Image courtesy of Flickr/Ravenz Shadow. Images (l to r) courtesy of Adobe Stock, USGS, USGS, NIAID, and Texas A&M AgriLife Research (photo by Kay Ledbette). Emerging contaminants include antibiotics, flame retardants, and personal care products. These compounds are commonly found in low concentrations in the wastewater, biosolids, and manure used in agricultural systems as irrigation and fertilizer. Emerging contaminants enter the environment as products that go down the drain in our homes, from industrial sources, and in the manure of livestock treated with antibiotics. Although increased monitoring has identified the presence of these compounds, little is known about how emerging contaminants move through the environment, or what impacts they may have over time. A special section recently published in the Journal of Environmental Quality (JEQ) titled, “Antibiotics in Agroecosystems: State of the Science,” summarized current knowledge, highlighted research gaps, and presented 16 research articles on this topic. Here we highlight three studies from this special section examining antibiotics. Although these papers are focused on antibiotics in agriculture, the questions and concerns over where these compounds are found, how they move through ecosystems, and what their impacts may be on plants, animals, and humans are relevant when evaluating the effects of emerging contaminants as a whole. Antibiotic resistance is commonly found in soil bacteria that live in feedlots and agricultural soils amended with manure, which is typically perceived to be the result of antibiotics used to treat livestock. However, antibiotic resistance does occur naturally in soils that have not been exposed to antibiotics, and determining these background levels could give researchers a baseline for comparison, according to ASA and SSSA member Lisa Durso, Research Microbiologist with USDA-ARS. “When you want to measure antibiotic resistance, [you're] generally measuring the drugs, the bugs, or the genes,” Durso explains. These three measures are used for both identifying the presence of antibiotic resistance and when thinking about ways to lower antibiotic resistance in agroecosystems, which is a common goal of researchers and farmers. But it is impossible to determine where antibiotic resistance is elevated or to set realistic targets for reduction without an understanding of the baseline antibiotic resistance in soils. In the article titled “Assessment of Selected Antibiotic Resistances in Ungrazed Native Nebraska Prairie Soils” (http://bit.ly/29EsXMG), Durso and colleagues were looking for the baseline “bugs” and “genes” in prairie soils. The first step was to find sites that had not been exposed to antibiotics through grazing or other agricultural practices. “I knew that prairies were the way to go,” Durso says, but finding ungrazed prairie sites in Nebraska took the researchers about a year. There is a long history of cattle grazing in the Great Plains, and even virgin prairies, which have never been plowed, have likely been grazed at some point in history. The time-consuming part of site selection was getting confirmation that prairie sites had not been grazed for the past 20 years. The team collected samples from 20 prairies across five counties in Nebraska. They analyzed the soil for standard physical and chemical properties, phenotypic resistance to selected antibiotics, and performed DNA isolations to serve as indicators for specific antibiotic resistance genes. Phenotypically, all 100 soil samples were resistant to tetracycline, a commonly used broad spectrum antibiotic, and cefotaximine, a third-generation cephalosporin antibiotic. Each bulk soil sample was also plated, and three isolates from each sample were subjected to disk diffusion assays of 12 antibiotic drugs. Durso and her colleagues found very few isolates resistant to ciprofloxacin (2% of isolates) and kanamycin (2% of isolates) while 43% of the isolates were resistant to ceftriazone. The phenotypic resistance to these 12 antibiotics was not statistically different when compared across all prairie sites, indicating that baseline antibiotic resistance is heterogeneous in this region. Test used by researchers to determine the number of generic bacteria in soil samples. Photo courtesy of the University of Nebraska. Taking samples from native prairie soils in Nebraska to isolate antibiotic-resistant bacteria. Photo courtesy of Lisa Durso. When looking at specific genes, antibiotic resistance genes were also common. Tetracycline genes (assays for 14 genes) were found at all sites, but not in all of the samples. The most common tetracycline genes were tet(D) and tet(A). Assays were also performed for two sulfonamide resistance genes; sul(I) was found at all 20 sites and sul(II) at 13 sites. This seeming abundance of resistance was not a surprise to the researchers. Durso's prior metagenomics research compared DNA in cattle fecal samples to DNA from a larger database, which included samples from the Sargasso Sea, Antarctic ice, and Kimchi fermentation. This earlier work revealed that all of the samples tested had antibiotic resistance in their genome. With researchers finding antibiotic resistance just about everywhere they look, it can be challenging to communicate their findings to a broad audience given the differences in the terminology between microbiologists and the medical community. “In human medicine, when you talk about resistance, you're talking about an infectious disease organism that's already making somebody sick, and then resistance is equivalent to treatment failure,” Durso says. “When you move into the environment, resistance isn't really defined in the soil…. Most of the organisms in the soil don't even have the capacity to be a pathogen to make people sick in the first place.” But the potential for increased antibiotic resistance in soil bacteria is still concerning from both a soil ecology and public health perspective. Understanding the background