Compound-specific stable hydrogen isotope (δ2 H) analyses of fatty acids: A new method and perspectives for trophic and movement ecology

Abstract

Rationale: Compound-specific stable isotope analysis (CSIA) is a powerful tool for a better understanding of trophic transfer of dietary molecules in and across ecosystems. Hydrogen isotope values (δ² H) in consumer tissues have potential to more clearly distinguish dietary sources than ¹³ C or ¹⁵ N values within and among habitats, but have not been used at the fatty acid level for ecological purposes.

Methods: Here we demonstrate a new online high-capacity gas chromatography-isotope ratio mass spectrometry technique (² H-CSIA) that offers accurate and reproducible determination of δ² H values for a range of fatty acids from organisms of aquatic food webs.

Results: We show that lipid extracts obtained from aquatic sources, such as biofilms, leaves, invertebrates, or fish muscle tissue, have distinctive δ² H values that can be used to assess sources and trophic interactions, as well as dietary allocation and origin of fatty acids within consumer tissue.

Conclusions: The new ² H-CSIA method can be applied to evaluate sources and trophic dynamics of fatty acids in organisms ranging from food web ecology to migratory connectivity.

FIGURE 1

Assessment of fatty acid δ ²H bias from preparative methylation. (A) Theoretical shifts between the derivatized FAME and original fatty acid of interest as a function of number of H atoms of the acyl chain as dependent on the isotope difference between methanol used for derivatization and the fatty acid in the sample. (B) Observed differences between FAME and fatty acid after correction for methyl group for data. Significant biases in δ ²H, as for LIN and ARA or ALA and EPA/DHA, may lead to data misinterpretation and reduce comparability between different laboratories

FIGURE 2

Impact of background correction on δ ²H values. (A) Individual background calculation resulted in peaks with high standard deviation regardless of peak intensity, while dynamic and BaseFit algorithm produced acceptable results if peak amplitude exceeded 1000 mV, with increase in standard deviation to about 10 ‰ at 250 mV and large increase below. (B) Background correction influences precision and the δ value of individual peaks. While background correction showed little effect, >2500 mV biases increased with reduced signal intensities. (C) Influence of varying signal intensities due to varying injection volumes on δ ²H values. BaseFit appeared to be most robust but required tuning for each setup and sample matrix. For peaks >500 mV of amplitude, dynamic background calculation performed equally well

FIGURE 3

(A) PCA of δ ²H values of fatty acids abundant in all species (C16:0, C18:0, LIN, ALA) revealed a distinction between allochthonous and autochthonous dietary sources, with fish clearly resembling the H isotopic values of insects, but not of terrestrial food sources. H isotopic values of Gammarus spp. matched those of biofilms more closely than leaves. (B–D) Site‐specific CSIA correlation analysis provided further indication that aquatic insects are the main food source for fish in these streams, with Gammarus spp. potentially being part of the diet of Cottus spp. and Salmo spp. despite a negative offset of the intercept, which could be due to isotopic fractionation of fatty acids during digestion. In contrast, leaf litter could be clearly excluded as a fatty acid dietary source for fish. δ ²H values of all fatty acids identified in both source and consumer have been used for analysis

FIGURE 4

Pooled compound‐specific δ ²H values of terrestrial leaves, stream biofilms, invertebrates, and fishes from all sampling sites (also see Table S3). Saturated and mono‐unsaturated fatty acid (SFA), n‐6 PUFA and n‐3 PUFA values were calculated by weight‐averaging all identified corresponding fatty acids for an individual sample. Omega‐3 PUFA isotopic values of fish closest resemble those of Ephemeroptera spp., indicating that this group is an important diet source of essential fatty acids for fish