APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1982, p.

1072-1075

Vol. 44, No. 5

0099-2240/82/111072-04$02.00/0

Bacterial Bioluminescence as a Bioassay for Mycotoxins

IDA E. YATES* AND JAMES K. PORTER
R. B. Russell Agricultural Research Center, Agricultural Research Service, U. S. Department of Agriculture,
Athens, Georgia 30613

Received 14 May 1982/Accepted 27 July 1982

The use of bacterial bioluminescence as a toxicological assay for mycotoxins

was tested with rubratoxin B, zearalenone, penicillic acid, citrinin, ochratoxin A,
PR-toxin, aflatoxin B1, and patulin. The concentrations of mycotoxins causing
50% light reduction (EC50) in Photobacterium phosphoreum were determined
immediately and at 5 h after reconstitution of the bacteria from a freeze-dried
state. Generally, less toxins were required to obtain an EC50 at 5 h. The effects of
the above mycotoxins on bioluminescence were determined after 5, 10, 15, and 20
min of incubation with the bacterial suspensions. The concentration of rubratoxin
B necessary to elicit an EC50 increased with time, whereas the concentration of
citrinin, penicillic acid, patulin, and PR-toxin necessary decreased with time.
There was very little change in the concentration of zearalenone, aflatoxin B1, and
ochratoxin A required to elicit an EC50 with time. The bacterial bioluminescence
assay was most sensitive to patulin and least sensitive to rubratoxin B.

Mycotoxins are toxic fungal metabolites

found as contaminants in many agricultural
products. These compounds cause deleterious
effects in biological systems and have been
implicated in carcinogenesis, toxicosis, and teratogenesis in mammalian populations. Mycotoxins derived from agricultural commodities lead
to insidious problems related to the reproduction, health, and growth performances of both
animals and humans. As a result, mycotoxins in
the food supply must be evaluated and closely
monitored (3, 5).
Various in vitro short-term biological assays
have been employed to screen for the presence
of several mycotoxins. Lompe and v. Milczewski (8) demonstrated that 30 ,ug of rubratoxin
B per ml was required to elicit cytocidal effects
in Girardi heart cells. All other cell lines required 125 ,ug/ml. Umeda (9) observed cytomorphological changes on cells cultured from rat
liver, kidney, and lung tissues. The hereditable
effects for several mycotoxins also have been
investigated with FM3A cells from mouse mammary carcinomas (10). Brine shrimp larvae have
been used to evaluate fungal toxins (4). The
concentrations of mycotoxins that could be detected in the chloroform extracts of fungal cultures ranged between 10 to 100 ,ug/ml for aflatoxin B1, ochratoxin A, and rubratoxin B and to
500 ,ug/ml for citrinin, penicillic acid, patulin,
and zearalenone. Zebra fish larvae were shown
to be very sensitive to several mycotoxins,
including aflatoxin B1, ochratoxin A, and patulin
(1). However, penicillic acid was not effective in
this system at a concentration of 5 ,ug/ml. Addi-

tionally, other in vitro systems have utilized

only one or two mycotoxins to study the biochemical and morphological changes produced
in cell systems. Problems encountered in these
various bioassays include the maintenance of
animals or cell lines and cultures, technically
complex procedures requiring extensive preparation or assay times or both, expensive materials, and subjective data analyses. The purpose
of this investigation was to determine the effects
of mycotoxins on bacterial bioluminescence.
Subsequently, such a procedure may serve as a
short-term assay for mycotoxins, which would
circumvent some of the disadvantages of bioassays currently used.
MATERIALS AND METHODS
Mycotoxins. Aflatoxin B1 was obtained from Calbiochem-Behring. Rubratoxin B, zearalenone, penicillic
acid, citrinin, ochratoxin A, PR-toxin, and patulin
were obtained from Sigma Chemical Co. All mycotoxins were dissolved in methanol except aflatoxin B,
which was dissolved in dimethyl sulfoxide and zearalenone which was dissolved in each of these solvent
vehicles. The purity and quantity of each mycotoxin
solution was determined by high-performance thinlayer chromatography (HPTLC) (7) and by Xmax (e) in
methanol with a Varian Cary model 15 UV spectrophotometer. Briefly, the mycotoxins were applied to
the HPTLC plates (10 by 10 cm; silica gel 60 F-254, E.
Merck) with either a Camag Nanoapplicator (0.2 ,ul) or
a capillary dispenser system (0.5 ,ul) in conjunction
with a Camag Nonomat. The HPTLC plates were
developed for 5 cm in a linear developing chamber
(Camag) with 1.5 ml of developing solvent in each
trough (toluene-ethylacetate-formic acid, 30:6:0.5

