Pollution Source Tracking Technologies

From Beachapedia

By Paula Sternberg Rodríguez, August 2022


Pollution source tracking is the study of the origin of contamination in the environment. A complete approach to source tracking will promote action to eliminate the contamination at its source to restore water quality conditions and improve human and environmental health. This article describes the most commonly used methods that are used to determine the sources of fecal pollution in recreational waterways, and includes a discussion of their general cost and accessibility.

Introduction


Pollution source tracking is typically performed in water bodies that support recreational uses and where pollution has the highest likelihood of causing human illness and impacts on the local economy. This is because an estimated four billion surface water recreation events occur annually in the United States, resulting in an estimated 90 million illnesses nationwide and costs of $2.2- $3.7 billion annually.[1] As part of the “ocean economy”, clean, healthy beaches drive extremely valuable coastal tourism industries and communities, as they support 2.5 million jobs and generate $157 billion annually in GDP.[2]

The risk of illness from exposure to polluted water reduces and can even negate the health and enjoyment people get from surface water recreation. In comparison to non-water recreators, water recreators in the U.S. have a higher incidence of acute gastrointestinal illnesses, and other illnesses that include respiratory, ear, eye, and skin symptoms[3]. The passage of the BEACH Act of 2000 established guidelines and federal funding assistance for water quality monitoring and notification programs to protect public health at the beach. Agency-run beach programs, as well as volunteer efforts like the Surfrider Foundation’s Blue Water Task Force, detect and measure concentrations of fecal indicator bacteria (FIB) as a measure of public health risk. The most commonly measured FIB, E. coli and enterococcus, are prevalent in the gut and digestive tract of warm blooded animals, including humans, birds, dogs, deer, rodents and other mammals.

High FIB concentrations indicate the presence of fecal matter and pathogens in the water that can make people sick. The testing methods used by most state and local beach programs, however, do not provide any information on which species is associated with those high FIB concentrations. For instance, high levels of enterococcus in coastal waters could be due to the presence of untreated human sewage, but it could also be due to a high density of marine birds defecating in the area.[4]

To truly improve human and environmental health conditions, pollution source tracking efforts must also be carried out in recreational waterways that experience problems with chronic or regular pulses of high levels of contamination. A wide variety of pollution source tracking methodologies exist, with some methods being more accessible, used, and further developed than others. While source tracking can tell us what species is causing fecal contamination, other methodology can be implemented to determine the specific source in the area (e.g. if it’s confirmed human-caused, where is the actual sewage coming from- a broken pipe, sewer misconnect, faulty septic system?) Because of this, there are other methods that help pinpoint exactly who is responsible for fecal contaminants and where they are coming from.

General limitations

As source tracking is a study that takes place in environmental systems, there are limitations associated with each method. Some of the general limitations for source tracking include spatial (geographic) and temporal (time-related) stability. This means some trackers, or parameters that are being measured, are regionally specific, may drift or travel, and may not persist over time since they can be metabolized or broken down. Changes related to seasons, climate, temperature, currents and natural variability in watersheds must also be considered, as these are often unpredictable and constantly changing factors that may affect both the spread and concentrations of trackers used to indicate pollution sources. Source tracking results will vary depending on the choice of tracker, location and time of sampling and analysis.

Types of Pollution Source Tracking


The predominant methods of pollution source tracking can be separated into two main types, microbial source tracking and chemical source tracking. There are also novel approaches, such as dog-sniffing, which will be touched on briefly in this article but are not used extensively.

Microbial Source Tracking


Microbial source tracking refers to the use of microbiology and genetic analysis to trace the origin of pollution. Microbial source tracking technologies have been developed to identify the sources of fecal pollution in waterways with the goal of guiding pollution solution strategies.[5] These techniques can be used to identify the exact species (i.e human, dog, or other animal) that are contributing fecal indicator bacteria to any given sampling site or waterbody. Microbial source tracking methods are able to detect and in some cases measure pollution trackers or markers such as microbes, bacteriophages, antibiotic resistant genes, and even entire communities of microbes. Different markers can be detected by taking samples of environmental DNA, or eDNA, which is DNA expelled from different organisms that accumulates in their surroundings. Environmental DNA can be collected from different types of environments including bodies of water, and can be analyzed using microbial source tracking technologies. There are a wide range of microbial source tracking approaches, with some of the most popular methods described below.

PCR Technologies


The most common microbial source tracking methods use PCR, or “polymerase chain reaction” analysis, to measure and characterize DNA. This is useful because each animal species, including humans, have different DNA markers that can be assessed and measured to determine what animal is actually contributing to the fecal pollution.

