New Low-Cost Protocol Speeds Up Microplastic Detection in River Sediments

Riverbed sediments are among the most important archives of plastic pollution on Earth. As rivers carry plastic debris from cities, industries and farmland toward the sea, fine particles settle into the streambed, where they accumulate over months and years. Reading that archive, however, has always been slow and expensive. A team of Italian researchers at the University of Florence, working with the water utility Publiacqua, has now optimized a sample preparation protocol that makes it far faster and cheaper to extract and identify microplastics from river sediments using Raman spectroscopy, opening the door to basin-scale monitoring programs that were previously impractical.

The scale of the problem the method addresses is enormous. Global plastic production has climbed from roughly two million metric tons in 1950 to nearly 391 million metric tons in 2021, and because most plastics do not biodegrade, discarded items persist and fragment. Macroplastics larger than 25 millimeters break down under sunlight and physical weathering into mesoplastics and then microplastics, defined as solid polymer-containing particles between one micrometer and five millimeters. These particles are classified as primary, when manufactured at microscopic sizes, or secondary, when produced by the fragmentation of larger debris, and they display enormous diversity in polymer type, shape, color and size.

Aquatic systems bear the brunt of this pollution. An estimated 4.8 to 12.7 million metric tons of plastic waste entered the oceans from land-based sources in 2015 alone, and rivers act both as sinks where particles settle and as major highways transporting them to marine environments. Once in the water, microplastics harm organisms that ingest them, disrupting feeding, energy metabolism and reproduction, and they can act as vectors for persistent organic pollutants and heavy metals that bioaccumulate up the food chain. Understanding where and when microplastics accumulate in river sediments is therefore essential for environmental risk assessment, yet no standardized, robust protocol for sampling and analysis in complex matrices has existed, meaning field studies often produce results that cannot be compared.

The difficulty lies in the sediment itself. Riverbed material is a mixture of light organic matter, such as plant detritus and locally produced biomass, and fine inorganic particles like silt and clay, all of which can trap microplastics and interfere with their chemical identification. Traditional protocols rely on sieving, density separation and filtration, often supplemented by chemical oxidation or enzymatic digestion. Some multi-step procedures applied to organic-rich sediments have required as long as 24 days per batch. Faster alternatives exist, including dye staining with Nile Red, electrostatic or magnetic separation, and oil-assisted extraction, but each carries drawbacks: staining produces false positives in organic-rich samples and adds fluorescence that overwhelms Raman signals, while olive oil residues left on filters proved fluorescent and blocked spectroscopic identification in the Florence team’s own tests.

To overcome these limitations, the researchers combined several established techniques into a streamlined workflow tailored for Raman analysis. Sediment collected from the Arno River in Florence and Pisa was dried, sieved, and sonicated to expose embedded particles. Fenton oxidation, using hydrogen peroxide with an iron catalyst at 35 degrees Celsius, then digested the organic matter over about a week. A density separation in saturated sodium chloride solution floated the low-density fraction, and finally a custom-made glass separator performed an affinity extraction in which microplastics preferentially migrate into cyclohexane, a non-polar solvent, while residual vegetable matter stays in the water phase. A small amount of sodium dodecyl sulfate was added to weaken the adhesion between particles and the sediment matrix, and the extraction was repeated three times to maximize recovery.

The choice of cyclohexane was the key innovation. Unlike olive oil, it does not stick to glassware, evaporates easily, and is optically transparent in the visible range because it is a saturated, symmetrical hydrocarbon with no conjugated electrons or heteroatoms. That means it generates no fluorescence background in Raman measurements, preserving the sensitivity needed to identify even weakly scattering environmental particles. It is also chemically inert, so free radicals left over from the Fenton step are unlikely to degrade it into fluorescing residues. Although cyclohexane is ranked as problematic from a safety and environmental standpoint, the protocol uses only about six milliliters per sample, and the custom glassware was designed to minimize solvent waste.

Validation was carried out on real Arno sediments spanning a wide range of conditions, from fine and medium silts to coarse gravel, with organic carbon contents between 0.14 and 2.2 percent. Samples were spiked with reference particles of five polymers covering a range of densities: polyethylene, acrylonitrile butadiene styrene, polyvinyl chloride, polyamide and polyethylene terephthalate. The overall recovery was 0.65, rising to 0.82 for low-density polyethylene and 0.72 for medium-density PVC, but dropping to 0.42 for high-density PET, which barely floats in the 1.2 grams per cubic centimeter sodium chloride solution. The team accepted this trade-off deliberately, since heavier salts such as sodium iodide or zinc chloride are costly, toxic or energy-intensive to recycle, and the vast majority of environmental microplastics have densities below the threshold. Only about 13 percent of global plastic production exceeds it.

The tests also revealed how strongly the sediment matrix itself influences recovery. Recovery was highest in medium and coarse sands but fell sharply in fine sands and coarse silts, where the greater surface area, cohesiveness and aggregation of small particles entrap microplastics and prevent them from detaching and floating. The cyclohexane extraction step alone achieved an overall efficiency of 0.89, and recovery correlated with polymer wettability: particles with a water contact angle above roughly 75 to 80 degrees were recovered almost completely, while more hydrophilic surfaces were harder to extract. Fibers proved slightly harder to recover than spheres or fragments. Crucially, a stability test confirmed that the Fenton oxidation did not alter the Raman spectra, dimensions or morphology of the reference particles.

When applied to unspiked Arno River samples, the workflow identified five polymers: polypropylene, polyester, polyethylene terephthalate, polycarbonate and polyethylene. Concentrations ranged from 0.030 items per gram of dry sediment in fine sand to 0.653 items per gram in coarse gravel, with a median of 0.151 and a mean of 0.217 items per gram. These values are lower than figures previously reported for emerged sediment bars on the Arno, which trap particles during floods and were measured with the hot needle method known to overestimate counts through false positives, but they align well with studies of smaller Tuscan rivers. Blank samples contained no microplastics above 20 micrometers, so no contamination correction was needed, thanks to plastic-free equipment, cotton clothing, filtered compressed air for the filters and rigorous glassware cleaning.

The researchers position the protocol as a practical compromise for large-scale monitoring, where throughput and cost matter more than perfect recovery of every high-density polymer. Compared with enzymatic digestion methods that offer high accuracy at the price of weeks of processing, or instrument-heavy approaches requiring expensive equipment and expert operators, the new workflow is fast, inexpensive and compatible with standard microRaman instruments using a 785-nanometer diode laser and the open-source OpenSpecy library for spectral identification. The authors suggest that testing alternative solvents could further improve effectiveness and eco-compatibility, and that a denser flotation salt could extend the method to specific high-density polymers when needed. As European regulators move toward harmonized basin-scale microplastic monitoring under the Water Framework Directive, tools like this one may determine whether such ambitions become routine practice.

Subject of Research: Optimization of sample pretreatment for Raman spectroscopic determination of microplastics in riverbed sediments

Article Title: Advances in the sample pretreatment for determination of microplastics in riverbed sediments by Raman spectroscopy

Article References: Dali, A., Mancini, M., Santianni, D., Solari, L., & Becucci, M. (2026). Advances in the sample pretreatment for determination of microplastics in riverbed sediments by Raman spectroscopy. Environmental Science and Pollution Research. https://doi.org/10.1007/s11356-026-38231-8

Image Credits: AI Generated

DOI: 10.1007/s11356-026-38231-8

Keywords: microplastics, Raman spectroscopy, river sediments, sample pretreatment, Fenton oxidation, cyclohexane extraction, Arno River, density separation, plastic pollution, environmental monitoring, polymer identification, water quality