was used as a probe to detect formation of ROS in Chlamydomonas reinhardtii cells. Metal-exposed cells were incubated with the fluorescent dye in a 96-well plate and monitored over 5.5 h. A dose-response of ROS formation was observed with Cu exposure in the range of 20-500 nM. Cu produced more ROS compared with either Zn or Cd (both nonredox active metals). The redox-active metal V produced increased ROS with increased concentration. The measurement of ROS may be a useful indicator of Cu toxicity, but the signal to noise ratio was better for the glutathione endpoint assay.

Relationships between surface-bound and internalized copper and cadmium and toxicity in Chlamydomonas reinhardtii.

In the present study, the adsorption and uptake of copper (Cu) and cadmium (Cd) in Chlamydomonas reinhardtii were examined to establish fundamental toxicity relationships to glutathione and cell-growth endpoints. Establishing these fundamental relationships of metal accumulation and toxicity metrics is necessary to subsequently implement an algal biotic ligand model. The glutathione response was similar to the response measured from growth endpoints for both internal and adsorbed Cu, indicating that glutathione may be a useful biomarker of toxicity. The glutathione response with Cd contrasted markedly with that observed with Cu and was therefore observed to be a metal-specific biomarker. The density of sites binding metals and the related stability constants for the algal cell surface were also determined. Short exposures to metals (2 h) were conducted, and we determined 6.0 × 10(-6) mol/g sites binding Cu and 2.0 × 10(-6) mol/g sites binding Cd and conditional stability constants as log K' = 7.2 and log K' = 6.7 for Cu and Cd, respectively. Experiments were also conducted to determine the effect on toxicity endpoints of varying nitrate concentrations and different humic acids (HA) in the exposure media. Varying nitrate concentrations did not have an effect on cell growth over 24 h. The surface-adsorbed Cu measurements from the experiments with HA depended on the type and concentration of HA.

Differential effects of copper and cadmium exposure on toxicity endpoints and gene expression in Chlamydomonas reinhardtii.

The toxicity of cadmium to aquatic organisms is well known, but the mechanisms of toxicity are not as clearly understood. In the present study, Cd bioassay experiments incorporating both traditional endpoints and novel thiol-based endpoints were conducted with Chlamydomonas reinhardtii. The results were compared with results from previous bioassay experiments to probe the apparent contrasting biochemical mechanisms of toxicity of copper and cadmium as expressed in cellular glutathione and the glutathione cycle. Total glutathione and reduced to oxidized glutathione ratio (GSH/GSSG) measurements were remarkably different in Cd- compared with Cu-exposed cells. Whereas total glutathione in cells decreased with increasing Cu concentration, Cd caused dramatic increases. Total glutathione increased by 4.5-fold with 80 nM Cd treatment over concentrations in Cd-free controls. Glutathione reductase (GR) enzyme activity was positively correlated (r(2) (Cu) = 0.96, r(2) (Cd) = 0.85) with glutathione concentrations for both metals. Measurements of mRNA for GR were increased 2-fold in response to Cd exposure (80 nM) and correlated well with GR enzyme activity. Glutathione concentrations and GR enzyme activity are useful endpoints for both Cu and Cd toxicity in algae, even though the metals elicit opposing responses. We conclude that Cu decreases glutathione concentrations by inhibiting GR enzyme activity. In contrast, Cd stimulates GR enzyme activity and increases glutathione concentrations as cells respond to Cd-induced stress by producing increased antioxidant capacity. The present study demonstrates that determining the glutathione response in cells is important for understanding the metal-specific mechanisms of toxicity and that these associated novel endpoints may be useful metrics for accurately predicting toxicity.

Analysis of glutathione endpoints for measuring copper stress in Chlamydomonas reinhardtii.

Glutathione (GSH) is the most abundant nonprotein thiol in eukaryotic cells and it protects cells by functioning as an antioxidant and a metal-binding ligand. Because glutathione readily undergoes oxidation-reduction reactions to combat oxidative stress, intracellular ratios of the reduced (GSH) to the oxidized (GSSG) forms of glutathione may serve as an important biomarker of exposure and effect of trace metals in eukaryotic cells. We compared sensitivity of glutathione ratios in the freshwater alga Chlamydomonas reinhardtii to the traditional endpoints of cell growth rates and chlorophyll a following exposure to Cu for periods of 6 and 24 h. A response of the GSH:GSSG ratio to Cu concentration was observed at Cu levels of 40 and 80 nM after exposure for both 6 and 24 h. The concentration of total GSH at 24 h was roughly half the value at 6 h after exposure to either 40 or 80 nM Cu. A response for cell growth rate was observed only at 24 h, whereby the average specific growth rate decreased from about 1.1 to 0.4 d(-1). The total Cu concentrations eliciting a cell response of 50%, effect concentrations (EC50s), after 24 h of exposure were similar (49.2, 49.8, and 38.2 nM Cu) and not significantly different for GSH:GSSG ratio, GSH levels, and specific growth, respectively. Total cell-associated Cu concentrations after exposure for 24 h were calculated from the EC50 endpoints and ranged from 13.3 to 17.0 fg/cell. Overall, thiol ratios were indicative of toxicity resulting from exposure to Cu, but precision may be greater for the cell growth rate endpoints.