Supporting Information Nanoplastic Affects Growth of S. obliquus and ...
Supporting Information
Nanoplastic Affects Growth of S. obliquus and Reproduction of D. magna
E. Besseling,†,‡,* B. Wang,† M. Lürling,† and A.A. Koelmans†,‡ †
Aquatic Ecology and Water Quality Management Group, Wageningen University, P.O. Box
47, 6700 AA Wageningen, The Netherlands ‡
IMARES – Institute for Marine Resources & Ecosystem Studies, Wageningen UR, P.O. Box
68, 1970 AB IJmuiden, The Netherlands
* Corresponding Author; e-mail: [email protected]
Page S2:
Details on the nano-PS concentrations in the bioassays
Page S3:
Pilot bioassay to assess sodium dodecyl sulphate (SDS) toxicity thresholds for Scenedesmus obliquus
Page S4:
Nile Red availability in the bioassays
Page S5:
Figure S1. TEM image of nano-PS
S1
DETAILS ON THE NANO-PS CONCENTRATIONS IN THE BIOASSAYS Scenedesmus bioassay. Scenedesmus obliquus were exposed to 44×10-1100 mg/L nano-PS, specifically being 4.4×101, 9.9×101, 2.2×102, 4.9×102 and 1.1×103 mg nano-PS/L. Daphnia bioassay. Pristine exposures were applied at ten microplastic concentrations in the range of 0.22 – 150 mg nano-PS/L, specifically being 2.2×10-1, 4.4×10-1, 8.8×10-1, 1.8, 3.5, 7.0, 1.4×101, 3.2×101, 7.0×101, 1.5×102 mg nano-PS/L. Pristine-kairomone exposures were at 8.8×10-1 and 1.8 mg nano-PS/L. The aged treatment contained 3.2×101 nano-PS/L and the aged-filtered treatment was pre-treated with 3.2×101 nano-PS/L, after which it was filtered as stated in the main paper.
S2
PILOT BIOASSAY TO ASSESS SODIUM DODECYL SULPHATE (SDS) TOXICITY THRESHOLDS FOR SCENEDESMUS OBLIQUUS Method. The test algae Scenedesmus obliquus SAG276/3a was obtained from the culture collection of SAG (Göttingen, Germany). It was grown in a 1 L chemostat on a modified WC medium in a temperature-controlled chamber at 20°C under continuous light (100 µmol/m2/s). Algae were collected from the chemostat and used in several assays run in 100 ml Erlenmeyer flasks containing 50 ml of medium. Flasks were incubated in a climate controlled chamber at 20 °C under continuous cool-fluorescent light at 175 µmol/m2/s for 48 h on a rotating shaking table (80 rpm). Algae were exposed to sodium dodecyl sulphate (SDS, Merck, Amsterdam, The Nederlands, 113760) at a log increase concentration range of 0.001-10 mg/L. A control without SDS was included and all treatments were performed in triplicate. Test dispersions were diluted 200×, after which cell densities and size distributions were determined by Coulter Multisizer II (Coulter Electronics, Luton, UK, capillary 100 µm orifice width). Growth rates (µ) of S. obliquus were predicted from the increase in biovolume (V) with the formula µ = (ln(Vend)-ln(Vstart)/dt, with t being the time in hours. Results. Growth rates were within a range of 1.3-1.5 day-1, and did not significantly differ among treatments (1-way ANOVA, p-value = 0.087). This implies SDS did not cause effects at least up to a concentration of 10 mg/L, which is 200 times higher than the maximum concentrations that occurred in the assays reported in the main paper.
