Supporting Information

Profiling of Selected Functional Metabolites in the Central Nervous System of Marine Medaka (Oryzias melastigma) for Environmental Neurotoxicological Assessments

Elva Ngai-Yu LEI, Man-Shan YAU, Chi-Chung YEUNG, Margaret B. MURPHY,

Ka-Leung WONG, Michael Hon-Wah LAM


Fish culturing, dietary exposure, tissue burden determination, QA/QC procedures and performance parameters of the overall analytical protocol, multivariate analysis and pathway analysis

Fish culturing

The 3-month-old marine medaka were obtained from culture stock originally purchased from Interocean Industries (Taiwan) and reared in the City University of Hong Kong for more than 30 generations. The culturing conditions and test conditions recommended by the OECD1 were adopted. Phenotypic sex was judged on the basis of secondary sex characteristics, namely, the shapes of the dorsal and anal fins. Prior to initiation of all experiments, the fish were acclimated in male-female pairs for 36 h (although we were only using female fish for our experiments), kept in fully aerated water at 25 ± 1 °C under a light-dark cycle of 14:10 h.

Bioencapsulation of BDE-47 in Artemia nauplius

The bioencapsulation of BDE-47 in Artemia nauplii was in accordance with the method reported by Muirhead et al.2 and Merwe et al.3 A solution of 825 μL of 10000 ppm BDE-47 was added to a 150 mL conical flask and evaporated to dryness over a gentle stream of dried nitrogen gas. Afterwards, a 100 mL of newly hatched Artemia nauplii (ca. 1500 nauplii/mL) was directly added to the conical flask. The Artemia nauplii culture was incubated under light aeration on a 12:12 h light:dark cycle for 24 h. After the 24 h incubation, Artemia nauplii were harvested, rinsed with artificial seawater thoroughly and resuspended in artificial seawater (ca. 1500 nauplii/mL). Subsequently, a 3 ´ 1 mL of this Artemia nauplii feed was used for the analysis of bioencapsulation of BDE-47, and the remaining feed was distributed to glass tubes in amounts required for each day of exposure. All feed tubes were kept frozen at -20 °C and defrosted each morning immediately prior to feeding to the marine medaka. Another batch of newly hatched Artemia nauplii were prepared in the same manner except with no BDE-47 added. This batch Artemia nauplii feed were feeding to the control marine medaka.

Dietary exposure of marine medaka to BDE-47

The 3-month-old marine medaka were randomly divided into three treatment groups: (i) control, (ii) low-dose (2.5 mg-BDE-47 kg-1 wet wt fish day-1), and (iii) high-dose (5.0 mg-BDE-47 kg-1 wet wt fish day-1). Each group contained 30 glass tanks, filled with 2.0 L of 30 PSU artificial seawater, with gentle aeration.

Individual fish was fed either with Artemia nauplii (controls) or BDE-47 bioencapsulated Artemia nauplii (treatment groups) daily. While feeding, glass dividers were put in place for 30 min to ensure that the same dose of BDE-47 was given to each fish over the course of the experiment. Fish were also fed to satiation twice daily with hormone-free flake fish food (AX5, Aquatic Ecosystems, USA). All the exposure were conducted in an animal holding room with ambient temperature controlled at 25 ± 1 °C and a light-dark cycle of 14:10 h. Half water in each tank was refreshed every other day and all solid waste was removed daily.

Sampling

On Day 5 and Day 21 of exposure, 15 fish (10 for neurometabolomic profiling; 5 for quantification of the bioaccumulation and biotransformation of BDE-47 in the brain and other body tissues) were removed from their tanks approximately 24 h after the exposure to BDE-47 bioencapsulated Artemia nauplii on the previous day. Each fish was euthanized immediately in iced Milli-Q water and dissected under a stereoscopic microscope to isolate the brain, gall bladder, gill, gonad, intestines and liver from the carcass. All the tissues and carcass (except those brain tissues for metabolomics profiling) were immediately freeze-dried for analysis.