PCR techniques are a continually developing science, and as such, variations of the traditional technique are now being used. Traditional PCR makes millions to billions of copies of a specific DNA segment, which allows scientists to take a very small sample of DNA and “copy”, or amplify it to a sufficiently large enough amount to study in detail. Traditional PCR has served as a base for the development of other PCR technologies like qPCR, also known as real-time PCR, which stands for “quantitative polymerase chain reaction”, and ddPCR, which stands for “droplet digital polymerase chain reaction”. All PCR technologies have the goal of identifying specific DNA strands through amplification, yet they have slight differences in their methodologies, precision, and how commonly they are used.

Traditional PCR methods are less precise as they measure the final amount of replicated DNA instead of the replication as it occurs (like qPCR). Traditional PCR methods also require processing after the reaction, have lower sensitivity, and can take 3-4 hours to complete. Advancements in PCR methodologies have resulted in improved precision and performance. For instance, unlike traditional PCR techniques, qPCR measures the quantity of nucleic acids in DNA, instead of just identifying the presence or absence of a particular segment of DNA, which provides higher precision. This method is also less time-consuming, has higher resolution, and in recent years qPCR has proven advantageous over traditional PCR techniques. Because of its advantages, qPCR is one of the primary techniques employed in microbial source tracking. Alternatively, ddPCR makes a discrete “digital” measurement by measuring individual DNA molecules that are separated into small droplets. When used properly, ddPCR can be even more precise than traditional PCR and qPCR techniques, and is currently being used to detect fecal indicator bacteria for rapid beach testing in the San Diego region.

A wide variety of organisms found in nature have DNA that can be detected by PCR techniques and used as trackers to identify sources of contamination. Different trackers such as microbes, bacteriophages, antibiotic resistant genes, and even entire communities of microbes found in bodies of water can be put through different PCR analyses. The results of those analyses can determine what species the samples are associated with. For instance, once the DNA has been identified and quantified, it is possible to determine whether certain fecal contamination is human in origin or if other animal species are contributing to the pollution including dog, cattle, bird, rodents, deer and other warm-blooded animals. Once the presence and concentration of a particular tracker is found, identifying the source of the contaminant is much easier. The decision to use PCR, qPCR, or ddPCR depends on the goals, budget, and tracker used for a particular study.

Conducting source tracking studies with qPCR analyses has become more and more accessible, with companies like Jonah Ventures offering source tracking services that use host-specific DNA. With this company, the cost per sample kit is $125 and samples environmental DNA to find marker genes of human, dog, cattle, pig, sheep, and poultry DNA. The sampling kit allows for any person who purchases the kit to collect water in a syringe, push it through a filter, inject a preservative, and mail the filter to Jonah Ventures in the supplied barcoded sample cup and return shipping envelope. For other qPCR analyses, costs can vary depending on the quantity of samples you have and the amount of “primer sets”, or different species, you want to test for. Since qPCR has a variety of applications, it is a tool that can be employed on different organisms and genetic trackers.

Despite ddPCR’s merits, it still holds some disadvantages, such as higher costs and less availability because fewer labs run this analysis than qPCR. It has been reported that ddPCR can cost up to twice as much as qPCR, and its lesser availability has prevented the distribution of ddPCR technology in developing countries20. However, as these technologies continue to progress, lower costs and higher accessibility is sure to follow.
In summary, PCR techniques are tools that can help us identify DNA fragments coming from different organisms or sources called markers or trackers. The following are common microbial source trackers that can be amplified and identified with the help of PCR technologies.

Host-specific Bacteria


Host-specific bacteria are some of the most studied and widely used source trackers. There are many bacteria that are host-specific, meaning that they can only be found within a certain species. Because host-specific bacteria are also often associated with fecal waste they are a good source of information regarding which species (human or animal) are contributing to fecal pollution in recreational waters. There are many bacteria that display host-specificity. Host-specific microbes that are used for source tracking usually have an associated marker gene that can serve as a unique ID-card for the microbe of interest. Source tracking techniques have been developed for Bacteroides, Bifidobacterium, Clostridium, and other microbes.[6]

One of the most commonly used host-specific microbial genus for source tracking studies is Bacteroides, which has a marker gene called HF183 and can be identified through different PCR assays. The Bacteroides’ HF183 marker gene is human-specific, and is frequently utilized in sewage-associated studies.[7] The most commonly used analysis method for host-specific bacteria is qPCR, as it provides rapid results without the need to culture the organisms of interest, and doesn’t require an extensive library of genetic data for comparison.[8] However, one particular study compared ddPCR and qPCR methodologies to see if ddPCR could improve sensitivity of microbial source tracking in Bacteroidales. The study found that both methods are suitable tools for microbial source tracking, but elements like cost-effectiveness should be considered for ddPCR assays.[9]

