Detection of Warfare Agents in Liquid Fo

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 LC50 was determined for each
analyte by plotting probits of mortality against analyte concentration (log10 ). 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 LC50 ) of analyte in a cup of milk (200 mL) consumed by a 6-y-old (20 kg)
was less than the respective published rat LD50 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

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 detection 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 others 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 extracts (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 principle 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 LC50 (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,
MS 20100662 Submitted 6/14/2010, Accepted 10/28/2010. Authors are with
and pesticides such as malathion, parathion, dichlorvos, diazinon,
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@ and dursban. Milk was chosen as a key food because of the high
vulnerability in the production chain (Wein and Liu 2005; Lui
umn.edu).
and Wein 2008; Poore 2010) while orange juice was chosen for its

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 immunodetection 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 specific 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 methods 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 toxicity in pharmaceutical and toxicology investigations. It uses the

T: Toxicology &
Chemical Food Safety

T16

Journal of Food Science r Vol. 76, Nr. 1, 2011

R

C 2010 Institute of Food Technologists

doi: 10.1111/j.1750-3841.2010.01966.x

Further reproduction without permission is prohibited

Detection of warfare agents in foods . . .
acidic pH. Since many of these compounds are poorly soluble in incubated at 28 ◦ C. Mortality (%) was determined after 24 h.
water, they were dissolved in DMSO to effect their easy dispersion Depending on the medium in which the analyte was dissolved in,
into water, milk, and orange juice.
50 µL of DMSO, milk, or orange juice alone were used as the
control.

Materials and Methods
Brine shrimp lethality assay
Brine shrimp eggs were purchased from Fisher Scientific Co.
(Pittsburgh, Pa., U.S.A.) while seawater, DMSO chemical toxins
were purchased from Sigma-Aldrich Inc. (St. Louis, Mo., U.S.A.).
Brine shrimp eggs were hatched in seawater at 28 ◦ C and used after
48 h. The analytes (toxins) were prepared by dissolution in DMSO
after which they were introduced into whole milk and orange
juice. The assays were performed in triplicate by introducing 50 µL

of analyte (dissolved in DMSO, milk, or juice) into vials containing
10 freshly hatched (48 h) brine shrimp nauplii. The volumes in
the vials were then made up to 5 mL with seawater, and the setups

A

Processing of results
Brine shrimp mortality (%) was converted to probits by using
Finney’s probit analysis table (Finney 1952), and a plot of probits
(average of 3 determinations) vs log concentration was made. A
probit of 8.09 corresponds to 100% mortality, and the concentration of analyte that corresponds to a probit of 5 is the LC50
(concentration of analyte in the 50 µL sample [µg/mL] that kills
half the shrimp population). Probit analysis is a type of regression used to transform a sigmoid binomial dose–response curve
into a linear distribution. Using the LC50 value, the hypothetical dosage (mg/kg body weight) of each analyte contained in a
200-mL cup of milk consumed by a 6-y-old (20 kg) was calculated
Figure 1–Mortality curve showing LC50 for
dursban dissolved in (A) DMSO and (B) whole
milk.

9

8

Probits of Mortality

7
6

y = 2.34x + 7.5698
= 0.8858

5
4
3
2

LC50 = Log-1 (-1.1)
= 79 µg/mL

1
0

-3.5

-3

-2.5

-2

-1.5

-1

-0.5

.5

Log Concentration (mg/mL)

B

10
9

7
y = 1.7429x + 6.0805
= 0.945

6
5
4
3
2

LC50 = Log-1 (-0.6)
= 251 µg/mL

1
0
-4

-3

-2

-1

2

Log Concentration (mg/mL)

Vol. 76, Nr. 1, 2011 r Journal of Food Science T17

T: Toxicology &
Chemical Food Safety

Probits of Mortality

8

Detection of warfare agents in foods . . .

for each agent. These were compared to published LD50 values better dispersion with DMSO as well as chemical interactions of
the agents with milk components.
(determined in rats by oral administration) of the analytes.
Based on the LC50 values, the hypothetical dosage of analytes
Results and Discussion
in a cup (200 mL) of milk consumed by a 6-y-old child of weight
Results of this investigation revealed the suitability of the brine 20 kg was calculated for each toxin (Table 1). These were comshrimp lethality assay for nonspecific detection of analytes at pared to published LD50 values (lethal dosage of analyte that kills
very low concentrations. The pesticides, dichlorvos, dursban, 50% of the population) of the respective toxins as determined
parathion, malathion, and diazinon, as well as T-2 toxin and in rats by oral administration. The LC50 for the pesticide durstrimethylsilyl cyanide were screened for relative toxicities in whole ban (in milk) was 251 µg/mL. Thus, the dosage of dursban in a
milk. Figure 1 shows the mortality curve for dursban in DMSO cup (200 mL) of milk consumed by a 6-y-old (20 kg) would be
(Figure 1a) and in whole milk (Figure 1b). LC50 values of the 2510 µg/kg body weight, which is considerably lower than the
toxins screened are given in Table 1. Of the analytes screened, T-2 LD50 value for dursban in rats (82000 µg/kg body weight) by
toxin was the most lethal whereas the least lethal was malathion the oral route (note that the LD50 values [rat, oral route] were
(in DMSO). Except for trimethylsilyl cyanide, the LC50 values for obtained from the respective material safety data sheets [MSDS]
analytes dissolved in whole milk prior to the assay were slightly supplied by the vendor [Sigma-Aldrich]). This difference was also
higher than values obtained for analytes dissolved in DMSO as true for the other analytes except for parathion, whose calculated
might be expected. It is believed that the reduction in lethality of dosage (7200 µg/kg body weight) in a cup of milk consumed
the analytes in milk was partially due to the nutrition provided to by a 6-y-old child was higher than the presumed human LD50
the shrimp by the milk components, whereas shrimp that had no (2000 µg/kg body weight). This is an indication that most of
access to milk, the control treatment, were more susceptible to the these analytes can be detected in milk at concentrations considerlethal effects of the toxins as indicated by the lower LC50 values ably below that which will cause harm in humans.

of the analytes dissolved in DMSO. It also may be in part by the
Results obtained for analytes dissolved in orange juice showed
an unusual trend (Table 2). The LC50 values were lower than those
Table 1– Concentration of toxic compounds needed to kill 50%
of test shrimp (LC50 values) in DMSO and whole milk.
a

Analyte
T-2 toxin
Dichlorvos
Diazinon
Dursban
Malathion
Parathion
Trimethylsilyl cyanide

LD50
(µg/kg
body
weight)
2700
17000
696000
82000
290000
2000
Not available

Table 2– Concentration of toxic compounds needed to kill 50%
LC50 (µg/mL)
of test shrimp (LC50 values) in DMSO and orange juice (extrapb
Dosage in olated values).
cup of milk
LC50 (µg/mL)
a
Whole (µg/kg body
LD50 (µg/kg
DMSO
milk
weight)
body
Orange
Analyte
weight)
DMSO
juice
4
5
50
66
45
79
3019
631
860

790
660
251
13804
724
670

7900
6600
2510
13804
7240
6700

a

LD50 values (rat, oral route) were obtained from the respective MSDS supplied by the
vendor (Sigma-Aldrich).
b
Hypothetical dosage of toxic compound in a cup (200 mL) of milk that would be
consumed by a 6-y-old (20 kg) based on the calculated LC50 values.

T-2 toxin
Dichlorvos
Diazinon
Dursban
Malathion
Parathion
Trimethylsilyl cyanide

y = 0.3196x + 6.8785
= 0.2421

Probits of Mortality

5.12 × 10−3
1.43 × 10−2