NOTE: Protocols beyood this aep are BOt withxa the eofltnaed S2S0X10 sunple. We will leek the achue of the 

 Coetraaing Officer prior to imdaiakiqg apy mrrrrrirug a^(, and woold reqoetl approval prior to aaalyus 

 Thuc procedum iDclude brevetosis radiobnaimoasuy, Fourier iraosforD infrared aaalyus, NMR, or bxmss 

 tpeOTcmtuy. 



NOTES 



|1] Bioauay: Gombiuia it^intf, or moiqmto fish, are ittcd as bioasuy ^Kdmea for aD tte|&. Assays 



■re cooduaed is 20 mL leawaier of 15% salinity uiiig 1 fish per 50 mL beaker. For lohitioas, cuspea toxxo 

  a <V*^^ in OJOl mL methaDoI at ooocemratioos equtvakst to 5% of the ongjnal sasi|^ extract. For thts-laycr 

 dirooatograpby plates, a 03 em wide oohuBD is ca b devdoped plates fron ongtn to soKeai from, and 1 cxs 

 taD fractioits are cut, crushed, and added to dni^icate iadividua] beakers mntaining fish. Lethahty is assessed 

 24 hr (or 48 hr) later aod toaddty (death '4* or bo death '-*) soled. Sensttivity (LDjd approximates 7D «tg/kg. 

 Assay at steps (5) and (7) are alinoct uniformly taadc, and may be dispensed with in favor of sample conservation. 

 Only '* ' samples proceed to the subsequent step. 



[2] Aberrant Samples: If any single sample is very oily at this stage, it is partitioned between aqueous 



methane^ and li^t petroleum by dissolvii^ the lamf^ in 100 mL 95% wrthannl and nnrarting with 3- 100 mL 

 portions of light pe&oleum, m a 500 mL separatory htnneL the three ii^ pctrolmm fractions are combined 

 in a fresh 500 mL separatory funnel and are back-extracted once with 100 mL 95% methanol/water. Both 

 nethano fraoions are combixked in a 500 mL round bonon flask and the sfdvent is removed by flasb-evqiaration. 

 The light petroleum fradioos are discarded in the solvenl f ca yv er y ( 



[i] TLC Plate Preparation for Toodcity Testing: Developed thin-layer plates are air-dried, followed 



by observation under short wave ohraviolet Ught. Using pencil, enorde in>>-absarbing bands. Remove lie plate 

 from nv Ughi and cot two grooves from origin to solvent front, placed 03 cm qaait, qiprooomately 10 cm in from 

 the sides of the plate. From origin to solvent fatrnt, cut 1 cm laments fatn the developed plate b et w uj i the 

 two parallel score marks, and cnuh each 03 cm x 1 cm silica gel rmanglr into a powder to be added tobioassay 

 beakers. 



f4] CAUTION: Brevetoxins are potent re^nratory irritants, especially when adsorbed oo siEca gel 

 particles. Take aO precau t i o ns necessary for respiratary irritants &at are riwrififd as Class I poisanous 

 ffuKtt"":* NIOSH certified panicle filtration masks may be worn in qiplicableiitnatiaas where expossre could 

 occur. Al 1 scraping operations and grinding of silica gel powd ers tbould be canied out in an ap pr o v ed 

 ventilation hood. 



120 



