Silver bioaccumulation dynamics and modeling in an estuarine invertebrate following aqueous exposure

Transcription

1 Supporting Information Silver bioaccumulation dynamics and modeling in an estuarine invertebrate following aqueous exposure to nanosized and dissolved silver Farhan R. Khan, Superb K. Misra, Javier García-Alonso, Brian D. Smith, Stanislav Strekopytov, Philip S. Rainbow, Samuel N. Luoma,, and Eugenia Valsami-Jones Number of pages: 6 Number of figures: 4 Experimental animals Estuarine mud snails Peringia ulvae were collected from the Blackwater Estuary, Essex, UK ( N E) at low tide. Snails were transported back to the laboratory and kept in their natural sediment. Two weeks prior to experimentation snails were removed from sediments by sieving through a 1.8 mm mesh under running water. Snails were maintained in 3.75 L tanks with synthetic estuarine water (17 salinity) at 10 ± 1 C with an approximate 16 h light: 8 h dark cycle. Snails were regularly fed on diatoms (Chaetoceros mulleri, CCAP, Oban, UK). Water was changed every day for the first few days and every 2-3 days thereafter. Two days before the start of each exposure, 48 adult snails (>2 mm) were randomly grouped and food was withheld during this period. Alongside all dissolved Ag and Ag NP experiments a control set of exposures were conducted with no added Ag to ensure that there was no inadvertent contamination from experimental conditions. In addition a sample of snails was sacrificed prior to each experiment to measure the initial background Ag burden. The background concentration was subtracted from the measured Ag concentration in each experimental snail sample.

2 Sample preparation and analysis To analyse Ag concentrations in P. ulvae soft tissue, snails were soaked in 1 % HCl (Aristar grade, VWR, UK) overnight at room temperature to soften the shell (particularly around the operculum), rinsed in ultrapure water and returned to the freezer. The soft tissue of partially thawed P. ulvae was removed through the operculum with superfine forceps under a light dissection microscope. From each group of 48 individuals, eight snail soft tissues were pooled into six samples. The soft tissue samples were dried to constant weight at 80 C, weighed (mean weight of 3.73 ± 0.82 mg, n=774 across all experiments) and digested in 100 µl concentrated nitric acid (Aristar grade, VWR, UK) at 100 C. Samples were made up to 5 ml with 2% nitric acid and the Ag concentration was determined by ICP-MS (Varian 810). Procedural blanks (HNO 3 only digests) and certified reference material NIST-1566b (oyster tissue from the National Institute of Standards and Technology, US Department of Commerce) were routinely analysed alongside samples. In each sample an internal standard solution (In) was added at 1% of the final solution volume for matrix-related quality assurance. All Ag concentrations are expressed as nmol g -1 (dw).

3 Figure S1. Mean dry weights of P. ulvae samples during long term experiments. Each sample is the pooled weight of 8 individuals and each data-point represents the mean from 6 replicates (mg ± SD). Control (no added Ag, open squares), dissolved Ag (closed circles) and Ag NP (open circles) treatments are shown for the following experiments; (A) 16 d efflux following 24 h exposure at 556 nmol L -1, (B) 16 d efflux following 24 h exposure at 928 nmol L -1, (C) 24 d efflux following 5 d exposure at 556 nmol L -1, (D) 21-d exposure at 186 nmol L -1 and (E) 21-d exposure at 464 nmol L -1. Mean sample weights did not change significantly within each experiment (one-way ANOVA). This led to the exclusion of growth (g) as a factor in our biodynamic models (see section Biodynamic modeling and membrane transport characteristics in the main text).

4 Figure S2. Characterization of citrate-capped Ag NPs after synthesis. Imaged particles (A) were determined to have a mean size of 16.5 ± 4.5 nm (n=200) with the size range distributed close to the mean (B). UV-VIS showed no change in absorbance peak height or intensity at day 0 (after synthesis, solid line) and 7 days later (prior to Ag NP use, dashed line) indicating that there was no change in nanoparticle size (C).

5 Figure S3. DLS of hydrodynamic size distribution of Ag NPs expressed by intensity (%) in deionised water (red line), estuarine water (17 salinity, green line) and marine water (33 salinity, blue line). Higher salinities caused the hydrodynamic size of the particles to increase demonstrating aggregation. There is a large increase between size in deionised water and estuarine water, but the further increase to full strength seawater does not further increase the effective size of the particles. 10 In ten s ity ( % ) Size (d.nm)

6 Figure S4. Accumulated Ag (i.e. after removal of background concentrations) in the soft tissue of P. ulvae (nmol g -1 (dw) ± S.D., n=6) during 21 day exposures to dissolved Ag (A, closed symbols) and citrate-capped Ag NPs (B, open symbols). Snails were exposed at concentrations of 186 nmol L -1 (circles) or 464 nmol L -1 (squares).