Microplastics, defined as solid polymeric particles smaller than 5 millimeters, have become an emerging concern worldwide. They arise either from deliberately manufactured microscopic materials, so-called primary microplastics, or from the progressive fragmentation of larger plastic objects exposed to physical, chemical, and biological weathering, known as secondary microplastics. Wastewater treatment plants sit squarely on the front lines of this pollution pathway. They receive enormous loads of plastic fibers and fragments from household laundry, cosmetic products, and urban runoff, yet they also act as concentrators, retaining a significant fraction of the incoming plastic in the sludge line. When that sludge is reused on land or improperly disposed of, it can become a secondary source of contamination for soils and waterways. Understanding exactly how many particles flow through these facilities—and what they are made of—depends on analytical methods that can reliably pry plastic particles out of a sea of organic matter without destroying the evidence in the process. That is precisely the challenge the new research set out to solve.

Led by Ivanilson da Silva de Aquino of the University of Brasília, together with colleagues at the UnB Planaltina Faculty and the Federal University of Jequitinhonha and Mucuri Valleys, the team built its methodological framework from the ground up. The first step was to manufacture reference microplastics from the six commodity polymers that dominate urban plastic waste streams: polyethylene terephthalate, high-density polyethylene, low-density polyethylene, polyvinyl chloride, polypropylene, and polystyrene. These laboratory-made particles, produced as irregular fragments ranging from 0.1 to 3.0 millimeters, served as benchmarks against which every subsequent processing step could be tested. Before any chemistry was attempted, the researchers confirmed by attenuated total reflectance Fourier-transform infrared spectroscopy—ATR-FTIR for short—that each batch of reference particles displayed the characteristic absorption patterns of its parent polymer, ensuring that the starting materials were spectroscopically unambiguous.

The heart of the new protocol is a digestion step based on Fenton’s reagent, a mixture of hydrogen peroxide and iron catalyst that generates highly reactive hydroxyl radicals capable of shredding organic matter. Wastewater and sludge samples are so loaded with organic material, microbial biomass, and inorganic debris that microscopic plastic particles would otherwise be invisible. The catch is that the digestion must be aggressive enough to clear the matrix but gentle enough to leave the plastics themselves chemically intact. If the polymers are oxidized too severely, their infrared fingerprints can shift, complicating identification, and in some cases particles can fragment or lose mass entirely. The team therefore subjected its six reference polymers to five different Fenton protocols spanning temperatures from 30 to 80 degrees Celsius and various reaction durations, then scrutinized the consequences using FTIR and a metric known as the carbonyl index.

The carbonyl index tracks the relative abundance of carbon-oxygen double bonds on polymer surfaces, a hallmark of oxidative degradation. Because polyolefins such as polyethylene and polypropylene naturally lack carbonyl groups, even small increases in the index reveal genuine surface oxidation. The results were revealing. At 30 degrees Celsius the reaction was too sluggish to digest organic matter effectively. Above 80 degrees Celsius oxidation became severe, with polyethylene terephthalate and polypropylene showing the largest changes in carbonyl absorption. But in the moderate window between 40 and 60 degrees Celsius, with a reaction time of about two hours, the Fenton treatment delivered the best of both worlds: organic matter was substantially degraded, while the carbonyl index of all tested polymers showed no significant changes. FTIR spectra recorded before and after digestion showed no meaningful spectral deviations, meaning the polymeric fingerprints needed for reliable identification survived the chemical assault intact.

With digestion optimized, the researchers turned to the second major hurdle: separating buoyant plastic particles from the remaining debris. Density separation exploits the simple physical fact that most common polymers are less dense than suitable salt solutions, so they float while heavier mineral and organic particles sink. The team built a custom separation device and systematically compared separation media. Plain water, with a density of 1.0 grams per cubic centimeter, performed poorly because more than half of the target polymers are denser than water and simply sank. Saturated sodium chloride solution, the cheap and inert classic, reached about 1.2 grams per cubic centimeter and successfully floated roughly two-thirds of the studied plastics—but it excluded the high-density polymers polyvinyl chloride, at 1.10 to 1.45 grams per cubic centimeter, and polyethylene terephthalate, at 1.37 to 1.45 grams per cubic centimeter. Relying on sodium chloride alone, the authors caution, could systematically underestimate microplastic abundance.

