Nano- and Micro-Plastics Preferentially Drive

Home , Emiliania huxleyi, Thalassiosira pseudonana

1 Cell size matters: nano- and micro-plastics preferentially drive

2 declines of large marine phytoplankton due to co-aggregation 3 4 Craig J. Dedman1, Joseph A. Christie-Oleza,1,2 Víctor Fernández-Juárez,2 Pedro Echeveste3,4 5 6 1 School of Life Sciences, Gibbet Hill Campus, University of Warwick, Coventry, CV4 7AL, United 7 Kingdom. 8 2 Department of Biology, University of the Balearic Islands, Ctra. Valldemossa, km 7.5. CP: 07122, 9 Palma, Spain. 10 3 Instituto de Ciencias Naturales Alexander von Humboldt, Universidad de Antofagasta, Antofagasta, 11 Chile 12 4 Instituto Milenio de Oceanografía, Concepción, Chile 13 14 15 Supplementary Information 16 17 SI.1 Flow cytometric analysis of natural phytoplankton communities. 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Figure SI.1. Gating of phytoplankton groups utilised for determination of respective cell 40 densities in response to nano- and microplastics exposure. Light green: 41 Picocyanobacteria; Dark green: Picoeukaryotes; Blue: Nanophytoplankton; Red: 42 reference beads used for calculation of cell density. 43 44 45 46 SI.2 72 h phytoplankton growth 47 48 Table SI.1. Summary of data obtained following 72 h exposures of phytoplankton to nano- (NPs) and 49 micro- plastic (MPs) particles, or copper (Cu). 50 *Significant alteration in cell density between treated and untreated cultures (two-way T-test, p<0.05). Cell % Change in % Change in EC50 Species Volume cell density cell density Cu (µm3) NPs MPs Prochlorococcus sp. MED4 0.9 -4.42% +112.05%* >0.05 Synechococcus sp. WH7803 3.1 +23.88% +0.41% >0.05 Ostreococcus tauri OTH95 9.2 +1.98% -6.43% 0.70 ± 0.04 Micromonas sp. CMP2709 33.5 -32.12% -41.67% 0.82 ± 0.57 Phaeodactylum tricornutum +67.68%* -29.80% 50.0 3.96 ± 2.82 CCMP2561 Thalassiosira pseudonana -69.68% -75.19% 268.1 6.65 ± 5.43 CCMP1335 Emiliania huxleyi CCMP 1516 523.6 -95.06%* -36.97% 2.69 ± 0.37 51 52

Figure SI.2. Cell density of phytoplankton grown in the presence of nanoplastics (NPs) or microplastics (MPs) added at a concentration of 0.001% w/v for a period of 72 h. A: Prochlorococcus sp. MED4 (Pro MED4); B: Synechococcus sp. 7803 (SYN 7803); C: Ostreococcus tauri OTH95 (Ostreo); D: Micromonas sp. CMP2709 (Micro); E: Phaeodactylum tricornutum CCMP2561 (Phaeo); F: Thalassiosira pseudonana CCMP1335 (Thal); G: Emiliania huxleyi CCMP1516 (E. hux). Data is presented as the mean ± standard deviation of triplicate samples. Stars indicate where two-way T- tests identified cell density to significantly vary between the untreated control and treated cultures at the 72 h timepoint (p≤0.05). 53 SI.3 Shotgun proteomic analysis supporting information. 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 Figure SI.3. Volcano plots (T-test; FDR=0.05, S0=0.1) of the cellular proteome of Emiliania 69 huxleyi exposed to A) nano- and B) microplastics (0.001% w/v). Red markers indicate proteins identified as significantly altering in abundance between control and treated samples (two-way 70 t-test, p≤0.05) 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 Figure SI.4. Relative abundance of protein groups identified in the cellular proteome of Emiliania 97 huxleyi exposed to nano- (NP) or microplastics (MP) at a concentration of 0.001% w/v. Markers 98 indicate where relative abundance of individual protein groups varies significantly from the 99 untreated control, as identified by two-way T-tests (p≤0.05). 100 101 102 SI.4 Emiliania huxleyi cell density following 72 h exposure to plastics during imaging 103 experiment. 104 105 106

Figure SI.5. Cell density of Emiliania huxleyi following 72 h exposure to nano- (NPs) or microplastics (MPs) at a concentration of 0.001% w/v (n=3). Data is presented as the mean ± standard deviation.