Detection in Antarctic Krill (Euphasia Superba Dana)
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POLAR RESEARCH, 2018 VOL. 37, 1457395 https://doi.org/10.1080/17518369.2018.1457395 RESEARCH ARTICLE Di(2-ethylhexyl) adipate (DEHA) detection in Antarctic krill (Euphasia superba Dana) Xiangning Han & Daicheng Liu Key Laboratory of Animal Resistance, College of Life Science, Shandong Normal University, Jinan, P. R. China ABSTRACT KEYWORDS In this reported study, a novel high-performance thin-layer chromatography (HPTLC) method toxicity; high-performance was developed for the detection and quantification of the toxic substance di(2-ethylhexyl) thin-layer chromatography adipate (DEHA) in Antarctic krill. This procedure was based on the extraction of DEHA by (HPTLC); ecotoxins; plastic ultrasonic solvent extraction with anhydrous ethanol, silica-gel column chromatographic particles; marine pollution; separation, HPTLC detection and quantification using petroleum ether/ethyl acetate/ acet- krill oil one/glacial acetic acid (29:1:0.5:2d*, v/v/v/v) as the developing solvent and bromine thymol ABBREVIATIONS blue solution as the chromogenic agent. The content of DEHA in freeze-dried Antarctic krill 2d: two drops; AU: was found to be ca. 0.63 ± 0.05 mg/g. The structure of DEHA in the Antarctic krill was absorbance unit; DEHA: subsequently determined by gas chromatography–mass spectrometry (GC-MS) and infrared di(2-ethylhexyl) adipate; FTIR: chromatography, which verified the presence of this compound in the krill. The HPTLC Fourier-transform infrared method exhibited excellent accuracy, with a recovery of 97.1–101.6% and good precision spectroscopy; GC-MS: gas with a relative standard deviation of 2.47–4.90%. The DEHA in Antarctic krill oil was extracted chromatography-mass by n-hexane and detected using the same method described above, which verified that DEHA spectrometry; HPLC: was also present in krill oil at a concentration of ca. 2.16 ± 0.08 mg/g. The presence of DEHA high-performance liquid in kill oil is very concerning because of its demonstrated harmful ecotoxicity, and since chromatography; HPTLC: high performance thin-layer Antarctic krill is the key link in the food chain in the Antarctic coastal marine ecosystem. chromatography; IR: infrared The adverse effects of DEHA on Antarctic krill and the source of DEHA will be explored in chromatography; RSD: future research. relative standard deviation; SD: standard deviation; TLC: thin-layer chromatography Introduction be released into the environment during its synthesis and distribution and in consumer use of finished DEHA is a suitable substitute for di(2-ethylhexyl) plastic products (Felder et al. 1986). phthalate in some applications (Felder et al. 1986). DEHA seriously inhibits the survival and growth This compound has been widely used in the plastics of algae and the planktonic crustacean Daphnia industry and other fields on account of its high boil- magna at very low concentration (Felder et al. 1986; ing point, good electrical properties, good weathering Bi et al. 2016). DEHA can induce remarkable histo- resistance and good thermal and light stability (Abdul pathological changes in gill tissue and can produce Rahman et al. 2009). It is primarily used as a plasti- hepatocellular changes in various species of fishes cizer in food wraps, vinyl blood bags and haemodia- (Üçüncü et al. 2010; Üreten & Üçüncü 2013). It has lysis bags (Yavan 1984). It is also added into plastic been reported that DEHA seriously damaged the liver toys and other toys for children (Rahman & Brazel of rats and mice by affecting lipid metabolism, caus- 2004). DEHA has been a component of cosmetic ing liver tumours and inducing DNA damage, among bases and solvents and it also has been widely used other negative effects (Bell 1983; Takagi et al. 1990; in the aerospace and automotive industries, as well as Lake et al. 1997; Dalgaard et al. 2003). Studies have under extreme working conditions, such as high-tem- found that DEHA can produce reproductive and perature environments, on account of its high ther- developmental toxicity by interfering with the estrous mal-oxidation stability, large specific heat capacity, cycle and increasing ovarian follicle atresia; it was good stickiness index and excellent thermal conduc- found to be harmful to both the mother and foetus tivity (Yavan 1984; Qian et al. 2016). DEHA has been (Miyata et al. 2006). DEHA has caused adrenal shown to migrate when packaging material is in weight decrease in adult male rats (Miyata et al. direct contact with high fat content foods, which 2006). All of these results strongly suggest that the can lead to high DEHA concentration and contam- toxicity of DEHA, particularly its ecotoxicity, should ination of the food (Badeka et al. 1999). DEHA can CONTACT Daicheng Liu [email protected] Key Laboratory of Animal Resistance, College of Life Science, Shandong Normal University, 88 East