Development of a Toxicity Identification Evaluation Protocol Using Chlorophyll-a Fluorescence in a Marine Microalga D. Strom Æ P. J. Ralph Æ J. L. Stauber Received: 29 November 2007 / Accepted: 31 March 2008 / Published online: 1 May 2008 Ó Springer Science+Business Media, LLC 2008 Abstract Growth inhibition bioassays with the microalga Nitzschia closterium have recently been applied in marine Toxicity Identification Evaluation (TIE) testing. However, the 48-h test duration can result in substantial loss of tox- icants over time, which might lead to an underestimation of the sample toxicity. Although shorter-term microalgal bioassays can minimize such losses, there are few bioas- says available and none are adapted for marine TIE testing. The acute (5-min) chlorophyll-a fluorescence bioassay is one alternative; however, this bioassay was developed for detecting herbicides in freshwater aquatic systems and its suitability for marine TIE testing was not known. In this study, a chlorophyll-a fluorescence bioassay using the marine microalga Isochrysis galbana was able to detect contaminants other than herbicides at environmentally relevant concentrations and tolerated the physical and chemical manipulations needed for a Phase I TIE. Phase I TIE procedures were successfully developed using this chlorophyll-a fluorescence bioassay and used to identify all classes of contaminants present in a synthetic mixture of known chemical composition. In addition, TIEs with both the acute fluorescence bioassay and the standard growth inhibition bioassay identified the same classes of toxicants in a sample of an unknown complex effluent. Even though the acute chlorophyll-a fluorescence end point was less sensitive than the chronic cell division end point, TIEs with the chlorophyll-a fluorescence bioassay provided a rapid and attractive alternative to longer-duration bioassays. Introduction Contaminants are often released into the environment as part of complex effluents that contain mixtures of com- pounds, only some of which might be toxic. Direct toxicity assessment (DTA) alone cannot identify the chemicals responsible for toxicity (US EPA 1991). However, DTA in combination with Toxicity Identification Evaluation (TIE) procedures, which manipulate the chemical and physical characteristics of the effluent, have been widely used to characterize toxicants in complex effluents (US EPA 1996). These combined methods of toxicant assessment have allowed the implementation of new measures to reduce the toxicity of effluents, such as alternative manufacturing processes, controlling or limiting inputs of the toxicants to wastewaters, or developing waste-treatment systems to remove toxic components (Ankley and Burkhard 1992). Toxicity identification evaluation involves three phases. In Phase I (Characterization), the physical and chemical nature of the toxic constituent is broadly characterized. In Phase II (Identification), the toxicants are identified using specific analytical techniques appropriate for the chemical classes identified in Phase I. Phase III (Confirmation) provides evidence to confirm that the suspected contami- nants are the true cause of toxicity (US EPA 1991). Toxicity identification evaluation protocols using a range of marine and freshwater aquatic and benthic species have been developed and applied to identify specific causes of toxicity in effluents, leachates, and sediments (Bailey et al. 1995; Burgess et al. 1995; Ho et al. 1997). Despite the D. Strom  P. J. Ralph Institute for Water and Environmental Resource Management, University of Technology, Sydney, P.O. Box 123, Broadway, NSW 2007, Australia D. Strom (&)  J. L. Stauber Centre for Environmental Contaminants Research, CSIRO Land and Water, Private Mailbag 7, Bangor, NSW 2234, Australia e-mail: David.Strom1@csiro.au 123 Arch Environ Contam Toxicol (2009) 56:30–38 DOI 10.1007/s00244-008-9174-x