1072

EFFECT OF MYCOTOXINS ON BIOLUMINESCENCE

VOL . 44, 1982

TABLE 1. EC50 for mycotoxins

ECS0 (>g/ml) at:
Mycotoxin

Patulin

Bacterial

type used

Fresha

Agedb
PR-toxin

Fresh

Aged
Penicillic acid

Fresh

Aged
Citrinin

Fresh

Aged
Zearalenone

Fresh

Aged
Ochratoxin A

Fresh

Aged
Aflatoxin B1

Fresh

Aged

Mean EC50
(>ig/ml) for
5,10, 15,
and 20 min

1073

EC50 %
change

from 5 to
20 min

5 min

10 min

15 min

20 min

7.53
6.17

3.87
3.45

2.67
2.36

2.16c

4.06
3.45d

-72%

1.82c

7.79
9.49

3.26
3.43

2.10
2.26

1.72c
1.80c

3.72

4.25d

-78%
-81%

15.95
14.67

10.65
9.79

8.72
7.60

7.44c

27.74
30.70

20.46
19.99

17.07
16.60

14.46c

20.05
20.44

-46%
-53%

14.37
11.59

13.70
11.59

13.21
11.37

12.29c
11.30

13.51

-11%

11.46d

-2%

18.49
18.53

16.60
16.40

16.17
16.39

16.27c
16.68c

16.88
17.00

-12%

21.97
24.79

21.19
24.43

19.44

20.35
25.87

20.73

-7%
+4%

25.66

5.91c

14.91c

-70%o

10.69

-53%

9.49d

-60%o

25.19d

-10%o

Fresh
31.79
33.36
35.17
34.73c
33.76
+9%o
26.68
Aged
31.09
31.82
32.82c
+23%
31.23d
a Freshly reconstituted bacterial suspension.
b Aged bacterial suspension.
c Significantly different from the 5-min value at the 0.05 level.
d Significantly different from the value for the freshly reconstituted bacterial suspension at the 0.05 level.

Rubratoxin B

[vol/vol], 30:14:4.5 [vol/vol], or both). Scanning of the

HPTLC plates was performed with a Camag photodensitometer (monochromator version) attached to a
Hewlett-Packard 3390A reporting integrator. The
amounts of mycotoxin present in each test solution
were determined immediately before the bacterial bioluminescence assay, and quantities reported were the
averages of triplicate analyses.
Bioluminescence assay. The protocol used for the
bacterial bioluminescence analyses for toxicity was
essentially that of Bulich and Isenberg (2). The freezedried bacteria (a strain of Photobacterium phosphoreum), reconstitution solution (ultrapure water), and
diluent (a solution containing 2% NaCI to provide
osmotic protection for the marine bioassay organism)
were from Beckman Instruments, Inc. The dilution
series for each mycotoxin was constructed so that the
highest concentration caused a 90%o light decrease and
the lowest concentration caused a 10% light decrease.
The concentrations of the solvent vehicles, DMSO and
methanol (,ul/ml of diluent), in the assay dilution
causing the greatest light decreases were: PR-toxin,
0.75; penicillic acid, 1.0; citrinin and zearalenone, 2.5;
ochratoxin A, 4.5; patulin and rubratoxin B, 5; and
aflatoxin Bl, 10. Bioluminescence determinations
were made with the Microtox Analyzer (Beckman
Instruments) at 5, 10, 15, and 20 min after addition of
toxin to the bacterial suspensions. Assays were performed at 15C immediately after bacterial reconstitution (freshly reconstituted suspensions), as recommended by Bulich and Isenberg (2). In addition,
bacterial suspensions maintained at 3C for 5 h after

reconstitution (aged suspensions) were tested for sensitivity to the mycotoxins.

Data analysis. The Microtox data reduction was
accomplished by the calculation of gamma (the ratio of
light lost to the light remaining) (6) by using a correction factor to accommodate the normal change of light
by the bacteria without added toxicant. The correction
factor was determined by dividing the diluent control
blank reading at the time point analyzed (i.e., 5, 10, 15,
or 20 min) by the zero time reading. The gammas for
each concentration of mycotoxin at a given time point
were subjected to linear regression and power curve
analyses with the algorithms developed by HewlettPackard for the HP41-C calculator. The power curve
was used to derive the concentrations (in micrograms
per milliter) causing 50 and 20%o light reduction (EC50
and EC20, respectively) for each mycotoxin and represents the mean of at least three separate experiments.
Analysis of variance was used to determine significant
differences between EC50 values for freshly reconstituted bacteria and aged suspensions and also for
significant differences between EC50 values for different incubation times.

