Influence of Phytoplankton on Fate and Effects of Modified Zerovalent Iron Nanoparticles

Nanoscale zerovalent iron (nZVI) and its derivatives hold promise for remediation of several pollutants but their environmental implications are not completely clear. In this study, the physicochemical properties and aggregation kinetics of sulfide/silica-modified nZVI (FeSSi) were compared in algal media in which <i>Chlamydomonas reinhardtii</i> had been cultured for 1, 2, or 11 days in order to elicit the effects of organic matter produced by the freshwater algae. Furthermore, transformation of FeSSi particles were investigated in <i>C. reinhardtii</i> cultures in exponential (1-d) and slowing growth (11-d) phases while monitoring the response of algae. We found evidence for steric stabilization of FeSSi by algal organic matter, which led to a decrease in the particles’ attachment efficiency. Transformation of FeSSi was slower in 11-d cultures as determined via inductively coupled plasma and X-ray analyses. High concentrations of FeSSi caused a lag in algal growth, and reduction in steady state population size, especially in cultures in exponential phase. The different outcomes are well described by a dynamic model describing algal growth, organic carbon production, and FeSSi transformations. This study shows that feedback from algae may play important roles in the environmental implications of engineered nanomaterials

Schematic of dynamic model of environmental feedback.

<p>Phytoplankton grow and produce DOC, which inactivates AgNPs and silver ions (Ag⁺). Both active and inactive AgNPs dissolve, introducing Ag⁺ into the environment. Environmental Ag⁺ is either made inaccessible to phytoplankton (inactivated Ag⁺) or bioaccumulated by the phytoplankton (entering the Ag⁺ quota). The bioaccumulated Ag⁺ and the still active AgNPs exert toxic effects on the phytoplankton.</p

Model predictions with inactivation mechanisms of DOC on AgNP and Ag⁺ separately <i>and</i> in unison.

<p>Model simulations demonstrate the significance of DOC inactivation of AgNPs <i>and</i> Ag⁺ (red lines). The simulations suggest that DOC mitigation of nanotoxicity provides a much stronger feedback than mitigation of ionic toxicity: while the model without ionic mitigation (black lines) generally follows the observations and only predicts the second dip slightly sooner, the model without AgNP inactivation (blue lines) radically departs from the observations, with the population going extinct by day three of the exposure.</p

3.5 µg/L Ag⁺ has little to no effect on late stages of batch culture growth.

<p>3.5 µg/L Ag⁺ in the form of AgNO₃ was introduced to batch cultures in the same way as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074456#pone-0074456-g001" target="_blank">Figure 1</a>. This concentration caused initial toxicity to cultures in fast growth phase, but these cultures were able to recover to the level of the control cultures in fast growth phase. This concentration had no visible effect on cultures in slowing and stationary growth phases. The data points are averages from three replicate cultures and the error bars reflect their standard error. The lines just connect the data points and help differentiate between culture in fast (solid), slowing (large dashes), and stationary (short dashes) growth phases.</p

Simple dynamic model describing the feedbacks.

<p>Subscripts used are N:silver nanoparticles; I: ionic silver; U: non-bioavailable (inactivated) silver. In the balance equations, denotes the rate of transformation of silver in state <i>n</i> to state <i>m</i>. Parameters are defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074456#pone-0074456-t002" target="_blank">Table 2</a>. Note: the notation [<i>x</i>]₊ means use the value of x if it is positive, otherwise set to zero.</p

Citrate-AgNPs are more toxic to cultures in earlier stages of growth than in later stages and our dynamic model captures the algal dynamics.

<p>5/L citrate-coated AgNPs were introduced to <i>C. reinhardii</i> batch cultures at three different stages of growth: fast growth (solid lines), slowing growth (large dashes), and stationary growth (small dashes). The dynamic model developed through analyses of these data captures the algal dynamics well with a single parameter set (lines). Batch cultures in slowing and stationary growth phases had grown for one and two weeks, respectively, prior to the start of the experiment and before the introduction of AgNPs. Time in this figure is represented as the absolute day of growth of the culture – all cultures were exposed on the same day of the experiment but on different days of growth (cultures in fast growth phase were dosed with AgNPs on day 1 of growth, cultures in slowing growth phase were dosed on day 6 of growth, and cultures in stationary growth phase were dosed on day 13 of growth). AgNPs caused complete mortality of cultures in fast growth phase within two days of introduction. For these cultures, chlorophyll measurements were below detectable limits (denoted by x) by day 3 but the culture was sampled through day 6. We measured concentrations of chlorophyll a to indicate algal cell viability and response to AgNPs because we empirically confirmed that chlorophyll a/cell ratios remain constant after day 5 of growth in algal cultures grown in the light and temperature environments used in this experiment. However, AgNPs had an initial toxic effect on cultures in slowing and stationary growth phases from which the cultures were able to recover until they declined again on days 8 and 10 of the experiment (days 13 and 15 of growth for cultures in slowing phase and days 20 and 22 of growth for cultures in stationary growth phase). The data points are averages from three replicate cultures and the error bars reflect their standard error.</p

Initial conditions and parameter values for the model fits shown in Figures 1 and 5.

<p>Parameters were estimated from the growth curves of the control cultures. Parameters and were calculated from measured DOC values. Other parameters were minimizing the residual sum of squares between model output and chlorophyll a data for all three treatments simultaneously.</p

Removal of DOC from algal cultures in stationary growth restores the toxicity of AgNPs.

<p>AgNPs cause complete mortality of cells in stationary growth (red) after removal of organic material and resuspension of algal cells in media without nutrients. Control cultures (green), which were also centrifuged and resuspended in media without nutrients, persisted for at least 5 days. This pattern of toxicity is very similar to the rate of decline of cultures in fast growth phase exposed to 5 mg/L AgNP (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074456#pone-0074456-g001" target="_blank">Figure 1</a>). This finding indicates that the difference in toxicity observed between growth stages of the algae is most likely due to differences in the extracellular environments of the growth stages. The data points are averages from three replicate cultures and the error bars reflect their standard error.</p