## 3 Links between the Chemistry and Biochemistry and Toxicology of CTXs
### Stability of CTXs  
The mouse bioassay has determined that P-CTXs levels are not affected by freezing, cooking at 120 \({}^{\circ}\)C in water, drying, or salting [166]. When diluted in olive oil, 100% of the toxicity was retained at <200 \({}^{\circ}\)C [94]. Furthermore, in water, pyridine and acetic acid P-CTXs solutions were stable at 100 \({}^{\circ}\)C for at least 10 min [167]. Few studies have evaluated the stability of CTXs in various temperature or acid/basic conditions. [PERSON] and [PERSON] [59] confirmed that rapid cold alkali treatment for P-CTXs, which was used to facilitate rapid defatting of extracts, did not affect the toxicity in mice or the preparative thin layer chromatographic behaviour. Alkaline stability was later confirmed using purified P-CTX-1 [94; 167]. However, \(\sim\)50% of C-CTXs were lost upon alkaline treatment, making this rapid defatting step unsuitable for C-CTX extraction or quantitation. Based on the organic solvents used during purification, P-CTXs and C-CTXs are stable in acetone, diethyl ether, chloroform, benzene, and ethanol, with methanol preserving ciquadoxins for months [167] and most stable forms for years ([PERSON] and [PERSON], unpublished results). C-CTX-1 remained unchanged after six months of storage at \(-10\)\({}^{\circ}\)C in 50% aqueous acetonitrile, or after 4 h in 50% acetonitrile/5% trifluoracetic acid [35]. However, C-CTX-2 was converted into C-CTX-1 after six months of storage at \(-10\)\({}^{\circ}\)C in 50% aqueous acetonitrile [35]. In an acidic medium, (1 M HCl) at ambient temperatures, C-CTXs were stable [94], while the activity of P-CTXs decreased by >50% at room temperature and was completely lost at 100 \({}^{\circ}\)C [167]. [PERSON] [168] assayed C-CTXs with specific reagents and reported a total disappearance of activity when targeting -OH or a vicinal diol moiety with \(\text{HIO}_{4}\) in acetic acid for 2 h at 25 \({}^{\circ}\)C, or with acetic anhydride in pyridine 1 h at 100 \({}^{\circ}\)C. A similar loss of toxicity was observed when targeting double bonds between two adjacent carbons, with the addition of OsO\({}_{4}\) for 3 h at 25 \({}^{\circ}\)C, the addition of the oxidants KMnO\({}_{4}\) for 30 min at 25 \({}^{\circ}\)C, or H\({}_{2}\)O\({}_{2}\) into CH\({}_{3}\)COOH for 2 h at 85 \({}^{\circ}\)C, while the activity of C-CTXs in the controls remained unaffected [168]. Thus, these experiments suggest that primary or secondary hydroxyl(s) and carbon double bonds are major structural determinants of C-CTX activity. These key features are consistent with the structures of the major CTXs found in fish [35; 98; 99; 125]. The instability of C-CTX-1 in strongly acidic conditions was recently demonstrated using methanol (or acetonitrile) and a C56 methoxy congener, C-CTX-1-Me, which was structurally characterized using its mass fragmentation pathways in liquid chromatography-tandem mass spectrometry and high-resolution mass spectrometry [121; 131]. Thus, using strongly acidic conditions during sample pre-treatment for C-CTX analysis is expected to adversely affect the ability to quantitatively determine the levels of C-CTX-1 in extracts of fish or dinoflagellates [121].