S3
NILE RED AVAILABILITY IN THE BIOASSAYS Fluorescent dyes have been used before in living systems, without effects reported.1–8 In the nano-PS used in this bioassay, the fluorescent hydrophobic dye Nile Red was included in the polymer matrix for visualisation purposes. The nano-PS contained 0.01% on mass basis of Nile Red. Due to its hydrophobicity, Nile Red becomes practically completely incorporated in the polymer matrix during synthesis of the nano-PS. During the bioassay, leaching of Nile Red from the nano-PS is negligible. As the glass transition temperature of PS is ~100 °C,9 PS is a glassy polymer with extremely low intrapolymer diffusivities at the temperatures in the bioassay of respectively 20 °C and 21 °C. However, as Wu et al.10 report an effect of Nile Red on the endpoint chlorophyll fluorescence of the alga Botryococcus braunii at a total concentration of 1 mg/L, we calculated the maximum exposure to Nile Red in our bioassays. Even if all Nile Red would have been released from the nano-PS, the maximum total concentrations in respectively our algae and Daphnia bioassays would have been a factor 9 – 64 below the concentration reported by Wu et al. The previous calculation is worst case, assuming all Nile Red is in the water, which is not realistic. A more realistic maximum Nile Red exposure concentration for our bioassays can be estimated assuming release of Nile Red from the nano-PS only until thermodynamic partitioning equilibrium between nano-PS, water and algae has been established (equilibrium partitioning theory). Combination of: - the mass balance equation: CW = Mtot / (VW + knano-PS×Mnano-PS + kalgae×Malgae),11 in which CW is the aqueous Nile Red concentration in mg/L, Mtot the total Nile Red mass in mg, VW the water volume in the bioassay in L, Mnano-PS and Malgae the masses of respectively nanoPS and algae in the bioassay in kg and knano-PS and kalgae the partitioning coefficients of Nile Red to respectively nano-PS and algae in L/kg; - the partitioning relationships and coefficients logKOW = 5,8,12 knano-PS = 6.5 derived from the relationship between KOW and Kd of the nano-PS that was used in our bioassay13 and kalg ≈ KOW*flip, with flip being the lipid fraction of the algae of 0.075;14 - Nile Red, nano-PS and algal masses at the effect thresholds in the bioassays, yields an aqueous equilibrium concentration of 3.1×10-5 and 3.2×10-5 mg/L in the Daphnia and the algae bioassay, respectively. These concentrations are a factor 1.5×104 below the toxic aqueous concentration of 0.47 mg/L, which we calculated from the total concentration reported by Wu et al.10 based on partitioning between water and algae. Even taking uncertainty into account, by calculating the aqueous concentration with an initial total amount of Nile Red a hundred times as high as in our bioassay, the aqueous concentration would still be below 3.2×10-5 mg/L due to the high hydrophobicity of Nile Red. We therefore argue that the observed toxicity in our bioassay cannot be explained by the Nile Red concentration. Furthermore, as mentioned in the main manuscript, a radical increase in malformation occurrence was observed in the aged treatment, in contrast to the pristine treatment, which implies that these malformations were not due to any initially present cocontaminant like styrene, SDS or Nile Red.
S4
500 nm
Figure S1. The TEM image from Velzeboer et al. 2014 confirms the nominal size of ~70 nm of the primary nano-PS in freshwater.13
S5
References (1)
Cedervall, T.; Hansson, L.-A.; Lard, M.; Frohm, B.; Linse, S. Food chain transport of nanoparticles affects behaviour and fat metabolism in fish. PLoS One 2012, 7, e32254.
(2)
Cole, M.; Lindeque, P.; Halsband, C.; Galloway, T. S. Microplastics as contaminants in the marine environment: A review. Mar. Pollut. Bull. 2011, 62, 2588–2597.
(3)
Greenspan, P.; Mayer, E. P.; Fowler, S. D. Nile Red: A selective fluorescent stain for intracellular lipid droplets. J. Cell Biol. 1985, 100, 965–973.
(4)
Kashiwada, S. Distribution of Nanoparticles in the See-through Medaka (Oryzias latipes). Environ. Health Perspect. 2006, 114, 1697–1702.
(5)
Rao, J. P.; Geckeler, K. E. Polymer nanoparticles: Preparation techniques and sizecontrol parameters. Prog. Polym. Sci. 2011, 36, 887–913.
(6)
Rosenkranz, P.; Chaudhry, Q.; Stone, V.; Fernandes, T. F. A comparison of nanoparticle and fine particle uptake by Daphnia magna. Environ. Toxicol. Chem. 2009, 28, 2142–2149.