Figure 1. Bacteriophage image sourced from Mansour 2017.
Host-specific Bacteriophages


Bacteriophages, also known as phages, are viruses that infect bacteria. Phages consist of a nucleic acid molecule surrounded by a protein coat called a capsid (Figure 1). Phages are more abundant than bacteria in most environments, which makes them useful for source tracking.[10] Similar to host-specific bacteria, bacteriophages are usually specialized, meaning they can only infect certain bacteria. Once inside their bacterial host, phages are able to replicate and take over the cell.

Many phages are able to survive a variety of natural and human stressors, including wastewater treatment. In addition to lasting a long time in nature (even more than some anaerobic bacteria), their resistance to heat, UV radiation, and chemical disinfectants also makes them useful source trackers.[11] One source tracking study analyzed Bacteroides-specific phages through qPCR. Since the bacterial genus Bacteroides is already host-specific to humans, phages that strictly infect Bacteroides can also be evidence of human sources of fecal pollution. To quantify phages, host-bacteria needs to be grown under anaerobic conditions and analyzed in a laboratory10. Once the host bacteria has been cultivated properly, phages are extracted and qPCR analyses can determine what animal or human host they’re associated with. Using ddPCR for bacteriophage enumeration is not as common, although it has been suggested as a valuable new tool in bacteriophage research.[12]

Microbial Community Fingerprinting


Microbial community fingerprinting is a form of microbial source tracking that focuses on entire microbial communities instead of just one species of microorganism. Microbial community source tracking is based on the theory that pollution sources have a unique microbial community composition, or “fingerprint”. Not only do different sources of pollution in a given watershed have unique fecal microbial communities, but there are location-based, regional differences in similar sources. For example wild hogs in Maui would have a unique microbial community in their digestive system, than wild hogs who live in the Carolinas. In order to identify a microbial community of a particular source in any location, a sample from the polluted recreational water body of interest would need to be compared to local contamination sources, like wastewater treatment plants. This means that both contaminated wastewater and contamination sources must be sampled so they can be directly compared with each other.[13] [14] To identify microbial fingerprints, an extensive library of genetic information from pollution sources is also needed. With this information, contaminated waterway samples can then be accurately matched with their local or regional pollution sources.

Microbial communities can be complex and contain hundreds to thousands of microbial taxa3. To analyze and enumerate these communities, qPCR is most commonly used. Once enumerated, the information for community-based microbial source tracking can be very data heavy. Therefore, it is important to have computational algorithms and programs capable of determining potential sources of fecal contamination waterways.[15] [16] Programs like the SourceTracker software, allow for huge amounts of data to be processed quickly. The software was designed for microbial-community analyses and determines what source different microbial taxa come from. In one study, SourceTracker was 91% accurate in identifying five different contamination sources in recreational freshwater based on microbial community fingerprinting.[17]

Antibiotic Resistant Genes (ARGs)


Any substance that stops the growth and replication of a bacteria or kills it outright can be called an antibiotic. Antibiotics have always been a natural element of microbial ecosystems, and they are developed as a defense-response to other competing microbes. For example, let’s pretend A-microbes were constantly stealing food from B-microbes. Then, B-microbes start producing antibiotics and kill A-microbes… now B-microbes have food again! In comparison, antibiotic resistance is a defense that some bacteria have developed as a response to their antibiotic-producing neighbors.[18] For example, if B-microbes are now killing A-microbes with antibiotics, A-microbes might develop antibiotic resistance… now you can’t kill A-microbes as easily! Antibiotic resistance is created through antibiotic resistant genes (ARGs), which can be inherited or acquired through mutations.[19]

When antibiotics were discovered for the purpose of disease control, it sparked a medical revolution that led to the production and development of natural and synthetic antibiotics. As of 2015, it’s estimated that around 100,000 metric tons of antibiotics are produced every year.[20] Now, bacteria are beginning to fight back, causing antibiotics to become less and less effective against bacteria that develop antibiotic resistance.[21] In addition to the growing concern in the medical field, there is now speculation that the environment can contribute to the development of ARGs outside of the clinical realm. ARGs may have the potential to cause negative ecological and/or human health effects and could be a contaminant of concern.[22] It’s also been observed that the amounts of ARGs increase the closer you sample to human dominated environments – that include but are not limited to – wastewater treatment plants, pharmaceutical factories, livestock farms, and impacted sediment or water sources.[23]

