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9 protocols using benz a anthracene

1

Quantitative Analysis of Polycyclic Aromatic Hydrocarbons

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PAH calibration standards, including five types of isotopically labeled internal standards and 16 types of PAHs, were used for quantification. The PAH mix (2000 µg/mL) was purchased from Supelco and included acenaphthene, dibenz[a,h]anthracene, anthracene, chrysene, benz[a]anthracene, benzo[b]fluoranthene, fluorene, benzo[k]fluoranthene, naphthalene, benzo[a]pyrene, phenanthrene, benzo[ghi]perylene, pyrene, fluoranthene, acenaphthylene, and indeno [1,2,3-cd]pyrene. The internal standards (2000 µg/mL) were also purchased from Supelco and included benz[a]anthracene-d12 solution, chrysene-d12 solution, naphthalene-d8 solution, perylene-d12 solution, and phenanthrene-d10 solution.
Five-point standard calibration solutions were prepared to ensure the accuracy and precision of the PAHs for quantitative and qualitative analysis. The standard calibration solutions were measured at levels ranging from 1.25 to 25 µg/mL and internal standards of 10 µg/mL. Each analytical series included one blank test. The 16 PAHs and five internal standards in the samples were defined by the precursor ions and retention time, as shown in Table 1.
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2

Polycyclic Aromatic Hydrocarbons Analysis

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Thirteen polycyclic aromatic hydrocarbons were analysed in this study. Certified reference material EPA 525 PAH Mix B (SupelCo, Bellefonte, PA, USA) was acquired as an internal standard, containing: acenaphthylene (ACNY), anthracene (ANTH), benz[a]anthracene (B[a]A), benzo[b]fluoranthene (B[b]F), benzo[k]fluoranthene (B[k]F), benzo[ghi]perylene (B[ghi]P), benzo[a]pyrene (B[a]P), chrysene (CHRY), dibenz[a,h]anthracene (D[ah]A), fluorene (FL), indeno[1,2,3-cd]pyrene (I[123-cd]P), phenanthrene (PHEN), and pyrene (PYR).
Sodium chloride (NaCl), magnesium sulfate (MgSO4), and hexane (C6H14) (HPLC grade) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Hypergrade acetonitrile (ACN) was purchased from Merck (Kenilworth, NJ, USA). Sample clean-up was performed using Supel QuE Z-Sep tubes (SupelCo). Preparation of samples was performed using polypropylene tubes. Glass tubes were used during extraction to minimise interference of the samples.
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3

Comprehensive PAH Analysis Using Standard Mix

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Analytical grade reagents were used throughout the analysis. Standard mixture (QTM PAH Mix standard) containing sixteen PAHs, including naphthalene (NAP) acenaphthylene (ACY), acenaphthene (ACE), fluorene (FLU), phenanthrene (PHE), anthracene (ANT),fluoranthene (FLT), pyrene (PYR), benz(a)anthracene (B[a]A), chrysene (CHR), benzo(b + k)fluoranthene (B[b+k]F), benzo(a)pyrene (B[a]P), indeno(1.2.3.cd)pyrene (IND), dibenz(a.h)anthracene(DbA), benzo(g.h.i)perylene (B[g]P) and an alkylated PAH 2-bromonapthalene (BNAP) was purchased from Supelco Sigma-Aldrich Pty. Ltd (NSW, Australia).
All the reagents and reference standards used are the same as those reported earlier by Duodu et al. [1] (link).
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4

Polycyclic Aromatic Hydrocarbon Analysis

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BcFE (Fig. 1) was obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Deuterated pyrene (Pyr-d10), an internal standard, was purchased from Wako Pure Chemicals (Osaka, Japan).
EPA610 PAH Mix, a mixture of naphthalene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene (Flu), pyrene (Pyr), benz[a]anthracene (BaA), chresene (Chr), benzo[b]fluoranthene (BbF), benzo[k]fluoranthene (BkF), BaP, DBA, benzo[ghi]perylene (BghiPe), and indeno[1,2,3-cd]pyrene (IDP), was purchased from Supelco (Bellefonte, PA, USA). Other reagents were analytical grade.
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5

