Wastewater influent is notoriously difficult to analyze. Between overcoming the limits of commercial solid-phase extraction (SPE) cartridges and mitigating ion suppression at the detector, analytical chemists face steep technical hurdles when isolating ultra-trace chemicals of emerging concern (CECs).
In this expert feature, we delve into insights from Dr. Diana Aga, a State University of New York (SUNY) Distinguished Professor and Director of the Research and Education in eNergy, Environment, and Water (RENEW) Institute at the University at Buffalo. As a Fellow of the American Chemical Society, current associate editor of the Journal of Agricultural and Food Chemistry, and a former editor for the Journal of Hazardous Materials, Dr. Aga brings decades of experience in environmental monitoring to the table. Her laboratory's pioneering research spans the complex realms of non-targeted analysis, antimicrobial resistance, and the ongoing pursuit of characterizing and degrading the elusive forever chemicals (PFAS).
Mastering the Matrix: Extraction Strategies
Dr. Aga’s laboratory analyzes an incredibly diverse array of matrices, ranging from clean drinking water to highly complex matrices such as manure, wastewater, urine, blood, and fish. Because no single method yields 100% recovery across the entire spectrum of highly polar to highly hydrophobic compounds, labs are forced to compromise.
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“Ideally, you want to capture everything, but it's not possible to have 100% recovery of all compounds,” notes Aga.
To build an effective overarching strategy, Dr. Aga describes the following extraction approaches:
- The default foundation: "We have just defaulted to using hydrophilic-lypophilic balance (HLB)," Dr. Aga remarks, explaining that it captures the broadest baseline of compounds.
- Targeted additions: When extracting PFAS, the lab includes an ion-exchange step in addition to the HLB cleanup.
- Matrix-specific tweaks: For uniquely challenging matrices such as fish tissue, graphitized carbon black (GCB) is incorporated into the cleanup process.
By strategically layering these techniques, the lab can maximize chemical recovery rates across a wide variety of challenging sample types.
Municipal vs. Agricultural Wastewater
Not all wastewater is created equal. The challenges inherent to testing municipal influent differ vastly from those of agricultural waste, requiring distinct analytical approaches:
- Agricultural wastewater: According to Aga, this matrix is exceptionally dirty, consisting of a slurry of mixed urine and manure. However, analytically, the target list is narrower; scientists typically look for very specific compounds, such as hormones and veterinary antibiotics.
- Municipal wastewater: While municipal wastewater is relatively physically cleaner due to settling, grit filtration, and biological treatments that remove natural organic matter, it is chemically far more complex. Analysts must screen for a wide range of pharmaceuticals, personal care products, and industrial chemicals.
"It's more complex in terms of analytes, but in terms of matrix, it's less complex compared to animal manure," explains Aga. Understanding these distinct matrix compositions is crucial for analysts when developing and optimizing their extraction and detection methodologies.
The Threat of Antimicrobial Resistance (AMR)
A major focus in agricultural wastewater analysis is tracking veterinary antimicrobials before manure slurry is spread onto fields. Constant environmental exposure to low levels of these antibiotics creates conditions conducive to the emergence of antibiotic-resistant bacteria.
Because these agricultural antimicrobials currently lack strict regulations or maximum limits, analytical labs are forced to establish their own testing baselines to properly characterize the threat.
"There are no regulations, so there's really no limit," Dr. Aga asserts, regarding current testing thresholds. "So I guess the goal is to attain limits that are as low as possible."
Navigating Chemical Cocktails and the EPA's Draft CCL 6 Chemicals
When evaluating the combined toxicity of chemical mixtures or "cocktails" in wastewater, Dr. Aga clarifies that the primary bottleneck is not analytical capability. Modern LC-MS instruments can easily analyze over 100 target compounds in a single run when optimized properly. Instead, the real challenge is deciding which compounds to target when there are no regulatory guidelines dictating testing lists.
However, the regulatory landscape is showing signs of a historic shift. Driven by a new "Make America Healthy Again" governmental priority, the U.S. Environmental Protection Agency (EPA) recently released the Draft Candidate Contaminant List 6 (CCL 6). For the first time in almost 30 years, since the occurrence of many pharmaceuticals in surface water has been reported, EPA proposes to include pharmaceuticals as a group in CCL to further prioritize research and information needed to identify which specific pharmaceuticals are occurring in drinking water, and may be of greatest public health concern.
"We have been studying pharmaceuticals for 30 years, and this class of compounds has never made it onto the candidate list; now, unexpectedly, they have," states Dr. Aga. "For us, this is a significant development. It suggests a recognition of their environmental relevance."
