Tyler Barkich Grade 9 Pittsburgh Central Catholic High School.

Pesticide Runoff Effects on Microbial Life

Tyler BarkichGrade 9

Pittsburgh Central Catholic High School

Any chemical, antibacterial, biological agent, or other similar substance used to kill or repel unwanted species.

Used commonly in agriculture and around homes

877 million pounds of pesticides were used agriculturally in 2007.

Bayer Pesticide Active Ingredients:β-cyfluthrin and imidacloprid.

Pesticides

Research suggests over 100 active pesticide ingredients can cause cancer or birth defects.

Pesticides have been linked to endocrine disruption.

Pyrethroids, a type of ingredient found in the pesticide used in this experiment, are toxic to most forms of life.

An estimated 98% of sprayed insecticide and 95% of herbicide are not used on target species.

Previous Studies

Rod shaped cells, usually 2 nanometers in length

Prokaryotic Gram-negative Found in the intestines of many mammals Commonly used model Reproduces rapidly, usually within thirty

minutes Many strains, most non-pathogenic

Escherichia coli (E. coli)

Bacteria that is mostly harmless and lives normally on skin and mucous membranes of humans

Gram-positive

Many forms are considered non-pathogenic

Pathogenic forms can be lethal

Staphylococcus epidermidis

Gram+

• Most pathogenic• Simple cell wall.• Antibiotics work against

the formation of the cell wall.

• Staphylococcus epidermidis

Gram-

• The cell wall is a thin layer of lipopolysaccharide, which adds extra protection.

• This layer protects the cell from certain antibiotics.

• Escherichia coli

Gram+ vs. Gram- Bacteria

Do pesticides have an effect on the microbial populations in natural ecosystems?

Question

To determine if the Bayer Advanced Pesticide in different concentrations will affect the survivorship or Escherichia coli or Staphylococcus epidermidis.

Purpose

Null Hypothesis: The pesticide will not significantly reduce the survivorship of Escherichia coli or Staphylococcus epidermidis.

Alternative Hypothesis: The pesticide will significantly reduce the survivorship of Escherichia coli or Staphylococcus epidermidis.

Hypotheses

Materials Sterile test tubes Micropipettes Bayer Pesticide

(0.015% β-cyfluthrin, 0.012 % imidacloprid)

Vortex Ethanol Spreader LB Agar Plates LB Media (0.5% yeast

extract, 1% tryptone, 1% sodium chloride)

Incubator Labeling Tape Marker

Escherichia coli (DH5-Alpha)


Test tube rack Sidearm flask Sterile filters Klett Spectrophotometer Pipette tips Sterile Dilution Fluid

(100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl)

Bunsen Burners Matches Latex gloves

1. Escherichia coli and Staphylococcus epidermidis were grown overnight in sterile LB media.

2. A sample of those cultures were added to media in a sidearm flask.

3. The cultures were incubated until a density of 50 Klett spectrophotometer units was reached. This represents a density of 108-109 cells/ml.

4. This cultures were diluted in sterile dilution fluid to a density of 105 cells/ml.

5. The pesticide was sterile filtered and was diluted to concentrations of 0%, 0.01%, 0.1%, and 1% in sterile dilution fluid.

Procedure

0% Pesticide

0.01% Pesticide

0.1% Pesticide

1% Pesticide

SDF 9.9 mL 9.899 mL 9.89 mL 9.8 mL

Pesticide 0 mL 0.001 mL 0.01 mL 0.1 mLE. Coli/Staph 0.1 mL 0.1 mL 0.1 mL 0.1 mL

Total 10 mL 10 mL 10 mL 10 mL

Chart of Liquid Concentrations

6. 0.1 mL of the E. coli culture was added to 4 tubes of each concentration of pesticide and 0.1 mL of the Staphylococcus was added to 4 tubes of each concentration of pesticide. This produced a final volume of 10 mL, and a cell density of 103 cells/ml.

7. After vortexing to evenly suspend cells, 0.1 mL aliquots from each tube was spread evenly on LB agar plates.

8. The plates were incubated at 37 °C overnight.

9. The resulting colonies were counted. Each colony is assumed to have risen from one cell.

Procedure (cont.)

1. Sterilized pesticide was spread evenly over LB agar plates in two concentrations, a stock concentration (200 µL of pesticide) and a 10% concentration (20 µL of pesticide and 180 µL of sterile dilution fluid.)

2. The plates were incubated at 37 °C for two hours.

3. Escherichia coli and Staphylococcus epidermidis were grown overnight in sterile LB media.

4. 100uL aliquots of Control cell suspension were spread onto the infused plates.

