Supplementary materials

Acute effects of polyethylene microplastic on the organ toxicity of

adult zebrafish (Danio rerio)

Chu Wa Mak, Kirsten Ching-Fong. Yeung, King Ming Chan

S1. GC-MS Operating Conditions for PAH mix measurement.

The GC operating conditions for PAH measurement are as follows:

Column HP-5MS 5% Phenyl Methyl Silox

Injection mode Split

Split flow 21.2 ml/min

Septum purge flow 21.3 ml/min

Carrier mode Constant Flow

Carrier gas Helium (99.99 %)

Flow rate 1.0 ml/min

Injection Volume 1 μl Temperature Program Rate (oC/ min) Value (oC) Hold Time (min)

20 175 0

5 200 0

2 250 0

10 325 5

Run Time 50.25 min

Transfer Line Temp. 230 oC

The MS operating conditions for PAH compounds are as follows:

Ion source EI

Ion source Temperature 230 oC – 250 oC

Scan Type MS1 Scan in range m/z 35 to 550; Scan

Time: 200 ms

S2. Cumulative Time in each exhibited behavior during microplastics exposure experiment; Six

zebrafishes’ exhibited behavior type and corresponding cumulative time in each tank were record. Data are

shown as means and standard deviations in triplicate.

Behavior TypeCumulative Time on each exhibited behavior

(minute, second)

Erratic movement 6 min 1 sec ± 5 min 12 sec

Darting 4 sec ± 4 sec

Nibbling 7 sec ± 12 sec

Biting 0 sec

Body color change 0 sec

Chasing 2 sec ± 3 sec

C-bend 2 min 26 sec ± 3 min 25 sec

Diving 2 sec ± 3 sec

Shoaling 6 min 8 sec ± 1 min 56 sec

Seizure 6 min 1 sec ± 10 min 25 sec

Resting 4 min 9 sec ± 3 min 10 sec

Wobbling 3 sec ± 6 sec

Tail bent upward / downward 7 min 57 sec ± 6 min 53 sec

Labored breathing 10 min 23 sec ± 9 min 4 sec

Sickness behavior 3 min 32 sec ± 3 min 13 sec

S3. Risk assessment matrix in each exhibited behavior; Likelihood of occurrence in each exhibited behavior (20 min video recording, n = 3) were classified

in five categories depend on the cumulative time which zebrafish exhibited this behavior: Rare = ≤ 2 min.; Unlikely = ≤ 4 min.; Possible = ≤ 6 min; Likely = ≤

8 min; Almost certain = > 8 min. The reason for aforementioned categories because i) zebrafish exhibited specific behavior in more than 40 % of recording time

which was considered as high probability to be happened; The severity of consequences was categorized to five levels: Without impairment = not significant

(1); Temporary impairment = Minor (2); Permanent impairment = Moderate(3); Permanent impairment with behavior changes = Major(4); Related to fatality =

Severe(5). Three considered factors for aforementioned categories were i) whether impairment can be recovered, ii) whether impairment might induce severe

behavior and iii) whether the behavior was related to mortality. Each exhibited behavior was elevated based on likelihood of occurrence and severity of

consequences to obtain a risk matrix score: Score 1-4: Level 1; Score 5-8: Level 2; Score 9-12: Level 3; Score 13-16: Level 4; Score17-25: Level 5; 20-25; The

risk matrix scores were used to categorize into different risk level for formulation of “sickness scale in microplastic exposed zebrafish.

Severity of Consequences

Without

impairment

Temporary

impairment

Permanent

impairment

Permanent impairment

with severe behavior

changes

Related to

fatality

Not significant

(1)Minor (2) Moderate (3) Major (4) Severe (5)

Likelihood of

occurrence

(cumulative time)

> 8 min Almost certain

(5)5 10 15 20 25

≤ 8 min Likely (4) 4 8 12 16 20

≤ 6 min Possible (3) 3 6 9 12 15

≤ 4 min Unlikely (2) 2 4 6 8 10

≤ 2 min Rare (1) 1 2 3 4 5

S4. Sequences of oligonucleotide pairs used in RT-PCR assay. Nucleotide sequences listed in the order 5’ to 3’.

Oligos Accession Number Sequence (5'->3')Tm

Fragment

size (bp)Forward Reverse

ef1α NM_131263.1 ACCTACCCTCCTCTTGGTCG GGAACGGTGTGATTGAGGGA 64.9 164

rp18 NM_200713.1 TGGTGTGGCTATGAACCCTG TGGACGGTCTTTGTTCCTCG 65.0 159

gapdh NM_001115114.1 TATAGCGAGAGGGACCCAGC ACCCATGACAAACATGGGGG 65.7 174

β-actin NM_131031.1 ACGAGAGATCTTCACTCCCCT TGCCAACCATCACTCCCTGA 65.1 189

hprt NM_212986.1 ATGGACCGAACTGAACGTCT CTGTCATGGGAATGGAGCGA 64.3 163

vtg 1 NM_001044897.2 CTGCGTGAAGTTGTCATGCT GACCAGCATTGCCCATAACT 63.6 510

Cyp 1a NM_131879.1 GCATTACGATACGTTCGATAAGGAC GCTCCGAATAGGTCATTGACGAT 64.3 147

S5. The TIC chromatograph of standard PAH mix and tested microplastics (second figure) by GC.MS.

Standard PAH mix

Under the current setting, peaks of few standard compounds in PAH mix, including Anthracene, Benzo[b]fluoranthene, Chrysene, 1-Methylnaphthalene, 2-

Methylnaphyhalene were not revealed.

Tested Microplastics

S6. Identified chemicals in used microplastics. Concentrations of PAHs on MPs were below the detection

limit (LOD).

Retention Time (min) Identified Chemical

5.381 Hexane

5.416 Nonane

6.010 2-Undecene

6.046 3-Undecene

7.022; 7.263; 7.328 Dodecane

7.657 1-Tetradecene

7.781 1-Ethyl-2,2,6-trimethyl-Cyclohexane

7.840; 7.904 Cyclohexane

8.516 Tetradecane

12.774 p-Terphenyl (Internal standard)

S7. The physical attributes characterization of microplastic before and after the exposure experiments; The sizes, shapes, colors and

number of ingested microplastics before and after the exposure experiment were shown.

Individual size range of MP Mixed size range of MP

MP

Concentration2 mg/L NC EC MC HC

Organs Gills Intestine Feces Gills Intestine Gills Intestine Gills Intestine Gills Intestine

Size none All individual size of microplastic none none none none none212-250

µm

212-250

µm

212-250

µm

ShapeSpherical, except 500-600µm yellow MP will slightly

deformation Spherical

ColorNo change of color compared to control and original

microplasticClear

Number of

ingested MPnone N.A. none none none none none 0.5 ± 0.5 0.4 ± 0.5 0.9 ± 0.5

S8. Microplastic characterization in their size, shape and color before and after

exposure experiments.

S9. Gene stability analysis on five reference genes in male adult zebrafish exposed to

(A) Microplastics, (B) Benzo(a)pyrene (BaP) and (C) Bisphenol A (BPA) using the

online tool RefFinder.

S10. PCR efficiency determination of housekeeping genes and target genes used in

this study. (A) Flowchart of the efficiency test. (B) Graphic result of PCR efficiency

test of 2 housekeeping gene candidates. (C) Summary of PCR efficiency of target 2

genes used in this study.