(7)
Rothen-Rutishauser, B.; Mühlfeld, C.; Blank, F.; Musso, C.; Gehr, P. Translocation of particles and inflammatory responses after exposure to fine particles and nanoparticles in an epithelial airway model. Part. Fibre Toxicol. 2007, 4, 1–9.
(8)
Küchler, S.; Abdel-Mottaleb, M.; Lamprecht, A.; Radowski, M. R.; Haag, R.; SchäferKorting, M. Influence of nanocarrier type and size on skin delivery of hydrophilic agents. Int. J. Pharm. 2009, 377, 169–172.
(9)
Rieger, J. The glass transition temperature of polystyrene - Results of a round robin test. J. Therm. Anal. 1996, 46, 965–972.
(10)
Wu, H.; Volponi, J. V; Oliver, A. E.; Parikh, A. N.; Simmons, B. A. Supporting Information - In vivo Lipidomics Using Single-Cell Raman Spectroscopy. PNAS 2011, 108, 3809–3814.
(11)
Koelmans, A. A.; Besseling, E.; Wegner, A.; Foekema, E. M. Plastic as a carrier of POPs to aquatic organisms. A model analysis. Environ. Sci. Technol. 2013, 47, 7812– 7820.
(12)
Wang, J. D.; Douville, N. J.; Takayama, S.; ElSayed, M. Quantitative analysis of molecular absorption into PDMS microfluidic channels. Ann. Biomed. Eng. 2012, 40, 1862–1873.
(13)
Velzeboer, I.; Kwadijk, C.; Koelmans, A. A. Supporting Information - Strong sorption of PCBs to nanoplastics, microplastics, carbon nanotubes and fullerenes. Environ. Sci. Technol. 2014, 48, 4869–4876.
S6
(14)
Lürling, M.; De Lange, H. J.; Van Donk, E. Changes in food quality of the green alga Scenedesmus induced by Daphnia infochemicals: biochemical composition and morphology. Freshw. Biol. 1997, 38, 619–628.
S7
Nanoplastic Affects Growth of S. obliquus and Reproduction of D. magna
E. Besseling,†,‡,* B. Wang,† M. Lürling,† and A.A. Koelmans†,‡ †
Aquatic Ecology and Water Quality Management Group, Wageningen University, P.O. Box
47, 6700 AA Wageningen, The Netherlands ‡
IMARES – Institute for Marine Resources & Ecosystem Studies, Wageningen UR, P.O. Box
68, 1970 AB IJmuiden, The Netherlands
* Corresponding Author; e-mail: [email protected]
Page S2:
Details on the nano-PS concentrations in the bioassays
Page S3:
Pilot bioassay to assess sodium dodecyl sulphate (SDS) toxicity thresholds for Scenedesmus obliquus
Page S4:
Nile Red availability in the bioassays
Page S5:
Figure S1. TEM image of nano-PS
S1
DETAILS ON THE NANO-PS CONCENTRATIONS IN THE BIOASSAYS Scenedesmus bioassay. Scenedesmus obliquus were exposed to 44×10-1100 mg/L nano-PS, specifically being 4.4×101, 9.9×101, 2.2×102, 4.9×102 and 1.1×103 mg nano-PS/L. Daphnia bioassay. Pristine exposures were applied at ten microplastic concentrations in the range of 0.22 – 150 mg nano-PS/L, specifically being 2.2×10-1, 4.4×10-1, 8.8×10-1, 1.8, 3.5, 7.0, 1.4×101, 3.2×101, 7.0×101, 1.5×102 mg nano-PS/L. Pristine-kairomone exposures were at 8.8×10-1 and 1.8 mg nano-PS/L. The aged treatment contained 3.2×101 nano-PS/L and the aged-filtered treatment was pre-treated with 3.2×101 nano-PS/L, after which it was filtered as stated in the main paper.