Zinc chloride proved to be the decisive upgrade. At concentrations reaching 1.6 and 1.8 grams per cubic centimeter, zinc chloride solutions kept every polymer tested in suspension, allowing effective recovery of even the heaviest particles. In the validation experiments, the densest solution ensured that all target polymers remained in the floating fraction, minimizing losses during sample processing. The researchers note, however, that the analytical superiority of zinc chloride comes with trade-offs: its toxicity, higher operational cost, and disposal requirements must be weighed when designing routine monitoring programs, and reuse of the solution can help mitigate cost and waste.

The true test came when the optimized protocol was unleashed on real samples from a full-scale facility, the Brasília North wastewater treatment plant. The researchers deliberately chose two matrices representing opposite ends of the processing complexity spectrum: raw wastewater, with a chemical oxygen demand averaging 882.7 milligrams of oxygen per liter, and dewatered sewage sludge, whose chemical oxygen demand averaged a staggering 30,600 milligrams of oxygen per liter—roughly thirty-five times higher. Applying the optimized Fenton protocol cut chemical oxygen demand by 81.3 percent in the raw wastewater and 82.3 percent in the sludge, leaving values of 165.4 and 5,420 milligrams of oxygen per liter respectively. Visually, the transformation was dramatic: dark, turbid samples became noticeably clearer and more translucent after treatment, although sludge samples, given their enormous organic load, sometimes required a second digestion cycle to remove residual suspended material.

Following digestion, density separation with zinc chloride at 1.8 grams per cubic centimeter produced striking stratification in the custom unit, with a floating fraction enriched in microplastics accumulating at the top and residual organic matter and denser inorganic particles settling at the bottom. Spiked reference particles of every polymer type were successfully recovered from both matrices, including the notoriously difficult polyethylene terephthalate and polyvinyl chloride. The researchers then applied the validated workflow to hunt for native microplastics—particles that arrived in the samples from the real world rather than from the laboratory. Stereomicroscopy revealed a rich menagerie of fragments, films, and fibers varying widely in color, size, and surface texture, exactly the heterogeneity expected from the diverse plastic sources entering urban sewer networks.

ATR-FTIR analysis of the recovered native particles confirmed the method’s analytical power. Polyethylene emerged as the dominant polymer in both wastewater and sludge, identified through its characteristic absorption bands near 2915, 2845, 1462, and 717 wavenumbers, corresponding to carbon-hydrogen stretching, methylene bending, and rocking vibrations. A subtle band near 1377 wavenumbers even allowed the team to distinguish low-density from high-density polyethylene. Crucially, the spectra of environmental particles retained all the features needed for identification, confirming that the upstream pretreatment had not compromised the spectroscopic evidence. The predominance of polyethylene aligns with the ubiquity of packaging films and containers in urban life and echoes findings from previous studies around the world.

The significance of this work extends well beyond a single treatment plant in the Brazilian capital. By demonstrating that Fenton digestion, zinc chloride density separation, stereomicroscopy, and ATR-FTIR can operate as a coherent, validated pipeline across both liquid and solid waste streams, the study provides exactly the kind of methodological scaffolding that the field has lacked. The authors emphasize that their framework can support future monitoring programs aimed at evaluating the occurrence, transport, and fate of microplastics throughout wastewater treatment systems, and that it contributes to the ongoing effort to establish standardized protocols for microplastic analysis in complex environmental matrices. As regulators and researchers worldwide push to quantify how much plastic flows from drains to rivers to oceans—and how much lingers in the sludge spread across farmland—reliable, reproducible measurements will be the foundation of every policy decision. This new protocol offers a tested blueprint for generating them.