The illegal practice of using cyanide (CN) as a stunning agent to collect fish for both the marine aquarium and live fish food trades has been used throughout the Indo-Pacific for over 50 years. CN fishing is destructive to all life forms within the coral reef ecosystems where it is used and is certainly one of many anthropogenic activities that have led to 95% of the reefs in the Indo-Pacific being labeled at risk for degradation and loss. A field-deployable test for detecting fish caught using CN would assist in combating the use of this destructive practice, however, no reliable and robust test exists. Further, there is little toxicokinetic data available on marine fish to support the development of such a test, yet such data is critical to establishing the concentration range and time scale over which such a test would be viable. This study presents the first direct measurement of the half-life of the metabolite thiocyanate (SCN) after pulsed exposure to CN in a marine fish. SCN was measured in the plasma of Amphiprion ocellaris after exposure to 50 ppm CN for three exposure times (20, 45, and 60 s) using HPLC-UV and a C30 column pre-treated with polyethylene glycol. Plasma SCN levels observed are dose-dependent, reflecting a longer time for conversion of CN to SCN as the dose of CN increases. SCN plasma levels reached a maximum concentration (1.2–2.3 ppm) 12–20 h after exposure to CN. The half-life for the elimination of SCN was 1.01 ± 0.26 days for 45 s exposure and 0.44 ± 0.15 days for 20 s exposure. Fish were also directly exposed to SCN (100 ppm for 11 days) and the observed half-life for SCN elimination was 0.35 ± 0.07 days. Plasma SCN levels did not return to control levels, even after 41 days when exposed to CN but did return to control levels after 48 days when exposed to SCN. The similar half-lives observed for CN and SCN exposure suggests that SCN exposure can be used as a proxy for measuring the rate of SCN elimination following CN exposure. In order for plasma SCN to be used as a marker for CN exposure, these results must be extended to other species and endogenous levels of SCN in wild caught fish must be established.

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 study 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 . Fish were pulse exposed to 50 ppm CN for 20 or 45 seconds or chronically exposed to 100 ppm SCN for 12 days. Blood plasma 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. After exposure to CN, depending on the duration of exposure, SCN plasma levels reached a maximum concentration (300–470 ppb) 0.13–0.17 days after exposure, had a 0.1 to 1.2 day half-life, and often did not return to baseline levels. The half-life of plasma SCN after direct exposure to SCN was found to be 0.13 days, similar to the CN exposure, and that SCN in the holding water would often drop below detection. Finally, we observed that when a fish, never exposed to SCN, was placed in aquarium water spiked with SCN, there was a steady decrease in aqueous SCN concentration over 24 hours until it could no longer be detected. This pattern was repeated with a second sequential dose. These results demonstrate that A. clarkii do not excrete SCN after CN exposure, but in fact can absorb low concentrations of SCN from water, refuting several publications. It appears that A. clarkii exhibit a classic two compartment model where SCN is rapidly eliminated from the blood plasma and is distributed throughout the tissue but not excreted in their urine. This study demonstrates that SCN may be used as a marker of CN exposure only if fish are tested shortly after exposure. There is species specific variability in response to CN, and studies of other taxa need to be performed before this test can be deployed in the field.