Kavya Gupta & Dagny Whall
San Diego, California
Regional Stockholm Junior Water Prize (ISEF affiliate award) | 1st place award for Environmental Sciences and Management Category (Senior Division) at the Greater San Diego Science & Engineering Fair | ASM Materials Most Outstanding Exhibit in Materials Science Award | Professional Engineers in California Government (STAGE) | Selected for CSEF (California Science & Engineering Fair)
The research and solution address the chemical GenX, a toxicant present in major water lines across the United States due to factory spillage and carless removal. This chemical is used in many daily products such as the coating of non-stick cookware, plastic cups and even clothes. Currently, the United States does not have a system to detect the presence of GenX in home water systems because the effects were unknown until now which is why a simple test kit for home consumers to apply for their home water systems was designed. A colorimetric indicator approach was used to isolate the main function groups and molecules in the unique structure of GenX. Data showed how GenX is even more harmful to our metabolism; therefore, as a solution this project was done to develop a simple product for home consumers to test whether GenX is present in their water sample. By analyzing two genes’ protein expression in the C. elegans when exposed to GenX, gene expression was measured using qRT-PCR (a method which measures protein folds or “density”). Since a downregulation in the activity of the metabolism related genes was shown, it was concluded that GenX also has detrimental effects on organisms. In the future, plans are to improve the design of the indicator by creating a “strip-like” single paper test that can effectively detect GenX in aqueous solutions and to create a system to filter out the toxicant from the waters.
Key Words: GenX, Hexafluoropropylene oxide dimer acid, per- and polyfluoroalkyl substances (PFAS), Caenorhabditis elegans (C. elegans), Escherichia coli, Polymerase chain reactions, Carboxylic acids, pxmp-4, nhr-4
INTRODUCTION
GenX is a cousin of per- and polyfluoroalkyl substances (PFAS) man-made chemicals used in the production of many consumer products like cookware, food packaging, and stain repellent (Tribune News Service, 2019). Hexafluoropropylene oxide dimer acid (HPFO) dimer acid, also known as GenX, when released to the freshwater environment will dissociate to the HFPO carboxylate anion and H3O+. The ammonium salt will dissolve to the HFPO carboxylate anion and the ammonium cation (NH4+). Both have high solubilities in water and are expected to remain in water with low sorption to sediment or soil. GenX is a trade name for a technology that is used to make high-performance fluoropolymers (e.g., some nonstick coatings) without the use of perfluorooctanoic acid (PFOA). HFPO dimer acid and its ammonium salt are the major chemicals associated with GenX technology. Due to chemical spills and improper disposal, these toxicants have been found in surface water, groundwater, finished drinking water, rainwater and air emissions in some areas (Tribune News Service, 2019).
PFAS chemicals are carcinogenic to humans (State of North Carolina Department of Health and Human Services Division of Public Health, 2017). There are many other PFAS, including GenX chemicals and PFBS in use throughout our economy. A study, published in the journal International Journal of Environmental Research and Public Health, found that the most well-studied PFAS compounds – PFOA, formerly used by DuPont to make Teflon, and PFOS, formerly an ingredient in 3M’s Scotchgard – exhibit up to five key carcinogenic characteristics(State of North Carolina Department of Health and Human Services Division of Public Health, 2017). Research has shown that PFAS impacts biological functions linked to an increased risk of cancer (State of North Carolina Department of Health and Human Services Division of Public Health, 2017). This is worrisome, given that all Americans are exposed to PFAS mixtures on a daily basis, from contamination in water, food and everyday products. It was also shown that factory workers working in factories that processed PFAS chemicals were starting to develop tumors and different types of cancers. Since these groups of chemicals are known as the “forever chemicals” the exposure is very likely to move onto the next generation as well. (Woolverton, 2020) This was proven with factories that use PFAS chemicals where the factory worker’s children were born with misshapen faces and tumors because of the mothers who were exposed to PFAS chemicals (DuPont Lawsuits (Re PFOA Pollution in USA) | Business & Human Rights Resource Centre, n.d.).
According to a Q&A session held by the government, currently, the EPA is in the process of making the draft toxicity assessments of GenX but there are no regulations (State of Rhode Island: Department of Health, n.d.). The Q&A answered by the government proves that they are not making any effort to change the status quo. Currently, the only way for homeowners to test their water systems for GenX is through a lab process by sending their water samples to a government facilitated lab which takes around 6 to 7 weeks(Culligan Waters, n.d.), it is costly and requires previous experience in order to operate the machines at the labs since it uses the EPA recommend method for testing for GenX. Currently, there is no easy and cheap method for home consumers to test their waters for GenX chemicals and homeowners have no option for filters nor home testing kits.
