Flush with Data

For most of us, the artifacts of our morning constitutionals are out of sight, out of mind. But for scientists in the College of Agricultural Sciences, human waste is worthy of attention, especially when a community's "creations" are considered collectively. That's because these byproducts brim with valuable information about human well-being and ecosystem health.

"Wastewater monitoring can give a snapshot of what's happening in a community at any given moment--from the level of illness to the degree of exposure to industrial chemicals," said Andrew Read, director of the Huck Institutes of the Life Sciences and Evan Pugh Professor of Biology and Entomology. "We can use this information to make decisions that protect human and ecosystem health."

According to Read, wastewater surveillance has become an important tool for monitoring disease outbreaks and environmental contamination. With deep expertise in infectious disease and emerging contaminants, and with access to state-of-the-art facilities and instruments, he and other scientists in the college are at the forefront of this increasingly sophisticated area of scientific study.

Viral Pathogens

In the summer of 2020, when COVID-19 was spreading rapidly throughout the United States, Read--along with Tom Richard, professor of agricultural and biological engineering; Heather Preisendanz, associate professor of agricultural and biological engineering and associate director of research for the Institute for Sustainable Agricultural, Food, and Environmental Science (SAFES); and other colleagues across Penn State--knew that a wastewater surveillance program could not only advance science by contributing valuable information about how

the SARS-CoV-2 virus spreads but it also could alert University decision makers to potential outbreaks several days before individuals exhibit symptoms of infection.

The research team--which included members of Penn State's Office of the Physical Plant and staff at the local State College wastewater treatment facility--quickly implemented a daily schedule of wastewater testing for SARS-CoV-2. The team screened the samples for the virus's genetic material and used it to track the concentrations of the virus over time. The researchers also conducted novel studies to understand fluctuations in wastewater flow volumes, speed, and mixing of water from each individual flush and how they influenced concentrations of the virus.

According to Read, the sampling efforts were among the most intensive in the country for SARS-CoV-2. "Sampling every day during the height of the pandemic was really important for us to understand the variation in the viral concentrations as students returned to campus, left for holidays, and came back again," he said. "I think it gave us one of the clearest pictures of what was happening anywhere in the country."

Currently, the team is continuing its monitoring for SARS-CoV-2 by sampling three times per week. In addition, they also now are monitoring for influenza.

Meanwhile, with funding from the U.S. Food and Drug Administration, another researcher in the college, Edward Dudley, professor of food science, is monitoring wastewater for SARS-CoV-2 in rural areas.

"During the pandemic, we saw a lot of disruptions in our food supply," he said. "As employees got sick, there weren't sufficient people working in the fields or in processing facilities. If you're counting on having a workforce of one thousand people come in to do the harvest, you need to be prepared for the possibility that large numbers of them could get sick. Wastewater monitoring in rural agricultural areas could serve as a sentinel for rising cases and enable employers to make alternative plans."

Dudley was selected by the FDA to do this work because of his lab's participation in the agency's GenomeTrakr Network--a group of public health and university laboratories that collects and shares genomic and geographic data on foodborne pathogens. Dudley's expertise in whole-genome sequencing enables him not only to detect the presence of the SARS-CoV-2 virus in wastewater, but also to identify specific variants.

"It's been a really powerful way to demonstrate that in these rural communities, just like in the larger studies, we can also track which of the variants are present over time," he said.

Foodborne Pathogens

Viruses aren't the only pathogens that can be identified in wastewater; bacteria such as Salmonella and E. coli are also present. Dudley believes that wastewater surveillance can play an important role in monitoring for foodborne illnesses.

"Most people who have a foodborne illness never go to the doctor," said Dudley. "As microbiologists, we estimate that for every case of foodborne illness that we hear about, there are probably thirty to fifty that we don't hear about."

Dudley explained that since foodborne illnesses are underreported, a program of wastewater surveillance could give a much clearer picture of the presence of these bacteria in the community.

"With wastewater surveillance, even if there aren't any reported cases of illness, if we detect pathogens in the water, we could alert hospitals and physicians' offices that people may soon need treatment," he said. "By simultaneously sampling tens to hundreds of thousands of individuals, wastewater provides a much more sensitive way of understanding what's going on in community health."

Dudley noted that because foodborne pathogens are transmitted to food by humans, outbreaks often originate in agricultural settings. "A lot of our produce in this country is harvested by hand," he said. "For a grower who has one thousand workers who are involved in picking produce, wastewater monitoring could help identify if any of those workers might be carrying some of these organisms and whether interventions, such as better education about and access to handwashing stations, could reduce transmission to the greater population."