levels of antibiotic resistance is an important step in determining which types of resistance traits and genes are the result of agricultural practices and developing methods to reduce antibiotic resistance in agroecosystem soils. As Durso says, “There's lots of resistances out there, so let's prioritize those that are the most important.” Identifying the priorities and developing action plans will require the expertise of microbial, soil, and human health scientists. The methods used in this study are widely available, and Durso thinks that documenting baseline antibiotic resistance in conjunction with other soil research could advance the understanding of antibiotic resistance in soils. “We start to see correlations between soil physical and chemical parameters and types of antibiotic resistance bacteria or genes.” Suggesting that different soil types may influence the soil bacterial community present necessitate different methods to reduce elevated levels of antibiotic resistance and the best management practices to minimize antibiotic exposure. As the list of emerging contaminants grows and their presence in the environment increases, researchers need methods to identify which compounds are of greatest concern to human health. Kuldip Kumar, Senior Environmental Soil Scientist with Metropolitan Water Reclamation District of Chicago, became curious about plant uptake of emerging contaminants while investigating the fate and transport of antibiotics in soils from corn fields receiving swine manure as a source of nutrients. Finding that not all antibiotics were taken up by the corn, cabbage, and onion in a greenhouse study conducted at the University of Minnesota in Saint Paul, Kumar started reading medical and pharmaceutical literature about antibiotics. In his reading, Kumar found research describing how three properties—lipophilicity, polarity, and molecular weight—regulate the permeability of compounds across a lipid membrane in mammalian cells. Thinking these parameters made sense for uptake in plants too, where antibiotics and other emerging contaminants must also cross membranes, Kumar decided to investigate further. “Whenever you apply manure, biosolids, or recycled water to the soil, they have to go through the root membranes, and then they have to be transported aboveground,” Kumar notes. His investigation of this mechanism was the basis for the paper he was the lead author on titled, “A Framework to Predict Uptake of Trace Organic Compounds by Plants” (http://bit.ly/29Ez3LR), which can be used as a starting point for identifying emerging contaminants most likely to be taken up by plants exposed to manure, biosolids, or any industrial or agricultural by-product. Kumar found the existing model for mammalian tissues, known as the “Rule of 5,” which stated compounds with lipophilicity, polarity, and molecular weights less than specific factors of 5 (e.g., molecular weight < 500) were more permeable. Lipophilicity, or how well a compound will dissolve in fat or oil, is important because compounds with higher lipophilicity (measured as log Kow) are less likely to cross a lipid membrane. Polarity, expressed in terms of H-bonding, matters because a greater number of H-bond donors or acceptors impairs permeability of compounds. And compounds with a larger molecular weight (as a measure of molecular size) are less likely to pass through a membrane via diffusion. Kumar used the Rule of 5 as a starting point and reviewed the existing research on plant uptake of emerging contaminants. Based on the literature available at the time, he determined that a “Rule of 3” and “Rule of 3–5” were more appropriate for plant uptake of chemical compounds. The Rule of 3 states that uptake of a compound is more likely when the molecular weight < 300, log Kow < 3, the number of H-bond acceptors < 6, and H-bond donors < 3. The Rule of 3–5 suggests limited uptake by plants when the molecular weight is 300–500, log Kow 3–5, H-bond acceptors 6–10, and H-bond donors 3–5. Kumar points out that recent research on the uptake of emerging contaminants has supported this general rule. In developing this framework, the researchers were focused on the emerging contaminant compounds that are of greatest concern for human exposure via a plant-based diet. But there are broader ecological considerations, and Kumar points out that exposure levels and risk are different for other organisms exposed to wastewater, biosolids, and manure. To address this ecological question, Kumar says he's working on a similar framework looking at “what kind of properties the compounds will have that will accumulate more in aquatic organisms, or soil terrestrial organisms, like earthworms.” The issue of limiting exposure to emerging contaminants is much broader than evaluating uptake by food crops since emerging contaminants exist in many household products. The issue of limiting exposure to emerging contaminants is much broader than evaluating uptake by food crops since emerging contaminants can accumulate in other organisms (e.g., earthworms). Kumar also wonders about the risk of exposure to emerging contaminants via plant consumption compared with other sources of exposure. There are emerging contaminants in products we sometimes use on a daily basis like shampoo, toothpaste, plastic water bottles, and clothing treated with flame retardants. From this perspective, the issue of limiting exposure to emerging contaminants is much broader than evaluating uptake by food crops, but the questions of how low levels of emerging contaminants may effect human health and how to limit ecological impacts as these compounds enter aquatic and terrestrial systems remain the same. Alison Franklin, Ph.D. student in soil science and biogeochemistry at Penn State, collecting samples of wastewater treatment plant effluent that is being spray-irrigated at the university's Living Filter. Corn irrigation at Penn State's “Living Filter.” Photo by Emily Woodward/Penn State. by