Testing for Chemical Toxicity Using Bacteria An Undergraduate Laboratory Experiment H. Brouwer
Redeemer College. Ancaster, ON Canada L9G 3N6 The impact of chemicals on people, animals, and the environment in general has become a major concern of scientists in recent years. Many new analytical methods have been develooed to detect trace levels of hazardous chemicals. and a wide variety of biological screening tests are being employed in testing chemicals and environmental samples. Yet most laboratory courses tend to omit toxicity studies since many of the tests are expensive and time-consuming. In this laboratory activity a relatively simple and inexpensive screening test usinga bacterial culture is described for determining chemical toxicity. Chemicals may be harmful to an organism in a variety of ways. Acute toxicity refers to the short-term effect of a toxic material on an organism and is usually measured by the symbol L D a or ECm, This is the lethal dose (LD) causing deathin 50%of the test organisms or the effective concentration (EC) causing a 50% decrease in a specific effect or response. The former is often measured in milligrams of the chemical per kilogram of body weight, whereas the latter may be measured in milligrams per liter (ppm) of solution ( I ) . Acute toxicitv is determined from the mortality rate when the test organism is exposed to the chemical o;environmental sample for a prescribed period of time. Chronic toxicitv, on the other hand, involves exposing the test organism to low levels of the chemical for a longer period of time, sometimes for several generations, to determine if the chemical has any adverse effects. Since living organisms may respond differently toward chemicals, a battery of screening tests involving different organisms is normally performed to evaluate the toxicity of a sample. Due to the high costs associated with common laboratory animals, microbes are often employed in screening tests since their costs arelow, they have ashort life cycle, and a large number of individuals will be exposed to the test
chemical. Two testa in particular have received wide application. The Ames teat detects the presence of reverse mutations in a special bacterial culture upon exposure to a test chemical. The Microtox test measures the decrease in light emission from photobacteria when exposed to the sample. Numerous other methods have been develoued for measuring the inhibition of microbial activity (2,3).. The orocedure described in this oaDer is a modification of a screehing test developed by D. ~i;and co-workers (4-8). The test employs an electron acceptor dye, resazurin, which changes color in the presence of dehydrogenase enzyme activity resulting from microorganisms actively growing in a culture medium. In the presence of an active bacterial culture, resazurin changes color from blue to pink, as follows:
RESAZURIN (BLUE 610 nm)
RESORUFIN (PINK 580 nm)
The visible spectra of resazurin (blue) and ita reduced product. resomfin (oink). are shown in Fieure 1. If bacterial growth is inhibi'ied, reduction of the r&azurin occurs, and such a sample would remain blue. If the tent chemical has no
Volume 68 Number 8 August 1991
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Table 1. Percent lnhlbltlon ot Dehydmgenase Actlvlty by Organlc Chemlcals at 500 ppm Uslng Methanol as Solvent Test chemical
%Inh
chloroform carbon tetrachloride benzene t01"ene wlene (mixed isomers) naphthalene chlorobenzene bromobenzene +dichlorobenzene pdichlonrbenzene pchlomtoluene phenol mchiorophen~l pchlorophen~l
3 66 2
89 98 5
95 100 95 50 95 7
95 50
Figure 1. Visible spectra of reserurin and resarufin (obtained using a Spec tronlc 20 specnophotometer). Table 2.
effect on the bacteria, then the resazurin is reduced t o the pink color. Since resorufin absorbs onlv weaklv at the waveiength giving the maximum absorba&e for iesazurin, the decrease in resazurin concentration mav be measured usine a Spectronic 20 or similar instrument,-and, by varying th; concentration of the test chemical, the EC5o -~ value for that chemical may then he estimated. The bacterial culture used in this toxicity test may be any culture that is convenientlv o r e ~ a r e dA . oure culture is oreferred t o allow compariso&*wiih other data using t h s k ture. The bacterium used in this oaoer. Bacillus cereus. was readily cultured and maintained: *
Sample Data Uslng Cu2+
Test Tube
Absorbance
A (reagentcontrol)
0.62 0.083
B (cell control)
C (test chemical): 500 ppm CuZt 100 ppm Cue+
0.29 0.58 0.345 0.245 0.124
10 ppm Cu2+ 1 ppm Cu2+
0.1 ppm Cu2+ 'See
%kh
39* 92
48 30 8
ted f a explanation.
should be repeated using dilutions of the test chemical in order to determine the concentration at which the chemical causes a 50% decrease in bacrerisl actirity. Fur organir cumpounds, tw