ARG source tracking is most often studied through the detection of specific marker-genes processed through qPCR, which allows for the quantification of target genes.[24] In one study, ddPCR and qPCR technologies were compared for the detection of ARGs. While qPCR is more commonly used, the study found that ddPCR is also an effective method for ARGs.[25] Source tracking of ARGs can also be performed through metagenomics. Metagenomics-based approaches look at how ARGs change in entire environmental microbial communities depending on different levels of exposures to contamination sources.[26]

Other Microbial Source Tracking Methods


Microbial source tracking is a continually developing field. As such, new technologies and markers to identify sources of fecal pollution are constantly being developed and sought. Technological advances in the genetic field allow for more robust and precise analyses to be performed. Among these emerging methods are subtractive hybridization and microarray analyses.

Microarray analyses are tools that allow thousands to hundreds of thousands of target genes to be assayed at once. Microarrays have the potential to overcome the limitations of current culture and qPCR based assays and are of increasing interest in the research realm.[27] One study incorporated common waterborne pathogens, previously published MST marker genes and organisms, antibiotic resistance genes, fecal indicator bacteria, and more for their microarray analysis.[28] This makes microarrays incredibly powerful tools for microbial source tracking, allowing for a massive amount of information to be processed at once. However, not many laboratories are equipped to perform microarray analyses, so they currently remain largely inaccessible and expensive.

Subtractive hybridization is a technique that can be used to isolate a DNA segment that is missing from another sample of DNA. Subtractive hybridization can be used in source tracking to identify bacterial gene sequences that are found exclusively in a particular host species or group.[29] Subtractive hybridization is relatively low in cost, but lacks specificity and is often used to supplement the information provided by already-established traditional source tracking methodologies.

Chemical Source Tracking


Chemical source tracking offers potential advantages over microbial source tracking. Chemicals are generally faster to prepare and analyze, they are considered more specific to their original source since they do not replicate or grow in the environment, and they can be more stable geographically and over time. However, chemical source tracking methods haven’t received the same amount of attention and scrutiny, and they require specialized equipment. It’s also important to mention that a lot of chemicals that are specific to human waste can be diluted in the environment, and that they may experience differences in spread, transport and persistence in water when compared to pathogens and other fecal indicator bacteria.[30] Below are some of the most commonly used chemical markers for source tracking.

Caffeine


Caffeine is a common pharmaceutical that is usually present in surface water contaminated with domestic wastewater. Approximately 3% of the amount of caffeine ingested is excreted in urine. Additionally, unconsumed caffeinated drinks that are dumped down the drain like coffee, tea, soft drinks, cocoa, chocolate drinks and energy drinks enter water bodies through domestic wastewater. For these reasons, caffeine has been detected often in surface water systems throughout the United States.[31]

Because caffeine is regularly consumed and excreted by humans, it is a chemical generally associated with domestic wastewater contamination and is used as a chemical tracker for fecal wastewater contamination.[32] In addition to helping track fecal contamination, it has been shown that caffeine can negatively affect the overall function of microbial environments like algae, which can have negative effects and consequences on aquatic life and ecosystem functions. A study in North Carolina recommended that caffeine should be regularly monitored to help with health evaluations at a local creek.

Source tracking with caffeine requires liquid chromatography - mass spectrometry analysis which can be expensive. Costs can range from $50 - $200 per sample depending on the amount of samples required. Samples can be taken by volunteers but must be shipped to a professional lab to be prepared and analyzed. Costs include processing and analysis.

Optical (Laundry) Brighteners


Optical brighteners are dyes present in many laundry detergents, which attach to fibers in clothing to make them appear whiter. Greywater from laundry is part of most home’s wastewater, so if optical brighteners are detected in recreational waterways, it is a good indicator that human sewage is also present. Optical brighteners persist for a long time in the environment, and have been used to detect sewage discharges or misconnects into storm drains.

Identifying optical brighteners in water is relatively simple, as they glow under black (ultra violet) light despite being invisible to the naked eye. When a high concentration of optical brighteners is detected in a surface waterbody, it is likely there is some source of wastewater discharging or leaching into the watershed.