Joy Drohan If our food crops are spray-irrigated with treated wastewater, are we taking in minute doses of antibiotics and other emerging contaminants when we eat those crops? ASA, CSSA, and SSSA member Alison Franklin and her colleagues set out to answer that question in the article “Uptake of Three Antibiotics and an Antiepileptic Drug by Wheat Crops Spray Irrigated with Wastewater Treatment Plant Effluent” (http://bit.ly/29Fb01L). With antibiotic resistance much in the news, Franklin, a Ph.D. student in soil science and biogeochemistry at Penn State, wanted to begin to understand whether spray irrigation of crops with treated wastewater could be a problem long term. Her experiment is among the first to quantify the problem under field conditions using spray-irrigated effluent. Irrigation of croplands with treated effluent is increasing worldwide as water supplies tighten. In Israel, for instance, a significant amount of food crops are irrigated with wastewater. Forty-four percent of reclaimed wastewater projects in southern Europe have a predominantly agricultural use.1 Franklin's study examined uptake of three antibiotics and an anti-epileptic drug by wheat on the Living Filter, Penn State University's wastewater reuse system. The site receives about 5 cm of spray-irrigated effluent per week, at 12-hour intervals, year-round. The effluent first receives primary and secondary treatment at the University Park Wastewater Treatment Plant where the permitted capacity is 4 million gallons of influent per day. Spray irrigation provides tertiary treatment and recharges groundwater. Franklin chose four compounds to study that are meaningful to health after speaking with the pharmacy director at Penn State's University Health Services. Sulfamethoxazole and trimethoprim are typically prescribed together to treat ear or urinary tract infections or bronchitis. This combination, commonly known as Bactrim, is still generally effective against methicillin-resistant Staphylococcus aureus (MRSA), so doctors want to preserve its efficacy. Ofloxacin is a stronger version of the more commonly prescribed ciprofloxacin (Cipro). Carbamazepine is an antiepileptic drug that alters brain chemistry and persists in the environment. Although concentrations of carbamazepine in effluent were very low, its effects and behavior make it an important chemical to study. Franklin and her colleagues chose wheat as the study plant because it is the third most commonly grown cereal grain worldwide and the fourth most common agricultural crop in the United States. The wheat in this study was destined for animal consumption. The team sampled effluent in spring, summer, and fall to determine variation in the target compounds throughout the year. The population of University Park is highest in spring and fall, when classes are in session, and drops during the summer when most students leave. Concentrations of the target compounds reflected these population swings and the higher use of antibiotics in spring. Concentrations of sulfamethoxazole in spring were 22 µg/L, whereas in summer, they were only 580 ng/L. Maximum concentrations of trimethoprim and ofloxacin were much lower, 1 and 2.2 µg/L, respectively, in spring. The maximum concentration of carbamazepine was much lower still—23 ng/L. Wheat samples were collected three weeks before harvest and at harvest. Samples were rinsed with methanol to remove any of the target compounds adhering to the outside of the plant. Methanol was used because some of the compounds are not entirely water soluble. Straw and grain were extracted and analyzed separately via liquid chromatography–tandem mass spectrometry analysis. With spray irrigation of effluent, these pharmaceuticals and personal care products do cling to plant surfaces and are taken up by wheat. Ofloxacin was found at higher concentration in the straw (10 ng/g straw) than in the grain (2 ng/g). Trimethoprim was found only on the grain surface. Carbamazepine and sulfamethoxazole were concentrated in the grain (1.9 ng/g and 0.6 ng/g, respectively). The target compounds behaved differently because of their chemistry. The more hydrophobic a compound is, such as carbamazepine, the more likely it is to be bound with organic matter in wheat and soil. “The Living Filter is effective in trapping these compounds in soil,” says coauthor and ASA and SSSA member Clinton Williams, USDA soil scientist. This is important to know as land application of treated wastewater becomes more common. The concentrations in wheat are a million times lower than a typical adult dose (400–800 mg) of these drugs, assuming the USDA average daily wheat consumption of 166 g. To ingest the lowest typical dose of sulfamethoxazole, a person might have to consume 50–100 kg (100–200 lb) of this grain, says coauthor and SSSA member Jack Watson, a crop and soil scientist at Penn State. “But we don't know the long-term impacts,” Franklin cautions. “Could these compounds continue to accumulate in the environment and affect our health? We should either make sure they aren't getting into the environment or figure out the possible health effects of these low concentrations.” Still, Williams is encouraged that “the majority of the emerging contaminants are in a place where we can deal with them”—on the outside of the plants—and there was little contamination inside the plants. Franklin is continuing to explore this issue with investigations into whether microbial populations are affected by these compounds. She'll also look for antimicrobial resistance and, to help us decide whether effluent for irrigation of croplands requires additional treatment and/or testing, assess whether the compounds singly or in combination may produce negative biological impacts. J. Drohan, contributing writer for CSA News magazine View the articles from the special section in JEQ “Antibiotics in Agroecosystems: State of the Science” here: http://bit.ly/29wbOYF.
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