Changes on cylindrospermopsin concentration and characterization of decomposition products in fish muscle (Oreochromis niloticus) by boiling and steaming
Introduction
Cyanotoxins, produced by cyanobacteria, are increasingly perceived as a global water-quality issue growing in scope and persistence (Loftin et al., 2016). Potential human illness and mortality may occur following direct consumption or indirect exposure to contaminated organisms or toxins in the environment (Jasim & Saththasivam, 2016). In this group, an important emergent toxin is Cylindrospermopsin (CYN), produced by several cyanobacteria such as Cylindrospermopsis raciborskii (Ohtani, Moore, & Runnegar, 1992), Umezakia natans (Harada et al., 1994), Anabaena bergii, Aphanizomenon ovalisporum, Aphanizomenon flos-aquae and Raphidiopsis curvata (Falconer, 2006, p. 279). Structurally, CYN (C15H21N5O7S, M = 415.43) is an alkaloid consisting of a tricyclic guanidine moiety combined with hydroxymethyluracil (Ohtani et al., 1992). It has hepatotoxic, general cytotoxic and neurotoxic effects, affecting plants, several aquatic organisms and mammals with different degrees of damage (Gutiérrez-Praena et al., 2014, Guzmán-Guillén et al., 2015a, He et al., 2016, Prieto et al., 2011, Puerto et al., 2011). In the normal pH range of natural waters, it is a zwitterion, making it highly water-soluble, as much as 90% of total CYN is found outside the cells (He et al., 2016). The relatively high stability of CYN to light and over a wide range of pH and temperature (21–100 °C), although decomposition of CYN occurs after incubation at high temperatures combined with alkaline pH, might have significant consequences for aquatic environments, (Adamski et al., 2016a, Chiswell et al., 1999).
The high levels and persistence of CYN in waters can potentiate its accumulation in a wide range of aquatic animals (Freitas et al., 2016, Gutiérrez-Praena et al., 2013). CYN has been detected in lake samples showing extracellular superficial values ranging from 2.6 to 126 μg/L, exceeding the recommended safety value of 1 μg/L in drinking water, and in tissues from Salmo trutta trouts (up to 2.7 ng/g) (Messineo, Melchiorre, Di Corcia, Gallo, & Bruno, 2010). CYN was also found to contaminate crayfish (Cherax quadricarinatus, up to 4.3 μg/g) and fish (Melanotaenia eachamensis, up to 1.2 μg/g) from a small Australian aquaculture pond (Saker & Eaglesham, 1999), as well as freshwater mussels (Anodonta cygnea, up to 2.52 μg/g) in a 16-day exposure study (Saker, Metcalf, Codd, & Vasconcelos, 2004). Berry, Jaja-Chimedza, Dávalos-Lind, and Lind (2012) showed accumulation of CYN (0.09–1.26 μg/kg) in several species of finfish (Oreochromis aureus, Dorosoma mexicana, Bramocharax cabelleroi, Heterandria jonesii, Vieja sp., Cichlasoma sp. and Rhamidia sp.) caught and consumed locally in the Catemaco Lake. Therefore, this fact constitutes a serious concern, especially if the organisms can be destined to human consumption (Messineo et al., 2010).
Based on the potential risks for human health, a provisional tolerable daily intake (TDI) of 0.03 μg CYN/kg of body weight has been proposed by Humpage and Falconer (2003). Most evaluations of the risk associated with ingestion of seafood products are performed on the product uncooked, as sold or captured (Chiocchetti et al., 2016, Domingo, 2011). However, fish is normally processed and/or cooked before consumption, and these practices can alter the concentration of CYN available in food (Freitas et al., 2016). In addition, one important concern for public health is that cyanotoxins-contaminated fish, either raw or cooked, look normal and undetectable by taste, appearance and smell (Wong, Hung, Lee, Mokb, & Kam, 2009). In this sense, studies of the influence of cooking on food are required to achieve a more accurate knowledge of the actual intake and reducing the risk for the consumer in the last step of the food chain.
With respect to the effects of cooking on the increase or decrease of contaminants in food (especially fish and meat), the studies are mainly focused on a number of chemical pollutants, such as metals (Laparra, Vélez, Montoro, Barberá, & Farré, 2003), polychlorinated biphenyls, polycyclic aromatic hydrocarbons -PAHs-, hexachlororobenzene (HCB) (Perelló, Martí-Cid, Llobet, Castell, & Domingo, 2009), dioxins (Hori et al., 2005), as has been reviewed by Domingo (2011) and Chiocchetti et al. (2016). It seems that these changes depend mainly on the cooking conditions including time, temperature, and medium of cooking, with a clear general tendency to increase metal concentrations after cooking (Domingo, 2011). Concerning cyanotoxins, studies are scarce, and they have shown different patterns of variation for microcystins (MCs), nodularin (NOD), paralytic shellfish toxins (PSP-toxins) and CYN in clams, mussels, scallops, prawns and fish (Bruno et al., 2009, Freitas et al., 2014, Freitas et al., 2016, Guzmán-Guillén et al., 2011, Morais et al., 2008, Van Buynder et al., 2001, Wong et al., 2009, Zhang et al., 2010).
