Natural abundance δ¹³C constraints on the detection of microplastic-derived carbon in freshwater environments

Microplastics (MPs) are increasingly recognized as emerging pollutants in freshwater systems. Detecting and tracing MP-derived carbon in aquatic food webs, however, remains unresolved, limiting our understanding of ecological impacts. Here, we evaluate the potential and limitations of natural abundance stable carbon isotope measurements (δ¹³C) as a tool to identify MP signals in freshwater ecosystems. For this purpose, two freshwater algae, Chlorella vulgaris and Chlamydomonas reinhardtii, were exposed under controlled laboratory conditions to one non-biodegradable polymer, low-density polyethylene (LDPE), and two biodegradable polymers, polylactic acid (PLA) and polybutylene adipate-co-terephthalate (PBAT), to assess isotope composition and growth. Laboratory data were complemented by particulate organic carbon (δ¹³C_POC) measurements from seasonal Danube River campaigns (2023-2024) with modeled predictions based on dissolved organic carbon (δ¹³C_DIC). MP exposure did not inhibit algae growth, but C. vulgaris exhibited significant (p <0.05) δ¹³C enrichment (+4 to +5 ‰), whereas C. reinhardtii showed no isotopic response. These shifts were unrelated to polymer isotope values and likely reflect indirect physiological stress rather than assimilation of polymer-derived carbon. Complementary binary mixing experiments further confirmed that measurable isotopic shifts occur only at unrealistically low algae-to-MP ratios (≤10:1), underscoring the limited sensitivity of isotope mass balances. Field surveys revealed pronounced seasonal δ¹³C_POC variability in the Danube, spanning 7.4 ‰ annually. Yet deviations from modeled expectations were inconsistent with MP inputs and instead reflected natural drivers such as productivity and remineralization. Overall, while natural abundance δ¹³C can capture subtle algae responses to MP exposure under laboratory conditions, its diagnostic power for tracing MP-derived carbon in complex freshwater systems appears limited.