Multiple stable isotopic signatures corroborate the predominance of acetoclastic methanogenesis during CH4 formation in agricultural river networks.

• Three isotopic methodologies were used to quantify the biogenic CH 4 production pathway. • AM processes predominant the CH 4 production in the agricultural river network. • Long-term measurements provide the new understanding of atmospheric CH 4. Methane (CH 4) emitted from river networks provides a globally significant, yet poorly constrained barrier for the refinement of national greenhouse gas (GHG) inventories and the global CH 4 budget. This study employed three isotopic methodologies, i.e., a simple mixing model, apparent fractionation factor, and dual isotopes (δ13C−CH 4 and δD−CH 4) to identify the biogenic CH 4 production pathway (BMPP) in a typical agricultural river network in subtropical China. The results indicated that the contribution of acetoclastic methanogenesis (AM) to BMPP in the Tuojia River network (TRN) ranged from 85 to 99%, with the first reach (S1) supporting the most biogenic CH 4 production. Furthermore, the α C value ranged from 1.00 to 1.039 and increased spatially over the four reaches (S1 to S4), while the α D value varied from 1.393 to 1.394, with S1 having the lowest value. The dual isotopes of δ13C and δD reflected a mixed BMPP. Clearly, the isotopic composition of CH 4 corroborated the predominance of AM in the BMPP in the TRN. Additionally, there was a remarkable spatiotemporal variation in δ13C−CH 4 and δD−CH 4 , as well as α C and α D. In combination with the correlation between the two paired isotopic signatures (antipathetic and positive, p < 0.05, respectively), this suggested a change in the BMPP in the TRN. The findings of our study implied that there was great impact of agricultural production and residential activities on environmental behavior of stable isotopes. Consequently, we recommend that multiple isotopic measures, based on long-term in situ isotopologue monitoring, should be applied when the uncertainties of CH 4 emissions from river networks are addressed. [ABSTRACT FROM AUTHOR]