Mercury and explosive compound 4-aminodinitrotoluene (4-ADNT) in dab (Limanda limanda) caught at munition dumping site Kolberger Heide in the Kiel Bight, Baltic Sea

Dumped munitions contain various harmful substances which can affect marine biota like fish. One of them is mercury (Hg), included in the common explosive primer. Another is 4-aminodinitrotoluene (4-ADNT), an explosive-metabolite. 251 individual dab (Limanda limanda L.) caught at the dump site Kolberger Heide a and nearby reference sites in 2017 and 2018 were analysed. The table contain individual data on Hg, 4-aminodinitrotoluene, age, length, weight, sex and condition factor. Mercury at AAS For sample preparation, portions of muscle samples were freeze dried using a lyophilizer and subsequently homogenized using an agate mortar or an ultra turrax tube drive dispenser (IKA, Staufen, Germany) equipped with glass grinders respectively to obtain a dry sample powder suitable for Hg analysis. Total Hg was determined by atomic absorption spectrometry using a Direct Mercury Analyzer (DMA-80, MLS, Leutkirchen, Germany). Known amounts (20–30 mg) of each sample were weighted into the boat containers (precleaned with nitric acid) of the DMA-80. Direct analysis for total Hg content was performed using a 10-level calibration with standards in 0.5 M nitric acid. The accuracy of the procedure was determined by analysis of Certified Reference Material (DORM-3 and DORM-4, both fish protein homogenates) obtained from the National Research Council (NCR) in Canada which was taken through the same analytical procedure as the samples. Details on reference materials are given in Table 1. All samples were analysed in triplicate. External quality assurance was done by successfully participation in laboratory proficiency tests (z-score 0.7) conducted by QUASIMEME (www.wepal.nl) designed for marine environment analytics. The limit of detection (LD) and the limit of quantification (LQ) were calculated from a standard curve according to DIN 32,645 (DIN, 1994) with a confidence level of 99%. Considering the sample preparation, a LD of 0.080 µg/kg wet weight (w. w.) and a LQ of 0.230 µg/kg w. w. were determined for Hg. No values below these limits were found in any sample under investigation. Recovery of the method was 100.3% and precision was 8.20%. Explosives at LC-MS/MS 5 μL of the final bile extract were injected on a column in an Agilent 1290 Infinity High-Performance-Liquid-Chromatograph coupled to an AB Sciex QTrap 5500 Triple Quadrupole/Ion-Trap Mass Spectrometer (HPLC-MS). Gradient separation was achieved using an Acclaim Explosives E2 Column (Thermo Fisher Scientific) kept at 22 °C. Ionization was conducted in negative atmospheric pressure chemical ionization (APCI) mode. Explosives and selected metabolites were detected via a multiple reaction monitoring (MRM) mode based on characteristic MS/MS transitions, previously optimized using commercially available standard substances. Characterisation of unknown compounds was achieved in scan-mode of the linear ion trap by selecting nitroaromatic compounds using a neutral loss scan targeting the loss of nitro-groups (m/z 46), followed by an enhanced product ion scan, producing a sensitive mass spectrum of the nitro-containing molecule. Abundances of explosive compounds were quantified via the internal standard 1,4-DNB and corrected for response factors. Response factors were determined using an external calibration covering the expected range of concentrations. The method used is described in detail by Koske et al. (2019). Limits of detection (LOD) and limits of quantitation (LOQ) for explosive compounds were calculated according to DIN 32645 (2008). LOD 4-ADNT: 2.8 ng/ml bile. LOQ 4-ADNT: 18.8 ng/ml bile.