S2
PILOT BIOASSAY TO ASSESS SODIUM DODECYL SULPHATE (SDS) TOXICITY THRESHOLDS FOR SCENEDESMUS OBLIQUUS Method. The test algae Scenedesmus obliquus SAG276/3a was obtained from the culture collection of SAG (Göttingen, Germany). It was grown in a 1 L chemostat on a modified WC medium in a temperature-controlled chamber at 20°C under continuous light (100 µmol/m2/s). Algae were collected from the chemostat and used in several assays run in 100 ml Erlenmeyer flasks containing 50 ml of medium. Flasks were incubated in a climate controlled chamber at 20 °C under continuous cool-fluorescent light at 175 µmol/m2/s for 48 h on a rotating shaking table (80 rpm). Algae were exposed to sodium dodecyl sulphate (SDS, Merck, Amsterdam, The Nederlands, 113760) at a log increase concentration range of 0.001-10 mg/L. A control without SDS was included and all treatments were performed in triplicate. Test dispersions were diluted 200×, after which cell densities and size distributions were determined by Coulter Multisizer II (Coulter Electronics, Luton, UK, capillary 100 µm orifice width). Growth rates (µ) of S. obliquus were predicted from the increase in biovolume (V) with the formula µ = (ln(Vend)-ln(Vstart)/dt, with t being the time in hours. Results. Growth rates were within a range of 1.3-1.5 day-1, and did not significantly differ among treatments (1-way ANOVA, p-value = 0.087). This implies SDS did not cause effects at least up to a concentration of 10 mg/L, which is 200 times higher than the maximum concentrations that occurred in the assays reported in the main paper.
S3
NILE RED AVAILABILITY IN THE BIOASSAYS Fluorescent dyes have been used before in living systems, without effects reported.1–8 In the nano-PS used in this bioassay, the fluorescent hydrophobic dye Nile Red was included in the polymer matrix for visualisation purposes. The nano-PS contained 0.01% on mass basis of Nile Red. Due to its hydrophobicity, Nile Red becomes practically completely incorporated in the polymer matrix during synthesis of the nano-PS. During the bioassay, leaching of Nile Red from the nano-PS is negligible. As the glass transition temperature of PS is ~100 °C,9 PS is a glassy polymer with extremely low intrapolymer diffusivities at the temperatures in the bioassay of respectively 20 °C and 21 °C. However, as Wu et al.10 report an effect of Nile Red on the endpoint chlorophyll fluorescence of the alga Botryococcus braunii at a total concentration of 1 mg/L, we calculated the maximum exposure to Nile Red in our bioassays. Even if all Nile Red would have been released from the nano-PS, the maximum total concentrations in respectively our algae and Daphnia bioassays would have been a factor 9 – 64 below the concentration reported by Wu et al. The previous calculation is worst case, assuming all Nile Red is in the water, which is not realistic. A more realistic maximum Nile Red exposure concentration for our bioassays can be estimated assuming release of Nile Red from the nano-PS only until thermodynamic partitioning equilibrium between nano-PS, water and algae has been established (equilibrium partitioning theory). Combination of: - the mass balance equation: CW = Mtot / (VW + knano-PS×Mnano-PS + kalgae×Malgae),11 in which CW is the aqueous Nile Red concentration in mg/L, Mtot the total Nile Red mass in mg, VW the water volume in the bioassay in L, Mnano-PS and Malgae the masses of respectively nanoPS and algae in the bioassay in kg and knano-PS and kalgae the partitioning coefficients of Nile Red to respectively nano-PS and algae in L/kg; - the partitioning relationships and coefficients logKOW = 5,8,12 knano-PS = 6.5 derived from the relationship between KOW and Kd of the nano-PS that was used in our bioassay13 and kalg ≈ KOW*flip, with flip being the lipid fraction of the algae of 0.075;14 - Nile Red, nano-PS and algal masses at the effect thresholds in the bioassays, yields an aqueous equilibrium concentration of 3.1×10-5 and 3.2×10-5 mg/L in the Daphnia and the algae bioassay, respectively. These concentrations are a factor 1.5×104 below the toxic aqueous concentration of 0.47 mg/L, which we calculated from the total concentration reported by Wu et al.10 based on partitioning between water and algae. Even taking uncertainty into account, by calculating the aqueous concentration with an initial total amount of Nile Red a hundred times as high as in our bioassay, the aqueous concentration would still be below 3.2×10-5 mg/L due to the high hydrophobicity of Nile Red. We therefore argue that the observed toxicity in our bioassay cannot be explained by the Nile Red concentration. Furthermore, as mentioned in the main manuscript, a radical increase in malformation occurrence was observed in the aged treatment, in contrast to the pristine treatment, which implies that these malformations were not due to any initially present cocontaminant like styrene, SDS or Nile Red.