Enrichment and Isolation of PAH-Degrading Strains

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M9 minimal medium (Na2HPO4 6.0 g, KH2PO4 3.0 g, NaCl 0.5 g, NH4Cl 1.0 g, CaCl2 0.01 g, FeCl3 0.002 g, and MgCl2 0.25 g/L, pH 7.2) was used for the enrichment and isolation of PAH-degrading strains. Luria–Bertani medium (LB, tryptone 10.0 g, yeast extract 5.0 g, and NaCl 8.0 g/L, pH 7.2) was used for strain culture. Solid plates were prepared by supplying agar (15.0 g/L) into liquid medium. Different aromatic chemicals (phenanthrene, naphthalene, anthracene, pyrene, dibenzothiophene, 9-bromophenanthrene, benz[a]anthracene, benz[b]anthracene, catechol, salicylic acid, pathalic acid, protocatechuate, xylene, and biphenyl, > 99% purity, HPLC grade) were purchased from Sigma-Aldrich (Allentown, PA, USA). HPLC-grade methanol and acetonitrile (> 99.5% purity) were purchased from EMD (Gibbstown, NJ, USA). All of the other chemicals were of analytical grade.
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6

Polycyclic Aromatic Hydrocarbons and Omega-3 Fatty Acids Protocol

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Benzo(a)pyrene (BP), benz(a)anthracene (BA), chrysene, benzo(b)fluoranthene (BbF), benzo(k)fluoranthene (BkF), and dibenz(a,h)anthracene (DBA) were procured from Sigma-Aldrich (St. Louis, MO, USA). Indeno(1,2,3-c,d)pyrene (IP) was obtained from Absolute Standards (Hamden, CT, USA). Fish oil (from menhaden) was from Sigma-Aldrich (St. Louis, MO, USA). Omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were from Cayman Chemical (Ann Arbor, MI, USA). Materials for DNA extraction [31 (link),32 (link),33 (link),34 (link)], RNA isolation [35 (link)], and 32postlabeling methods [31 (link),36 (link),37 (link)] have been published previously.
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7

Benz[a]anthracene Extraction and Analysis

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Benz[a]anthracene was purchased from Sigma-Aldrich (St. Louis, MO, USA). Isopropyl-d-1-thiogalactopyranoside (IPTG), T4 DNA ligase, and the restriction enzymes were purchased from TaKaRa. An RNA Extraction Kit was purchased from Promega (Promega Corporation, Madison, WI, USA). Random primers, ribonuclease inhibitor, dNTP mixture, recombinant DNase I, and SYBR® Premix Ex Taq™ (Tli RNaseH Plus) were purchased from TransGen Biotech (TransGen Biotech, Beijing, China).
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8

Polycyclic Aromatic Hydrocarbon Analysis in Refinery Effluents

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Acenaphthylene, anthracene, acenaphthene, Benz[a]anthracene, phenanthrene, fluoranthene, Benzo[a]pyrene, chrysene and pyrene of high-purity grade were purchased from Sigma Chemical Company. Solvents were in analytical grade (Merck Laboratory Supplies). Fungal cultivation media and their components were obtained from Difco Laboratories. Refinery effluents were collected from three different refining areas in Egypt including Mostorod (the north of Capital Cairo), Ameria (the west north of Alexandria) and Tanta (Delta region) refineries at a discharge point, transported to laboratory in ice tank and preserved at 4 °C until analysis.
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9

Comprehensive PAH Analysis Protocol

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All chemicals and reagents were of the highest purity available and were used without further purification unless otherwise stated. ORBO 43 Supelpak-20 specially treated Amberlite XAD-2 (20/40) sorbent tubes (100/50 mg), naphthalene, phenanthrene, anthracene, pyrene, fluoranthene, benz[a]anthracene, chrysene, perylene, and benzo[a]pyrene standards were acquired from Sigma-Aldrich (St. Louis, MO). HPLC grade methanol and acetonitrile were obtained from Tedia (Fairfield, OH). Deionized water was further produced by a Pall Cascada laboratory water purification system (Port Washington, NY). Face masks with bacteria filtration efficiency ≥ 95%, ≥ 98%, and ≥ 99% were purchased from a local pharmacy.
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