While the research community is excited by this overdue attention, Dr. Aga notes there is still widespread skepticism about whether these monitoring efforts will eventually result in enforceable regulations, or if the chemicals will simply drop off the list when the five years of nationwide monitoring are up.
The Critical Role of Non-Targeted Analysis (NTA)
Routine labs typically test for known parent compounds, but this approach has a dangerous blind spot during treatment.
"The parent compound can disappear, but it doesn't mean it's removed," warns Dr. Aga. "It could just be that it was transformed to a different byproduct that may still have bioactivity, or sometimes may even be more toxic than the parent compound."
To combat this, academic and government research labs utilize high-resolution mass spectrometry for non-targeted analysis (NTA). Instead of looking for every single degradation product, NTA aims to identify which unknown byproducts are environmentally persistent so researchers can subsequently test their toxicity. Commercial labs rarely perform NTA because the unpredictability of the findings makes it difficult to guarantee results or price the service effectively for customers.
Capturing Elusive Ultrashort-Chain PFAS
The industry standard for PFAS analysis is LC-MS, but researchers know this method frequently misses highly polar, mobile, and potentially toxic ultrashort-chain chemicals.
"Supercritical fluid chromatography (SFC) is something that can readily separate these polar compounds," Dr. Aga says. "Why haven't we used it a lot when it's possible? I think it's because traditionally they are not readily available in laboratories because the application is very specific."
However, as the scientific community recognizes how many polar compounds are going undetected, SFC adoption is likely to grow.
Bioremediation and the Fluorine Mass Balance
In the pursuit of eliminating PFAS, Dr. Aga’s team studies the ability of the Labrys portucalensis (F11) bacterium to metabolize PFAS by breaking carbon-fluorine bonds. Tracking this biodegradation poses three chromatographic challenges:
- The missing fluorine: Researchers struggle to close the "fluorine mass balance". The amount of fluorine present in the initial PFOS does not match the measured fluorine left after incubation, leading scientists to suspect the creation of volatile byproducts that evade detection.
- Ultra-low concentrations: The parent compounds may degrade into byproducts at concentrations so low they cannot be detected, even by non-targeted analysis. 3. Isomer chaos: PFOS exists as a mixture of linear and many branched isomers. Removing even a single fluorine atom during degradation produces exponentially more isomeric byproducts, resulting in coeluting chromatographic peaks that are difficult to separate.
Overcoming these three hurdles remains a critical focus for researchers attempting to definitively prove the efficacy of bacterial PFAS remediation.
The Gap Between Academia and Commercial Testing
Translating ultra-sensitive academic workflows into rugged, high-throughput commercial methods highlights a core tension in the industry. Academic labs have the flexibility to instantly tweak flow rates, swap solvents, and optimize methods for highly specific matrices. Commercial labs, however, are bound to follow strict, established procedures (such as EPA 1633) exactly as written.
"To standardize reporting, commercial laboratories need to follow the validated procedure that's been established," Dr. Aga notes of the commercial testing environment. "But sometimes the established method only works for limited applications and certain sample matrices. So you have to modify some parameters when a new matrix is involved."
A Wishlist for the Future: Universal Databases & NMR
To make environmental characterization and non-targeted analysis more manageable over the next five years, Dr. Aga points to two major innovations:
- Universal, data-rich databases: Currently, NTA is severely bottlenecked because unknown compounds lack physical standards, forcing analysts to rely heavily on database matching. However, databases are often vendor-specific and prohibitively expensive. A universal, open-access database is needed that goes beyond simple mass and fragmentation matching, incorporating robust physicochemical data such as collision cross-section (CCS) values and retention times at defined chromatographic conditions.
- Highly sensitive NMR: Because fluorine only has one stable isotope (unlike chlorine or bromine), identifying unknown PFAS via MS fragmentation and isotopic patterns is particularly difficult. On the other hand,19Fluorine nuclear magnetic resonance (19F-NMR) can be very valuable in identifying and quantifying fluorinated organochemicals even without standards based on the chemical shifts and intensity of the fluorine atoms of a compound. However, current NMR hardware lacks the sensitivity of LC-MS. In our laboratory, we need to concentrate 2 liters of surface water samples down to just 1 mL to detect PFAS signals at typical environmental concentrations —a step that is impractical for large number of samples. NMR is also not useful for low-volume matrices such as human blood.
Developing and integrating these two complementary analytical approaches in the workflow would dramatically accelerate the environmental testing community's ability to identify and quantify emerging organofluorine pollutants.