Determination of tissue distribution of BDE-47 and their biotransformation products

Freeze-dried tissues and carcass samples (ca. 16 mg) were grounded with 1.5 g of anhydrous sodium sulfate and spiked with 50 ppb of 10 μL 13C-labeled BDE-47 surrogate for 3 days prior to sample preparation. The sample extraction & workup procedure was modified from Wan et al.4 Prepared samples were extracted by accelerated solvent extraction using a Dionex ASE-350 accelerated solvent extractor (Sunnyvale, CA). A mixture of n-hexane and dichloromethane (DCM) (1:1) was used as the first extraction solvent at 100 ºC. Samples were then extracted by a mixture of n-hexane and methyl butyl ether (MTBE) (1:1) at 60 ºC. Two cycles (10 min each) were performed for each solvent system per sample, and the two extraction fractions were combined for subsequent cleanup.

The combined extracts were rotary evaporated to dryness and reconstituted in 1000 μL n-hexane. The reconstituted extract was then split into two portions: one was 150 μL, another 850 μL. The 150 μL portion was further split into three portions of equal volume as triplicates for lipid content determination using the micro-colorimetric sulfo-phospho-vanillin (SPV) analysis method modified from Lu et al.5 In brief, the extract was first evaporated to dryness over a gentle stream of dried nitrogen gas, then, 50 µL concentrated sulfuric acid was added and the mixture was vortexed and heated for 10 minutes at 100 ºC on a heating block. After that, 450 µL of the colorimetric SPV reagent (120 mg vanillin, 20 mL Milli-Q water, 80 mL phosphoric acid) were added, followed by thorough vortex mixing. After incubating at room temperature for 30 min, the absorbance at 525 nm was measured using a microplate reader (Molecular devices SpectraMax M2e, Sunnyvale, CA). A calibration curve was prepared using menhaden fish oil standard and the absorbance was measured as the sample extracts. The lipid contents in the extracts were estimated from the calibration curve.

The second portion (800 μL) of the extract was mixed with 400 µL of 0.5 M potassium hydroxide in 50% ethanol. Phenolic compounds in the extract were retained and back-extracted by 3 ´ 800 µL of fresh n-hexane. This fraction of organic extract was collected and regarded as the “neutral fraction”. After that, the aqueous layer was acidified with 150 µL of 2.0 M hydrochloric acid and phenolic compounds were extracted with 3 ´ 800 mL of fresh n-hexane/MTBE (9:1 v/v). This fraction of organic extract was regarded as the “phenolic fraction”.

The “neutral fraction” was concentrated into approximately 100 µL over a gentle stream of dried nitrogen gas, and then directly applied to a multi-layer column chromatography, previously conditioned with 3 mL of n-hexane. The stationary phase of multi-layer column was acidified silica gel (10 g of silica gel mixed with 5.4 mL of concentrated sulfuric acid) on top of neutral alumina. The column was packed in a glass wool plugged glass Pasteur pipette using dry method in the follow order from top to bottom: 250 mg of anhydrous sodium sulfate, 400 mg of acidified silica, 400 mg of neutral alumina and 300 mg anhydrous sodium sulfate. After application of the sample, the column was eluted with 1 mL of n-hexane and 1 mL of DCM. The eluate was evaporated to dryness and reconstituted in 150 µL n-hexane for GC-MS analysis. Quantification of the various BDE congeners and their methoxylated metabolites was conducted via standard addition. A 50 ppb of 10 μL 13C-labeled BDE 77 and 138 were used as internal standards.

The “phenolic fraction” was also evaporated to dryness. A 480 µL of a solvent mixture comprising acetonitrile, methanol, water and pyridine in 5:2:2:1 ratio was added, followed by 40 µL of neat derivatizing agent ethyl chloroformate (ECF). The reaction mixture was vortexed and reacted at room temperature for an hour, followed by quenching with 1.2 mL of Milli-Q water. The aqueous solution was extracted with 3 ´ 6 mL of n-hexane, and the extracts were combined and concentrated to approximately 100 µL over a gentle stream of dried nitrogen gas. The concentrated extract was cleaned up by column chromatography as described above for the “neutral fraction”. The eluent used was 1.5 mL of n-hexane and 1.5 mL of DCM. The eluate was evaporated to dryness and reconstituted in 50 µL n-hexane for GC-MS analysis. Quantification of the various BDE hydroxylated metabolites was conducted via external calibration.

