SUPPLEMENTARY MATERIAL
, Oleh Koshovyi The essential oil (EO) yield and chemical composition of Thuja occidentalis L. (Cupressaceae) growing in Estonia were investigated to assess intraspecific variability, chemotypic stability, and the consistency of toxic potential associated with thujone content. Fresh leaf material was collected from several geographically distinct populations (n = 12), together with dried leaves and fruits (n = 5), and analysed by hydrodistillation and gas chromatography‒mass spectrometry (GC–MS). Fresh leaf EO yields ranged from 0.73% to 1.32% (mean 1.04%), whereas dried leaves yielded up to 2.98%. In total, 105 volatile constituents were identified. Across all fresh leaf samples, the EO composition was consistently dominated by oxygenated monoterpenes, particularly fenchone (12.08%–29.99%), α-thujone (10.75%–22.74%), and β-thujone (5.13%–17.17%). Their combined proportion ranged from 28% to 70% of the total EO composition (mean 52%). The α-/β-thujone ratio ranged from 0.84 to 2.50. Fresh and dried plant materials did not differ qualitatively in their chemotypic profiles, as both retained the same fenchone–thujone dominance, although drying increased the essential oil yield quantitatively. Fruit EOs differed quantitatively from leaf oils, exhibiting markedly lower proportions of fenchone and both thujone isomers, together with substantially higher levels of monoterpene hydrocarbons, particularly sabinene. No significant geographical trends in total thujone content were observed across growing locations. Given the well-documented neurotoxicity of thujones, the observed chemical uniformity indicates that T. occidentalis in Estonia has geographically independent toxic potential. The absence of thujone-poor chemotypes and the consistently high proportion of thujones suggest a persistent toxicological potential, based on literature data on thujone toxicity. These findings are relevant for risk assessment and the safety evaluation of Thuja-derived materials.
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