Wenhua Road, Jinan 250014, P. R. China. © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 X. HAN & D. LIU receive great attention. Unfortunately, there is little batch of Antarctic krill used in this study (35–55 mm in available scientific data on the concentration of body length) was caught in the first quarter of 2015 DEHA in Antarctic krill. from the waters surrounding the Chinese Great Wall Antarctic krill (Euphausia superba), a marine plank- Antarctic Station (48.1–48.3 zone in the Antarctic). A tonic crustacean that lives in schools, is the key compo- DEHA standard (≥99%) was obtained from Aladdin nent of the food webs in the Antarctic coastal marine Chemical (Shanghai, China). All the other reagents ecosystem (Kokubun et al. 2015). This abundant species used in this study were analytically pure. An FUD- comprises an estimated biomass of 400–1550 million 1200 freeze-dryer was obtained from Tokyo Rikakikai metric tonnes, which may be the largest biomass of any Co., Ltd (Tokyo, Japan). The ultrasonic cleaning device multicellular species on the planet (Gigliotti et al. 2011). used in this study was purchased from Kun Shan Antarctic krill contains high-quality lipids, and krill oil Ultrasonic Instruments Co., Ltd (Jiangsu, China). A contains high levels of omega-3 polyunsaturated fatty rotavapor-3 rotating evaporator was obtained from acids (n-3 PUFA) such as eicosapentaenoic (EPA, Buchi (Flawil, Switzerland). A silica gel chromatogra- 20:5n3) and docosahexaenoic (DHA, 22:6n3), which phy column (3 × 60 cm) was obtained from Shandong are easily absorbed by humans (Gigliotti et al. 2011; Research Institute of Chemical Industries (Shandong, Ali-Nehari et al. 2012). The content of antioxidant China). Pre-coated silica gel GF254 high-performance astaxanthin in Antarctic krill is extremely high; astax- TLC plates (10 × 10 cm2) and silica gel (200–300 mesh) anthin can effectively scavenge oxygen free radicals in were purchased from Haiyang Chemical (Qingdao, the body (Ali-Nehari et al. 2012). The lipids in Antarctic Shandong, China). A chromatography development krill are beneficial to health in the areas of obesity, chamber (10 × 12 × 5 cm3, consisting of a twin trough coronary heart disease, inflammation and brain func- glass chamber) was obtained from Shanghai Xinyi tion, among others (Maki et al. 2009;Fosshaugetal. Instrument Co., Ltd (Shanghai, China). A TLC 2011; Wibrand et al. 2013). These features, together Scanner 3 equipped with the Wincats 1.4.1 software with others, make Antarctic krill an outstanding food was purchased from CAMAG (Muttenz, Switzerland). source and medical resource. The Fourier transform infrared spectrometer used for Methods for detecting DEHA have been presented in this study (Bruker Tensor II) was purchased from numerous reports. For instance, Di Bella et al. (2014) Bruker Instruments (Karlsruhe, Germany). None of determined DEHA residues in moka pots, coffee pods the experimental instruments and materials was com- and coffee capsules made from polypropylene and in posed of plastic to avoid any hint of contamination. polypropylene with a micro-perforated film of low-den- sity polyethylene lids by GC-MS. Lo Turco et al. (2015) Preparation of sample solution and standard studied plasticizer residues in tea by solid phase extrac- solution tion–GC-MS. Fromme et al. (2013)usedaGC-MS method to study the daily intake of DEHA by infants Krill samples (ca. 200 g) were cut from the middle through their standard diet by sampling for detection of section of a chunk of frozen krill – to avoid sampling DEHA in urine and proved that GC-MS was an excellent krill in contact with the plastic packaging – and were analytical method with good accuracy and high sensitiv- freeze-dried at −46°C to produce 40 g of dry Antarctic ity. Wang detected DEHA in mixed plasticizer by HPLC krill. This dry Antarctic krill sample was extracted three (Wang 1996). times with anhydrous ethanol (1:15, w/v) using ultra- Although there is a wide choice of chromatographic sound for 0.5 h, at 35°C, 350 w, 40 kHz. The extracts methods for plasticizer analysis (GC, HPLC), the tech- were combined, filtered (after flushing with anhydrous nology of HPTLC has gained greater acceptance as an ethanol) and evaporated to dryness (50°C). The residue analytical procedure because of its low operation cost was dissolved in anhydrous ethanol and the volume of and high sample throughput in recent years (Rezić the mixture was accurately set to 10 ml in a volumetric et al. 2005;Heetal.2013;Lietal.2016). As there has flask. This original DEHA sample (Sample A) was used been no detailed HPTLC detection method for DEHA for analysis and refinement. for krill and similar animals, the aim of the present Standard solutions of DEHA were prepared by dis- study was to detect the presence of DEHA in Antarctic solving accurate quantities of DEHA (≥99.5%) in metha- krill using an HPTLC detection method, determine its nol.