In terms of a new device or method to detect GenX, the field is rather undeveloped. Besides the standard EPA method, which takes an abundance of time and costly equipment, there are a couple of methods being worked on. One of which is using a chip. The “chip method” is still rather inconvenient because of the expensive equipment that it requires (Fang et al., 2019, p. 167). A second method is the “voltage method”. The voltage method works by applying current through very small electrodes in hope to create a reaction (Ranaweera et al., 2018). The science behind this method is based on the idea that GenX weakens water’s surface tension, making bubbles form more easily. This theoretically would be extremely useful with the exception of the nano electrodes, which are extremely costly.
The goal of this project is to prove how GenX may also be as deadly to humans as PFAS chemicals are. Caenorhabditis elegans (C. elegans) are microscopic translucent worms about smaller than a grain of rice mainly used as model organisms. Around 80% of the C. elegans share similar genes to humans which make them good model organisms for research.
By focusing on metabolism-related genes in C. elegans research can determine whether or not GenX affects the metabolism of the organism. According to the Public Comment Draft: Human Health Toxicity Values for GenX Chemicals, previous research on toxicokinetic was performed on rats and mice and the HPFO dimer acid was shown to be absorbed from the gastrointestinal tract at levels that were proportional to the dose given.(EPA 18) Gene expression is an extremely common way of measuring changes in various organisms. By exposing C. elegans through their food, Escherichia coli, a type of bacteria that C. elegans feed on and live, it is possible to extract its RNA for analysis. Now the effects of GenX on the C. elegans genes can be related to how humans’ bodies react to the GenX. This is because the genes analyzed have human orthologs, meaning that humans have similar types of genes except in a larger scale that help operate even more organs in the body system. Extracting a segment of the gene’s RNA and amplifying it using polymerase chain reaction (PCR) can show measurable differences compared to other genes or genes in other organisms. qRT-PCR involves with quantifying/counting how much of the section has been collected through a fluorescent aid. In this case, one can compare the data found from this experiment to how humans’ bodies are reacting to GenX with quantitative data.
Once the gravity of the problem is figured out through analysis of the gene expression, the goal is to create an efficient device to detect GenX in an aqueous solution. This leads to the second part of the project, focusing on the properties of GenX. By creating indicators to detect different groups in GenX’s aqueous form, a detection system can be created. With the use of simple day to day objects, the cost of many of the materials can be cut down. Using things like pH paper (for the Carboxylic Acid, a type of function group/organic compound present in HFPO acid that contains a carboxyl group (R-COOH)) and Ammonia Strips for the Ammonium group in HPFO dimer Acid help create a cheap and affordable device.
MATERIALS & METHODS
First, all the materials were gathered to perform both the experiment and test the prototype detector. These materials included: ammonia strips, blue litmus paper, fluorite powder, test tubes with stands, syringe, contaminated water samples, distilled water, fluorite, C. elegans, NDM plates, LB, E. coli, a Trizol kit, and RNase free materials.
To test the C. elegans, the organism was first isolated at L1 life stage using a common bleaching technique. When bleach was poured on the plate, only the eggs would remain alive due to their shell outing. Then, 48-72 hours were given to let the worms hatch and grow into the latter life stages. After, to expose the C. elegans, one plate is streaked with E. coli and one with E. coli and GenX in order expose the E. Coli to GenX. This was performed with a common crossing streaking technique for E. coli to ensure proper growth of E. coli. For the exposed sample, a solution containing 1% GenX was pipetted on top of the sample and grew for 5 days. To place the bleached C. elegans on the plate, a pipette is used to place them on each respective plate.
To perform the PCR, reverse transcriptase was done on the RNA so that the RNA can be turned into DNA. This is crucial because the conversion from RNA to DNA allows PCR to occur, which would otherwise not be possible. This specific way of analyzing measures the gene’s activity in the mRNA, or messenger RNA, state. After 2 hours, RNA extraction is performed. This was done using the Trizol method with RNase/nuclease-free materials. Once the processes has been completed, Nanodrop is used to analyze the results and create a heat graph and melt curve to show how the results look graphically.
The beginning of design process for the chemical Indicators and Test Kit was done by using a model that applies voltage to water, expecting a different appearance between distilled and GenX water. Using a gel box, both distilled water and GenX water was tested with the same amount of voltage. After testing, there was no difference between the two water samples when the voltage was applied.
