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.

Abstract-In whole effluent toxicity tests, organisms are exposed to various effluent concentrations for a specific time period to estimate the potential effects of an effluent on a receiving stream. Laboratories typically have good success performing valid chronic toxicity tests. However, some difficulty in conducting valid chronic whole effluent toxicity tests with Pimephales promelas (fathead minnow) has been encountered as a result of sporadic control mortality. Some investigators report an interference that causes anomalous patterns of survival in chronic fathead minnow tests. This interference has been termed sporadic mortality phenomenon. Characteristics of sporadic mortality phenomenon include high variability among replicates, nonmonotonic dose responses, mortality beginning on or about day 4 of the test, and fungal growths often observed on the larvae. Histopathologic examinations often indicate bacterial and/or fungal infections on fish exhibiting symptoms of sporadic mortality phenomenon. The most plausible explanation of sporadic mortality phenomenon is a naturally occurring pathogen or pathogens that interfere with the test method. This interference may invalidate tests or falsely indicate toxicity. Sporadic mortality phenomenon can be reduced or eliminated by sample treatments intended to inactivate (heating, antibiotics, or ultraviolet light) or remove (filtration) pathogenic microorganisms. These methods must be used with forethought because of their potential to alter the toxicity of a sample.

Whole effluent toxicity testing is used to evaluate the discharge of materials that may be harmful to indigenous aquatic life. Unlike most environmental analyses, receiving water (the water body into which the effluent is discharged) often is used as dilution water in whole effluent toxicity tests to simulate the aquatic environment into which the effluent is introduced. In approximately 26% of whole effluent toxicity tests conducted by Wisconsin (USA) certified labs between 1988 and 1998, a pathogenic effect caused failure of the receiving water controls during the fathead minnow chronic test (i.e., > 20% mortality). We performed microbiological work to isolate pathogenic organisms from receiving waters, the fish, and their food. We found pathogenic organisms such as Flexibacter columnaris, Aeromonas hydrophila, and Flavobacter spp. to be ubiquitous and were not able to remove them from the test (e.g., through decontamination of the fish food and carefully following sterility procedures). To eliminate the pathogenic effect, we evaluated manipulations of the sample and the test method including filtering receiving water, irradiating receiving water, using older fish (48 h), using clean test beakers each day of the test, and using smaller test beakers (30 ml) with two fish per beaker. In samples demonstrating the pathogenic effect, most of these manipulations significantly reduced mortality. The use of smaller tests cups was significantly better at reducing the effect than all of the other sample and method manipulations. These results indicate that a simple method modification to the fathead minnow chronic test will improve test reliability when diluting effluents with receiving waters.

Abstract-In whole effluent toxicity tests, organisms are exposed to various effluent concentrations for a specific time period to estimate the potential effects of an effluent on a receiving stream. Laboratories typically have good success performing valid chronic toxicity tests. However, some difficulty in conducting valid chronic whole effluent toxicity tests with Pimephales promelas (fathead minnow) has been encountered as a result of sporadic control mortality. Some investigators report an interference that causes anomalous patterns of survival in chronic fathead minnow tests. This interference has been termed sporadic mortality phenomenon. Characteristics of sporadic mortality phenomenon include high variability among replicates, nonmonotonic dose responses, mortality beginning on or about day 4 of the test, and fungal growths often observed on the larvae. Histopathologic examinations often indicate bacterial and/or fungal infections on fish exhibiting symptoms of sporadic mortality phenomenon. The most plausible explanation of sporadic mortality phenomenon is a naturally occurring pathogen or pathogens that interfere with the test method. This interference may invalidate tests or falsely indicate toxicity. Sporadic mortality phenomenon can be reduced or eliminated by sample treatments intended to inactivate (heating, antibiotics, or ultraviolet light) or remove (filtration) pathogenic microorganisms. These methods must be used with forethought because of their potential to alter the toxicity of a sample.

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