5. The plates were incubated at 37 °C overnight. Resulting colonies counted.

Agar Infusion Procedure

0.00% 0.01% 0.10% 1.00%0

50

100

150

200

250

300

350309.5

290.25275

239

Colonies

[Pesticide]

Nu

mb

er

of

Colo

nie

sPesticide Effects on E. coli

SurvivorshipP value: 8.19E-06

0.00% 0.01% 0.10% 1.00%0

50

100

150

200

250228.25

191

154.5138.5

Colonies

[Pesticide]

Nu

mb

er

of

Colo

nie

sPesticide Effects on Staphylococcus

epidermidis SurvivorshipP value: 2.23E-06

Dunnett’s Tests

Pesticide concentration

T value Interpretation

0.01% 2.47 Not significant

0.1% 4.43 Significant

1% 9.06 Significant

T critical = 3.5Alpha = .05





1% 9.72 Significant

Escherichia coli


𝑡𝑑=𝑀𝑖−𝑀𝑐

√ 2𝑀𝑆𝐸𝑛h

0.00% 0.01% 0.10% 1.00%0

102030405060708090

100

E. coliStaph

[Pesticide]

Perc

en

t S

urv

ivors

hip

Pesticide Effects on E. coli and Staphylococcus Survivorship

Pesticide Effects on E. coli and Staphylococcus Survivorship: Infused Plates

E. Coli Staph0

50

100

150

200

250

300

350

400

198.375

333.875

178.5

288.625

146.125

248.75

Control Low Concentration High Concentration

Nu

mb

er

of

Colo

nie

s

P-value:1.8E-09

P-value:1.07E-08

Dunnett’s Tests: Infused



Low 4.16 Significant

High 10.93 Significant

T critical = 2.67Alpha = .05



Low 5.3 Significant

High 9.97 Significant

Escherichia coli


𝑡𝑑=𝑀𝑖−𝑀𝑐

√ 2𝑀𝑆𝐸𝑛h

A. Liquid Exposure• The null hypothesis that pesticide will not affect E.

coli or Staphylococcus epidermidis survivorship was rejected for all of the concentrations of pesticide, except for the 0.01% exposure to E. coli.

• Evidence suggests that the pesticide significantly reduces the survivorship in both species.

B. Agar Infusion• The null hypothesis was rejected.• Evidence suggests that the pesticide significantly

reduces the survivorship in both species.

Conclusions

Limitations

It was difficult to exactly synchronize the plating for the microbes, possibly contributing to the varying colony counts.

Infusion of pesticide into LB agar was not exact.

Extensions and Future Studies

Higher and lower concentrations of pesticide Different types of pesticides More species of bacteria More replicates Liquid agar infusion Vary pesticide exposure time Perform a growth rate experiment

Limitations, Extensions, and Future Studies

http://www.epa.gov/opp00001/pestsales/07pestsales/market_estimates2007.pdf

http://www.epa.gov/pesticides/about/ http://www.green-networld.com/facts/

pollution.htm http://www.beyondpesticides.org/

pesticides/factsheets/Synthetic%20Pyrethroids.pdf

http://www.docstoc.com/docs/9271628/Effect-of-pesticides-on-environment

References

SUMMARYGroups Count Sum Average Variance

0% 4 1238 309.5 1270.01% 4 1161 290.25 80.250.1% 4 1100 275 1241% 4 956 239 153.3333

ANOVASource of Variation SS df MS F P-value F crit

Between Groups 10686.19 3 3562.063 29.4031 8.19E-06 3.490295Within Groups 1453.75 12 121.1458

Total 12139.94 15

E. coli ANOVA

SUMMARYGroups Count Sum Average Variance

0% 4 913 228.25 188.250.01% 4 764 191 270.66670.1% 4 618 154.5 203.66671% 4 554 138.5 19.66667


Between Groups 19226.19 3 6408.729 37.57408 2.23E-06 3.490295Within Groups 2046.75 12 170.5625

Total 21272.94 15

Staphylococcus ANOVA

SUMMARY

Groups Count Sum Average Variance

Control 8 1587 198.375 114.2679

Low 8 1428 178.5 57.14286

High 8 1169 146.125 102.4107


Between Groups 11128.58 2 5564.292 60.96263 1.8E-09 3.4668

Within Groups 1916.75 21 91.27381

Total 13045.33 23

E. coli ANOVA: Infused

Groups Count Sum Average Variance

Control 8 2671 333.875 166.4107

Low 8 2309 288.625 304.8393

High 8 1990 248.75 403.3571

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 29023.58 2 14511.79 49.77706 1.07E-08 3.4668

Within Groups 6122.25 21 291.5357

Total 35145.83 23

Staphylococcus ANOVA: Infused

Tyler Barkich Grade 9 Pittsburgh Central Catholic High School.

Documents