Figure 1: The 3D model that was created first on a software, Maya Autodesk.
Figure 2: This is the unique chemical structure of GenX where we have circled the main groups that we indicate using simple tests in our kit.
This led to a more chemical design approach. In this approach, pH strips and ammonia strips were used based on the knowledge of carboxyl groups and ammonia groups in GenX. As shown in Figure 2. HFPO dimer acid has three main functional groups, fluorine, ammonia and carboxyl. Since GenX’s chemical structure is so unique tests are found for each specific group then other possible options of substances can be eliminated to be present in the water sample. Isolating the fluorine group was focused on, ammonia and carboxylic acid. For each of these groups, a simple color test was used to see whether that substance is present in the water sample or not. Since carboxylic acid is an acid, a pH of around 2 or 3 indicated on a pH strip means that there is acid present in the water sample. The same goes with detecting ammonia through an ammonia strip. Then for fluorine, a mineral called fluorite was used which was supposed to have a small bubble reaction with a GenX solution. The fluorite, however, did not produce bubbles when mixed with the aqueous solution of GenX. To construct the device prototype, first three different test tubes were obtained and stood upright and labeled to show which tubes continued which indicator. For example, the tube with the pH strip would be labeled with a piece of tape with writing showing “pH strip.” . In two tubes containing 1% GenX one ammonia strip was placed in one and one pH strip was placed in the second tube. In the third distilled water was poured in containing no GenX to act as a control. Since it was observed that yes, there was the presence of carboxylic acid and ammonia with the strip tests, it could be concluded that the water sample is contaminated with GenX. A simple test kit idea was then formed which included a box, and a small kit with all of the necessary testing strips. For the box, a 3D printed version of the designed model which was created. The wells and spout were designed to be clearly defined for the consumer to be able to put in their water sample and test whether or not GenX is present. Figure 3 shows the 3D printed prototype of the detection kit. As the labels describe, there is the initial spout where the water sample will enter and then move into the four wells containing the tests for each group, including one that is designed to be a control with no test present in the well.
Figure 3: A prototype of the 3D model was created for the test kit with the help from a 3D printing lab from Qualcomm.
RESULTS
Gene Expression Quantified through qRT-PCR
Figure 4: Calculated using the information from the heat map, the figure shows the increased protein expression in the two genes when the C. elegans food is GenX contaminated. The blue and orange bars represent the relative expression for each gene. Using a 2-ΔΔCt threshold value the bar graphs were created to show the relative expressions between the GenX contaminated and control worm’s gene expression. This data was confirmed with a melt curve.
Figure 5: Figure 5 shows the increased folds/protein expression in the genes, again there is an increase in folds in the C. elegans contaminated by GenX. The red indicates a higher expression of the gene, while the green demonstrates a lower. In the figure, the left column represents the control and the right column represents the GenX.
Figures 6 & 7: Figures 6 and 7 show the pH strips and Ammonia strips after dipped in regular distilled water and the GenX aqueous solution. The pH of the GenX is approximately 1.3 and the pH of the distilled water is 7. Ammonia in the GenX water is approximately 3mg/L while control has 0mg/L.
DISCUSSION
Based on the analysis of gene expression, expression of the C. elegans, GenX significantly impacts its body systems. The two focus genes for this experiment, nhr-49 and pxmp-4 showed significant change between the control and experimental groups. In the experimental groups, nhr- 49 and pxmp-4 downregulated by a 4.3-fold and 1.8-fold. Nhr-49 and pxmp-4 both contribute to metabolism. Since the two genes were downregulated, it can be assumed that metabolism will be slowed down since the genes are not as active. This is because nhr-49 is a regulator for metabolism and pxmp-4 is also expressed in metabolism. Also, the decreased expression suggests decreased demand for peroxisome membrane proteins, specifically impacting lipid metabolism. Once metabolism is affected, every system is drastically impacted because the organs are not able to acquire the necessary amount of energy to maintain regular function. This clearly proves how GenX affects organisms just like PFAS chemicals. The effects that we see on the C. elegans metabolism is the same to what is happening to humans and their systems.
For the indicator test, the results went as predicted. The pH was extremely low proving that the carboxylic acid in the GenX solution makes it extremely acidic. The clear ammonia strips also had a difference where the GenX contaminated solution strip has a bluish tint to it, showing a higher level of ammonia in the GenX solution. The fluorite mineral in the aqueous solution did not show significant change compared to the control meaning fluorite does not cause a chemical reaction with the fluorine in the HPFO dimer acid solution. However, the data provided only is applicable for 1% concentrated GenX in water solutions, higher concentrations may provide different results.
