Supplementary Information for:
Environmental mercury concentrations in cultured, low
trophic level fish using an alternative feed (i.e. food waste)
strategy
Zhang Cheng a,b, Wing Yin Mo c, Yu Bon Man a,c, Cheung Lung Lam c, Wai Ming Choi c,
Xiang Ping Nie d, Yi Hui Liu e, Ming Hung Wong a,c*
a
Consortium on Health, Environment, Education and Research (CHEER), and
Department of Science and Environmental Studies, Hong Kong Institute of Education,
Tai Po, Hong Kong P.R. China;
b
College of Resources and Environment, Sichuan Agricultural University, Chengdu, P.R.
China;
State Key Laboratory in Marine Pollution – Croucher Institute for Environmental
c
Sciences, Hong Kong Baptist University and City University of Hong Kong, Hong Kong,
P.R. China;
d
Institute of the Hydrobiology, Jinan University, Guangzhou 510632, P.R. China;
e
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences,
Guangzhou, 510380, P.R. China;
* Corresponding author: minghwong@ied.edu.hk; Tel.: +852-2948-8706; Consortium on
Health, Environment, Education and Research (CHEER), and Department of Science and
Environmental Studies, Hong Kong Institute of Education, Tai Po, Hong Kong P.R. China
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S1.Method
S1.1 Chemical analysis on fish carcass and feed
S1.1.1 Feed protein solubility in potassium hydroxide (KOH)
The protein solubility was determined according to the procedure of Araba and Dale
(Araba &Dale 1990). Feed sample (~1.5g) was added to a 0.2% KOH solution (75 mL)
with a magnetic stirrer for 20 min (in duplicate). Then the sample was centrifuged at
3000 rpm for 15 mins and the supernatant was filtered and the total nitrogen (15 mL of
extracts each, determined in triplicate) was determined by the Kjeldahl method. The
solubility of the protein, expressed as a percentage, was calculated by dividing the protein
content of the KOH extracted solution by the protein content of the fish feed sample.
S1.1.2 Protein analysis
Samples of fish feed and feces were analyzed for nitrogen content with a CHN
analyzer (Carlo Erba NA 1500, CE Instruments, Thermoquest Italia, Milan, Italy), the
protein content (%) was equal to the nitrogen content of sample (%) multiplied by 6.25.
S 1.1.3 Moisture and ash content determination
In brief, the moisture content and ash, of the experimental diets, fish carcass were
determined according to the procedures of the Association of Official Analytical Chemist
(AOAC) (1990a, b). For the moisture determination (AOAC 1990b), the sample (fish
feed or carcass) (~0.5g) was weighted (W sample initial) and dried at pre-weighted porcelain
crucible with cover at 100 ± 2° C for 24 hr. Then the crucible were dried at desiccators
for 2 hr and weighted to nearest 0.1 mg immediately (W
sample final).
The crucible was
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previously dried at 100 ± 2° C for 2 hr and weighted (Wc) as mentioned. The Percent
Total Dry Matter (Total DM, %) was calculated as following:
% Total DM = 100 x (W sample final – Wc)/ (W sample initial – Wc)
W sample final = sample and crucible weight after drying
W sample initial = sample and crucible weight before drying
Wc = dried crucible weight
For the ash determination (AOAC 1990a), the sample (fish feed or carcass) (~0.5g)
was weighted (W ash initial) and combusted at pre-weighted porcelain crucible with cover at
600 ± 2° C for 2 hr. Then the crucible were dried at desiccator for 2 hr and weighted to
nearest 0.1 mg immediately (W
sample final).
The crucible was previously dried at 100 ± 2°
C for 2 hr and weighted (Wc) as mentioned.
% Total ash = 100 x (W ash final – Wc)/ (W ash initial – Wc)
W ash final = sample and crucible weight after combustion
W ash initial = sample and crucible weight before combustion
Wc = dried crucible weight
S1.1.4 Lipid determination
Ultrasound-assisted extraction with modifications was adopted for lipid content
determination in fish feed and fish carcass (Metherel et al. 2009). 0.1 g of sample was
extracted with 3:2 v/v hexane: isopropanol mixture (12 mL) for 20 min of 40KHz
sonication bath, Branson Bransonic 5510. The organic layer was collected after
centrifuged, the extraction was then repeated for 2 times and the organic layer were
pooled and dried in a pre-weighted glass tube under nitrogen and the tube was keep at
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102°C for 30 mins. The tubes were weighted again after kept in desiccators for 3 hrs.
S1.1.5 Crude Fibre determination
Crude fibre was determined by the loss on ignition (AOAC 1984) of defatted feeds
(2g) after digestion with 200 ml of 0.255N H2SO4 and then 0.313 NaOH for 30 min each.
The residue were filtered and washed with hot distill water at least twice. The residue was
washed with 10ml of acetone twice after all digestions. The weight of residue was
determined after 105 ºC (24 hours) and 550ºC (3 hours).
The percentage fibre was determined as following:
% Fibre= Weight of residue after 105 ºC - Weight of ash after 550ºC x 100 / Weight of
sample
S1.1.6 Nitrogen-free extract determination
Nitrogen-free extract (carbohydrates) was calculated by subtracting sum of
(moisture % + crude protein % + crude fat % + crude fibre % + ash %) from 100 (Castell
&Tiews 1980).
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Figure S1 Concentrations of MHg and THg in water, SPM and sediment in experimental ponds, which using fish feeds from food
waste.
Note: The results were tested by one way ANONVA; Error bars represented the standard deviation of samples (n=3)
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Reference:
AOAC (1984): Association of Official Analytical Chemists. Fibre (crude) in animal feed (962.06).
In Official methods of analysis, Association of Official Analytical Chemists, 14th Edition.
Washington, DC 160–162
AOAC (1990a): Ash of Animal Feed. (942.05) Official methods of Analysis. Association of
Official Analytical Chemists, 15th Edition
AOAC (1990b): Moisture in Peat. (967.03) Official Methods of Analysis. Association of Official
Analytical Chemists. 15th Edition
Araba M, Dale NM (1990): Evaluation of KOH solubility as an indicator of overprocessing
soybean meal. Poultry Sci 69, 76–83
Castell J, Tiews K (1980): Report of the EIFAC, IUNS and ICES Working Group on
standardization of methodology in fish nutrition research.(Hamburg, Federal Republic of
Germany, 21-23 March 1979)
Metherel AH, Taha AY, Izadi H, Stark KD (2009): The application of ultrasound energy to
increase lipid extraction throughput of solid matrix samples (flaxseed). Prostag Leukotr
Ess 81, 417-423
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