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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.