In this study, we incubated 103 animals specimens equivalent to 19 macrofaunal species to quantify holobiont-associated methane (CH4) fluxes and metabolic processes [oxygen (O2) - respiration and ammonium (NH4+) - excretion rates]. The specific goals were to quantify holobionts CH4 production/uptake and to establish correlations between CH4 fluxes and environmental factors (e.g., salinity). Invertebrates were collected in 4 coastal systems and incubated in 22 mL glass microcosms filled with 0.22 µm twice-filtered in situ water. Individual and Mass-standardized CH4 Production Rates (IPR and MPR, respectively), O2 Respiration Rates (IRR and MRR) and NH4+ Excretion Rates (IER and MER) were measured in 103 animals’ incubations. 1. IRR were calculated from linear regression analysis of the solute (O2) versus time equation: \(IRR=(Reg.Slope × V)/N\) where IRR (µmol O2 ind.−1 day−1) is the respiration of the chemical species O2; Reg.Slope is the slope of the regression (µmol O2 L−1 day−1); V (L) is the water volume in the glass microcosm; N is the number of incubated animals per microcosm. 2. IER and IPR were calculated from the difference in concentrations (NH4+ and CH4) in the water using the equation: \(IER and IPR =((C_f-C_i )×V)/(N×t)\) where IER and IPR (µmol ind.−1 day−1 and nmol ind.−1 day−1) are the excretion or production of the chemical species (NH4+ or CH4); Cf and Ci (µmol or nmol L−1) are the final and initial concentrations of the chemical species; V (L) is the water volume in the glass microcosm; N is the number of incubated animals per microcosm; and t (days) is the incubation time. Positive values represent productions while negative values represent uptake. Same equations were used to calculated mass-standardized rates, but instead of N the total animal biomass (gdw) was used. Animals’ biomass was determined as dry weight (DW) or as dry weight shell free (DWSF) for bivalves, after the desiccation at 70°C until constant mass. Water temperature and salinity were measured in situ with a multiple probe (556 MPS, YSI). Rates are reported as average ± standard error.

This work was supported financially by project INBALANCE funded by the European Social Fund (Grant No. 09.3.3-LMT-K-712-01-0069). Stefano Bonaglia was additionally supported by the Swedish Research Council Formas (Grants No. 2017-01513 and No. 2022-00546).