Ocean acidification by anthropogenic carbon dioxide emissions is projected to depress metabolic and physiological activity in marine calcifiers. To evaluate the sensitivity of marine organisms against ocean acidification, the assimilation of nutrients into carbonate shells and soft tissues must be examined. We designed a novel experimental protocol, reverse radioisotope labelling, to trace partitioning of nutrients within a single bivalve species under ocean acidification conditions. Injecting CO2 gas, free from radiocarbon, can provide a large contrast between carbon dissolved in the water and the one assimilated from atmosphere. By culturing modern aquifer organisms in acidified seawater, we were able to determine differences in the relative contributions of the end members, dissolved inorganic carbon (DIC) in seawater and metabolic CO2, to shell carbonate and soft tissues. Under all pCO2 conditions (463, 653, 872, 1,137 and 1,337 μatm), radiocarbon (Δ14C) values of the bivalve Scapharca broughtonii shell were significantly correlated with seawater DIC values; therefore, shell carbonate was derived principally from seawater DIC. The Δ14C results together with stable carbon isotope (δ13C) data suggest that in S. broughtonii shell δ13C may reflect the kinetics of isotopic equilibration as well as end‐member contributions; thus, care must be taken when analysing end‐member contributions by a previous method using δ13C. The insensitivity of S. broughtonii to perturbations in pCO2 up to at least 1,337 µatm indicates that this species can withstand ocean acidification. Usage of radioisotope to dope for tracer experiments requires strict rules to conduct any operations. Yet, reverse radioisotope labelling proposing in this study has a large advantage and is a powerful tool to understanding physiology of aquifer organisms that can be applicable to various organisms and culture experiments, such as temperature, salinity and acidification experiments, to improve understanding of the proportions of nutrients taken in by marine organisms under changing environments.

In order to allow full comparability with other ocean acidification data sets, the R package seacarb (Gattuso et al, 2020) was used to compute a complete and consistent set of carbonate system variables, as described by Nisumaa et al. (2010). In this dataset the original values were archived in addition with the recalculated parameters (see related PI). The date of carbonate chemistry calculation by seacarb is 2020-11-30.