1. 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.

2. 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.

3. Under all pCO₂ conditions (463, 653, 872, 1137, and 1337 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 analyzing end-member contributions by a previous method using δ¹³C. The insensitivity of S. broughtonii to perturbations in pCO₂ up to at least 1337 µatm indicates that this species can withstand ocean acidification.

4. Usage of radioisotope to dope for tracer experiments requires strict rules to conduct any operations. Yet, reverse radioisotope labeling 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.