In his research, which began in January, Dudley and his colleagues are collecting two samples per week from two Pennsylvania locations. "We have found E. coli and Salmonella, which isn't surprising, but it is too early to make any conclusions," he said.

Pharmaceuticals

Not only can wastewater provide information about the pathogens that are present in the community, but it also can reveal the extent to which people are using medications to treat the associated illnesses. For example, with the novel coronavirus infecting millions of people worldwide, therapeutic agents were urgently needed to treat COVID-19 patients. Medications such as remdesivir and dexamethasone were used to treat hospitalized patients; antibiotics were prescribed for associated bacterial infections; and over-the-counter pain and fever-reducing medications were recommended to patients who were experiencing mild COVID-19 symptoms.

Preisendanz, who long has studied the fate of pharma-ceuticals in wastewater, noted that some medications are excreted by humans, and many are known to persist through wastewater treatment plants and enter into nearby waterways, where they can negatively affect aquatic organisms.

"This knowledge spurred concerns that increased use of pharmaceuticals during the pandemic could also lead to increased concentrations of these drugs in wastewater treatment plant effluent and potentially harm aquatic life," she said.

Preisendanz and her colleagues--including scientists from the U.S. Department of Agriculture--collected weekly influent (incoming untreated) and effluent (outgoing treated) samples from two wastewater treatment plants in central Pennsylvania between May 2020 and May 2021. One of the sites included a hospital in its service area. The other included Penn State's Living Filter, which provides a second level of filtration beyond what is provided by typical wastewater treatment plants.

Next, they tested the samples for two over-the-counter fever reducers/pain relievers (acetaminophen and naproxen); five antibiotics (ampicillin, doxycycline, ofloxacin, sulfamethoxazole, and trimethoprim); and two COVID-19 therapeutic agents (remdesivir and dexamethasone, which is used to reduce severe upper respiratory inflammation in patients on ventilators).

"We thought the concentrations of these pharmaceuticals would increase with increasing concentrations of SARS-CoV-2," said Preisendanz. "By analyzing wastewater for SARS-CoV-2 and various medications, valuable information regarding the well-being of entire communities may be gained without the need to interview, survey, or test individuals."

The team found that remdesivir concentrations were correlated with the number of hospitalized COVID-19 patients, while dexamethasone concentrations were associated with the number of hospitalized patients on ventilators. Influent to the wastewater treatment plant servicing the hospital had detectable concentrations of remdesivir and dexamethasone in 28 and 31%, respectively, of samples collected during the study period while the average removal efficiencies by the wastewater treatment plant for these drugs were 39 percent and 56 percent, respectively.

"The virus concentrations alone couldn't tell us whether we would see those medications; rather, it was really tied back to who was in the hospital and who was on ventilators," said Preisendanz.

According to Preisendanz, although risk to aquatic organisms from remdesivir could not be calculated, as no research had yet been done to determine the concentrations that could pose a risk, dexamethasone was detected in quantities that could pose a low risk to fish.

Among the antibiotics tested, the team found that trimethoprim concentrations could pose a low to medium risk to aquatic life, while sulfamethoxazole could pose a high risk, specifically to algae, which is a food source for many organisms.

In addition, the team found that although acetaminophen and naproxen were present at concentrations much higher than all the other pharmaceuticals of interest, no correlations were observed between virus concentrations and influent concentrations of either drug, suggesting that they are not indicators of the prevalence of COVID-19 in the community. However, naproxen concentrations detected in effluent were at levels that could pose a low to medium risk to aquatic organisms.

"While the concentrations we calculated considered the individual risks that each drug could pose to aquatic life, these calculations do not account for the potential risks that could come from the synergistic effects of these drugs in a mixture, which could be much higher," said Preisendanz. "Importantly, our study highlights the opportunity that wastewater surveillance provides to understand the effects of human health on water quality and ecological health."

Industrial Chemicals

In addition to tracking the concentrations of pharma-ceuticals in wastewater effluent, Preisendanz also is interested in understanding how well treatment plants filter industrial chemicals. For example, in a recent study, she and her colleagues tracked PFAS (per- and polyfluoroalkyl substances), a group of more than 4,700 fully synthetic compounds that are widely used in industrial and manufacturing processes and found in many consumer products, such as nonstick cookware, stain-resistant clothing, textiles, cleaning products, and food packaging.

"PFAS are so pervasive that they have been found in the blood of people all over the world," said Preisendanz. "Unfortunately, these compounds have been shown to negatively affect human health; particularly, they can affect development in children, increase risk of cancer, contribute to elevated cholesterol levels, interfere with women's fertility, and weaken immune systems."

