## 7 Conclusions  
The recent finding that C-CTX-5, produced by _G. silvae_ and _G. caribeus_, is the likely algal precursor of fish C-CTX and undergoes biotransformation to C-CTX-1/-2 in fish liver microsomes fills a significant knowledge gap in understanding the origins of Caribbean C-CTXs. This finding provides a coherent link to C-CTX-1, which typically dominates ciguotoxic fish from the Caribbean. Importantly, C-CTX-5 from _G. silvae_ was confirmed to be a voltage-gated sodium channel activator, indicating its potential to contribute to cigarette. Despite these advances, the relative potencies of new Caribbean ciguatoxins still remains to be determined using pure and/or calibrated standards. However, until these become available, an estimation of total CTX activity based on voltage-gated sodium channel activity (ideally human sodium channels) should be used to quantify ciguatera risk when multiple contaminating congeners are present. Recent developments in LC-MS/MS allow for confirmation of C-CTXs in fish from the Caribbean Sea and Northeast Atlantic. Quantitation of C-CTX-1 is dependent on the availability of reference materials, which continue to limit analytical approaches, including the development of new extraction and purification procedures. The recent use of high-resolution mass spectrometry also provides an important contribution to chemical and toxin profile analysis in fish and dinoflagellates. However, it is not appropriate for public health controls, especially in small, under-resourced islands where cigarette is a major public health problem. The lack of commercially available certified C-CTX reference materials is currently the most critical concern. This constitutes the main difficulty in developing an official LC-MS monitoring program for Caribbean CTXs. Finally, since the first studies in the 1950s, cigarette researchers initially investigated the origins, distribution, chemistry and detection of P-CTXs. Despite significant, more recent progress, Caribbean ciguatoxins remain less understood, in part due to the multiplicity and variability of the congeners involved. This is reinforced by the diversity of associated vectors, dinoflagellates, and fish species at risk, and by the variability of ciguatoxic effects and responses in humans. This complexity likely reflects differences in food chain-associated detoxification mechanisms, which are only now being studied. However, despite this complexity, the clinical features of Caribbean ciguatera have remained constant over the last 50 years, supporting the view that appropriate toxicity measures can accurately reflect cigarette risk. To conclude, the methodological approach used for monitoring ciguatoxins usually includes two-tiers, with a first tier using a biological assay screen and the second tier using analytical methods. Protocols that have been developed for the identification and/or quantification of CTXs were compared in this review, including basic (HPLC-MS) to more sophisticated (UHPLC-MS/MS or HRMS) approaches. Each approach has advantages, disadvantages, and contributions to the field discussed. However, to control fish toxicity within a public health framework, alternative bioassays can more simply account for the multiplicity of similar toxins that often contribute to human poisoning. This includes, for example, the use of the mouse bioassay to control the shellfish toxins (50 lipophilic toxins with a common unique backbone) responsible for paralytic shellfish poisoning (PSP), which was the reference biological method validated by the AOAC (Association of Official Analytical Chemists) [213] and used in Europe until January 2019 [214; 215; 216; 217]. Recently, the MBA was replaced by the HPLC-fluorescence method developed by [PERSON] et al., which is now the official reference method for PSP detection in the European Union [217].