## 6 Discussion and Perspectives
### Multiplicity of Caribbean Cgiuotoxins  
A key point emerging from the literature is the presence of multiple Caribbean ciquatoxins in fish from the Atlantic Ocean and Caribbean Sea. This multiplicity, reminiscent of P-CTX multiplicity [33; 119], could be due to strain-dependent variations in toxin content of dinoflagellates belonging to the genus _Gambierdiscus_, as well as differences in the metabolic activities of fish, especially in their microsomal detoxication capacities including glucuronidation [165]. For decades, research has focused on ciguatera fish poisoning in the Lesser and Greater Antilles, as well as Southern Florida. Since 2004, fish caught in the Northeast Atlantic (Canary Islands and Archipelago of Madeira) have been associated with ciguatera-type poisoning. So far, no P-CTXs have been identified in fish from this new ciguatera area. To date, C-CTX-1 (C\({}_{62}\)H\({}_{92}\)O\({}_{19}\), 1140.6 Da) and occasionally its isomer C-CTX-2 have been found in fish from the Caribbean Sea ([22; 23; 35; 37; 38; 39; 41; 42; 108; 172; 174; 200]), and now from the Canaries Islands and Madeira Archipelago [109; 110; 112; 130; 131; 155; 175; 176]. Only C-CTX-1 (1140.6 Da) and one of its isomers, C-CTX-2, have been structurally characterized using NMR. Their in vivo toxicity in mice is known, with lethal doses of 3.6 and 1 ug\(\cdot\)kg\({}^{-1}\), respectively, and their mode of action has been demonstrated. They target receptor site 5 of voltage-gated sodium channels. Few studies relating to the toxin concentrations of C-CTX-1 have been carried out with a calibration, using a standard of C-CTX-, due to limited amounts of C-CTX-1-purified material [23; 41; 42; 131; 200]. [PERSON] at al. estimated that \(\sim\)50% of the toxicity determined using the MBA was explained by the presence of C-CTX-1 in 30 fish from the Caribbean [200]. Thus, C-CTX-1 may not be the only biomarker that can confirm the presence of C-CTXs in fish flesh, as C-CTX-1 and other C-CTX analogues have been detected in fish from the Lesser Antilles [23; 37; 38; 39; 41; 42; 108] or the Northeast Atlantic ocean [109; 110; 131; 175]. By using HRMS combined with chemical reactions, some C-CTX congeners have been tentatively characterised (see Section 5.3.1). The congeners C-CTX-1159, C-CTX-3/-4, C-CTX-1157, and C-CTX-1127, were identified in fractions that showed toxic activity either using CBA-N2a or RBAs (Table 7). However, due to their similar structures, they are difficult to separate in reverse-phase LC, posing a challenge in confirming their toxicity with certainty. Finally, among the analogues, two groups of C-CTX congeners have been recurrently reported in fish from the Atlantic zone:  
* Compounds with [M+H]\({}^{+}\)_at_\(m/z\) 1143.6: isomers previously known as C-CTX-1143 showed no significant activity in radio-ligand binding assays [23; 41]. However, RBA may not be sensitive enough to detect low amounts in fish, and another study identified an analogue, C-CTX-1143, using LC-MS/MS in a positive fraction by CBA-N2a [108]. Two anomers, C-CTX-3 and C-CTX-4 (C\({}_{62}\)H\({}_{94}\)O\({}_{19}\)), have been characterised using HRMS as the reduced forms of C-CTX-1 [120; 131]. Their toxic potential and their role in fish total toxicity are yet to be clearly demonstrated due to their unstable hemiketal structure. * Compounds with [M+H]\({}^{+}\) at \(m/z\) 1157.6: two compounds with [M+H]\({}^{+}\) at \(m/z\) 1157.6 were recently identified using LC-HRMS, 17-hydroxy-C-CTX-1 ([M+H]\({}^{+}\)\(m/z\) 1157.6255) with a possible location of the -OH group in the E-ring, and putative 17-hydroxy-50,51-didehydro-C-CTX-3 ([M+H]\({}^{+}\)\(m/z\) 1157.6255) [131]. An analogue C-CTX-1157 was also reported in a CTX-positive fraction analysed using CBA-N2a. [108] and in a positive fraction analysed using RBA (unpublished data from [40]). C-CTX profiles may vary depending on the fish species and their regional distribution.