Supporting information. Toxicity of silver nanoparticles to Chlamydomonas reinhardtii
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1 0 0 0 Supporting information Toxicity of silver nanoparticles to Chlamydomonas reinhardtii Enrique Navarro,, Flavio Piccapietra, Bettina Wagner, Fabio Marconi, Ralf Kägi, Niksa Odzak, Laura Sigg and Renata Behra *. Eawag, Swiss Federal Institute of Aquatic Science and Technology, Überlandstrasse, P.O. Box, 00 Dübendorf, Switzerland. Instituto Pirenaico de Ecología (CSIC), Avda. Montañana 00, Apdo. 0, 000 Zaragoza, Spain Summary Effects of cysteine on the photosynthetic yield of C. reinhardtii (Figure S) page S Effects of ph on the size of AgNP (Figure S) page S Stability constants for Ag + concentration calculations (Table S) page S Effect of cysteine on the AgNP size (Figure S) page S DGT measurements of Ag in AgNP suspensions page S DGT-labile Ag from AgNP (Table S) page S Ratio of DGT-labile Ag to total Ag concentration (Table S) page S Measurements of dissolved Ag by centrifugal ultrafiltration (Table S) page S Measurements of Ag + by ISE in AgNP suspensions page S Calibration of silver ion-selective electrode (Figure S) page S Measured potential in diluted AgNP suspensions (Figure S) page S S
2 Effects of cysteine on the photosynthetic yield of C. reinhardtii 0 00 % Photosynthetic yield Cysteine (nmol L - ) Figure S. The effect of cysteine on photosynthetic yield was assessed after hour of exposure. Photosynthetic yield was not affected by the cysteine in the nm range (Dunnet test, MS = 0., df =.0). S
3 Effects of ph on the size of AgNP ( "M) AgNP diameter (nm) MOPS Nanopure water Acetate buffer 0 0 Figure S. AgNP diameter (nm) as a function of the ph in MOPS, acetate buffer or nanopure water. Aggregation was ph-dependent. Larger aggregates were formed at lower ph. The aggregation of AgNP results from reduction in absolute values of the Zeta potential (mv values are indicated on the symbols; error bars are smaller than symbols). Stability constants for Ag + concentration calculations Reaction log K Reference Ag + + Cys - + H + AgCysH. Ag + + Cys - + H + - Ag(CysH). Ag + + H O AgOH + H + - Ag + + H O Ag(OH) - + H + - Cys - + H + CysH - 0. Cys - + H + CysH. Cys - + H + + CysH 0. ()Adams, N. W. H.; Kramer, J. R. Potentiometric determination of silver thiolate formation constants using a Ag S electrode. Aquat. Geochem.,, -. () Martell, A. E.; Smith, R. M.; NIST Standard Reference Database, 00. Table S. Stability constants used to calculate the Ag + concentrations at equilibrium with cysteine. Constants for AgCysH and Ag(CysH) are calculated from () after J. Kramer (personal communication, 00). ph S
4 Effect of cysteine on the AgNP size 00 After 0. hours After hours AgNP diameter (nm) Cysteine (nmol L - ) Figure S. To assess whether AgNP were affected by cysteine, AgNP were exposed to 0.,, 0, 00 and 000 nm cysteine, and their size measured upon 0. and hours in 0 mm MOPS, ph.. The Kruskal-Wallis ANOVA on ranks performed at 0. (H (,0) =., p=0.) and hours (H (,) =0.0, p=0.) revealed no significant effect of cysteine concentrations on the size of AgNP. Error bars represent the SD of two samples (the value of every sample is the average of three measurements). Results obtained in absence of cysteine shows that particles display no significant increase in size with time, under conditions as those used for the toxicity experiments. S
5 0 0 0 DGT measurements of Ag in AgNP suspensions DGT performance to measure Ag + under controlled conditions (ph, T, ionic strength), was tested. Five different Ag + concentrations were prepared from stock solution using 0.0 M NaNO in L PP-Beakers and DGT devices deployed for hours at ºC, ph. and at a shake rate of 00 r min -. The results showed that DGT measured 00 % of total Ag available in the solutions with a very good correlation between ICP-MS and DGT measurements (r 0.). AgNP exposed to light (exposed to sunlight at the lab) and dark conditions for days in artificial water solution at constant temperature of ºC and ph. tend to agglomerate and adsorb onto the beakers walls. Total Ag concentration dropped by around 0 % in the beakers not exposed to light and more (around 0 %) in the beakers exposed to light (Tab. S, S). Conditions Average Ag + Maximum Ag + Total Ag Start Total Ag End Dark 0. ( 0.) 0..0 (.). ( 0.) Light 0. ( 0.0) 0.. (.).0 (.) Table S. DGT-labile Ag (mostly Ag + ) from AgNP exposed to light and dark conditions for days in synthetic medium at constant temperature of C and ph.. Maximum Ag + release values found among replicates are also shown. Errors represent standard deviation of four replicates. Conditions Ag + /Total Ag Start % Ag + /Total Ag End % Total Ag End/ Start % Average Dark Average Light Maximum Ag Table S. Ratio of DGT-labile Ag to total Ag concentration at the beginning and at the end of -day experiment Measurements of dissolved Ag by centrifugal ultrafiltration Factor Treatments Ag in filtrate % of total Ag 0. Time (h) Cysteine (nm) Algae, cells ml x 0 0. Table S. Influence of time, cysteine and algae on the dissolved Ag concentration in the AgNP suspensions ( MM in 0 mm MOPS, ph.) as percentage of the total silver. Silver in the filtrate was related to the Ag before centrifugation, as determined by ICP-MS. S
6 Measurements of Ag + by ISE in AgNP suspensions Measured potential [mv] y =.x +. R = log concentration Ag+ [M] Figure S: Calibration of silver ion-selective electrode in the range log [Ag + ] = -./-, with various added chloride concentrations for the range log [Ag + ] = -./ Potentilal (mv) AgNP log concentration (M) Figure S: Measured potential in diluted AgNP suspensions (in 0 mm MOPS, ph.). S
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