Supporting InformationCombined effects of polystyrene microplastics and natural organic matter on the

accumulation and toxicity of copper in zebrafishRuxia Qiao, Kai Lu, Yongfeng Deng, Hongqiang Ren, Yan Zhang*

State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu 210023, ChinaCorresponding author:Yan Zhang (yanzhang@nju.edu.cn)

This Supporting Information contains:Page S2: Characteristics of microplastics (MPs) used in this study. Figure S1Page S3: Aggregation of MPs. Figure S2Page S4: Influence of NOM on the accumulation of MPs in zebrafish tissues (liver,

gut, and gill). Figure S3Page S5: Influence of MPs and NOM on the accumulation of Cu in zebrafish tissues

(gill, gut, and liver). Figure S4Page S6: Fold change of differentially expressed genes (DEGs) in different treated

groups compared with the control groups. Figure S5Page S7: The mRNA expression of mt2 in zebrafish lives in different groups. Figure S6Page S8: Fluorescence values of digestion liquid of 5 mgC/L NOM, deionized water

and zebrafish tissue in control group. Table S1Page S9: Gene-specific primer sequences for RT-qPCR. Table S2Page S10: Pseudo-second-order kinetic parameters of adsorption kinetics for Cu

adsorption no MPs. Table S3Page S11: Freundlich Isotherm Parameters of Adsorption isotherms for Cu adsorption

no MPs, Table S4Page S12: Distribution of differentially expressed genes (DEGs) only shared by both

Cu + SMPs group and Cu + SMPs (N) group over different GO terms, Table S5

Page S14: RT-qPCR validation of gene expression detected by RNA-Seq analysis in the Cu+SMPs(N) group. Table S6

S1

Figure S1. Characteristics of microplastics (MPs) used in this study. (A) Morphological characterizations of microplastics detected by fluorescence microscope. (B) Morphological characterizations of microplastics detected by scanning electron microscope (C) FTIR spectroscopy characterizations of pristine microplastics.

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Figure S2. Aggregation of MPs. (A) Aggregation of 100 nm MPs detected by DLS. (B) Aggregation of 20 μm MPs detected by UV-vis spectrophotometer.

The sedimentation of 20 μm MPs was determined by time-resolved optical absorbency using a UV-vis spectrophotometer (Shimadzu UV-2700, Japan) at 260 nm. Optical absorbency was measured every 1 min for 260 min.When the microplastics aggregate and settle down in water, the solution optical absorbance declined rapidly. Optical absorbance changed with time was associated with the normalized microplastics concentration C/C0 (i.e. the absorbance measured with time / the initial absorbance at time 0), where C is the concentration in time, and C0 is the initial concentration. The sedimentation rate is then d (C/C0)/dt.

S3

Figure S3. Influence of NOM on the accumulation of MPs in zebrafish tissues (liver, gut, and gill) after exposed for 72 h. *, p < 0.05. ND, not detected.

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Figure S4. Influence of MPs and NOM on the accumulation of Cu in zebrafish tissues (gill, gut, and liver) after exposed for 72 h. Different letters over the bars indicate significant differences among groups (p < 0.05).

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Figure S5. Fold change of differentially expressed genes (DEGs) of zebrafish liver in different treated groups compared with the control groups.

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Figure S6. The mRNA expression of mt2 in zebrafish lives in different groups (*, p < 0.05).

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Table S1. Fluorescence values of digestion liquid of 5 mgC/L NOM, deionized water and zebrafish tissue in control group (excitation and emission wavelengths of 418 and 518 nm).Digestion liquid type Fluorescence values5 mgC/L NOM 0.21 ± 0.03Deionized water 0.42 ± 0.08Liver 0.64 ± 0.01Gut 1.11 ± 0.08Gill 1.72 ± 0.06

S8

Table S2. Gene-specific primer sequences for RT-qPCR.Gene symbol Primer sequence (5’ to 3’) Product length (bp)Loxa F: GGCGATGATGAAGTGATGGTTG 199

