The assessment of toxicity of chlorine was carried out on Escherichia coli (E. coli) using the ToxTrak toxicity test (DR/4000 Procedure). The chlorine plays many important roles in medical science. It is also used as disinfectant. This method is a colorimetric test based on the resazurin reduction by the respiration process of bacteria. The resazurin is a redox-active dye. It is blue color dye but when it is reduced, it shows pink color. The change in color from blue to pink is depends upon the bacterial respiration. The rate of reduction of resazurin can be inhibited by the toxic substances. The role of chemical accelerator was to speed up the rate of reaction (ToxTrak Test handout). The initial and final absorbance was measured using colorimeter at 603 wavelengths. Using the appropriate formula, the % inhibition (percent inhibition) for different tenfold solutions (100%, 10% and 1% solutions) was calculated. The sample 1 with 100% solution of chlorine shows 107.9% inhibition, the sample 2 with 10% chlorine solution shows 99.8% inhibition and sample 3 with 1% chlorine solution shows 70% inhibition. It shows increase in % inhibition with the increase of chlorine concentration. The result shows biocide effect of different concentration of chlorine toxicity on bacteria. The ToxTrak Test used to measure the toxicity of chemical compound on different species and organisms.
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Chlorine plays many important roles in medical science, as disinfectants, and it is also a constituent of medicines. The chloroform was the first anesthetic used during surgery. Chlorine affective against disease causing microorganisms that is the reason, that it is used as disinfectant in drinking water, swimming pool, and also in wastewater treatment plant. It is also used to disinfect surfaces by using bleach. Bleach consists of chlorine in the gaseous form which is dissolved in an alkali-solution. The mechanism of action of chlorine on microorganisms is to affect the bacteria and viruses on their chemical bonds in their molecules. The disinfectants consist of chlorine compounds that can exchange atoms with other compounds, for example, enzymes in bacteria. When chlorine comes in contact with enzymes, it replaces the hydrogen in the molecule and it results in change in the shape of molecule and ultimately it falls apart. The 0.2-0.4 mg/L of chlorine is enough to kill the bacteria. The factors affecting the affectivity of chlorine are Chlorine concentrations, duration of exposure, temperature, pH, and number and types of microorganisms that are exposed (Lenntech.com).
Table: 1 The disinfection time for different microorganisms with chlorinated water.
Chlorine concentration = 1 mg/L or 1 ppm, pH = 7.5 and T = 25 °C. (Lenntech.com)
Organisms Name Duration of disinfection
Hepatitis A virus
Escherichia coli 0157 H7 bacterium
Less than 1 minute
The reaction of chlorine on human body depends on various factors like, the concentration of chlorine, the duration and frequency of exposure to the chlorine compound. It can also depend on the health of a person and the environment during the exposure. The exposure to chlorine or chlorine compound can affect the respiratory system. The effects vary from coughing, chest pains, and accumulation of fluid in the lungs. It can also cause skin irritation and eye irritations upon exposure. The chlorine in the pure form can be very toxic. Even the small amount of pure chlorine can be deadly to the individual. The chlorine is much denser than the air. Chlorine gas affects the mucous membrane of nose, throat, and eyes. It is very damaging to mucous membranes as it dissolves them (Lenntech.com).
The WHO standards for drinking water set the amount of chlorine is 2-3 mg/L and it gives a satisfactory disinfection of water. The WHO set the maximum amount of chlorine that is used by people is 5 mg/L. The national drinking water standards set the amount of chlorine in residual area is 4 mg/L (Lenntech.com).
Materials and Method:
The ToxTrakáµ€á´¹ toxicity test was used to determine the % of inhibition (percent inhibition) of Escherichia coli after exposed to different tenfold concentrations of chlorine solutions. The household chlorine bleach was used during the experiment. One day before the experiment, the inoculum was prepared. The source culture of E. coli was transferred by using the sterilized dropper pipets of 1.0 mL into the lauryl tryptose broth tubes. The tubes were incubated at 35°C until the vial contents shows turbidity (bacterial growth). On the next day, the 10 mL of test tubes were labeled and prepared by adding the appropriate amount of deionized water, chlorine solution (household bleach), accelerator, reagent pillows and E. coli inoculums as shown in Table 2 ( DR/4000 Procedure handout).
Table 2 Preparation of test tubes:
Always on Time
Marked to Standard
After the preparation of tubes, the readings were recorded using the spectrophotometer at a wavelength of 603 nm and the absorbance was adjusted to 0.00 Abs. The initial absorbances for all four tubes were recorded and than all the tubes were allowed to react. The absorbance of control was checked frequently until it shows decreased in Abs to at least 0.06 than the previous recorded Abs. The decrease in the Abs usually takes 30-45 minutes. The reaction of resazurin reduction gives the decrease in absorbance. The resazurin is the redox dye and it gave blue color to control and samples in its oxidized form. The bacteria start using the nutrient from the broth and it convert the resazurin in its reduced form and ultimately it gave pink color to the tubes. The conversion of blue to pink shows the respiration process of bacteria and also the utilization of glucose in the beef extract reduced the resazurin compound. After 30 minutes of reaction, the absorbance was measured again. The spectrometer was adjusted to 0.00 using the blank and then the tubes were placed one by one and the absorbances were recorded for each tube (DR/4000 Procedure handout).
The % inhibition (percent inhibition) was calculated for control and the sample tubes using the below formula;
% I (percent inhibition) = [1 - âˆ†A sample ÷ âˆ†A control] - 100
Where, âˆ†A= Initial absorbance value- Final absorbance value.
The initial and final absorbances were recorded in the Table 3 and also the % inhibitions for samples and control was recorded. The results are as seen below in Table 3.
Table 3 Initial and final absorbance and % inhibition for Control and Sample tubes.
The graphical representation of the results is shown below;
Graph 1: It shows the initial and final absorbance differences in each tenfold chlorine sample solutions.
Graph 2: It shows the % inhibition of sample solutions.
The result shows the highest percentage inhibition in the sample 1 with 100% of chlorine solution (Table 3). The gradual decrease in the % inhibition can be seen in all the samples from sample 1 to sample 3 as seen in Graph 2. The graphical representation of results is easy to interpret the data and it shows that the result support the objective of the study.
The change in color from blue to pink in the sample and control solution was the conversion of resazurin in to its reduced form. The reagent pillow contains resazurin, beef extract, 4- morpholinepropane sulfonic acid, 4- morpholinepropane sulfonic acid sodium salt, peptone, and sodium acetate. The resazurin is the redox dye and it show blue color in its oxidized form. The bacteria oxidize the glucose and it reduces the resazurin. The resazurin reduced and form resafurin, which is pink. Further reduction of this compound gives colorless solution which has dihydroresofurin. The substances or chemicals that are toxic to the bacteria can inhibit the rate of their metabolism and thus inhibit the rate of reduction of resazurin. The accelerator is the solution of glutaric dialdehyde and water which increase the rate of reaction or reduce the reaction time of sample solutions and control. As expected, the sample 1, 2 and 3 shows positive percent metabolic inhibition in E. coli. The % inhibition of chlorine increases with the increase in the tenfold concentration of chlorine in the sample solutions (Result Table 3).
The results of the experiment of assessment of toxicity of chlorine by using the ToxTrak test method suggest that the chlorine solution (household bleach) is very effective for bacteria like E. coli. as biocides at very low concentration.