Urea and lipid extraction treatment effects on δ(15)N and δ(13)C values in pelagic sharks.

Rationale: Stable isotope analysis (SIA) provides a powerful tool to investigate diverse ecological questions for marine species, but standardized values are required for comparative assessments. For elasmobranchs, their unique osmoregulatory strategy involves retention of (15)N-depleted urea in body tissues and this may bias δ(15)N values. This may be a particular problem for large predatory species, where δ(15)N discrimination between predator and consumed prey can be small.

Methods: We evaluated three treatments (deionized water rinsing [DW], chloroform/methanol [LE] and combined chloroform/methanol and deionized water rinsing [LE+DW]) applied to white muscle tissue of 125 individuals from seven pelagic shark species to (i) assess urea and lipid effects on stable isotope values determined by IRMS and (ii) investigate mathematical normalization of these values.

Results: For all species examined, the δ(15)N values and C:N ratios increased significantly following all three treatments, identifying that urea removal is required prior to SIA of pelagic sharks. The more marked change in δ(15)N values following DW (1.3 ± 0.4‰) and LE+DW (1.2 ± 0.6‰) than following LE alone (0.7 ± 0.4‰) indicated that water rinsing was more effective at removing urea. The DW and LE+DW treatments lowered the %N values, resulting in an increase in C:N ratios from the unexpected low values of <2.6 in bulk samples to ~3.1 ± 0.1, the expected value of protein. The δ(13)C values of all species also increased significantly following LE and LE+DW treatments.

Conclusions: Given the mean change in δ(15)N(1.2 ± 0.6‰) and δ(13)C values (0.7 ± 0.4‰) across pelagic shark species, it is recommended that muscle tissue samples be treated with LE+DW to efficiently extract both urea and lipids to standardize isotopic values. Mathematical normalization of urea and lipid-extracted δ(15)N(LE+DW) and δ(13)C(LE+DW) values using the lipid-extracted δ(15)N(LE) and δ(13)C(LE) data were established for all pelagic shark species.