Does mill-grinding affect stable C and N isotopic analysis of animal tissues?

Abstract

Mechanical grinding is increasingly used in samples preparation for stable isotope analysis. However, plastic abrasion could contaminate samples and bias the isotopic analysis. Previous studies on plant material and large quantity of marine animal samples showed that this bias is limited. However, there is no evaluation of this potential contamination on smaller animal samples. We evaluate potential plastic contamination by analysing the C and N elemental composition and stable isotope values of ∼50 mg (dry weight) of samples of a freshwater crustacean hand-ground versus mill-ground for durations of 1, 2 and 5 min. We found no differences in %N among treatment groups, but an increase in the %C with ball milling time, with significant differences between samples hand ground and mill-ground for 2 min, hand ground and mill-ground for 5 min, and mill-ground for 1 min and mill-ground for 5 min. No significant differences in neither the δ¹⁵N nor δ¹³C were found among the four grinding methods. We estimated plastic abrasion to be 1.65 mg. Our results show that the use of ball milling for homogenising samples for C and N stable isotope analysis does not affect animal muscle samples with a commonly used amount of material (∼50 mg). Finally, we provided a graphical recommendation on the minimum amount of sample to be ground by milling without incurring in plastic contamination bias.