Hydrogen and oxygen stable isotope analysis of pollen collected from honey

Analyses of stable isotope ratios of pollen present within complex matrices may be useful in sample provenance or climate change applications. We developed a method for separating pollen from honey and preparing pollen for hydrogen and oxygen stable isotope ratio analysis. The method combined several treatments: ethanol dilution to separate pollen; hexane washing to remove wax; and acetolysis to digest all but the external layer of pollen granules. There were large isotopic effects associated with treatments but effects were consistent between replicate applications of the method. We used the method to separate pollen from 45 honey samples from across the United States. Pollen δ2H and δ18O isotope values were significantly positively correlated (P < 0.05), but there was little variation in δ2H values explained by δ18O values (r2 = 0.17). Pollen δ2H values were significantly correlated with δ2H values of precipitation predicted for hive locations (P < 0.05, r2 = 0.24), but there was no significant correlation between pollen and precipitation δ18O values (P = 0.34, r2 = 0.03). These results suggest hydrogen and oxygen stable isotope analysis of pollen may not be as useful as analysis of liquid honey or beeswax in investigating provenance.