R: CGTAGGTGGATGCTTGGATGTCox4i1l F: TCTGGATTCTGGAGGAGACTGT 161

R: TTCTTCTGTTCGGCACTGAGGMat2ab F: TGTTGGACGCTCACCTGAAG 133

R: TCTCGGACCACCTTCTGATAGTAbca12 F: AGTCCGTCCAGATGCTCAATG 102

R: CTAACTGCTGCTGCTGCTCTAAbcb5 F: CTGTGCTCTTCTCATTGCTGTG 344

R: CGGCGTTGACCAAGTAAGGAKif20bb F: ACGGACAGATGAGGAGGAATTG 173

R: GCTGCTGCCTGGTATGTTCTRad52 F: ACGGCACTTCTTGGCAACA 179

R: GCCTCCATCTCCTGTCTGAACLmx1ba F: ACTCTCAGAGACTCGGACAAGA 114

R: CGCTGCTCTCCATAGATACCAAMiox F: TGGCATTCAAACGGCGACTA 115

R: CTGGTAGTTCAGTGCTCTTGGTDhrs7cb F: CAACAGCAGCATGAAGGTGAAA 198

R: TAGCAGGTTCTGAACGGAAGAGβ-Actin F: TTGTGATGGACTCTGGTGATGG 388

R: TTGAAGGTGGTCTCGTGGATACNote: Ten differentially expressed genes due to Cu and microplastics exposure were selected. RT-qPCR analysis was conducted on the same total RNA samples used for RNA-seq. First-strand cDNA synthesis and RT-qPCR were performed using a Rever Tra Ace qPCR RT Kit (Toyobo, Osaka, Japan) and SYBRGreen PCR kit (Toyobo, Osaka, Japan), respectively. The procedures were in accordance with the manufacturer’s instruction. β-Actin was selected as an internal reference for normalization.

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Table S3. Pseudo-second-order kinetic parameters of adsorption kinetics for Cu adsorption on MPs.Type of MPs IR (mg·g-1·d-1) k (g·mg-1·d-1) qe (mg·g-1) R2

SMPs 16.55 0.05 18.19 0.95LMPs 16.44 0.09 13.27 0.99

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Table S4. Freundlich isotherm parameters of adsorption isotherms for Cu adsorption no MPs.Type of MPs Kf 1/n R2

SMPs 9.85 0.81 0.73LMPs 8.47 0.59 0.83

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Table S5. Distribution of differentially expressed genes (DEGs) only shared by both Cu + SMPs group and Cu + SMPs (N) group over different GO terms.GO ID GO term Symbol Log2Fold Change

Cu+SMPs Cu+SMPs(N)Metal ion transportGO:0046872 Metal ion binding Lmx1ba 2.52 ± 1.47 3.03 ± 1.16

Zfand5b 2.29 ± 0.39 1.02 ± 0.18Nrp2a 1.99 ± 0.27 1.51 ± 0.45Tshz2 1.12 ± 0.26 1.28 ± 0.19Lima1a 1.06 ± 0.50 1.07 ± 0.63Fynb -1.07 ± 0.52 -1.05 ± 0.80Pparg -1.26 ± 1.12 -1.10 ± 0.35Miox -1.53 ± 1.18 -1.31 ± 0.63Si:dkeyp-84f3.9 -1.61 ± 0.31 -1.18 ± 0.07Lhx9 -1.68 ± 0.39 -1.93 ± 0.89Dhh -2.14 ± 2.24 -1.34 ± 0.74Si:ch211-261d7.6

-2.33 ± 1.36 -1.45 ± 0.99

Ftr34 -2.39 ± 1.26 -1.35 ± 0.81Si:ch73-144d13.7

-2.48 ± 1.44 -2.47 ± 1.45

Ppm1lb -2.55 ± 2.13 -1.47 ± 0.43Cyp7a1 -3.22 ± 0.86 -1.73 ± 1.08

GO:0055085 Transmembrane transport

Trpm5 3.37 ± 1.29 2.86 ± 0.67Slc25a38b -1.26 ± 0.67 -1.02 ± 0.30Abcc12 -1.45 ± 0.37 -1.42 ± 0.32Zgc:114041 -1.56 ± 0.51 -1.80 ± 0.60Slc5a6a -1.98 ± 1.09 -1.16 ± 0.29

