LOCALIZATION OF OXYTETRACYCLINE IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE) 1 Roberta J. Garcia Marine Estuarine Environmental Sciences Program, University of Maryland Baltimore, Baltimore, Maryland 21201, USA Aquatic Pathobiology Center, VA-MD Regional College of Veterinary Medicine, Maryland Campus, 8075 Greenmead Drive, College Park, Maryland 20742, USA Andrew S. Kane Emerging Pathogens Institute, PO Box 100009, University of Florida, Gainesville, Florida 32610-0009, USA David Petullo Center for Veterinary Medicine, FDA, 7500 Standish Place, Rockville, Maryland 20855, USA and Renate Reimschuessel 2 Center for Veterinary Medicine, FDA, 8401 Muirkirk Road, Laurel, Maryland 20708, USA Oxytetracycline (OTC) is an important antimicro- bial used in aquaculture. However, residues of OTC have been isolated from nontarget aquatic organ- isms, sediments, and water located near aquaculture facilities. Identifying OTC in plant material is partic- ularly difficult due to interference from pigments and polyphenol substances but is important espe- cially for algae since they are a primary food source for fish in early life stages. In this study, we describe the effect of OTC (0.1, 1, 10, 25, 50, 100 lg Æ mL )1 ) on cell growth, and the localization of OTC (0, 1, 25, 100 lg Æ mL )1 ) in vacuoles of Chlamydomonas reinhardtii P. A. Dang. (wildtype, ATCC 18798). We also present a method for semiquantifying OTC in living cells using fluorescent microscopy and Adobe Photoshop. We exposed algal cells to OTC and sam- pled after 2 or 7 d exposure. On day 7, OTC signifi- cantly inhibited algal growth at 1, 10, 25, 50, and 100 lg Æ mL )1 . When viewed with fluorescent microscopy, cells exposed to the 25 and 100 lg Æ mL )1 contained yellow fluorescent areas, £1 lm in diameter that were easily discernable against the red fluorescence of the intracellular chl. The fluorescent areas corresponded to small spheri- cal vacuoles (i.e., polyphosphate bodies that contain calcium and magnesium complexed with polyphos- phate) seen in the cells by LM. Since OTC has a high affinity for divalent cations, we suggest that OTC is localized in these vacuoles. Key index words: Chlamydomonas reinhardtii; oxy- tetracycline; vacuoles Abbreviations: ATCC, American Type Culture Col- lection; CFR, Code of Federal Regulations; OTC, oxytetracycline; SAS, Statistical Analysis Software; U.S. EPA, United States Environmental Protection Agency; U.S. FDA, United States Food and Drug Administration OTC is currently one of four antibiotics that are approved by the United States Food and Drug Administration (U.S. FDA) for aquaculture, and it is typically administered to fish through medicated feed (Greenlees 1997, U.S. FDA 2006). OTC is poorly absorbed and mostly excreted in an active form. Residues of OTC that exceed daily tolerances set by the United States Environmental Protection Agency (U.S. EPA) and FDA for some regulated commodities have been identified in a number of nontarget organisms, including plants located near aquaculture facilities (40 CFR 180.337 2007, 21 CFR 556.500 2004, Capone et al. 1996, Pouliquen et al. 1996, Delepee et al. 2004, U.S. EPA 2006a). It is essential to understand the effects of OTC in aqua- tic ecosystems, since photosynthetic organisms form the basis for aquatic food webs (Lalli and Parsons 1997, WHO 1999). C. reinhardtii is a well-studied green alga that is a powerful model organism for studying cell and molecular biology, genetics, plant physiology, and biotechnology (Davies and Grossman 1998, Ghirardi et al. 2000, Raven and Girrard-Bascou 2001, Prosc- hold et al. 2005). It has also been used extensively in toxicity assays, including assays for many organic pol- lutants that are of environmental concern (pesticides, herbicides, heavy metals, etc.), as well as some that are particularly relevant to aquaculture (formalin, 1 Received 31 August 2007. Accepted 14 March 2008. 2 Author for correspondence: e-mail renate.reimschuessel@ fda.hhs.gov. J. Phycol. 44, 1282–1289 (2008) Ó 2008 Phycological Society of America DOI: 10.1111/j.1529-8817.2008.00574.x 1282