Effects of sample preparation on stable isotope ratios of carbon and nitrogen in marine invertebrates: implications for food web studies using stable isotopes

Abstract

Trophic ecology has benefitted from the use of stable isotopes for the last three decades. However, during the last 10 years, there has been a growing awareness of the isotopic biases associated with some pre-analytical procedures that can seriously hamper the interpretation of food webs. We have assessed the extent of such biases by: (1) reviewing the literature on the topic, and (2) compiling C and N isotopic values of marine invertebrates reported in the literature with the associated sample preparation protocols. The factors considered were: acid-washing, distilled water rinsing (DWR), sample type (whole individuals or pieces of soft tissues), lipid content, and gut contents. Two-level ANOVA revealed overall large and highly significant effects of acidification for both delta(13)C values (up to 0.9 per thousand decrease) and delta(15) N values (up to 2.1 per thousand decrease in whole individual samples, and up to 1.1 per thousand increase in tissue samples). DWR showed a weak overall effect with delta(13)C increments of 0.6 per thousand (for the entire data set) or decrements of 0.7 per thousand in delta(15) N values (for tissue samples). Gut contents showed no overall significant effect, whereas lipid extraction resulted in the greatest biases in both isotopic signatures (delta(13)C, up to -2.0 per thousand in whole individuals; delta(15)N, up to +4.3 per thousand in tissue samples). The study analyzed separately the effects of the various factors in different taxonomic groups and revealed a very high diversity in the extent and direction of the effects. Maxillopoda, Gastropoda, and Polychaeta were the classes that showed the largest isotopic shifts associated with sample preparation. Guidelines for the standardization of sample preparation protocols for isotopic analysis are proposed both for large and small marine invertebrates. Broadly, these guidelines recommend: (1) avoiding both acid washing and DWR, and (2) performing lipid extraction and gut evacuation in most cases.

Preservation methods alter stable isotope values in gelatinous zooplankton: implications for interpreting trophic ecology

“…Stable isotope analysis is a fundamental tool in food web studies yet comes with a number of well-documented caveats (Gannes et al 1997; Vander Zanden and Rasmussen 2001; Mateo et al 2008). For example, numerous studies have indicated how inconsistencies in 13 C and 15 N values for the same species can arise through diVerences in preanalytical protocols (Feuchtmayr and Grey 2003;Sarakinos et al 2002;Syväranta et al 2008).…”

“…For example, numerous studies have indicated how inconsistencies in 13 C and 15 N values for the same species can arise through diVerences in preanalytical protocols (Feuchtmayr and Grey 2003;Sarakinos et al 2002;Syväranta et al 2008). In this context, the need to standardise, or at least consider the eYcacy of protocols, is important for the success of future studies (Mateo et al 2008;Carabel et al 2009). Ideally, when only large medusae are to be considered, separating the bell into three equal components and subjecting each to a diVerent preservation might be appropriate, but for small species such as A. aurita this is not always possible.…”

“…Preservation methods employed prior to stable isotope analysis (SIA) have been shown to alter isotopic composition in a range of marine taxa ranging from algae (Kaehler and Pakhomov 2001;Carabel et al 2009) through to invertebrates (Bosley and Wainwright 1999;Kaehler and Pakhomov 2001;Carabel et al 2009) and higher vertebrates (Bosley and Wainwright 1999;Kaehler and Pakhomov 2001). Mateo et al (2008) additionally highlighted pre-analytical biases at the class level with the greatest eVects exhibited by Maxillopoda, Gastropoda and Polychaeta. Our knowledge of such eVects does not currently extend to gelatinous zooplankton, presenting a potential problem for future studies.…”

Trophic plasticity of Antarctic echinoids under contrasted environmental conditions

“…Following recommendations of Mateo et al (2008), animals were dissected to separate soft, non-metabolically active tissues. Due to differences in echinoids morphology, analyses were performed on the peristome for Echinidae, the stomach caecum for Schizasteridae and the spine muscles for Cidaridae.…”

“…Stable isotope ratios measurements were performed via continuous flow-elemental analysis-isotope-ratio mass spectrometry (CF-EA-IRMS) at University of Liège, using a Vario Micro Cube elemental analyser (Elementar Analysensysteme GmBH, Hanau, Germany) coupled to an Isoprime 100 mass spectrometer (Isoprime, Cheadle, UK). Since acidification is known to alter N isotopic ratios (Mateo et al 2008), acidified samples were analysed twice: once for C isotopic ratios, using decarbonated material, and once for N isotopic ratios, using native material. Isotopic ratios were expressed using the widespread d notation (Coplen 2011).…”