The analysis for optical brighteners is a simple procedure. Optical brightener-free cotton pads are used to monitor storm sewer outfalls or streams. These sample collection devices should be left in place for a period of 7-10 days (in non-tidally influenced locations) so the sample is representative for the period of interest. If the retrieved sample pad has collected optical brighteners, it will glow under an ultraviolet black light. The analysis can be performed with relatively low-tech equipment by viewing the sample pads under ultraviolet black lights and comparing them to controlled samples whose concentration is known. This type of testing is also extremely cost-efficient as no professional services need to be hired. Costs can be minimized to an ultraviolet light (approximately $250-350 for a good one) and collection materials like cotton pads, and control samples. Another option is to measure optical brighteners using a fluorometer. One source tracking study even used tampons to absorb and monitor for optical brighteners in water.

One drawback, however, to using optical brighteners as indicators of human sewage is that there is some organic matter naturally occurring in the environment that can fluoresce when exposed to UV light that could confound the results.[33] Additionally, source tracking studies using optical brighteners were not found to be widely documented in scientific literature within the last 10 years.

Sucralose


Sucralose is an artificial sweetener used to replace sugar. This chemical compound is the main ingredient in Splenda and can be found in a variety of products including diet soda, yogurt, sugar-free candy, and other snacks and beverages marketed as diet or low-calorie. In 2003, 300 tons of sucralose were produced in Europe and America for worldwide consumption.[34] By 2012, 25% of children and 41% of adults in the United States were consuming foods containing low calorie sweeteners, including sucralose.[35] A big reason behind the popularity of this artificial sweetener is that the body doesn’t recognize it as fuel, so it isn’t stored or used for energy in the body. Additionally, it doesn't release any calories or have a negative effect on dental plaque microflora, unlike regular sugar.[36] Since artificial sweeteners have become increasingly popular and 90% of sucralose is excreted from the human body unchanged, it is often found in surface waters that receive human waste.[37]

In addition to being commonly found in contaminated surface waters, sucralose is relatively stable and thus doesn’t degrade easily in the environment or in wastewater treatment plants. In order to detect sucralose in the environment, water samples must be taken so that the compound can be separated through a series of extraction processes. Later, the presence of sucralose is analyzed through liquid chromatography/mass spectrometry. Finally, the sample results are compared to known controls to determine concentrations. Since sucralose persists in the environment for long periods of time, it is commonly used as a source tracker that can be paired with other indicators that have shorter life-spans,[38] providing information on how recent a particular contamination event was. For instance sucralose can tell us how concentrated the contamination is, while another indicator with a shorter life-span, (see acetaminophen below) tells us how old the human waste is. This particular study used sucralose and acetaminophen as indicators, suggesting the co-analytes can help identify sewage leaks.

The cost of liquid chromatography/mass spectrometry analyses can be quite expensive, and the quickness of obtaining results will likely depend on the schedule of the analytical lab. Costs can range from $50 - $200 per sample depending on the amount of samples and the level of preparation required. Samples can be taken by volunteers but must be shipped to a professional lab to be prepared and analyzed. Costs include processing and analysis.

Acetaminophen (Pharmaceuticals)


Acetaminophen is a common, over-the-counter drug that can treat minor aches and pain and reduces fever. It is the active ingredient in Tylenol, and is hugely popular in the United States, with more than 60 million people consuming acetaminophen on a weekly basis. Since acetaminophen is toxic to many animals, its use in veterinary medicine is not frequent, making it strongly linked to human use.

When compared to other common chemical source trackers, acetaminophen experiences degradation in the environment, so its life-span is not as long. Acetaminophen is also effectively removed by wastewater treatment, so when it is detected in the environment, it indicates a more recent event of untreated sewage being released. This means that water bodies without the regular introduction of human waste should have lower concentrations of acetaminophen than water bodies with untreated sewage.[39] Since this compound degrades quickly, it is often used to indicate the age of contaminants in water bodies, making it an excellent co-analyte for trackers that have longer lifespans and can indicate contamination concentrations (see sucralose above).[40]

To detect acetaminophen in the environment, water samples must be taken and put through an extraction process. The presence of the pharmaceutical is then indicated through liquid chromatography and mass spectrometry analysis. Liquid chromatography/mass spectrometry can be expensive, and the quickness of obtaining results will likely depend on the schedule of the analytical lab. Costs can range from $50 - $200 per sample depending on the amount of samples and the level of preparation required. Samples can be taken by volunteers but must be shipped to a professional lab to be prepared and analyzed. Costs include processing and analysis.

