Effects of pre‐treatments on bulk stable isotope ratios in fish samples: A cautionary note for studies comparisons

Abstract

Rationale Stable isotope analysis (SIA) has revolutionised ecological studies over the past thirty years. One of the major fields where SIA is applied in the marine environment is related to the definition of ecosystem structure and function. With marine top predators such as sharks, SIA is a method of choice because tissue samples can be collected without the sacrifice of the animal. In elasmobranch research, the influence of compounds such as urea, trimethylamine oxide and lipids must be considered when using stable isotopes as ecological markers. Currently, a range of pre‐treatments are used to chemically remove these molecules prior to SIA. Methods This study investigated the impact of eleven commonly used pre‐treatments on carbon and nitrogen contents and C:N atomic ratio, as well as carbon and nitrogen SI ratios in elasmobranch tissues and its prey, measured by isotope ratio mass spectrometry. Three tissues were tested: blood and muscle of the ragged‐tooth shark Carcharias taurus, and muscle of one teleost species, the Cape knifejaw Oplegnathus conwayi. Results Compared with untreated samples, no trend or generalisation could be highlighted with the influence of pre‐treatments being species‐, tissue‐ and chemical‐element‐dependent. For the δ13C and δ15N values, differences among pre‐treatments were as high as 3‰, therefore potentially leading to erroneous ecological interpretation. Conclusions The chemical properties of compounds (e.g. urea, lipids) combined with the polarity of solutions (e.g. water, solvents) explained a large part of these observations. This study highlights that pre‐treatments need to be considered especially when comparing carbon and nitrogen stable isotope ratios between studies. The results of this study provide a call to all stable isotope researchers to make a concerted effort to standardise pre‐treatment methods. This is crucial as global reviews are becoming increasingly more informative.

Ontogenetic dependence of Nile crocodile (Crocodylus niloticus) isotope diet‐to‐tissue discrimination factors

“…Bias in δ 13 C that arises from lipids 25 can be avoided by chemically removing the lipids using one of a number of protocols, or it can be mathematically corrected using the C/N ratio as a proxy of the lipid content 25,51–53 . This assumes that the variability in the C/N ratio is driven by the lipid content and also that a demonstrable (calibrated) relationship exists between the Δ 13 C values and the C/N ratios.…”

Evolution of realized Eltonian niches acrossRajidaespecies

“…While the isotopic composition of white‐muscle tissue likely dampens short‐term variability, due to a long isotopic turnover rate, it is possible that detection of a phylogenetic signal in some ENCs only in a single scenario may be explained by sampling design and interannual baseline variation. Additionally, slightly different sample pre‐treatment methods between communities could be a source of variability in the stable isotope ratios (Connan et al 2019). However, the above factors are unlikely to introduce the variability necessary to bias overall ecological interpretation of Eltonian niche diversity in skates.…”

Determining the appropriate pretreatment procedures and the utility of liver tissue for bulk stable isotope (δ13C and δ15N) studies in sharks

“…Given the high concentrations of lipid in shark tissue, it is possible that (Connan et al, 2019;Sweeting et al, 2006). The strong polarity of chloroform-methanol may therefore effectively remove structural lipids in lipid-concentrated tissues such as liver, freeing low-weight amino acids (Mathew & Shamasundar, 2002;Murthy & Rajanna, 2011).…”