This research has proved how GenX, just like PFAS chemicals is harmful to our systems by affecting our metabolism. Companies are disposing these toxicants into major water sources which lead to the contamination of home water lines which is a vital reason why change is needed. Since the consequences of using GenX were proved through this project, GenX should have more regulations in order to ensure that cleaner water is provided to homes across the country. Governments need to be informed about the detrimental effects of GenX, to implement bans on the use of GenX. Since GenX is a newly introduced concept, the physical effects will appear in the coming years in the form of birth defects, tumors and even carcinogenic affects. Although GenX is a different type of PFAs chemicals, it is likely that it will cause similar problems to society. In order to stop even more future problems, GenX also needs to be removed from the system to save the lives of many humans. When the government was asked if the EPA has any regulations on GenX chemicals their response was no (EPA, 2018). This project has clearly proven how metabolism is affected by constant exposure to GenX which is why the government needs to take a stance with this.
The first step local citizens should take when realizing that GenX is everywhere around them is trying to see if they might be drinking it. As mentioned before, there are no easy and quick methods for home consumers to do so. (EPA, 2018) This is why the simple testing kit developed in this project would help local citizens be more aware about the toxicants in the water system and environment around them. The box works as a simple eye detection kit, immediately indicating the presence of two groups in the structure of GenX. Current work on this project is focusing on finding a way to have a simple indicator for the ether group. Finding more indicators and creating a bigger sort of “checklist” in for the kit will help remove false negatives that might come up and be more accurate. Due to the negative result of the fluorite testing, future plans include finding a simple indicator test for the ether group in the GenX structure. A quantitative method to identify the amount of GenX present in the water sample is also being developed within this project’s scope, so the home consumer knows whether or not the amount of toxicant present in their water sample is under the advised regulation of the EPA. Future work that many more researchers should focus on is creating a replacement for these chemicals, that way companies are able to make their products while the environment is kept safe.
CONCLUSION
GenX affects C. elegans through pxmp-4 and nhr-49 at the tested concentration
Nhr-49’s decreased expression indicates that GenX negatively impacted the organism’s energy metabolism and fat usage. Adult lifespan could be affected if the exposure did not stop at L4. pxmp-4’s slightly decreased expression suggests decreased demand for peroxisome membrane proteins, and potentially impacted on lipid metabolism (Worm base, C. Elegans). Potentially similar gene expression effects in humans, due to pxmp-4 and nhr-49 being also present in humans. Melt curve analysis shows there is a second amplification product for one technical replicate of pxmp-4 but taking out that data point will not affect our conclusion.
In the home consumer test kit, using Chemical indicators, proved to be successful. Both the results showed a distinct difference from water. If both of the indicators show that the functional groups are present in the water sample, then one can determine that their water sample has dissolved HPFO dimer acid in it. The final design, being the chemical indicator at-home kit, successfully meets the requirements set at the beginning of the project. The final product created is both affordable and effective in the detection of GenX. In the future, we would want to test fluorite’s ability to indicate the presence of fluorine to make an even more sound conclusion that the water is contaminated.
ACKNOWLEDGEMENTS
We would like to thank Jeanne Vu and Darcy Engelhart for all of their support throughout the process of experimentation and design. Additionally, Mrs. Holly Eaton for the generous mentorship she has provided. We would also like to thank the Boz Institute for providing us lab space, equipment, materials, and advice.
REFERENCES
Drinking Water Health Advisories for PFOA and PFOS. (2020, June 29). US EPA. https://www.epa.gov/ground-water-and-drinking-water/drinking-water-health-advisories-pfoa-andpfos#: %7E:text=EPA’s%20health%20advisory%20level%20for, and%20PFOS%20in%20drinking%20water.
Wormbase. “C. Elegans.” https://wormbase.org/species/c_elegans#401--9 (Accessed Feb. 10, 2020)
Ranjana Kishore, R. K. (n.d.). pxmp-4 (gene) - WormBase : Nematode Information Resource. Wormbase. Retrieved July 8, 2020, from https://wormbase.org/species/c_elegans/gene/WBGene00017574#0-9f-10
Tribune News Service. (2019, May 1). GenX found after Chemours water spill. News & Record.https://www.greensboro.com/ap/north_carolina/genx-found-after-chemours-water-spill/article_4 07aa20a-460d-530a-a60e-4e7793e458e6.html#:~:text=State%20officials%20started%20investig ating%20Chemours,private%20wells%20near%20the%20facility.