Other Pollution Tracking Methods


Dog-sniffing


Dog-sniffing is a unique and innovative approach to source tracking that has proven to be simple, fast, and inexpensive. The strong nose of a dog is highly effective and simple when compared to the amount of equipment, man-hours, and expenses associated with microbial or chemical source tracking. Dogs can be trained to detect human feces and surfactants, and are excellent at identifying sources of contamination. Dogs can also be trained to ignore distractive scents from other (cat, deer, dog) feces.[41] The benefits of adding sewage detection by dogs to the source tracking toolbox include real-time results, the ability to test a high number of sites per day, and low cost per sample.[42]

In one particular study, two dogs responded positively (70% and 100%) at sites for which sampled waters were confirmed as contaminated with human waste. When both dogs indicated a negative response, human waste markers were absent.[43] It is important to note that canine scent detection for fecal contamination is a qualitative approach, so it does not necessarily indicate the concentrations or age of the contaminants. In general, dog sniffing can be used as a complementary approach for sites with positive canine responses and can also be prioritized for sampling and more expensive analysis of human waste contamination.[44] The leading company using canine detection for pollution source tracking has historically been Environmental Canine Services, but as of January of 2022 has closed. If you are aware of a currently operating company, please let us know at kday@surfrider.org.

Emerging Methods in Development


As technology advances, new chemical trackers and technologies are constantly emerging. Other chemical source trackers that are currently available but are not used as commonly as the ones listed above, include fecal sterols and stanols, bile acids, surfactants, and fragrances.

Fecal stanols are compounds that are formed in the guts of animals when sterols (like cholesterol) are metabolized.[45] Sterols are a type of lipid that are typically associated with cell membranes, and once converted into stanols can serve as specific source trackers associated with human feces. The detection and use of fecal sterols and stanols as a means of identifying human-derived fecal pollution has received attention around the world, including the United States.[46]

Bile acids are primarily formed in the liver and are secreted to the intestine. Human feces can contain more than 20 different bile acids, which once excreted, are considered “secondary” bile acids. Identifying the source of pollutants in contaminated waterways is possible when looking at secondary bile acids, since they can be species-specific.[47] One controlled study looked at the potential for source tracking using a combination of fecal sterols and bile acids. The study found these markers could differentiate species responsible for fecal contamination, and could be potentially used to identify sources of fecal matter in water or sediments.[48] Real-world applications for source tracking with bile acids are limited.

Surfactants are a primary component of cleaning detergents, and help trap and remove dirt from a surface you’re cleaning. Since surfactants are a household chemical, they are often found in wastewater and can also be used as chemical source trackers. One study found that the degree of water treatment can be determined through surfactant concentrations.[49]

Fragrances are found in household products like soaps, detergents, cosmetics, perfumes, air fresheners and more. These synthetic fragrances are called polycyclic musks, and have also been assessed as potential human-waste markers. However, while polycyclic musks can be successfully detected, the low abundance in treated and untreated wastewater can be difficult for real-world source tracking applications.[50]

From “What” to “Who”: Pinpointing Pollution Sources


While the source tracking techniques discussed in this article can identify what is causing fecal contamination (whether human or other animal), not all of the techniques mentioned are able to attribute fecal contamination to a specific source. For example, if a study were to identify that most of the fecal contamination in an area belongs to cattle, then other techniques must be employed to identify exactly which farm(s) upstream are responsible for the contamination and where it’s coming from.

Microbial community fingerprinting is one of the previously mentioned techniques that can actually attribute fecal contamination to a specific source. Since samples are taken directly from suspected sources of contamination and from recreational bodies of water, they can be compared and eventually connected to their original source. Other methods that can detect where pollution is coming from are things like smoke testing, dye testing, or even computational modeling and satellite mapping.

Both smoke and dye detection methods can help identify sewer or storm drain misconnects in a specific area. Flushing dye or smoke into sewer or storm drain systems that are suspected of contaminating waterways can be useful to identify illicit connections. For example, fluorescent dye can be introduced into a stormwater system while the water in the collection system is monitored to determine whether an illicit connection is present. Smoke testing using zinc chloride smoke can also be injected into sewer lines. If the smoke emerges via vents on connected buildings or through cracks or leaks in the sewer line, it makes it easy to identify faults in the sewer system. Now we can tell where these contaminants are coming from, and how to fix the issue.

Additionally, mapping analyses performed through Geographic Information Systems (GIS) can map the watershed and identify potential contamination sources for additional follow up. Mapping can be fairly simple, or incorporate more complex models that include many factors and variables that affect the transport of contaminants. Soil and water properties, geologic and hydrologic characteristics, the layout of groundwater and watershed systems, and more can be included in GIS analyses for pollution source tracking.[51]

Computational modeling is a type of artificial intelligence that is designed to predict outcomes. Using historical data, machine learning technologies can predict values that inform us about where contamination originates. Computer models can examine the relationship between microbial sources, land cover, weather, and hydrologic variables in a watershed, making it a powerful tool to understand the sources and factors that influence pollution in water.[52]

It is evident that land use, weather, and hydrological factors play an important role in determining the sources of fecal contamination in water. Oftentimes, other microbial source tracking studies do not take into account the relationship between these factors and microbial sources. Computer modeling is therefore an important component to microbial source tracking, as it can predict major contamination sources. There are many types of algorithms that can be used, and among the most successful is XGBoost.[53] Source tracking through computer modeling is advantageous in that it is cheap, and requires few resources. However, expertise is required to run programs and interpret the results.

