Abstract-Biological assays using aquatic invertebrates and fish do not necessarily predict protection levels for primary producers such as algae and aquatic macrophytes. State regulatory programs may not be protecting the environment from many phytotoxic compounds. Recent modifications of the U.S. Environmental Protection Agency's algal test were evaluated for their potential use as a regulatory assay. Primary goals of this investigation were to downsize the algal assay and to evaluate various methods of automation. Disposable microplates with 2-ml sample wells were evaluated as an alternative testing chamber for the 96-h growth inhibition test with Raphidocelis subcapitata (formerly known as Selenastrum capricornutum). We compared the standardized Erlenmeyer flask test to the microplate test using CuCl 2 , NaCl, phenol, ZnCl 2 , and a surfactant. We noted improved control performance with the microplate test, whereas median inhibitory concentration values were similar for both methods. Other procedures we addressed included the use of EDTA, filtration of samples, and the effect of colored samples on algal growth. We also evaluated growth estimates by comparing manual cell counting to more automated growth estimates using fluorescence and absorbance endpoints. The use of fluorescence and absorbance measurements demonstrated reductions in replicate variability over manual counting and may offer time-saving alternatives for laboratory analysts.

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We studied the dynamic changes of cellular thiols and the extracellular release of glutathione (GSH) during growth of the marine diatom Thalassiosira weissflogii under varying levels of copper (Cu) addition in both metalbuffered (with EDTA) and unbuffered (without EDTA) media. The cell quotas of both thiols and pigments decreased with growth time; however, pigment-normalized cellular thiol concentrations at a given Cu exposure level were more or less conservative. In both media, specific growth rates of greater than 1 per day were obtained at total inorganic Cu concentrations of less than 80 nmol L Ϫ1; however, at higher Cu levels, cell growth was significantly suppressed. A dose-response relationship was observed between the phytochelatin-2 : GSH ratio and the Cu exposure level during the exponential growth period. GSH was released from the phytoplankton cells at similar concentrations in both media; therefore, the presence of a synthetic metal chelator does not affect thiol release, substantiating the premise that algae respond to inorganic species of Cu. GSH release was closely related to Cu-induced cell membrane damage. The extracellular GSH release rate was higher in normally grown cultures than in growth-limited cultures but lower than in growth-suppressed cultures. The excretion of GSH apparently reflects physiological conditions during algal growth rather than an enzymatic response of the algae to control trace metal speciation in the media. Therefore, the role of GSH in metal complexation in ambient waters is probably an inadvertent by-product of this process, although it could contribute a significant portion of Cu-complexing ligands in open ocean waters.

We investigated factors influencing the presence of the thiol glutathione (GSH) in estuarine waters. Our study addressed thiol phase-association, the biological release from algal cultures, and the role of copper in both thiol release and preservation. Our measurements in three diverse estuaries in the continental United States (San Diego Bay, Cape Fear Estuary, and Norfolk Estuary) show that dissolved GSH, present at sub-nanomolar levels, is preferentially partitioned into the ultra-filtrate fraction (<1 kDa) in comparison with dissolved organic carbon (DOC). Concentrations of GSH generally increased with increases in total copper (Cu)levels, although large variability was observed among estuaries. In 30-h exposure experiments, release of dissolved GSH from the diatom Thalassiosira weissflogii into organic ligand-free experimental media was a strong function of added Cu concentration. The released GSH increased from about 0.02 to 0.27 fmol/cell as Cu was increased from the background level (0.5 nM) to 310 nM in the modified Aquil media. However, excretion of GSH was lower (up to 0.13 fmol/cell) when cells were grown in surface waters of San Diego Bay, despite much higher total Cu concentrations. Experiments conducted in-situ in San Diego Bay water indicated that high concentrations of added Cu destabilized GSH, while both Mn(II) and natural colloids promoted GSH stability. In contrast, laboratory experiments in synthetic media indicated that moderate levels of added Cu enhanced GSH stability.

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