The translation of the original seawater signal (i.e., ambient temperature, salinity and δ18Osw) into distinct chambers of a single shell of a foraminifer during calcification can influence our interpretation of surface ocean conditions of the past, when based upon oxygen and carbon stable isotope geochemistry. Three different hypotheses related to: the size; the composition of the final chamber vs. the remaining shell; and species-specific offsets were tested to gain more insight into biological and ecological processes that influence the resultant composition of stable isotopes of oxygen (δ18O) in the shells of planktonic foraminifera. Shells of Trilobatus sacculifer, Globigerinoides ruber white and Neogloboquadrina dutertrei were picked from the top of the RETRO multi-core GS07-150-24, of Modern age, offshore of North-East Brazil (3°46.474' S, 37°03.849' W) and analysed for single shell and chamber stable isotope analysis. We show that there is a significant difference, of 0.203 ‰ ± 0.40 ‰ (1σ), in δ18O between the final chamber (δ18OF) and the test minus the final chamber (δ18O) of T. sacculifer. The formation of the final chamber approximately 1 °C cooler than the chambers formed prior, suggests both ontogenetic depth migration to deeper water and a potential offset from the surface signal. However, we show that there is no statistical difference in the δ18O values of shells of three different size classes, based upon measured size as opposed to sieve size, of T. sacculifer, although the pattern between the different size classes indicates depth migration during the life and growth of T. sacculifer. Comparison between T. sacculifer, G. ruber white and N. dutertrei suggests that G. ruber has a shallower depth habitat (~ 90–120 m) than the other species (~ 100–130 m). Disentangling depth versus seasonal habitat is complicated given the commonality between isotopes values from similar depths but different seasons, for instance the same average isotope value will have a shallower depth habitat in May than September. Calculation of seasonal-depth habitat was therefore tested. Our results highlight the complicated nature of interpreting oxygen isotopes even for the modern record.