DNA repairGO:0003677 DNA binding Xrcc2 3.20 ± 0.10 2.61 ± 1.77

Lmx1ba 2.52 ± 1.47 3.03 ± 1.16Zfand5b 2.29 ± 0.39 1.02 ± 0.18Si:dkey-23a13.22

1.78 ± 1.12 1.76 ± 0.80

Si:ch211-15d5.12

1.21 ± 0.61 1.12 ± 0.43

E2f8 1.21 ± 0.50 1.12 ± 1.37Etv2 1.14 ± 0.70 1.01 ± 0.18Tshz2 1.13 ± 0.26 1.28 ± 0.19Pparg -1.26 ± 1.12 -1.10 ± 0.35Lhx9 -1.68 ± 0.39 -1.93 ± 0.89

Cell cycleGO:0007264 Small GTPase Rab3db 1.60 ± 0.80 2.16 ± 0.85

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mediated signal transduction

Rnd2 1.18 ± 0.33 1.29 ± 0.11Rnd1b -1.69 ± 0.49 -1.18 ± 0.47Rapgef3 -1.81 ± 0.25 -1.10 ± 0.60

Oxidative stressGO:0055114 Oxidation-

reduction processZgc:154075 1.76 ± 0.19 1.19 ± 0.17Miox -1.53 ± 1.18 -1.31 ± 0.63Cyp7a1 -3.22 ± 0.86 -1.73 ± 1.08

OthersGO:0042981 Regulation of

apoptotic processZgc:153759 1.44 ± 0.40 1.10 ± 0.20Fynb -1.07 ± 0.52 -1.05 ± 0.80Inhbab -1.39 ± 0.44 -1.22 ± 0.61

GO:0008289 Lipid binding Star 2.32 ± 0.62 3.00 ± 0.99Apoeb 1.32 ± 0.55 1.08 ± 0.33Snx10a 1.91 ± 0.60 1.12 ± 0.58

GO:0008083 Growth factor activity

Gdf7 4.30 ± 0.93 2.86 ± 1.00Inhbab -1.40 ± 0.44 -1.22 ± 0.61Igf2b -1.82 ± 0.25 -1.22 ± 0.22

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Table S6. RT-qPCR validation of gene expression detected by RNA-Seq analysis in the Cu+SMPs(N) group.Symbol Gene name RNA-Seq analysis (n=3) RT-qPCR (n=3)Loxa Lysyl oxidase a 2.86 ± 0.91 1.85 ± 1.93Cox4i1l Cytochrome c oxidase

subunit IV isoform 1, like

1.95 ± 0.44 1.61 ± 0.55

Mat2ab Methionine adenosyltransferase II, alpha b

-3.80 ± 0.20 -1.72 ± 0.01

Abca12 ATP-binding cassette, sub-family A (ABC1), member 12

2.65 ± 0.31 1.43 ± 1.12

Abcb5 ATP-binding cassette, sub-family B (MDR/TAP), member 5

2.27 ± 1.80 3.33 ± 5.69

Kif20bb Kinesin family member 20Bb

3.01 ± 0.38 1.93 ± 1.25

Rad52 RAD52 homolog, DNA repair protein

3.79 ± 0.82 2.32 ± 2.33

Lmx1ba LIM homeobox transcription factor 1, beta a

3.03 ± 1.16 1.55 ± 0.42

Miox Myo-inositol oxygenase -1.31 ± 0.63 -1.39 ± 0.14Dhrs7cb Dehydrogenase/

reductase (SDR family) member 7Cb

2.89 ± 1.48 1.97 ± 2.81

Note: Reverse transcriptions were carried out in triplicate. The relative quantification values for each mRNA were calculated using the 2-ΔΔCT method. A statistical comparison of the relative mean expression level for each gene between treatment and control groups was performed using Student’s T-test, and p < 0.05 was considered a significant different.

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