Toxicokinetics of Cyanide and its Metabolites, and Related Gene Expression in Marine Fish: Implications Towards Combating Cyanide Fishing Through Detection

The illegal practice of cyanide fishing continues to damage coral reef ecosystems throughout the Indo-Pacific. To combat this destructive fishing method, a simple, reliable test to detect whether or not a fish has been captured using cyanide (CN) is needed. This thesis analyzed the toxicokinetics of acute pulsed CN exposure as well as chronic exposure to thiocyanate (SCN), the major metabolite of CN, in the clownfish species, Amphiprion clarkii. This work also addressed long-term gene expression post-exposure to CN in marine fish, Amphiprion ocellaris (common clownfish), Centropyge bicolor (Bicolor angelfish) and Chaetodon ephippium (Saddle butterflyfish). Fish were pulse exposed to 50 ppm CN for 20 or 45 seconds or chronically exposed to 100ppm SCN for 12 days. Finally, I tested SCN concentrations in aquaria water of exposed fish and SCN spiked aquaria water with non-exposed fish. Gene expression was quantified by real-time PCR amplifications carried out on a 7300 Real-Time PCR System (Applied Biosystems) by using QuantiTect® SYBR Green PCR +UNG Kit (Qiagen) along with species-specific primers for target and reference genes that were previously validated, following manufacturer’s protocols. Blood plasma and aquaria water levels of SCN were measured following derivatization to SCN-bimane using an Acquity UPLC I-Class and Q-Exactive hybrid Quadrupole-Orbitrap HRAM mass spectrometer or directly by HPLC-UV. This study demonstrates that both SCN and gene expression may be used as a marker of cyanide exposure only if fish are tested shortly after exposure. However, there may be species-specific variability in response to CN, and studies of other taxa need to be performed before a test can be deployed in the field