RESULTS AND DISCUSSION

Bacterial bioluminescence was inhibited by all
of the mycotoxins studied (Table 1), with the
sensitivity of freshly reconstituted bacteria after
5 min of incubation ranging from 31.79 txg/ml for
rubratoxin B to 7.53 ,ug/ml for patulin. Bacterial
suspensions aged for 5 h maintained sensitivity

1074

APPL. ENVIRON. MICROBIOL.

YATES AND PORTER

TABLE 2. Lower limits of detection (EC20) for

mycotoxins using bacteria immediately after
reconstitution
Incubation time
5 min

Mycotoxin
EC20

(>g/m1)

Patulin
PR-toxin
Penicillic acid

Citrinin
Zearalenone
Ochratoxin A
Aflatoxin B1
Rubratoxin B

20 min

2.56
3.55
5.34

11.08
9.35
12.61
4.54
19.69

Change
fromto
EC50
EC20

66%
57%
67%
60%
35%
32%
71%
38%

EC20 % Change
EC50
4Lg/ml) from
to EC20

0.89
0.92
3.14
7.00

9.69
12.56
3.61
26.36

59%
47%
58%
53%
21%
23%
82%
24%

to the mycotoxins. Patulin, penicillic acid, zea-

ralenone, and rubratoxin B were significantly

more potent on the aged bacterial suspensions
(Table 1) than on freshly reconstituted bacterial
suspensions. The increased sensitivity of aged
bacterial suspensions was not due to fewer cells
since cell counts revealed approximately the
same number of cells in both suspensions. The
effect of cell number on the EC50 for penicillic
acid was examined by using 20 ,u and the
standard 10 ,ul of aged bacteria per assay tube.
With this procedure, the increased number of
cells had little effect on the EC50 for penicillic
acid. There was no significant difference between aged and freshly reconstituted bacterial
suspensions for citrinin and ochratoxin A (Table
1). PR-toxin and aflatoxin B1 were less active on
the aged bacterial suspension than on the freshly
reconstituted bacteria (Table 1).
Reaction rates on the bioluminescence process were studied for each mycotoxin. The age of
the bacterial suspension did not significantly
affect the pattern of the response with time of
incubation for penicillic acid, citrinin, ochratoxin A, aflatoxin Bl, and rubratoxin B (Table
1). For example, the difference between the
EC50 values determined with freshly reconstituted bacteria and those determined with aged
bacterial suspensions remained constant from 5
to 20 min. There was a significant interaction at
the 0.05 level with regard to time of incubation
of toxin with the age of the bacterial suspension
for patulin, PR-toxin, and zearalenone. The inhibitory action of all mycotoxins on bacterial
bioluminescence approached the maximum effect by 15 min (Table 1). Generally, the greatest
change in the EC50 occurred between 5 and 10
min, with little change occurring between 15 and
20 min.
Except for aflatoxin B1 and zearalenone, the

change in the EC50 with time was significant for

all other mycotoxins studied. Three specific
categories could be established on the basis of
the change in the EC50 from 5 to 20 min. Patulin,
PR-toxin, penicillic acid, and citrinin constituted
one category. The amounts of these toxins required for an EC50 decreased by 46% or greater
from 5 to 20 min. The concentration of zearalenone, ochratoxin A, and aflatoxin B1 required
for an EC50 decreased by 12% or less. Rubratoxin B was the only mycotoxin analyzed that
required a significant increase in concentration
with time to elicit a 50% light reduction.
The EC20 values for each mycotoxin obtained
with freshly reconstituted bacteria at 5 and 20
min of incubation are shown in Table 2. These
concentrations would be the lower limits of
detection of these toxins by the bacterial bioluminescence assay. The reduction in the concentration of toxin required for an EC20 compared
to an EC50 was greatest for patulin, PR-toxin,
penicillic acid, citrinin, and aflatoxin B1. Likewise, these were the toxins (with the exception
of aflatoxin B1) which demonstrated the greatest
reduction in the EC50 from 5 to 20 min. The
EC20 for aflatoxin B1 may be a more definitive
expression of the sensitivity of bacterial bioluminescence for this toxin because the EC50 for
aflatoxin B1 must be extrapolated. The dilution
series analyzed for aflatoxin B1 ranged from
0.865 to 13.80 ,ug/ml. Higher concentrations
were not analyzed because of the limited solubility of this toxin. Consequently, concentrations
both higher and lower than the EC20 (4.54
,ug/ml) but none higher than the EC50 (21.97
,ug/ml) were analyzed. Since aflatoxin B1 has
limited solubility in methanol, DMSO was used
as the solvent vehicle for this toxin. Zearalenone
is soluble in both methanol and DMSO, and the
EC50 values obtained for this compound demonstrated that these solvent vehicles had little
influence on the toxin's action on the bioluminescence process.
The results obtained by the bacterial bioluminescent procedure have demonstrated a reliable
short-term method for assessing the toxicity of
mycotoxins. The order of toxicity determined by
bacterial bioluminescence parallels that reported
for mammalian cell cultures. Lompe and v.
Milczewski (8) used five mammalian cell lines:
Flow C II from porcine epithelia, AM II from
porcine kidney, Detroit-98 from human bone
marrow, Girardi from human heart, and FHL
from human lung. The range of concentrations
for mycotoxins to elicit cytocidal effects was
0.12 to 1.0 jig/ml for patulin, 0.2 to 2.5 ,ug/ml for
PR-toxin, 5.5 to 24 ,ug/ml for penicillic acid, 9 to
50 ,g/ml for aflatoxin B1, 10 to 33 ,ug/ml for
ochratoxin A, 30 to 125 ,ug/ml for rubratoxin B,
and 50 to 200 ,ug/ml for citrinin.

