Pyrrolizidine alkaloids in natural and experimental grass silages and implications for feed safety

• One of the first studies on PA-contamination of field samples of grass silage (n = 115).
• The levels of PA-contamination of the ensiled forage were low, however 18% positive samples from Bavaria, Germany.
• The PA-degradation during ensiling is compound dependent: the PA N-oxides (PANO) decomposed, however the PA free bases remained stable or modestly increased.
• Silage cannot be generally regarded as safe due to the stability of PA and the uncertainty as to which extent PANO are actually detoxified under field conditions.

Hepatotoxic 1,2-dehydro-pyrrolizidine alkaloids (PA) and their N-oxides (PANO) in feed are a potential threat for animal and human health. However, their risk assessment in preserved animal forage is difficult due to data gaps regarding their occurrence in field samples and contradictory results regarding their behavior during the ensilage process. In this study, 115 samples of grass silage originating from different districts in Bavaria (Germany) were analyzed for their PA and PANO contents. A sensitive LC–ESI-MS/MS method for the detection of 10 PA and 4 PANO was developed including a clean-up of the aqueous acidic extract by cation-exchange cartridges. The mean recoveries were between 70% for seneciphylline-N-oxide and 104% for senecionine-N-oxide. The limits of detection ranged between 0.14 μg senkirkine/kg dry matter (DM) and 1.3 μg retrorsine-N-oxide or monocrotaline-N-oxide/kg DM. Eighteen percent of the samples contained one or more of the analyzed compounds. The highest observed value, calculated as the sum of seneciphylline and senecionine, was 30 μg/kg DM. Other samples contained senecionine-N-oxide, seneciphylline-N-oxide, lycopsamine, intermedine, echimidine, and heliotrine in lower amounts. An accompanying laboratory scale ensilage trial showed a compound-dependent transformation of PANO during the fermentation process while PA-amounts remained stable or even increased. Impacts on safety of ensiled animal feed are discussed with regard to animal health.