Effects of acid treatment on carbon and nitrogen stable isotope ratios in ecological samples: a review and synthesis



  1. Stable isotopes of carbon and nitrogen are mainstay tracers in diverse fields of ecology, particularly in studies of food webs.
  2. Investigators are generally interested in tracing dietary C and N, and hence routinely remove non-dietary, inorganic C contained in calcified structures (e.g. shells, bones) by chemical dissolution of the carbonates. Acid treatment can, however, isotopically fractionate samples if part of the organic matter is lost or chemically modified, resulting in potentially altered δ13C and δ15N values.
  3. Here, we synthesize the effects of acid treatments on stable isotope analysis reported in the literature, showing that: (i) the method can change both δ13C and δ15N values; (ii) shifts in δ13C are generally, but not always, consistent with expectations of more depleted carbon ratios after the removal of the isotopically heavier inorganic carbonates; (iii) nitrogen ratios either decrease or increase in 15N content; and (iv) the majority (74–79% of comparisons) of reported changes to δ13C and δ15N values attributable to acid treatment are <1 ‰, but larger acid effects can occur.
  4. Acidification is needed if mechanical removal of calcified structures is unfeasible in carbonate-rich sample matrices containing low organic C and N, but should otherwise be very carefully considered before its use as a routine pre-treatment step of biological samples in isotope ratio mass spectrometry.