Contents lists available at ScienceDirect Marine Environmental Research journal homepage: www.elsevier.com/locate/marenvrev Tetrodotoxin and saxitoxin in two native species of puffer fish, Sphoeroides marmoratus and Lagocephalus lagocephalus, from NE Atlantic Ocean (Madeira Island, Portugal) Estefanía Pereira Pinto a , Susana Margarida Rodrigues a , Neide Gouveia b , Viriato Timóteo b , Pedro Reis Costa a,c,* a IPMA—Portuguese Institute of the Sea and Atmosphere, Av. Brasília, 1449-006, Lisbon, Portugal b Regional Fisheries Management—Madeira Government, DSI-DRP, Estrada da Pontinha, 9004-562, Funchal, Madeira, Portugal c CCMAR—Centre of Marine Sciences, University of Algarve, Campus of Gambelas, 8005-139, Faro, Portugal ARTICLE INFO Keywords: TTX STX Native puffer fish Chemical oceanography Marine toxins ABSTRACT The presence in EU waters of invasive tetrodotoxin (TTX) -harbouring puffer fishes has been receiving in- creasingly attention due to potential new threats posed by this potent neurotoxin. The present study investigates the occurrence of tetrodotoxin, saxitoxin (STX), and their analogues in two native puffer fish species from the NE Atlantic. High TTX content was detected by LC-MS/MS in several tissues of the Guinean puffer Sphoeroides marmoratus from Madeira Island (Portugal), reaching concentrations as high as 15 mg TTX kg -1 in the digestive tract of a male specimen and 7.4 mg TTX kg -1 in gonads of a female specimen. Several TTX analogues were also detected, including the 4-epi-TTX, 4,9-Anhydro-TTX, 5- 11- deoxyTTX and 6,11-dideoxyTTX. Although at low levels, STX was detected in liver of the Oceanic puffer Lagocephalus lagocephalus. Trace levels of dec- arbamoylsaxitoxin (dcSTX) were also observed in L. lagocephalus. This study reports the presence of TTX and STX in native fish from EU waters, highlighting the need for a proper understating of the origin, distribution and fate of these toxins in NE Atlantic. 1. Introduction Puffer fishes are marine, brackish and freshwater fishes that can be found mostly in tropical and subtropical waters, however some of them have been spotted in temperate waters such as the Guinean puffer fish Sphoeroides marmoratus and the Oceanic puffer Lagocephalus lagoce- phalus, being both naturally present in the NE Atlantic Ocean (Ribeiro et al. 2005; Vacchi et al. 2007). Most of puffer fish present a round body with small fins and large eyes and many species are characterized by the presence of a potent neurotoxin called Tetrodotoxin (TTX). Recognition of pufferfish as poisonous fish comes from antiquity. There are evidences to support that both the Egyptians and the Chinese from 2500–2800 BC were aware of the toxic effects of TTX from pufferfish and pufferfish eggs (Kao, 1966; Fuhrman, 1986). This natural occurring toxin can be found not only in puffer fishes, as it has been also described in several marine and terrestrial animals phylogenically unrelated, such as some species of starfish and frogs (Narahashi, 2008; Bane et al. 2014). The molecule of TTX is formed by a guanidinium moiety connected to a highly oxygenated carbon skeleton that possesses a 2,4-dioxaada- mantane portion which has five hydroxyl groups (Isbister and Kiernan, 2005). At least 26 TTX analogues have been described to naturally occur (Bane et al. 2014). TTX is a potent neurotoxin that blocks voltage- gated sodium channels in mammalian excitable neuronal and muscle cells causing paralytic poisoning and ultimately human fatalities due to respiratory and heart failure (Narahashi et al. 1967; Walker et al. 2012). Due to the presence of TTX in a wide range of animals, its origin remains debatable. In the case of puffer fish, the source of this toxin has been fixed in an endo-symbiotic bacterium, being TTX accumulated in their bodies via food chain and becoming potential toxin vectors (Yu et al. 2004; Noguchi and Arakawa, 2008; Bane et al. 2014). Although TTX occurrence and TTX content vary among species, when it is present in puffer fish it has been found to be spread among several tissues such as reproductive organs, liver, skin, muscle and in- testine (Jang and Yotsu-Yamashita, 2006; Jang et al. 2010). Never- theless, the accumulation of this toxin and its analogues along the puffer fish body has been proved to be dependent on the sex and marine https://doi.org/10.1016/j.marenvres.2019.104780 Received 20 June 2019; Received in revised form 12 August 2019; Accepted 2 September 2019 * Corresponding author. IPMA—Portuguese Institute of the Sea and Atmosphere, Av. Brasília, 1449-006, Lisbon, Portugal. E-mail address: prcosta@ipma.pt (P.R. Costa). Marine Environmental Research xxx (xxxx) xxxx 0141-1136/ © 2019 Published by Elsevier Ltd. Please cite this article as: Estefanía Pereira Pinto, et al., Marine Environmental Research, https://doi.org/10.1016/j.marenvres.2019.104780