State of Rhode Island: Department of Health. (n.d.). PFAS Contamination of Water: Department of Health. Retrieved July 8, 2020, from https://health.ri.gov/water/about/pfas/
State of North Carolina Department of Health and Human Services Division of Public Health. (2017). GenX Health Information. Department of Health and Human Services. https://files.nc.gov/ncdeq/GenX/GenX%20factsheet%20FINAL%2013Sep2017.pdf
Ranaweera, R., Ghafari, C., & Lou, L. (2018, June). Bubble-Nucleation-Based Method for the Selective and Sensitive Electrochemical Detection of Surfactants. National Library of Medicine
Fang, C., Wu, J., Sobhani, Z., Amin, Md. A., & Tang, Y. (2019). Aggregated-fluorescent detection of PFAS with a simple chip. Analytical Methods, 11(2), 163–170. https://doi.org/10.1039/c8ay02382d
Culligan Waters. (n.d.). GenX. Retrieved February 18, 2020, from https://www.culligannc.com/water-problems/genx
Hougue, C. (2018, February 12). What’s GenX still doing in the water downstream of a Chemours plant? Chemical & Engineering News. https://cen.acs.org/articles/96/i7/whats-genx-still-doing-in-the-water-downstream-of-a-chemours-plant.html
Woolverton, P. (2020, January 15). U.S. House Passes GenX/’Forever Chemical’ Water Contamination Bill. Fayetteville Observer. https://www.fayobserver.com/news/20200115/us-house-passes-genxlsquoforever chemicalrsquo-water-contamination-bill
DuPont lawsuits (re PFOA pollution in USA) | Business & Human Rights Resource Centre. (n.d.). Business & Human Rights Resources Center. Retrieved February 20, 2020, from https://www.business-humanrights.org/en/dupont-lawsuits-re-pfoa-pollution-in-usa
Waymer, J. (2018, November 15). Group says replacement non-stick chemical poses health risks. Floridatoday. https://eu.floridatoday.com/story/news/local/environment/2018/11/15/group-says-replacement-n on-stick-chemical-poses-health-risks/2010096002/
EPA. (2018, November). Fact Sheet: Draft Toxicity Assessments for GenX Chemicals and PFBS. Fact Sheet: Draft Toxicity Assessments for GenX Chemicals and PFBS Public Comment Draft: Human Health Toxicity Values for GenX Chemicals. https://www.epa.gov/sites/production/files/2018-11/documents/genx_public_comment_draft_toxicity_assessment_nov2018-508.pdf
BIBLIOGRAPHY
Bullis, K. (2015, December 1). Cheap Chemical Sensors. MIT Technology Review. https://www.technologyreview.com/2005/12/01/230011/cheap-chemical-sensors/
ChemistryViews.org. (2017, December 16). Easy Detection of Fluoride in Water :: News:: ChemistryViews. https://www.chemistryviews.org/details/news/10702136/Easy_Detection_of_Fluoride_in_Water.html
EPA PFAS Drinking Water Laboratory Methods. (2020, April 27). US EPA. https://www.epa.gov/pfas/epa-pfas-drinking-water-laboratory-methods
Farrell, B. L. (1974). Fluorine, a direct indicator of fluorite mineralization in local and regional soil geochemical surveys. Journal of Geochemical Exploration, 3(3), 227–244. https://doi.org/10.1016/0375-6742(74)90024-7Accessed Feb. 5, 2020).
Hiolski, E. S. T. C. (2019, May 13). New electrochemical method detects PFOS and PFOA. Chemical & Engineering News. https://cen.acs.org/environment/persistent-pollutants/New-electrochemical-method-detect s-PFOS-and-PFOA/97/web/2019/05
ABOUT THE AUTHORS
Kavya Gupta
I am Kavya Gupta, a high school sophomore in San Diego, California. I am an avid STEM enthusiast participating in many STEM-related activities around my community and school, being a WiSTEM ambassador/chapter founder for my school and being in county-wide women in STEM exposure program. During my free time, I love to play the piano, play tennis, and practice classical Indian dance. In the future, I am planning to go down a bioengineering/medical field. To further expand my interest in science, I try to work on different STEM problems that interest me whether it requires engineering or 3D imaging.
Dagny Whall
My name is Dagny Whall and I am a junior at La Jolla High School. In my future, I hope to pursue a career in STEM, potentially in computational biology. In my free time, I enjoy hiking, spending time with my friends, and learning about new topics. I have recently really enjoyed learning more about programming by exploring different languages as well as web development.