S4
500 nm
Figure S1. The TEM image from Velzeboer et al. 2014 confirms the nominal size of ~70 nm of the primary nano-PS in freshwater.13
S5
References (1)
Cedervall, T.; Hansson, L.-A.; Lard, M.; Frohm, B.; Linse, S. Food chain transport of nanoparticles affects behaviour and fat metabolism in fish. PLoS One 2012, 7, e32254.
(2)
Cole, M.; Lindeque, P.; Halsband, C.; Galloway, T. S. Microplastics as contaminants in the marine environment: A review. Mar. Pollut. Bull. 2011, 62, 2588–2597.
(3)
Greenspan, P.; Mayer, E. P.; Fowler, S. D. Nile Red: A selective fluorescent stain for intracellular lipid droplets. J. Cell Biol. 1985, 100, 965–973.
(4)
Kashiwada, S. Distribution of Nanoparticles in the See-through Medaka (Oryzias latipes). Environ. Health Perspect. 2006, 114, 1697–1702.
(5)
Rao, J. P.; Geckeler, K. E. Polymer nanoparticles: Preparation techniques and sizecontrol parameters. Prog. Polym. Sci. 2011, 36, 887–913.
(6)
Rosenkranz, P.; Chaudhry, Q.; Stone, V.; Fernandes, T. F. A comparison of nanoparticle and fine particle uptake by Daphnia magna. Environ. Toxicol. Chem. 2009, 28, 2142–2149.
(7)
Rothen-Rutishauser, B.; Mühlfeld, C.; Blank, F.; Musso, C.; Gehr, P. Translocation of particles and inflammatory responses after exposure to fine particles and nanoparticles in an epithelial airway model. Part. Fibre Toxicol. 2007, 4, 1–9.
(8)
Küchler, S.; Abdel-Mottaleb, M.; Lamprecht, A.; Radowski, M. R.; Haag, R.; SchäferKorting, M. Influence of nanocarrier type and size on skin delivery of hydrophilic agents. Int. J. Pharm. 2009, 377, 169–172.
(9)
Rieger, J. The glass transition temperature of polystyrene - Results of a round robin test. J. Therm. Anal. 1996, 46, 965–972.
(10)
Wu, H.; Volponi, J. V; Oliver, A. E.; Parikh, A. N.; Simmons, B. A. Supporting Information - In vivo Lipidomics Using Single-Cell Raman Spectroscopy. PNAS 2011, 108, 3809–3814.
(11)
Koelmans, A. A.; Besseling, E.; Wegner, A.; Foekema, E. M. Plastic as a carrier of POPs to aquatic organisms. A model analysis. Environ. Sci. Technol. 2013, 47, 7812– 7820.
(12)
Wang, J. D.; Douville, N. J.; Takayama, S.; ElSayed, M. Quantitative analysis of molecular absorption into PDMS microfluidic channels. Ann. Biomed. Eng. 2012, 40, 1862–1873.
(13)
Velzeboer, I.; Kwadijk, C.; Koelmans, A. A. Supporting Information - Strong sorption of PCBs to nanoplastics, microplastics, carbon nanotubes and fullerenes. Environ. Sci. Technol. 2014, 48, 4869–4876.
S6
(14)
Lürling, M.; De Lange, H. J.; Van Donk, E. Changes in food quality of the green alga Scenedesmus induced by Daphnia infochemicals: biochemical composition and morphology. Freshw. Biol. 1997, 38, 619–628.
S7