Other approaches that can be taken to hone in on where pollution is originating from in a watershed and to identify specific contamination sources include sanitary surveys, watershed assessments, stream segmenting and more. Stay tuned for a future Beachapedia article that will cover this component in more detail!

Discussion


Table 1 summarizes the key aspects and considerations of the microbial and chemical source tracking methods previously described. The associated cost, technical training required for sampling and analysis, and relative advantages and limitations are presented for each method. Of particular note, the major difference for practical applications of microbial and chemical methods is that chemical methods and markers are used to determine the presence or absence of human sources of fecal pollution, while qPCR-based microbial source tracking methods are able to detect and identify species - specific sources of pollution, i.e. human, dog, cattle, etc..

Final table.png


Currently, the most affordable and easiest tool for volunteers to use on their own are laundry brighteners. Laundry brighteners are easy to detect, do not require specialized laboratories or personnel and generally are low cost. While it's important to follow standard methods for sampling and interpreting results, laundry brightener studies are very low-tech and a great, volunteer-friendly, initial screening tool that can be used to convince local stakeholders and officials that more extensive investigation into the sources of pollution is warranted to protect public health from human sources of pollution.

Source tracking methods that require PCR technologies are more expensive than laundry brighteners, but are accessible through professional labs in multiple locations. PCR methodologies are versatile — trackers like bacteriophages, antibiotic resistant genes, or even indicator organisms are available depending on a specific study interest. The most accessible services are companies like Jonah Ventures that provide source tracking services using eDNA as a tracker for fecal contamination. This company provides the sampling kit and instructions, so volunteers can both take and prepare the samples for analysis. Once the samples have been shipped, the company will analyze them and a report will be sent over with results. Their sampling kit identifies up to 6 species that could be responsible for the fecal pollution in your sampling area.

Finally chemical source trackers like caffeine, sucralose, and acetaminophen require liquid chromatography / mass spectrometry analyses and have costs that can range between $50-200. In addition to requiring a professional lab, a careful extraction process is required before sending them off to be analyzed and requires the help of a specialist and specialized equipment.

Helping communities identify pollution sources provides them with the information they need to fix local pollution problems and restore clean water. Pollution source tracking is a valuable tool that is used to protect and restore water quality across the US. By providing data and information on where pollution is coming from, local and state initiatives that work towards beach health can take further action to ensure we have clean water and healthy beaches.