Qualitative and quantitative determination of all target compounds was performed using an Agilent Technologies 7890A GC, with a split/splitless injector, interfaced to a 5975C inert XL EI/CI mass spectrometer (GC-MSD). Chromatographic separation was achieved on a DB5-MS column (30 m × 0.25 mm × 0.1 mm; Agilent, Carlsbad, CA) with helium as the carrier gas at a flow of 1.2 mL/min. The MSD was operated in the Selected Ion-Monitoring (SIM) mode. The temperature of ion source was kept at 230 °C and that of quadrupole was kept at 150 °C. Details of GC-MSD parameters are shown in the Table S1. Ions monitored for PBDEs, methoxy PBDE congeners, derivatized hydroxyl PBDE congeners and dibromophenol (BRP) in the chromatogram are shown in the Table S2 & S3. The method-performance parameters were shown in the Table S4.

Quality control and quality assurance of chemical analyses

Throughout all determination of BDE congeners and their hydroxylated and methoxylated metabolites, samples were kept in dark to avoid photo-degradation. At least two method blanks and two laboratory-fortified blanks were prepared and analyzed with every preparation batch. Calibration control standards and instrument blanks were analyzed after every ten chromatographic analyses of samples. Surrogates (0.5–3.0 ng) were spiked to every sample, and the average surrogate recovery for all sample matrices fell between 70 and 120 %. Standard addition method was used to quantify the concentration of PBDEs and its potential biotransformation products in fish tissues and the performance of this method (accuracy, precision, method detection limit [MDL] and method quantitation limit [MQL]) was shown in Table S5. All of the calibration curves showed significant linearity with correlation coefficients ≥ 0.995.

For the classical neurotransmitters profiling, fish brains were dissected out rapidly over ice within 1 min and snap-frozen in liquid nitrogen. Throughout the analysis, samples were kept over iced Milli-Q water to avoid post-extraction changes at the metabolite level. At least two method blanks and two laboratory-fortified blanks were prepared and analyzed with every preparation batch. Moreover, calibration control standards and instrument blanks were analyzed after every ten samples injections. Triple solvent extraction for each sample was performed in order to ensure good recovery of the extracted species. Surrogate spikes (8 ng) were added to every sample, and the average surrogate recovery for all samples fell between 60 and 130 %. Results from triplicate of each authentic standard were plotted and analyzed by unweight linear least squares regression analysis. All of the calibration curves showed significant linearity with correlation coefficients ≥ 0.995.

Concentration of classical neurotransmitters and their related precursors and metabolites were quantified using an external calibration curve. The calibration consisted of preparing triplicate authentic standard solutions at fifteen concentration levels spreading from 0.005 to 400 ng, and replicating over three days. The linear dynamic range for ESI-MS/MS measurement of each dansylated analyte was different. The calibration curve of each dansylated analyte consisted of a minimum of five concentrations in the range of expected concentration. All of calibration curves showed strong linearity with correlation coefficients ≥ 0.995, and deviation of each data point from the regression line was less than 20% from its theoretical value.

As we were not able to identify any certificated reference material (CRM) to valid the presence analytical method, spiked samples (spiking authentic standards into samples) were used as references. Trueness was calculated as the percentage of the known amount of a spiked analyte recovered during the analytical procedure. Precision measured under repeatability condition, it was calculated as the maximal difference between two results, obtained under repeatable conditions, expected with a probability of 95%.

MDL was calculated by multiplying the standard deviation of the result obtained by compensation factor (t) from the Student’s t-Table with a confidence interval of 95%. MQL was calculated by multiplying the standard deviation of the result obtained by 10.

