Freezing prevents ⁶⁰Co‑γ irradiation artefacts in DOM concentration, composition and δ¹³C

Gamma (γ)-irradiation is often used to sterilise environmental water samples for international transport and biosecurity compliance. However, its impact on the molecular and isotopic integrity of dissolved organic matter (DOM) remains poorly understood. Here, we assess the effects of γ-irradiation (50 kGy), a commonly used sterilisation technique for water samples arriving into Australia, on dissolved organic carbon (DOC) concentration, DOM composition, and stable isotopic signatures (δ¹³C, δ¹⁵N, δ¹⁸O, δ²H) in natural waters and Milli-Q water mixed with organic matter standards, comparing frozen-irradiated and unfrozen-irradiated samples. Irradiation, when applied to unfrozen water, led to significant (p < 0.05) DOC loss, oxidative fragmentation of humic-like fluorophores, and significant (p < 0.05) δ¹³C enrichment of residual DOC, consistent with preferential ¹²C oxidation. Low-molecular-weight acids and microbial protein-like components increased, while aromaticity declined. In contrast, frozen samples exhibited no significant changes in DOC, DOM composition, and δ¹³C, indicating that immobilisation of DOM in ice suppresses its oxidation. Gamma irradiation also caused acidification (ΔpH ≈ -0.5 to -0.8 units) and altered concentrations of SO₄ and NO₃, with a significant increase in SO₄ likely resulting from the oxidation of dissolved organic sulfur. These results demonstrate that freezing samples prior to γ-irradiation effectively preserves both molecular and isotopic integrity of DOM and DOC concentration and therefore should be considered as an effective technique to maintain the integrity of environmental water samples during sterilisation in biogeochemical studies.