Further Reading


References

  1. ↑ DeFlorio-Barker, S., Wing, C., Jones, R. M., & Dorevitch, S. 2018. Estimate of incidence and cost of recreational waterborne illness on United States surface waters. Environmental Health, Vol. 17, No.1
  2. ↑ National Ocean Economics Program. 2019. Tourism and Recreation Economic Sector, Ocean Economy.
  3. ↑ DeFlorio-Barker, S., Wing, C., Jones, R. M., & Dorevitch, S. 2018. Estimate of incidence and cost of recreational waterborne illness on United States surface waters. Environmental Health, Vol. 17, No.1
  4. ↑ Mathai, P. P., Staley, C., & Sadowsky, M. J. 2020. Sequence-enabled community-based microbial source tracking in surface waters using machine learning classification: A review. Journal of Microbiological Methods, Vol. 177
  5. ↑ Harwood, V. J., Staley, C., Badgley, B. D., Borges, K., & Korajkic, A. 2014. Microbial source tracking markers for detection of fecal contamination in environmental waters: relationships between pathogens and human health outcomes. FEMS microbiology reviews, Vol. 38, No. 1
  6. ↑ Kinzelman, J., Kay, D., & Pond, K. 2011. Relating MST results to fecal indicator bacteria, pathogens, and standards. In Microbial Source Tracking: Methods, Applications, and Case Studies. Pp. 337-359
  7. ↑ Ahmed, W., Payyappat, S., Cassidy, M., & Besley, C. 2019. A duplex PCR assay for the simultaneous quantification of Bacteroides HF183 and CPQ_056 marker genes in untreated sewage and stormwater. Environment international. Vol. 126
  8. ↑ Griffith, J. F., Weisberg, S. B., & McGee, C. D. 2003. Evaluation of microbial source tracking methods using mixed fecal sources in aqueous test samples. Journal of Water and Health. Vol. 1, No. 4
  9. ↑ Nshimyimana, J. P., Cruz, M. C., Wuertz, S., & Thompson, J. R. 2019. Variably improved microbial source tracking with digital droplet PCR. Water research. Vol. 159
  10. ↑ Weinbauer, M. G. (2004). Ecology of prokaryotic viruses. FEMS microbiology reviews. Vol. 28, No. 2
  11. ↑ Jofre, J., Blanch, A. R., Lucena, F., & Muniesa, M. 2014. Bacteriophages infecting Bacteroides as a marker for microbial source tracking. Water research. Vol. 55
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  16. ↑ Knights, Dan, Justin Kuczynski, Emily S. Charlson, Jesse Zaneveld, Michael C. Mozer, Ronald G. Collman, Frederic D. Bushman, Rob Knight, and Scott T. Kelley. 2011. Bayesian community-wide culture-independent microbial source tracking. Nature methods. Vol 8, No. 9
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  24. ↑ Li, L. G., Huang, Q., Yin, X., & Zhang, T. 2020. Source tracking of antibiotic resistance genes in the environment—Challenges, progress, and prospects. Water Research. Vol. 185
  25. ↑ Park, S., Rana, A., Sung, W., & Munir, M. 2021. Competitiveness of quantitative polymerase chain reaction (qPCR) and droplet digital polymerase chain reaction (ddPCR) technologies, with a particular focus on detection of antibiotic resistance genes (ARGs). Applied Microbiology. Vol. 1, No. 3
  26. ↑ Corno, G., Yang, Y., Eckert, E.M., Fontaneto, D., Fiorentino, A., Galafassi, S., Zhang, T. and Di Cesare, A. 2019. Effluents of wastewater treatment plants promote the rapid stabilization of the antibiotic resistome in receiving freshwater bodies. Water research. Vol. 158
  27. ↑ Li, X., Harwood, V. J., Nayak, B., Staley, C., Sadowsky, M. J., & Weidhaas, J. 2015. A novel microbial source tracking microarray for pathogen detection and fecal source identification in environmental systems. Environmental science & technology. Vol. 49, No. 12
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  29. ↑ Dick, L. K., Simonich, M. T., & Field, K. G. 2005. Microplate subtractive hybridization to enrich for Bacteroidales genetic markers for fecal source identification. Applied and Environmental Microbiology. Vol. 71, No. 6
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  31. ↑ Kolpin, D. W., Furlong, E. T., Meyer, M. T., Thurman, E. M., Zaugg, S. D., Barber, L. B., & Buxton, H. T. 2002. Pharmaceuticals, hormones, and other organic wastewater contaminants in US streams, 1999− 2000: A national reconnaissance. Environmental science & technology. Vol. 36, No. 6
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  33. ↑ Hartel, P. G., McDonald, J. L., Gentit, L. C., Hemmings, S. N., Rodgers, K., Smith, K. A., ... & Schröder, E. CHartel, P.G., McDonald, J.L., Gentit, L.C., Hemmings, S.N., Rodgers, K., Smith, K.A., Belcher, C.N., Kuntz, R.L., Rivera-Torres, Y., Otero, E. & Schröder, E.C. 2007. Improving fluorometry as a source tracking method to detect human fecal contamination. Estuaries and Coasts. Vol. 30, No. 3
  34. ↑ Bahndorf, D., & Kienle, U. 2004. World market of sugar and sweeteners. International Association for Stevia Research.
  35. ↑ Sylvetsky, A. C., Jin, Y., Clark, E. J., Welsh, J. A., Rother, K. I., & Talegawkar, S. A. 2017. Consumption of low-calorie sweeteners among children and adults in the United States. Journal of the Academy of Nutrition and Dietetics. Vol. 117, No. 3
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  37. ↑ Oppenheimer, J., Eaton, A., Badruzzaman, M., Haghani, A. W., & Jacangelo, J. G. 2011. Occurrence and suitability of sucralose as an indicator compound of wastewater loading to surface waters in urbanized regions. Water research. Vol. 45, No. 13
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  41. ↑ Van De Werfhorst, L. C., Murray, J. L., Reynolds, S., Reynolds, K., & Holden, P. A. 2014. Canine scent detection and microbial source tracking of human waste contamination in storm drains. Water Environment Research. Vol. 86, No. 6
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