VOL. 44, 1982

EFFECT OF MYCOTOXINS ON BIOLUMINESCENCE

One major advantage of the bacterial bioluminescence assay over other currently used shortterm toxicity assays is that the instrumentation
reduces the chance of human error and conjecture inherent to many of these cell systems in
attempting to determine the extent of cellular
alteration. The expressions of toxicity used in
the bioluminescence assay varied by only 10%
or less. The data output is a mechanical function, and pipetting is the principal laboratory
manipulation to introduce error. Furthermore,
the system is efficient, inexpensive and simple to
perform. For absolute values of toxicity, the
time of incubation of the toxin with the bacteria
and the age of the bacteria themselves are critical for most of the mycotoxins. However, the
bacteria do maintain sensitivity to the mycotoxins even after 5-h reconstitution. Thus, in
screening many samples for mycotoxin activity,
the original bacterial suspension can be used for
an extended time period. In addition the bioassay presents many possibilities for a more complete understanding of the mechanism of action
of mycotoxins. Future studies with model compounds may provide significant correlations between the structures and their toxicities to the
bioluminescence process.
ACKNOWLEDGEMENTS
We thank Joyce L. Lanier, Gary M. Adcock, and Charles J.
Herndon for their technical assistance.

1075

LITERATURE CITED
1. Abedi, A. H., and P. M. Scott. 1969. Detection of toxicity
of aflatoxins, sterigmatocystin, and other fungal toxins by
lethal action of zebra fish larvae. J. Assoc. Off. Anal.
Chem. 52:963-969.
2. Bulich, A. A., and D. L. Isenberg. 1981. Use of the
luminescent bacterial system for the rapid assessment of
aquatic toxicity. Instrum. Soc. Am. Trans. 20:29-33.
3. Ciegler, A., H. R. Burmeister, R. F. Vesonder, and C. W.
Hesseltine. 1981. Mycotoxins: occurrence in the environment, p. 1-50. In R. C. Shank (ed.) Mycotoxins and Nnitroso compounds: environmental risks, vol. 1. CRC
Press, Inc., Boca Raton, Fla.
4. Harwig, J., and P. M. Scott. 1971. Brine shrimp (Artemia
salina L.) larvae as a screening system for fungal toxins.
Appl. Microbiol. 21:1011-1016.
5. Hesseltine, C. W. 1979. Introduction, definition, and history of mycotoxins of importance to animal production, p.
3-18. In Interaction of mycotoxins in animal production.
National Academy of Sciences, Washington, D.C.
6. Johnson, F. H., H. Eyring, and B. J. Stover. 1974. The
theory of rate processes in biology and medicine. John
Wiley & Sons, New York.
7. Lee, K. Y., C. F. Poole, and A. Zlatkis. 1980. Simultaneous multi-mycotoxin determination by high performance thin-layer chromatography. Anal. Chem. 53:837842.
8. Lompe, A., and K.-E. v. Milczewski. 1979. Ein zellkulturtest fur den Nachweis von Mykotoxinen. Z. Lebensm.
Unters. Forsch. 169:249-254.
9. Umeda, M. 1971. Cytomorphological changes of cultured
cells from rat liver, kidney and lung induced by several
mycotoxins. Japan. J. Exp. Med. 4:195-207.
10. Umeda, M., T. Tsutsui, and M. Saito. 1977. Mutagenicity
and inducibility of DNA single-strand breaks and chromosome aberrations by various mycotoxins. Gann 69:619625.