Suprasolv

Deionized water was of the highest purity possible. Ethanol absolute was purchased from VWR Chemicals (VWR Internationals, LLC, Llinars del Vallès, Barcelona, Spain). Sodium carbonate was from Honeywell Riedel-de Haën (Honeywell Specialty Chemicals, Seelze, Germany). 3-Chloroperbenzoic acid (mCPBA), dichloromethane (SupraSolv^®, purity ≥ 99.8%, Merck KGaA, Darmstadt, Germany), n-hexane (SupraSolv^®, purity ≥ 98%, Merck KGaA, Darmstadt, Germany), and sodium thiosulfate were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). MOSH–MOAH standards (reference 31070) and retention time standards (reference 31076) were from Restek (Restek SRL, Madrid, Spain).

Bees were thawed for 4 min prior to scent extraction and individually rinsed for 2 min in 1 mL n-pentane (SupraSolv, 99.9%, Supelco) to extract compounds from their cuticle surface. Before chemical analyses, solvent extracts were concentrated to a final volume of 300 μl by using a gentle stream of nitrogen. As an internal standard, 10 μl dodecane (C12) was added (99%, Sigma, Germany, stock solution: 100 μg/mL in n-hexane) for quantitative analysis.
All chemical analyses were performed on a gas chromatograph (Agilent 7890A, Agilent Technologies, Waldbronn, Germany) with a DB-5 capillary column (30 m × 0.25 mm inner diameter, J&W) and a flame ionization detector (FID). Hydrogen at a constant flow of 2.0 mL/min was used as a carrier gas. One microliter of the respective extract was injected splitless into the gas chromatograph at an injector port temperature of 310°C. After an initial time of 1 min at 50°C, the oven temperature increased continuously by 10°C/min to a final temperature of 310°C and held at that temperature for 35 minutes resulting in a total working time of 62 minutes.

We analysed the cuticular chemical profiles of workers because of its key role in intracolonial communication [51 (link)]. Bees from batch B2 (n = 289, 29 colonies) were thawed at room temperature for 4 min and rinsed in 1 ml n-pentane (SupraSolv, 99.9%, Supelco) for 1 min to extract cuticular chemical compounds. The extracts were concentrated to a final volume of 500 µl under a gentle nitrogen stream. We then added 10 µl of an internal standard (dodecane; 99%, Sigma, Germany, 100 µg ml⁻¹ in n-pentane) for quantitative analysis. Samples were stored at −20°C. Chemical analyses were performed by means of a gas chromatograph (Agilent 7890A, Agilent Technologies, Waldbronn, Germany) with a DB-5 capillary column (30 m × 0.25 mm inner diameter, J&W) and a flame ionization detector. Hydrogen was used as a carrier gas at a constant flow of 2.0 ml min⁻¹. One microlitre of extract was injected splitless into the gas chromatograph at an injector port temperature of 310°C. After an initial time of 1 min at 50°C, the splitter was opened and the oven temperature increased continuously by 10°C min⁻¹ to a final temperature of 310°C at which temperature was held for 35 min, resulting in a total run time of 62 min. Compounds were identified based on analyses of reference substances and earlier studies [52 (link),61 (link)].

For chemical analyses, EOs were diluted in cyclohexane MS SupraSolv^® for gas chromatography (Sigma-Aldrich, Steinheim, Germany). For enantiomers identification, the analytical standards of (−)-menthol, (+)-menthol, (−)-menthone, (+)-menthone, (−)-α-terpineol, (+)-α-terpineol, (−)-limonene, (+)-limonene, (−)-terpinen-4-ol, (+)-terpinen-4-ol, (−)-trans-caryophyllene, (−)-carvone, (+)-carvone, (−)-linalool, (±)-linalool, (−)-menthyl acetate, (+)-menthyl acetate, (−)-dihydrocarvone, (+)-dihydrocarvone, (−)-borneol, and undecane-2-one (all Sigma-Aldrich, Steinheim, Germany) were used.

Certified standard solutions (100–1000 μg/mL) of n-alkanes (C14-C32, only even + pristane/phytane), PAHs, alkylated PAHs, heteroaromatics and deuterated PAHs were purchased from Chiron AS (Trondheim, Norway). Spike and calibration standards were prepared by dilution in n-hexane or, for body burden analysis, in a matrix extract of unexposed haddock and cod eggs [31 (link)]. Details on all analytes are compiled in S1 Table. The deuterated internal standard used as a surrogate spike contained naphthalene-d8, biphenyl-d8, acenaphthylene-d8, anthracene-d10, pyrene-d10, perylene-d12 and indeno[1,2,3-cd]pyrene-d12. Dichloromethane (DCM) and n-hexane were of GC Suprasolv® analytical grade and supplied by Merck (Oslo, Norway).

A two‐step transesterification method was performed according to ISO 12966‐2:2017 with slight modifications. Briefly, aliquots of the oils were mixed with 200 µL 0.2 M sodium methoxide (Alfa Aesar, Thermo Fisher Scientific, Heysham, UK) and incubated at 60°C for 45 min under shaking. After the addition of 70 µL of 1 M methanolic sulfuric acid, samples were incubated a further 30 min at 60°C. FAMEs were extracted by addition of 600 µL saturated sodium chloride and 500 µL n‐hexane (Suprasolv, Merck KGaA, Darmstadt, Germany). Pentadecanoic acid (Sigma–Aldrich, St. Louis, MO, USA) was used as a recovery standard. GC‐MS analysis was performed as described previously.^[34] Full scans from m/z 40 to 400 were recorded and SIM scans at m/z 55, 67, 74, and 79 were used for quantification with the Supelco 37 Component FAME Mix (Sigma–Aldrich, St. Louis, MO, USA) as an external standard.

Analytical grade acetone (SupraSolv), petroleum ether, and acetonitrile (hypergrade) were obtained from Merck (Darmstadt, Germany). Ethanol and Tris(hydroxymethyl)aminomethan (TRIS) (≥ 99.9%) were provided by Carl Roth (Karlsruhe, Germany) and formic acid (99% ULC/MS) by Biosolve (Valkenswaard, Netherlands). Hydrochloric acid for pH value adjustment was purchased from Merck (Darmstadt, Germany). Cholesterol esterase from Pseudomonas sp. for enzymolysis was obtained from MP Biomedicals (Eschwege, Germany). All-E-astaxanthin standard in its free form (SML0982, ≥ 97%, 3S,3’S, from Blakeslea trispora) was provided by Sigma-Aldrich (Taufkirchen, Germany) and astaxanthin monopalmitate (1017, 3RS,3’RS) by CaroteNature (Münsingen, Swiss).