T: Toxicology & Chemical Food Safety Detection of Warfare Agents in Liquid Foods Using the Brine Shrimp Lethality Assay Stephen E. Lumor, Francisco Diez-Gonzalez, and Theodore P. Labuza Abstract: The brine shrimp lethality assay (BSLA) was used for rapid and non-specific detection of biological and chemical warfare agents at concentrations considerably below that which will cause harm to humans. Warfare agents detected include T-2 toxin, trimethylsilyl cyanide, and commercially available pesticides such as dichlorvos, diazinon, dursban, malathion, and parathion. The assay was performed by introducing 50 μL of milk or orange juice contaminated with each analyte into vials containing 10 freshly hatched brine shrimp nauplii in seawater. This was incubated at 28 ◦ C for 24 h, after which mortality was determined. Mortality was converted to probits and the LC 50 was determined for each analyte by plotting probits of mortality against analyte concentration (log 10 ). Our findings were the following: (1) the lethal effects of toxins dissolved in milk were observed, with T-2 toxin being the most lethal and malathion being the least, (2) except for parathion, the dosage (based on LC 50 ) of analyte in a cup of milk (200 mL) consumed by a 6-y-old (20 kg) was less than the respective published rat LD 50 values, and (3) the BSLA was only suitable for detecting toxins dissolved in orange juice if incubation time was reduced to 6 h. Our results support the application of the BSLA for routine, rapid, and non-specific prescreening of liquid foods for possible sabotage by an employee or an intentional bioterrorist act. Keywords: brine shrimp lethality assay, mortality, pesticides, toxins, warfare agents Practical Application: The findings of this study strongly indicate that the brine shrimp lethality assay can be adapted for nonspecific detection of warfare agents or toxins in food at any point during food production and distribution. Introduction Bioterrorism is regarded as a possible threat to the food supply mostly due to the ease with which biological and chemical agents can be acquired, and the vulnerabilities along the food supply chain. Protection of consumers from this threat depends largely on timely detection of these agents in the event of an attack on the food supply. Several analytical methods based on chromatography, mass spectrometry, nuclear magnetic resonance, and immunode- tection are currently in use to detect the presence of specific agents or toxins. However, the wide diversity of toxic compounds poses a challenge to timely detection, and it is infeasible to use several spe- cific methods to adequately protect consumers on a routine basis. Moreover, most of these methods would be extremely expensive if used on a routine basis. The Dept. of Homeland Security has called for the development of novel, rapid, and nonspecific meth- ods for the detection of toxic compounds before the intentionally adulterated food enters the retail chain. These methods would also be suitable for routine prescreening of food products for possible sabotage by a disgruntled employee. The brine shrimp lethality assay (BSLA) is a rapid nonspecific assay that has found use for preliminary assessment of acute tox- icity in pharmaceutical and toxicology investigations. It uses the MS 20100662 Submitted 6/14/2010, Accepted 10/28/2010. Authors are with Dept. of Food Science and Nutrition, Univ. of Minnesota, 1334 Eckles Ave., Saint Paul, MN 55108, U.S.A. Direct inquiries to author Labuza (E-mail: tplabuza@ umn.edu). mortality of brine shrimp nauplii in the presence of toxins or bioactive compounds as a measure of toxicity. It was proposed by Michael and others (1956) and has been utilized for the detec- tion of fungal toxins (Harwig and Scott 1971; Harwig and others 1979; Kumarasamy 2003), plant extract bioactivity (Meyer and others 1982; McLaughlin and others 1991; Pisutthanan and oth- ers 2004), cyanobacterial toxin (Metcalf and others 2002), and pesticides (Barahona and Sanchez-Fortun 1999). It has also been used for the assessment of pharmacological activity of marine ex- tracts (Carballo and others 2002) as well as toxicity of heavy metals in the marine environment (Saliba and Krzyz 1976; MacRae and Pandey 1991; Martinez and others 1999). The underlying princi- ple of the BSLA is that the dosage or concentration of a chemical that may be therapeutic in humans would be toxic to the brine shrimp because of its small size. Thus the test would indicate the presence of a chemical that may be harmful to humans in a higher dosage. However, this method has rarely been used for the detection of chemical contaminants in processed foods or food systems. The objective of this study was to determine relative toxicities of toxic chemicals dissolved in whole milk and orange juice to brine shrimp by calculating LC 50 (concentration of analyte that kills half of the shrimp population) and comparing results to those of toxins dissolved in dimethyl sulfoxide (DMSO). Compounds screened in this study included T-2 toxin, trimethylsilyl cyanide, and pesticides such as malathion, parathion, dichlorvos, diazinon, and dursban. Milk was chosen as a key food because of the high vulnerability in the production chain (Wein and Liu 2005; Lui and Wein 2008; Poore 2010) while orange juice was chosen for its C  2010 Institute of Food Technologists R  T16 Journal of Food Science  Vol. 76, Nr. 1, 2011 doi: 10.1111/j.1750-3841.2010.01966.x Further reproduction without permission is prohibited