Lab # 5: Rapid Screening for Toxicity of Water
(Agar Plate Method)
Performed by: Jonathan Medina
Partners: Deep Shickha Jasra, Sean O’neil
Performed on: November 1st, 2012
Submitted on: November 8th, 2012
Instructor: Mr. Daniel Wilson
Course: Environmental Biology and Toxicology BIOL CY502
Jonathan Medina
Lab 5: Rapid Screening for Toxicity of Water
8th, November 2012
Executive Summary
The rapid screening for toxicity of water was carried out by using potassium chromate as the
solute, but with the intent of assessing the effect of Cr (II) in bacteria growth. An initial solution of
984.815 ppm (theoretical = 1000 ppm) in 1.0 M KOH was prepared; and then, a series of dilutions were
also performed in order to have 500, 200, 50, and 10 ppm solutions of potassium chromate.
By using the uv spectromenter, a solution containing the Bacillus Cerus bacteria was prepared
for a final concentration of 0.1 A at 625 nm. The bacteria were then placed into the petri dishes in
triplicates to ensure a good result. The petri dishes were then put into the oven to dry and then a cuple
of droplets of each of the dilutions of Cr(II) were added clock wise into spots previously labeled with the
concentration. Petri dishes were incubated 24 hours at 35 degrees Celsius, and then removed for
analysis.
The results were collected and reported in table 1. The minimum concentration at which the
bacteria growth was inhibit was 200 ppm, or 200 mg/L of solution. This value represent the
concentration of Cr(II) that can cause harm to living organisms and human health.
A dose/response plot was done by using the mean values calculated and reported in table 1. A
high correlation of concentration (dose) and diameter of “clear spots” (response) was observed,
meaning that as the concentration of Cr(II) increases, the inhibition of bacteria growth increases,
therefore the higher the risk for human health as well.
BIOL CY502
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Jonathan Medina
Lab 5: Rapid Screening for Toxicity of Water
8th, November 2012
Purpose
In this experiment the student will learn how to screen a water sample for toxicity using the algar plate
method.
Introduction
This method was designed for the rapid toxicological screening of water, waste water and river
or lake sediments extracts. It can also be used as qualitative test for ranking the toxicity of chemicals.
This method has been used to evaluate polluted surface and ground water, domestic and industrial
waste waters and water soluble chemicals.
The toxicity data generated by this method is qualitative and semiquantitive in nature. However,
it can be used for ranking toxicological potential of various chemicals.
Because this experiment uses the bacteria Bacillus Cereus it is important that all the safety
measures for microbiology are followed.
Procedure
Refer to Environmental Biology and Toxicology Lab Manual, Experiment 5, pages 51 – 54
Results
Table 1. Inhibition of Bacteria Growth in a Seeded Agar Plate by Potassium Chromate (K2CrO4)
Concentration
Sample One:
Sample Two
Sample Three:
Mean
Standard
(ppm)
Diameter (mm)
(Sean’s):
Diameter
Diameter
Deviation
Diameter (mm)
(mm)
(mm)
(mm)
0 (control)
/
/
/
/
/
10
/
/
/
/
/
50
/
/
/
/
/
200
0.5
0.5
/
0.5
0.0
500
0.7
0.6
0.5
0.6
0.1
1000
1.0
0.7
0.7
0.8
0.17
BIOL CY502
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Jonathan Medina
8th, November 2012
Lab 5: Rapid Screening for Toxicity of Water
Response: non-growth spots diameter (mm)
Graph 1. Response / Dose Plot for Potassium Chromate (K2CrO4) Inhibition
of Bacteria Growth
1.2
1
0.8
0.6
Sample One
0.4
Sample Two
Sample Three
0.2
0
0
50
100
150
200
250
Dose: K2CrO4 concentration (ppm)
Respose: Non-Growth Spots. Diameter (mm)
Graph 2. Response / Dose Plot for Potassium Chromate (K2CrO4)
Inhibition of Bacteria Growth, using mean values
BIOL CY502
0.9
y = 0.0004x + 0.4194
R² = 0.9978
0.8
0.7
0.6
Mean Diameter
0.5
0.4
200
300
400
500
600
700
800
900 1000
Dose: K2CrO4 Concentration (ppm)
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Jonathan Medina
Lab 5: Rapid Screening for Toxicity of Water
8th, November 2012
Questionnaire
1. The mean values of diameter and standard deviation for each of the concentrations are
presented in table 1.
2. Extract from Chapter 3.4.2 Discharges to Sanitary and Combined Sewers; and Table 3.6 Limits on
Discharges to Sanitary and Combined Sewers; The Handbook of Environmental Compliance in
Ontario; pages 72, 102.
Discharges to storm sewers: the 1998 model By-Law contains numerical limits for a few
parameters. These include temperature (<40 oC), pH of 6 – 9, suspended solids of not more than
15 mg/L, limits for few metals, and limits of not more than 200 fecal coliform colonies per 100
mL.
The following table contains the rank of the chemicals listed in the laboratory manual as to their
toxicological potential, based on the minimum allowable discharge according to MOE:
1
2
3
4
5
6
Metals
Cadmium
Zinc
Nickel
Copper
Chromium
Manganese
MOE Model By-Law
0.7 mg/L
3 mg/L
3 mg/L
3 mg/L
5 mg/L
NA
City of Toronto
0.7 mg/L
2 mg/L
2 mg/L
2 mg/L
2 mg/L
5 mg/L
3.
In order to test sediments of a river or lake using the procedure indicated in this lab, it is
necessary to obtain the samples in the first place by using correct methodology for sediments
sampling, storage and preparation of the sample (vacuum or non vacuum probes methods) that
will not be discussed for the matter of this report; and a concentration of the sample to ensure
proper growth of the bacteria by the Continuous Flow Centrifugation method, which is suitable
to concentrate protozoa, bacteria and viruses present in water samples.
4.
Graph two represents the dose / response plot for the inhibition of bacteria growth
caused by potassium chromate, being the response the diameter of inhibition. The minimum
concentration at which bacteria stopped growing was 200 ppm which is equal to 200 mg of Cr
(II) per litre of solution. this value is reasonably similar, with the difference in units, to the LD 50
= 180 mg/kg found in literature for potassium chromate
Potassium chromate Toxicological data (MSDS):
http://www.sciencelab.com/msds.php?msdsId=9927403
BIOL CY502
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Jonathan Medina
8th, November 2012

