The toxicity of the greater blue-ringed octopus Hapalochlaena lunulata, whose bite is fatal to humans, was examined to better understand and prevent deaths from accidental bites. Living specimens were collected from tide pools on Ishigaki Island, Okinawa Prefecture, Japan, in November and December of 2015, 2016, and 2017. The specimens were examined for the anatomical distribution of the toxicity, which was expressed in terms of mouse units (MU), by the standard bioassay method for tetrodotoxin (TTX) in Japan. Paralytic toxicity to mice was detected in all of the soft parts. The posterior salivary glands exhibited the highest toxicity score with a maximum level of 9276 MU/g, which was classified as “strongly toxic” (more than 1000 MU/g tissue) according to the classification of toxicity established by the Ministry of Health, Labor and Welfare of Japan, followed by the hepatopancreas (21.1 to 734.3 MU/g), gonads (not detectable to 167.6 MU/g), arms (5.3 to 130.2 MU/g), and other body areas (17.3 to 107.4 MU/g). Next, the toxin from the salivary glands was partially purified by a Sep-Pak C18 cartridge and an Amicon Ultra Centrifugal Filter with a 3000-Da cut-off, and analyzed by liquid chromatography-mass spectrometry (LC-MS) equipped with a φ2.0 × 150-mm (5 μm) TSKgel Amide-80 column (Tosoh, Tokyo, Japan) with a mixture of 16 mM ammonium formate buffer (pH 5.5) and acetonitrile (ratio 3:7, v/v) as a mobile phase. This study aimed to clarify the toxicity and the composition of TTX and its derivatives in this toxic octopus. The main toxin in this toxic octopus was identified as TTX, along with 4-epi TTX, 4, 9-anhydroTTX and 6-epi TTX. Further, the toxicity of this species is also significant from a food hygiene point of view.

From September 2007 to January 2008, a total of 66 specimens of 7 gastropod species, Nassarius (Alectrion) glans (n=22), Bufonaria rana (n=11), Ficus subintermedia (n=10), Stellaria (Onustus) exutus (n=8), Tonna luteostoma (n=7), Hemifusus tuba (n=4) and Semicassis bisulcata persimilis (n=4), were collected from Tachibana Bay, Nagasaki Prefecture, Japan, and their toxicity was determined by mouse bioassay. Among the gastropods tested, all N. glans specimens were toxic, whereas no other species showed toxicity of more than 5 MU/g. The toxicity scores of N. glans were very high; 48-2,730 MU/g(775+/-615 MU/g)in the muscle, and 16-10,200 MU/g(1,490+/-2,530 MU/g)in the viscera, including digestive gland. Interestingly, toxin was localized in the muscle in 13 of 22 specimens, where the total toxicity of the muscle (725-9,860 MU/individual) was 5.9-110 times higher than that of the viscera. LC/MS analysis demonstrated that the toxin of N. glans consisted mainly of TTX, which accounting for about 60-65% of the total toxicity. As for the remaining toxicity, participation of 11-oxoTTX was suggested. No paralytic shellfish poison was detected in HPLC-FLD analysis.