Aquatic Pollution Problems
Run-off: Debris Dirt Chemicals
Sewage and other components
Cleaning chemicals
Pharmaceuticals
Pharmaceutical Contamination
Recent India Study
Drugs: ciprofloxacin, treatments for hypertension, heart disease, chronic liver ailments, depression, gonorrhea, ulcers and other ailments
Estrogen
Chemical steroid.
Functions as the primary female sex hormone.
Promotes the growth of secondary female sex characteristics, accelerates metabolism, reduces muscle mass, and decelerates height growth.
Estrogen As a Pharmaceutical
Treatments: OsteoporosisSymptoms of menopause
Used in hormone replacement therapy and birth control pills.
How Estrogen Enters Water
a) Flushed down drains in the form of urine
b) The discarding of estrogen pills
c) Accidently dumped by industries
Estrogen Contamination
High levels – Improper disposal
Near-impossible to remove
Florida Museum: “When adult male fish are exposed to short term and low concentrations of a synthetic estrogen, their fertility can drop by as much as 50%, according to a study by scientists at the Department of Energy's Pacific Northwest National Laboratory (PNL).”
Escherichia coli
Large and diverse group of gram (-) bacteria
Free living, symbionts, or pathogens
U.S. infections > 70,000 per year
Large outbreaks rare, sporadic food infestations
Serves as a common prokaryotic cell model.
E. coli and Estrogen
E. coli and other coliforms are common in rivers across the world.
Estrogen is a growing problem in many of the same rivers and tributaries.
Does estrogen affect E. coli populations?
Hypotheses
Null hypothesis: Estrogen does not affect E. coli survivorship in vitro.
Alternative hypothesis: Estrogen does affect E. coli survivorship in vitro.
• Labeling tape• Ethanol• Latex gloves• E. coli DH5 Alpha• Micropipettes• Micro rack• Ten micro tubes• LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride).•Mortar and Pestle
Materials
• Estrogen (0.5 mg pills)• Sterile Dilution Fluid (100mM
KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl)
• Turntable• 40 Luria Broth agar plates • Bunsen burner• Spread bar• Incubator• Klett Spectrophotometer• Matches
Procedure
1. E. coli was grown overnight in sterile LB media.
2. A sample of the overnight culture was added to fresh media in a sterile sidearm flask.
3. The culture was placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 108 cells/mL.
4. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL.
5. An estrogen suspension was mixed with the appropriate amount of SDF to create estrogen concentrations of 1%, 0.1%, and 0.01%.
Procedure6. 100 µL of cell culture was then added to the estrogen solutions,
yielding a final volume of 10 mL and a cell density of approximately 103 cells/mL.
7. The solutions were vortexed and allowed to sit at room temperature for 15 minutes.
8. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on LB plates.
9. The plates were incubated at 37 C for 24 hours.
10. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.
Chart of Concentration
0% Concentration
0.01% Concentration
0.1% Concentration
1% Concentration
Microbe 0.1mL 0.1 mL 0.1 mL 0.1 mL
SDF 9.9mL 9.899 9.89mL 9.8mL
Estrogen Solution
0mL 0.001mL 0.01mL 0.1mL
Total 10mL 10mL 10mL 10mL
102 cells103 cells/mL
105 cells/mL with estrogen
108 cells/mL (E. coli)
100 uL100 uL
105 cells/mL (E. coli)
100 uL
Estrogen Effects on E. coli Survivorship
0
10
20
30
40
50
60
70
80
90
100
Concentrations of Estrogen
P > .05
P > .05
P < .05
Co
lon
y C
ou
nt
0% 0.01% 0.1% 1%
P-value
2.59E-08
Variable Concentration
T value Interpretation
0.01% Estrogen 1.50 Not Significant
0.1% Estrogen 1.19 Not Significant
1% Estrogen 6.19 Significant
Dunnett’s Test Analysis
T Critical = 2.76 (significant)
Alpha = .05
Conclusions
The null hypothesis that Estrogen does not affect E. coli survivorship in vitro was rejected for the concentration of 1% estrogen.
The null hypothesis could not be rejected for the lower concentrations of 0.01% and 0.1% estrogen.
The results indicate that 1% estrogen did negatively influence E. coli survivorship.
Limitations, Extensions, and Future Studies
The E. coli plating was not exactly synchronized, possibly contributing to varying colony counts.
The estrogen was quite insoluble, requiring the use of a suspension.
Larger sample sizes will be employed in the next round of testing.
Higher and lower concentrations of estrogen will be tested.
Directly infusing estrogen into the LB agar for prolonged cell exposure.
Limitations
Extensions and Future Studies
References Estrogen Makes Fish Too Feminine To Reproduce
http://www.flmnh.ufl.edu/fish/InNews/estrogen2007.html
Short Term Exposure to Estrogen Cuts Fish Fertilityhttp://www.flmnh.ufl.edu/fish/InNews/fishfert2003.htm
Estrogen knocks out fish in whole-lake experimenthttp://pubs.acs.org/subscribe/journals/esthag-w/2003/aug/science/jp_lake.html
http://www.ninemilerun.org/resources/scientific_reports/Bacteria%20&%20Detergent%20Results%20August%202006.pdf
http://www.nmenv.state.nm.us/swqb/wps/San_Juan/SanJuanRiver-2004BacteriaSamplingReport.pdf
http://www.cdc.gov/nczved/dfbmd/disease_listing/stec_gi.html
Results
0% Estrogen 0.01% Estrogen 0.1% Estrogen 1% Estrogen
56 80 104 4
86 80 70 18
86 94 54 4
76 104 70 38
86 98 52 44
100 92 72 6
68 94 74 100
86 94 68 28
70 90 70 32
74 80 60 28
Colony Counts
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