Exposure Limits:
TWA: 0.05 (mg(Cr)/m) from ACGIH (TLV) [United States]Consult local authorities for
acceptable exposure limits.



Routes of Entry: Absorbed through skin. Inhalation. Ingestion.
Toxicity to Animals: Acute oral toxicity (LD50): 180 mg/kg [Mouse].
Carcinogenic effects: Classified A1 (Confirmed for human.) by ACGIH, 1 (Proven for
human.) by IARC.
Mutagenic effects: Mutagenic for mammalian somatic cells. Mutagenic for bacteria
and/or yeast. May cause damage to the following organs: kidneys, lungs, liver, upper
respiratory tract, skin, eyes.
Other Toxic Effects on Humans:
Very hazardous in case of skin contact (irritant), of ingestion. Hazardous in case of skin
contact (sensitizer), of inhalation (lung irritant). Slightly hazardous in case of skin contact
(corrosive, permeator), of eye contact (corrosive).


5.
Lab 5: Rapid Screening for Toxicity of Water
Bacteria are a very important ally of humans when it comes to testing, in this case, for
water toxicity. Some of the characteristics that make bacteria such an important element of
investigation are: The ease to handle, short life cycle, adaptability to changes, and relatively
inexpensive.
Toxicity of chemicals in waters is determined by the capacity of a substance to inhibit
the growth of bacteria. The outcome of such methods or assessment is qualitative and
semiquantitative, and translates to the concentration at which a compound may cause adverse
effects to living organisms and more specifically human health.
Discussions
The minimum concentration at which the bacteria growth was inhibited was 200 ppm according
to the results in table 1. By simple conversions this result can be expressed in terms of milligrams per
littler of solution: 200 mg/L. This value represent the concentration at which Cr(II) may represent a risk
of hazard for living organisms and human health, and can be compared with the LD50 = 180 mg/kg.
A visual representation of the results is the dose/response plot (graph 2). Such plot, shows that
there is a strong correlation between the concentration (dose) and the diameter of “clear spots” based
on the R2=0.9978. The meaning of this is that at a higher concentration of Cr(II) in the water, the more
bacteria die due to Cr(II) contamination, which translates to, the higher the risk of adverse effects for
human health.
BIOL CY502
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Jonathan Medina
Lab 5: Rapid Screening for Toxicity of Water
8th, November 2012
Source of error
Several sources of error may be identified for this experiment. The first one and more obvious is
an error in the calculation of the amount of potassium chromate needed to prepare a 1000 ppm
solution. Note that the mass of Cr (II) required is nearly ¼ the mass of potassium chromate.
Another factor that introduces error to the experiment is the incorrect preparation of the bacteria
solution, and this easily can occur if the spectrometer is not properly calibrated before using it to
determine the concentration, or if the wavelength is not the one stipulated for the procedure.
A third source of error to mention is always the human error. Lack of aseptic procedures, incorrect
manipulation of instruments, petri dishes and essentially, not following the procedure may generate a
possible interference with the correct development of the experiment.
Conclusion
Toxicity of chemicals in waters is determined by the capacity of a substance to inhibit the
growth of bacteria. The outcome of such methods or assessment is qualitative and semiquantitative,
and translates to the concentration at which a compound may cause adverse effects to living organisms
and more specifically human health.
BIOL CY502
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Jonathan Medina
BIOL CY502
Lab 5: Rapid Screening for Toxicity of Water
8th, November 2012
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Jonathan Medina
BIOL CY502
Lab 5: Rapid Screening for Toxicity of Water
8th, November 2012
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Jonathan Medina
BIOL CY502
Lab 5: Rapid Screening for Toxicity of Water
8th, November 2012
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Jonathan Medina
BIOL CY502
Lab 5: Rapid Screening for Toxicity of Water
8th, November 2012
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