Novel Fluorometric Method for the Determination of Production Rate and Steady-State Concentration of Photochemically Generated Superoxide Radical in Seawater Using 3',6'-(Diphenylphosphinyl)fluorescein.

Superoxide radical (O2(•-)) is an important reactive oxygen species in seawater. Measurements of its production rates and steady-state concentrations generated by photochemical processes have been a Herculean task over the years. In this study, a probe - 3'6'-(diphenylphosphinyl)fluorescein (PF-1) - was used to trap photochemically generated O2(•-) in seawater, thereby yielding fluorescein. The fluorescein produced was measured by an isocratic fluorescence HPLC at excitation/emission wavelengths of 490/513 nm, respectively. The reaction rate constant of PF-1 with O2(•-) (kPF-1) was pH-dependent: (3.2-23.5) × 10(7) M(-1) s(-1) at pHTOT 7.65-8.50. By applying appropriate equations, both the production rate and the steady-state concentration of O2(•-) generated by photochemical reactions in the seawater were quantified. Under the optimized experimental conditions, fluorescein standards (3-50 nM) exhibited linearity in the seawater by HPLC. The photoformation of fluorescein, due to the reaction of PF-1 with the O2(•-) photochemically produced in the seawater, was linear within the 20 min irradiation. The detection limit of the fluorescein photoformation rate was 0.03 pM s(-1), defined as 3σ of the lowest standard fluorescein concentration per 20 min irradiation. Using this value, the yield of fluorescein, and the fraction of O2(•-) that reacted with PF-1 in the seawater, the detection limit of the O2(•-) photoformation rate was 1.78 pM s(-1). Superoxide measurements using the proposed method were relatively unaffected by the potential interfering species in seawater. Application of the proposed method to ten (10) seawater samples from the Seto Inland Sea, Japan, resulted in measured O2(•-) photoformation rates of 3.1-8.5 nM s(-1), with steady-state concentrations ranging (0.06-0.3) × 10(-10) M. The method is simple, requires no technical sample preparation, and can be used to analyze a large number of samples.

Photoformation rate, steady-state concentration and lifetime of nitric oxide radical (NO·) in a eutrophic river in Higashi-Hiroshima, Japan.

Monthly measurements (January-December 2013) of the photoformation rate, steady-state concentration and lifetime of nitric oxide radical (NO·) in the Kurose River in Higashi-Hiroshima City, Japan, were obtained. Each month, river water samples were collected at six different stations (upstream to downstream). NO· was quantified using 4, 5-diaminofluorescein-2 (DAF-2) as a probe and triazolofluorescein (DAF-2T) as a standard. Results show that NO· photoformation rate ranged from 0.01 to 35.4 (×10(-10) M s(-1)). The radical steady-state concentration in the river ranged from 0.02 to 68.5 (×10(-11) M). There was a strong correlation (r(2)=0.95) between NO· photoformation rate and the nitrite concentration in the river suggesting that this anion is a major NO· precursor. On average, 98% of the photoformed NO· came from river nitrite, and this was calculated using the photoformation rate constant {5.7×10(-5) M(NO·)s(-1) M(NO2(-))(-1)} of NO· from the anion concentration found in the study. The NO· lifetime ranged from 0.05 to 1.3 s in the river and remained fairly stable in the upstream and downstream samples. The ·OH radical, which was quantified during the study, had a photoformation rate of 0.01-13.4 (×10(-10) M s(-1)) and a steady-state concentration of 0.04-119 (×10(-16) M) with a lifetime that ranged from 0.3 to 22.5 (×10(-6) s). ·OH only accounted for ⩽0.0011% of the total NO· scavenged, showing that it was not a major sink for river NO·.