It has been previously demonstrated that boiling fish muscle was able to reduce levels of unconjugated MCs (MC-LR, -RR and -YR) in cooked tilapia fish in a range between 34% and 59% (Guzmán-Guillén et al., 2011). On the contrary, MCs mean concentrations were significantly higher in muscle of bighead carp after boiling, and in clams boiled for 5 and 15 min, compared to unboiled controls (Freitas et al., 2014, Zhang et al., 2010). Concerning CYN, only Freitas et al. (2016) carried out a recent study on changes on CYN concentration in edible mussels after boiling or steaming, showing no significant alterations in toxin concentration due to the cooking processes, although the toxin was found in the cooking water. Therefore, the exact incidence of different cooking and eating practices on cyanotoxin levels and exposure remain uncertain (Testai et al., 2016).
Previous studies concerning cooking processes of aquatic organisms have determined MCs by ELISA (Bruno et al., 2009, Morais et al., 2008), High Performance Liquid Chromatography-Diode-Array (HPLC-DAD) (Metcalf & Codd, 2000), or Liquid Chromatography-Mass Spectrometry (LC-MS) (Freitas et al., 2014, Guzmán-Guillén et al., 2011, Zhang et al., 2010), as LC-MS has been proved suitable for determination of MCs in natural blooms, cyanobacterial strain cultures and biological samples (Cameán et al., 2004, Ruíz et al., 2005). PSP-toxins have been analyzed by HPLC-fluorescence (Wong et al., 2009), and NOD and CYN by LC-tandem Mass Spectrometry (LC-MS/MS) (Freitas et al., 2016, Van Buynder et al., 2001). However, these have not been conducted by Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), which has become the technique of choice for analysis of different cyanotoxins (several variants of MCs, NOD, CYN, anatoxin-a, homo-anatoxin and domoid acid) in water and complex matrices, and offers excellent specificity and sensitivity for their detection and quantification (Adamski et al., 2016a, Adamski et al., 2016b, Greer et al., 2016, Oehrle et al., 2010, Pekar et al., 2016).
Besides, in most of the above-mentioned studies, the structure and properties of the toxin decomposition products have not been described. Only Adamski et al., 2016a, Adamski et al., 2016b have investigated the stability of CYN from a C. raciborskii culture under the influence of pH, temperature and irradiation, characterizing its forming products by UPLC-MS/MS. Considering the relevant toxic properties of CYN, it would be of interest to investigate the mechanism of its decomposition in intoxicated fish after cooking. For this purpose, orbitrap can provide a much higher mass resolution and mass precision compared to triple quadrupole mass analyzers (MS/MS) (Gerssen, Mulder, & de Boer, 2011).
In view of these reports, the aim of the present study was to investigate for the first time the influence of common practices for cooking of fish meat and obtention of fish soups around the world, such as boiling and steaming, on CYN concentration in muscle of fish (Oreochromis niloticus) contaminated under laboratory conditions, assaying two different periods of time (1 and 2 min). Moreover, a precise characterization of CYN by-products in fish samples under the influence of cooking was conducted.
Section snippets
Chemicals and reagents
Cylindrospermopsin standard (purity 95%) was supplied by Alexis Corporation (Lausen, Switzerland). Standard solutions of CYN were prepared in Milli-Q water (100 μg/mL) and diluted as required for their use as working solutions (1–100 μg/L). All chemicals and reagents used in this study were analytical grade materials. HPLC-grade methanol, dichloromethane, acetonitrile, and trifluoroaceticacid (TFA) were purchased from Merck (Darmstadt, Germany). Deionized water (418 MΩ/cm resistivity) was
Results
Concerning UPLC-MS/MS, the LOD and LOQ obtained for the muscle were 1.6 and 3.4 ng/g dw, respectively, compared to the former 2.5 and 7.3 ng/g dw obtained by LC-MS/MS (Guzmán-Guillén et al., 2015b). In the case of waters, LOD and LOQ were 0.36 and 0.40 μg/L, respectively, compared to the former 0.5 and 0.9 μg/L obtained by LC-MS/MS (Guzmán-Guillén, Prieto, Gónzalez, Soria-Díaz, & Cameán, 2012). This contributes to a better sensitivity of the analysis by UPLC-MS/MS compared to LC-MS/MS. The
Discussion
Generally, estimations of exposure to cyanotoxins as food contaminants have assumed that the available toxin concentrations in raw and cooked food is similar, which is not true (Freitas et al., 2014). Ibelings and Chorus (2007) suggest that in industrially processed seafood, the removal of parts in which cyanotoxins can accumulate prior to processing could be effective in avoiding hazards. But this is impracticable in case of CYN accumulation in muscle of fish, as this is the edible fraction,
Conclusions
In conclusion, this study sheds new light on the issue of the influence of some cooking practices (boiling and steaming) on the levels of unconjugated CYN in edible organisms, being the first one conducted in fish. The results show that concentrations of CYN in fish are dependent on the cooking method, being the steaming for 2 min the most suitable, followed by boiling for 2 min, for a significant reduction of CYN levels in the fish muscles. Moreover, the decrease in CYN levels in cooked
Acknowledgements
The authors would like to acknowledge the Ministerio de Economía y Competitividad of Spain (AGL2015-64558-R, MINECO/FEDER, UE) for its financial support.
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