Superoxide (O 2 2 ) and hydrogen peroxide (H 2 O 2 ) concentrations ranging from 87 to 1120 pmol L 21 and 5 to 107 nmol L 21 , respectively, were measured in samples of surface water from the Great Barrier Reef (GBR) lagoon in the absence of photochemistry. Nonphotochemical, particle-associated net production rates of O 2 2 ranging from 1 to 16 pmol L 21 s 21 were also determined and calculated to be similar in magnitude to the likely abiotic photochemical O 2 2 production rates in GBR surface waters. Manipulative experiments using 0.22-mm filtration and addition of biological inhibitors demonstrated that the majority of this particle-associated production was biological and likely driven by photosynthetic organisms. Pseudo-first-order O

Significant production of superoxide, a known reductant of both inorganic and organically complexed iron(III), occurs in natural systems by both biotic and abiotic pathways. We have investigated the generation of superoxide by Chattonella marina (Subrahman.) Y. Hara et Chihara, a phytoplankton taxon known to produce high levels of this reactive oxygen species, and examined the role of superoxide in the acquisition of iron by this organism. Additionally, a generalized model for iron acquisition by C. marina has been developed, which includes three pathways of iron acquisition from organically complexed iron(III): nondissociative reductive uptake, dissociative reductive uptake, and nonreductive dissociative uptake. The model is shown to be particularly useful in ascertaining the relative importance of these various iron-uptake pathways as a function of solution parameters including concentration and iron-binding strength of the organic ligand and superoxide concentration. Our results suggest that superoxide can participate in the C. marina ironuptake process when iron is complexed to weak ligands, such as citrate, but plays only a minor role when iron is bound to a strong ligand. It thus appears that facilitation of iron acquisition is not the sole purpose of superoxide production by these organisms.

A new rapid and highly sensitive microplate-based chemiluminescence method for the detection of extracellular production of superoxide by phytoplankton cultures has been developed. Replicates of the sample, blank, and three standards were placed into 96-well plates and the chemiluminescence was detected with a microplate luminometer. The method was tested on Trichodesmium erythraeum cultures using two superoxidespecific chemiluminescent probes, MCLA and the closely related compound red-CLA, which emit light at 460 and 610 nm, respectively, in the presence of superoxide. Calibration of the chemiluminescent signal is performed individually for each sample using the xanthine/xanthine oxidase system by adding a fixed concentration of xanthine (50 µM) and variable concentrations of xanthine oxidase (0.001-0.5 units L -1 ). The method is selective for superoxide, and the detection limits are as low as 1.41 pmol/s for MCLA and 76 fmol s -1 for red-CLA, with limits of quantification of 4.70 pmol/s for MCLA and 253 fmol s -1 for red-CLA. Application of the new method to the determination of extracellular superoxide production by the prolific superoxide-producing phytoplankton Chattonella marina yielded results comparable to those obtained using an existing flow injection analysis method. The use of microplates offers several advantages over existing methods, including a short analysis time of 10 min for triplicates of blank, sample, and standards; good reliability of signals; and use of small sample volumes.