Trueness (spike recovery), precision (repeatability), MDL and MQL data were acquired from 5 replicate analyses on spiked samples at 1-3 spike levels over 2-50 ng regarding to the target neurotransmitters and are tabulated in Table S7. Spike recovery was successfully determined for most of the target analytes, with the exception of g-aminobutyric acid (GABA), glutamine (Gln), glutamate (Glu) and taurine (Tau), whose native concentration were close to upper limit of linear dynamic range. The spike recoveries for all the target analytes (except Gln, Glu, and Tau) were between 50 to 130%. Repeatability, MDL and MQL were successfully determined for all the target analytes. The repeatability for all the target analytes were in the range 0.6–21.3%.

Table S1. Details of GC-MSD parameters for PBDEs, MeO-PBDEs, ECFO-PBDEs and ECFO-BRP analysis.

Injector Information
Mode / Pulsed Splitless
Heater / 280 °C
Pressure / 10.42 psi
Total Flow / 54.2 mL/min
Septum Purge Flow / 3 mL/min
Gas Saver / 20 mL/min
Injection Pulse Pressure / 20 psi until 0.5 min
Purge Flow to Split Vent / 50 mL/min at 2 min
Column Information
Column / DB-5MS with 30 m Length, 0.25 mm ID, 0.1 mm Film Thickness
Flow Rate / Constant Flow at 1.2mL/min
Oven Information
Equilibration Time / 0.5 min
Temperature Program / Step 1: 60 °C for 2 min
Step 2: Ramp 15 °C/min to 250 °C for 0 min
Step 3: Ramp 5 °C/min to 280 °C for 5 min
Step 4: Ramp 30 °C/min to 290 °C for 20 min
Thermal Aux (MSD Transfer Line) Information
Heater / 290 °C

Table S2. Ion monitored for GC-MS determination of PBDEs and MeO-PBDEs in tissue extracts.

Group No.
(Start Time, min) / Rt, min / m/z
(Dwell Time, msec) / m/z Type / m/z Formula / Substance
1
(11.5) / 12.31 / 248 (200)
250 (200) / M
M + 2 / 12C12H979BrO
12C12H979Br81BrO / BDE-3
(4-MoBDE)
2
(14.3) / 14.50 / 326 (200)
328 (200)
330 (200) / M
M + 2
M + 4 / 12C12H879Br2O
12C12H879Br81BrO
12C12H881Br2O / BDE-15
(4, 4’-DiBDE)
3
(15.8) / 16.20 / 404 (200)
406 (200)
408 (200) / M
M + 2
M + 4 / 12C12H779Br3O
12C12H779Br281BrO
12C12H779Br81Br2O / BDE-28
(2, 4, 4’-TrBDE)
4
(17.5) / 18.29 / 484 (150)
486 (150)
488 (150) / M
M + 2
M + 4 / 12C12H679Br4O
12C12H679Br381BrO
12C12H679Br281Br2O / BDE-47
(2, 2’, 4, 4’-TeBDE)
18.29 / 496 (100)
498 (100)
500 (100) / M
M + 2
M + 4 / 13C12H679Br4O
13C12H679Br381BrO
13C12H679Br281Br2O / 13C-BDE-47
19.08 / 496 (100)
498 (100)
500 (100) / M
M + 2
M + 4 / 13C12H679Br3O
13C12H679Br381BrO
13C12H679Br281Br2O / 13C-BDE-77
5
(19.2) / 19.58 / 514 (200)
516 (200)
518 (200) / M + 2
M + 4
M + 6 / 12C13H879Br381BrO2
12C13H879Br381Br2O2
12C13H879Br81Br3O2 / 6-OMe-BDE-47
6
(19.9) / 20.15 / 341 (200)
356 (200)
516 (200) / M-CH379Br81Br
M - 79Br81Br
M + 2 / 12C12H579Br2O2
12C13H879Br2O2
12C13H879Br381BrO2 / 5-OMe -BDE-47
7
(25.0) / 25.80 / 654 (200)
656 (200)
658 (200) / M + 4
M + 6
M + 8 / 13C12H479Br481Br2O
13C12H479Br381Br3O
13C12H479Br281Br4O / 13C-BDE-138

Table S3. Ion monitored for GC-MS determination of ECFO-BPR and ECFO-PBDEs in tissue extracts.