The effects of preservation methods, dyes and acidification on the isotopic values (δ¹⁵N and δ¹³C) of two zooplankton species from the KwaZulu‐Natal Bight, South Africa

Abstract

Stable isotope measurements are an important tool for ecosystem trophic linkage studies. Ideally, fresh samples should be used for isotopic analysis, but in many cases organisms must be preserved and analysed later. In some cases dyes must be used to help distinguish organisms from detritus. Since preservatives and dyes are carbon-based, their addition could influence isotopic readings. This study aims to improve understanding of the effects of sample storage method, dye addition and acidification on the δ(15)N and δ(13)C values of zooplankton (Euphasia frigida and Undinula vulgaris). Zooplankton was collected and preserved by freezing, or by the addition of 5% formalin, 70% ethanol, or 5% formalin with added Phloxine B or Rose Bengal, and stored for 1 month before processing. Samples in 5% formalin and 70% ethanol were also kept and processed after 3 and 9 months to study changes over time. Formalin caused the largest enrichment for δ(13)C and a slight enrichment for δ(15)N, while ethanol produced a slight depletion for δ(13)C, and different effects on δ(15)N depending on the species. In formalin, dyes depleted the δ(13)C values, but had variable effects on δ(15)N, relative to formalin alone. Acidification had no significant effect on δ(15)N or δ(13)C for either species. Long-term storage showed that the effects of the preservatives were species-dependent. Although the effects on δ(15)N varied, a relative enrichment in (13)C of samples occurred with time. This can have important consequences for the understanding of the organic flow within a food web and for trophic studies. .

Freezing and fractionation: effects of preservation on carbon and nitrogen stable isotope ratios of some limnetic organisms

“…Several authors have attributed freezing‐related fractionation to mechanical damage to cells and tissues during freezing, and the subsequent loss of fluids that are isotopically enriched or depleted upon thawing . This effect appears to be compounded by multiple freeze/thaw cycles . It has also been suggested that flash freezing reduces mechanical damage and fluid loss, but in our study the relative effects of freezing and flash freezing differed between Mysis and zooplankton, questioning whether one method should be favored over the other .…”

“…[11,16] It has also been suggested that flash freezing reduces mechanical damage and fluid loss, but in our study the relative effects of freezing and flash freezing differed between Mysis and zooplankton, questioning whether one method should be favored over the other. [1,16] Research to examine whether fluids released from freezing/thawing differ in their elemental or isotopic composition from the parent material, and how this effect varies with taxa, tissue types, and freezing temperature and duration, could provide insights that can prevent freezing-related biases in the future. Furthermore, few studies have reported how long it took samples to become frozen but it seems plausible that this factor could affect sample decomposition and therefore isotopic fractionation prior to freezing.…”

“…This effect appears to be compounded by multiple freeze/thaw cycles . It has also been suggested that flash freezing reduces mechanical damage and fluid loss, but in our study the relative effects of freezing and flash freezing differed between Mysis and zooplankton, questioning whether one method should be favored over the other . Research to examine whether fluids released from freezing/thawing differ in their elemental or isotopic composition from the parent material, and how this effect varies with taxa, tissue types, and freezing temperature and duration, could provide insights that can prevent freezing‐related biases in the future.…”

“…The available studies suggest that freezing effects in macroinvertebrates are highly taxon‐dependent. We found no studies examining freezing effects on Mysis , but de Lecea et al . studied a krill species, marine malacostracans, which is anatomically similar to Mysis .…”

“…Some studies have shown significant elevation or reduction in δ 15 N and δ 13 C values, while others have seen no effects. [1,[8][9][10][11]13,[16][17][18][19][20][21][22] The effects of freezing on isotopic ratios and elemental values differed according to the taxa examined, but such effects have never been examined for many organisms. Further, there have been few studies designed to compare the effects of freezing temperature.…”