Because of their wide variety of uses, PFAS enter wastewater treatment plants from both household and industrial sources, she said, noting that the chemical structures of PFAS are difficult to degrade, causing them to persist in wastewater effluent. Given that PFAS have been shown to be taken up by crops and enter the food chain when the crops are consumed, Preisendanz decided to study PFAS concentrations after passage through Penn State's wastewater treatment plant and Living Filter and track their potential movement into the crops that are grown at the Living Filter site as a food source for livestock.

She explained that Penn State's Living Filter is a unique wastewater system that sprays treated water onto roughly six hundred acres of agricultural and forest land. This provides additional filtration as the water slowly percolates through the soil profile and into the groundwater. Eventually this groundwater makes its way into Spring Creek.

The team identified ten PFAS compounds present across the Living Filter site. "The Living Filter site seems to do a pretty good job of providing additional treatment for pharmaceuticals, but that isn't what we're seeing for PFAS," said Preisendanz. "We're seeing levels that are pretty much on the same order of magnitude in the influent, the effluent, and the groundwater across the sites. It means the compounds are persisting all the way down to the groundwater. That's not because the Living Filter isn't doing its job; rather, these chemicals are just so ubiquitous and persistent."

Unfortunately, she said, the state Department of Environmental Protection recently released health advisories for two of the most important PFAS--PFOA (perfluorooctanoic acid) and PFOS (perfluorooctanesulfonic acid)--such that "any detectable level is considered a risk to human health, presenting potential challenges for beneficial reuse of wastewater residuals, including treated wastewater and the biosolids."

Preisendanz noted that the groundwater at the Living Filter is not used for drinking; therefore, it is not likely to pose a risk to human health in that regard. However, the team did find several PFAS compounds in crop tissue samples collected at both irrigated and nonirrigated portions of the site.

"This suggests that PFAS may enter the food chain when these crops are fed to livestock," Preisendanz said, adding that future research is needed to determine potential risks to livestock health and the potential implications of PFAS presence in meat and dairy products, including milk. "Our study results have important implications to ensure that beneficial wastewater reuse activities achieve desired goals to reuse water and nutrients, while simultaneously ensuring PFAS levels are safe from a human health perspective."

A New-ish Paradigm for Public Health

Keeping people safe--from chemicals, drugs, and pathogens--is the ultimate goal of Preisendanz, Dudley, Read, and all of their collaborators, and wastewater provides the raw material for research that can do just that.

"Wastewater surveillance isn't new; it's been used in the past to monitor things like polio," said Read. "But given the advanced technologies, including genetic sequencing and mass spectrometry, that we have today, wastewater surveillance is likely to be a critical part of public health monitoring now and in the future."

The Living Filter

"Wastewater Reuse: 50-plus Years of Research, Management, and Lessons Learned" celebrates the science of wastewater filtration.

More than a half-century of research on the use of treated wastewater for irrigation, groundwater recharge, and research on emerging contaminants was the focus of a three-day conference hosted by the College of Agricultural Sciences in April. The conference highlighted Penn State's Living Filter, a year-round spray irrigation system that recycles the University's treated effluent.

"Discharging effluent from wastewater treatment plants directly to waterways such as streams and rivers is a common practice in water-rich regions such as Pennsylvania," said Heather Preisendanz, associate professor of agricultural and biological engineering. "Yet reusing water is an important way to reduce pollution in waterways and reduce the demands on freshwater use."

For nearly four decades, Penn State has reclaimed all of its wastewater effluent for forest and crop irrigation and groundwater recharge through its Living Filter system. Among the benefits it provides, the Living Filter enhances the treatment of water as it slowly percolates through the soil, naturally recharges the underlying aquifer, reduces the impacts of drought conditions, and improves the growth and yield of agricultural crops.

Conference sessions covered nutrient management, emerging concerns such as PFAS (per- and polyfluoroalkyl substances) and pharmaceuticals, soil health, land management, and wastewater treatment, among other topics. The last day of the conference included a tour of the Living Filter site, with numerous stops to highlight research conducted at the site and operational challenges that needed to be addressed throughout the history of the site.

The conference featured speakers from Penn State and other universities, state and federal government agencies, and private hydrogeology firms, and was intended for utility managers, watershed planners, researchers, students, consultants, and regulatory personnel engaged in innovative water resources management for the twenty-first century.

"The conference served as an important outlet for the decades of research--both basic and applied science--that has occurred at the Living Filter site," said Preisendanz. "The close connections between the research and operation branches of the University provide a unique opportunity to use the site as a living laboratory and facilitate engaged scholarship opportunities for graduate and undergraduate students. Many of these former students were among the invited speakers and demonstrated the profound impacts that conducting research at the Living Filter has had on their careers."

By Sara LaJeunesse