The effect of 2-Butoxyethanol on microbial life Alex Kochman ‘21 Central catholic high school

2-butoxyethanol Primary alcohol Clear, colorless, sweet smelling Flammable Used mainly in paints, solvents, inks, cleaning products and surface cleaners Usually used in amounts less than or equal to 700 ppm Recommended 25-50ppm/8 hour day General population usually exposed through household cleaners

Toxicity of this chemical Has negative effects on the following: Liver Reproductive health Lungs Skin DNA Hemoglobin levels Chemical balance in the blood Various cancers?

Microbial flora Various eukaryotes and prokaryotes; mostly bacteria Flora effects on human health is a hot area for research Positive micro flora effects- nutrition, digestion, immune regulation, and anti-foreign pathogens Most chemicals ingested by humans aren’t evaluated for their effects on microbial flora

Gram positive vs. gram negative Cell walls contain an extra layer of lipopolysaccharides for extra protection Outer membrane protects against local environments Most pathogenic bacteria in humans are Gram (+) organisms Simple cell wall Some antibiotics work against the formation of the cell wall

Escherichia coli (e.coli) Microbial flora commonly found in the human intestines and stomach Gram-negative Non-pathogenic (most strains) Prevents colonization of the intestine with pathogenic bacteria

Staphylococcus epidermidis (staph) Part of normal human flora, typically skin flora, and less commonly in mucosal flora Gram-positive Non-pathogenic Helps protect skin from pathogenic bacteria

Purpose To determine if 2-butoxyethanol has a significant impact on the survivorship of E.coli and Staph

Hypotheses Null: 2-butoxyethanol will not have a significant impact on the survivorship of E.coli and Staph Alternative: 2-butoxyethanol will significantly reduce survivorship among E.coli and Staph

Materials Micro pipettes Sterile pipette tips Sterile spreader bars Ethanol Bunsen burner Vortex Labeling tape 2-butoxyethanol E.coli (DH5 Alpha) Staphylococcus epidermidis (Ward’s Science) Sterile dilution fluid(10 mM KH2PO4, 10 mM K2HPO4,1 mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl) Sterile test tubes Luria broth agar plates (1% Tryptone, 0.5% Yeast extract, 1% NaCl) Pipettes Incubator

Procedures (liquid pulse) 1. The E.coli and Staph were grown overnight in sterile LB media at 37° Celsius 2. The cultures were diluted until certain concentrations of cells were reached (approximately 10⁵ cells/ml for both E.coli and Staph) 3. Used the following ratios and a 10% 2- butoxyethanol stock solution

Concentration chart 9.9mL 0mL Concentration 9.89mL 9.8mL 8.9mL 0.01mL 0% 0.01% 0.1% 1% Sterile Dilution Fluid 9.9mL 9.89mL 9.8mL 8.9mL Stock of 2- Butoxyethanol 0mL 0.01mL 0.1mL 1mL Microbe Final volume 10mL

Procedures (liquid pulse Continued) 4. Cell suspensions were vortexed 5. Tubes were incubated at room temperature for 10 minutes 6. 0.1mL was transferred and spread onto each plate (8 replicates for each concentration) 7. Plates were incubated for 24 hours, and then colonies were counted 8. Statistics were performed to check for significant variation

2-butoxyethanol effect on e.coli (liquid pulse) E.Coli Surviorship P-value= 2.32E-07 -10.8% 252.5 225.3 -28.1% 181.6 -49.2% Average Surviving Colonies 128.3 0% 0.01% 0.1% 1% Concentration

Dunnett’s test e.coli (T-crit= 3.03) Concentration T-value Significance 0.01% 1.72 Insignificant 0.1% 4.48 Significant 1% 7.86

2-butoxyethanol effect on staph (liquid pulse) Staph Surviorship 840.3 P-value=1.69E-09 Average Surviving Colonies -47.5% 441.3 -70.0 -68.5% 251.8 264.3 0% 0.01% 0.1% 1% Concentration

DunneTt’s test staph (t-crit= 2.21) Concentration T-value Significance 0.01% 6.96 Significant 0.1% 10.27 1% 10.05

Procedures (agar infusion) 1. 200 microliters of the 10% 2-butoxyethanol stock was spread directly onto plates to create a high concentration 2. 20 microliters of stock and 180 microliters of pure water were spread onto other plates to create a low concentration 3. Plates were incubated for one hour 4. 0.1mL from the liquid pulse control tube was spread onto plates 5. Plates were incubated for 24 hours

Combined survivorship graph (agar infusion) E. Coli P-value= 0.000195 Staph P-value= 6.24E-05 840.3 831.8 Average Surviving Colonies 367.5 252.5 223.3 173.5 Control Low High Concentration E.Coli Staph

Agar infusion Dunnett’s test (e.coli=3.03/Staph=2.21 Concentration T-value Significance E.Coli Hi 5 Significant E.Coli Lo 1.85 Insignificant Staph Hi 8.25 Staph Lo 0.15

Conclusions 2-butoxyethanol had a significant impact on the survivorship of microbial flora Data suggests that the null hypothesis was rejected for E.coli in all cases except 0.01% and the low concentration agar infusion test Data also suggests that the null hypothesis was also rejected for Staph in all cases but the low concentration agar infusion test

Limitations Only used a maximum of 8 replicates Didn’t analyze where or how the agent damaged the cell Only one exposure time Only liquid pulse and agar infusion tests were performed Reproduction was not examined Unknown concentration of 2-Butoxyethanol in body

Sources of possible error Small measurement errors Synchronization errors during plating processes Small differences in exposure time

Extensions Add more replicates or concentrations Perform other types of tests (E.g. disk assay) Perform a detailed analysis of cell damage Add other types of flora

References https://www.atsdr.cdc.gov/phs/phs.asp?id=345&tid=6 1 https://www.ewg.org/guides/substances/152484#.Wh HjFLpOmhA https://pubchem.ncbi.nlm.nih.gov/compound/8133

Raw data 0% (control) 0.01% 0.10% 1% Hi Lo 272 235 190 124 160 230 228 180 126 156 172 213 260 231 188 121 200 212 233 236 112 162 238 241 254 211 296 216 219 268 222 Staph 752 357 245 279 396 790 898 496 355 878 702 352 317 295 407 763 1009 560 214 223 312 896

Anova (e.coli) Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 8 2020 252.5 658.8571429 Column 2 6 1352 225.3333333 640.6666667 Column 3 1090 181.6666667 1224.266667 Column 4 4 513 128.25 368.25 Column 5 694 173.5 339.6666667 Column 6 893 223.25 164.9166667 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 52660.70833 5 10532.14167 16.54079894 2.32722E-07 2.586790087 Within Groups 16555.16667 26 636.7371795 Total 69215.875 31

Anova (staph) Groups Count Sum Average Variance Column 1 4 3361 840.25 19570.91667 Column 2 1765 441.25 10720.91667 Column 3 1007 251.75 2052.916667 Column 4 1057 264.25 960.9166667 Column 5 1470 367.5 1869.666667 Column 6 3327 831.75 4245.583333 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 1456161.208 5 291232.2417 44.32655549 1.69072E-09 2.772853153 Within Groups 118262.75 18 6570.152778 Total 1574423.958 23