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Mid-ocean ballast water exchange is mandatory for ships discharging foreign ballast in US territorial waters in order to reduce the risk of biological invasions. However, a reliable tool for determining whether the procedure took place is lacking. We investigated chromophoric dissolved organic matter (CDOM) fluorescence as a tracer of mid-ocean exchange on nine research cruises out of Asia, Europe, and the USA, focusing on challenging source conditions (high salinity, low CDOM). Using parallel factor analysis, we identified nine independent fluorescent components present in varying concentrations in the ocean and in ballast water. One component was sufficient for predicting the coastal vs oceanic source of most ballast water samples. Across nine cruises, thresholds (1.7 and 0.7 ppb quinine sulfate equivalent units) at two fixed wavelength pairs (lambda(ex)/lambda(em) = 320/414 and 370/496 nm, respectively) discriminated coastal from oceanic ballast water in >95% of samples (N = 514). Our results suggest that single- and dual-channel fluorometers could be optimized for verifying ballast water exchange.