Detection of Domoic Acid in Northern Anchovies and California Sea Lions Associated with an Unusual Mortality Event² Kathi A. Lefebvre, 1 Christine L. Powell, 2 Mark Busman, 2 Gregory J. Doucette, 2 Peter D. R. Moeller, 2 Joel B. Silver, 1 Peter E. Miller, 3 Margaret P. Hughes, 4 Sara Singaram, 4 Mary W. Silver 4 and Ronald S. Tjeerdema 5 1 Biology Department, University of California at Santa Cruz (UCSC), CA, USA 2 Marine Biotoxins Program, NOAA/National Ocean Service, Charleston, SC, USA 3 Monterey Bay Aquarium Research Institute, Moss Landing, CA, USA 4 Institute of Marine Science, UCSC, Santa Cruz, CA, USA 5 Department of Environmental Toxicology, University of California at Davis, CA, USA ABSTRACT The occurrence of an unusual mortality event involving California sea lions (Zalophus californianus) along the central California coast in May 1998 was recently reported. The potent neurotoxin domoic acid (DA), produced naturally by the diatom Pseudo-nitzschia australis and transmitted to the sea lions via planktivorous northern anchovies (Engraulis mordax), was identi®ed as the probable causative agent. Details of DA analyses for anchovy tissues and sea lion feces are described. Domoic acid levels were estimated in anchovy samples by HPLC-UV, and in sea lion feces using the same method as well as a microplate receptor binding assay, with absolute con®rmation by tandem mass spectrometry. The highest DA concentrations in anchovies occurred in the viscera (223  5 mg DA g 1 ), exceeding values in the body tissues by seven-fold and suggesting minimal bioaccumulation of DA in anchovy tissue. HPLC values for DA in sea lion fecal material (ranging from 152 to 136.5 mg DA g 1 ) required correction for interference from an unidenti®ed compound. Inter-laboratory comparisons of HPLC data showed close quantitative agreement. Fecal DA activity determined using the receptor binding assay corresponded with HPLC values to within a factor of two. Finally, our detection of P. australis frustules, via scanning electron microscopy, in both anchovy viscera and fecal material from sea lions exhibiting seizures provides corroborating evidence that this toxic algal species was involved in this unusual sea lion mortality event. Copyright  1999 John Wiley & Sons, Ltd. Key words: amnesic shell®sh poison (ASP); high performance liquid chromatography (HPLC); tandem mass spectrometry (LC-MS/MS); California sea lion (Zalophus californianus); Northern anchovy (Engraulis mordax); diatom (Pseudo-nitzschia); receptor binding assay; trophic transfer; domoic acid INTRODUCTION Domoic acid (DA) is a potent neuroexcitatory amino acid naturally produced by diatoms of the genus Pseudo- nitzschia. During toxic diatom blooms DA has been found to accumulate in the digestive tracts of filter- feeding marine species such as northern anchovies (Engraulis mordax), blue mussels (Mytilus edulis) and razor clams (Siliqua patula) (Quilliam and Wright, 1989; Wekell et al., 1994; Altwein et al., 1995). These species may subsequently act as vectors of the toxin to Natural Toxins Nat. Toxins 7: 85±92 (1999) *Correspondence to: K. A. Lefebvre, Biology Department, University of California at Santa Cruz (UCSC) CA 95064, U.S.A. Sponsors: California Sea Grant; Dr Earl Myers and Ethel Myers Oceanographic and Marine Biology Trust. ² The National Ocean Service (NOS) does not approve, recom- mend, or endorse any product or material mentioned in this publication. No reference shall be made to NOS, or to this publication furnished by NOS, in any advertising or sales promotion which would indicate or imply that NOS approves, recommends, or endorses any product or material mentioned herein or which has as its purpose any intent to cause directly or indirectly the advertised product to be used or purchased because of NOS publication. CCC 1056±9014/99/030085±08 $17.50 Copyright  1999 John Wiley & Sons, Ltd. Received 13 April 1999; Accepted 26 April 1999. RESEARCH ARTICLE