JACK LEECHPITTSBURGH CENTRAL CATHOLIC

HIGH SCHOOLGRADE 11

2010

Pesticide Effects on Yeast Survivorship

Pesticides

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

Grouped in other categories, such as bactericides, fungicides, herbicides, rodenticides, and insecticides.

Insecticides are grouped under other titles such as ovicides, larvicides, and adulticides.

Concern Insect Killing Soap

Main ingredients include potassium salts of fatty acids which pierce the cell membrane, causing the contents to leak out and dehydrate.

Works by penetrating and destroying the outer shells of soft-bodied insect pests, resulting in dehydration and death within hours of contact.

Concentration of 1.03mL (3.5oz) per gallon of water.

Ortho Rose Pride

This insecticide uses acephate as its main active ingredients.

Acephate prevents the neurotransmitter acetylcholine from being degraded and destroys the nervous system.

Safer Brand Yard and Garden

Contains pyrethrins and also potassium salts of fatty acids.

Pyrethrins attack the insect’s nervous system by depolarizing a normally negatively charged cell, causing the nerve cell to constantly trigger action potentials and keep firing.

Potassium salts weaken the insect’s protective outer shell.

Non-Target Effects of Pesticides

Potassium Salts of Fatty Acids: Slightly toxic through skin exposure, mildly toxic to plant leaves which

hold the soap to its surface. Highly toxic to aquatic invertebrates. Soil half-life of one day.

Acephate: Very low toxicity to most animals, but extremely toxic to beneficial

insects (honey bees). Soil half-life of about half a day.

Pyrethrins: Highly toxic to aquatic vertebrates that use skin as a primary means of

absorption. Soil half-life of 12 days.

Yeast

Saccharomyces cerevisiae. Easy to manipulate in the laboratory.Most commonly studied cell.Very similar in structure and

biochemistry to more complex eukaryotes, including human cells.

Used as a eukaryotic cell model to mimic non-target cells.

Buds in single colonies, can be easily counted.

Purpose

Determine if any of the pesticides will have a significant effect on the survivorship of yeast colonies.

Hypothesis

Null-the pesticides used will have no significant effect on yeast survivorship.

Alternative-the pesticides will have a significant effect on yeast survivorship.

Materials

YEPD agar plates (1% yeast extract, 2% glucose, 1.5% agar)

YEPD media (1% yeast extract, 2% peptone, 2% glucose) Sterile capped test tube sterile dilution fluid (SDF) (10

mM KH2PO4, 10 mM K2HPO4, 1 mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl)

Concern Insect Killing Soap Ortho Rose Pride Insecticide Safer Brand Yard and Garden Spray Micropipette Permanent marker Plate spreader Ethanol Bunsen Burner Klett Spectrophotometer Incubator Sidearm flask

Procedure (Continuous Contact Exposure)

1. S.c. yeast was grown overnight in sterile YEPD media.2. A sample of the overnight culture was added to fresh

media in a sterile sidearm flask.3. The culture was placed in a incubator (30 C) until a

density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 107 cells/mL.

4. 0.1 mL of each variable at concentration of 1X (recommended dosage) was pipetted onto five plates per variable.

5. The plates were labeled and the agar was allowed to absorb the variable for thirty minutes.

6. The cell culture was diluted in sterile dilution fluid to a concentration of approximately 103 cells/mL.

7. After vortexing to evenly suspend cells, 0.1mL aliquots were removed from the tubes and spread on YEPD plates.

8. The plates were incubated at 30 C for 48 hours.9. The resulting colonies were counted. Each colony is

assumed to have risen from one cell.

Procedure (Pulse Exposure)

1. S.c. yeast was grown overnight in sterile YEPD media.2. A sample of the overnight culture was added to fresh

media in a sterile sidearm flask.3. The culture was placed in a incubator (30 C) until a

density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 107 cells/mL.

4. The cell culture was diluted in sterile dilution fluid to a concentration of approximately 103 cells/mL.

5. Each of the variables was pipetted into the SDF tube to reach the concentration of 1X, where it remained for thirty minutes.

6. 0.1 mL of the yeast/variable concentration was pipetted onto five plates per variable.

7. The plates were incubated at 30 C for 48 hours.8. The resulting colonies were counted. Each colony is

assumed to have risen from one cell.

Yeast Survivorship After Continuous Contact Exposure

Control

Conce

rn S

oap

Ortho

Ros

e Pr

ide

Safe

r Bra

nd0

50100150200250300

Survivorship After Indirect Exposure

Survivorship Af-ter Indirect Ex...

p=7.65 ^-10

Resulting Colonies

Groups of Variables

alpha: p = .05

Yeast Survivorship After Pulse Exposure

0

50

100

150

200

250

300

Survivorship After Direct Exposure

Survivorship After Direct Ex...

p=3.39 ^-11

Resulting Colonies

Groups of Variables

alpha: p = .05

Dunnett’s Test

If t-value is > than the t-critical (3.29), significant variation.

Dunnett’s Tests Results for Continuous Contact Exposure

Variables Used Results

Concern Soap

Ortho Rose Pride

Safer Brand

t-value(9.25)>3.48(Significant)

t-value(14.32)>3.48(Significant)

t-value(3.49)>3.48(Significant)

Not significant if cut-off of .01 is used. (t-critical of 4.98)

Dunnett’s Tests Results for Pulse Exposure

Variables Used Results

Concern Soap

Ortho Rose Pride

Safer Brand

t-value(13.14)>3.48(Significant)

t-value(18.44)>3.48(Significant)

t-value(9.58)>3.48(Significant)

Variance Between Similar Variables

Pulse Exposure

Concern Soap- Average: 173.4

Ortho Rose Pride-Average: 130.8

Safer Brand-Average: 202

Continuous Contact Exposure

Concern Soap-Average: 204.8

Ortho Rose Pride-Average: 164.2

Safer Brand-Average: 251

Single Factor ANOVA Results

Concern Soap: p value = .008846 (significant)

Ortho Rose Pride: p value= .008151 (significant)

Safer Brand: p value= .000129 (significant)

Interpretation

Pulse exposure appeared to have a more significant effect on yeast survivorship when compared to the continuous contact exposure based upon the statistical analysis.

The continuous contact exposure of Safer Brand resulted in a significant t-value when the t-critical in the Dunnett’s test was 3.48, but not 5.22. These t-critical values represent .05 and the more stringent alpha of .01, respectively.

Conclusions

Null hypothesis rejected for all trials in both the pulse and continuous contact exposures.

However, the result found in the Safer Brand continuous contact trial, although significant when compared to the standard cut-off of .05, the result would not be significant with the more rigid value of .01.

Limitations and Inconsistencies

Although no visible contaminants, environmental contamination could have been a factor.

Other methods of destruction from the pesticides could have effected the yeast growth aside of the main active ingredients.

Plating was not exactly synchronized, which could possibly result in varying colony counts.

Continuations

Use a wider range of pesticides in addition to insecticides.

Test the specific ingredients of the pesticide instead of the entire product.

Test different concentrations of each variable instead of the listed recommended concentration.

A combination of pesticides could be used simultaneously (synergetic exposures).

Works Cited

www.planetnatural.com

www.saferbrand.com

www.npic.orst.edu

www.epa.gov