Fattorini R, Egan PA, Rosindell J, Farrell IW, Stevenson PC. Grayanotoxin I variation across tissues and species of Rhododendron suggests pollinator-herbivore defence trade-offs. Phytochemistry. 2023;212:113707. DOI: 10.1016/j.phytochem.2023.113707.
EFSA Panel on Contaminants in the Food Chain (CONTAM), Schrenk D, Bignami M, Bodin L, et al. Risks for human health related to the presence of grayanotoxins in certain honey. EFSA Journal. 2023;21(3):7866. DOI: 10.2903/j.efsa.2023.7866.
Fattorini et al. was published online on 4 May 2023 and is open access under CC BY 4.0. The authors are at Imperial College London, the University of Liverpool, the Swedish University of Agricultural Sciences, the Royal Botanic Gardens, Kew and the University of Greenwich. They measured grayanotoxin I in seven Rhododendron species growing at Wakehurst Place, West Sussex, in 2016; the seven do not include R. ponticum or R. luteum, the main sources of Turkish mad honey. The EFSA opinion is a scientific risk assessment by the European Food Safety Authority’s contaminants panel. Figures for Turkish, Iberian and Irish populations come from it, and other figures on this page come from the sources cited where they appear.
Of the more than 1,000 Rhododendron species recognised by botanists, not all have been found to contain grayanotoxins, and among those that do, concentrations vary considerably from one species to another and also within and between populations of the same species (EFSA 2023). Understanding this distribution helps in judging which plants may pose a honey toxicity risk, and in understanding grayanotoxin’s role in plant defence (Fattorini et al. 2023).
Comparative surveys, which measure the same compound across several species and tissues, are one way of documenting this variation. Fattorini et al. (2023), for example, measured grayanotoxin I in the leaves, petals and nectar of seven Rhododendron species growing at Wakehurst Place, West Sussex, sampled between April and June 2016. Grayanotoxins are restricted to the Ericaceae, the family to which Rhododendron belongs, and around 300 natural grayanoids have now been isolated from Ericaceae plants (Fattorini et al. 2023; EFSA 2023). However, knowledge of how grayanotoxin I is distributed across the genus, and between different plant tissues, is still limited (Fattorini et al. 2023).
Key Takeaways
- Of more than 1,000 Rhododendron species, a documented subset is known to contain grayanotoxins. In Turkey, R. luteum and R. ponticum are the most common toxic species, and Nepalese mad honey appears to differ in grayanane composition from Turkish honey (EFSA 2023).
- GTX concentration varies between species, between populations of the same species and between plant tissues, so species identity alone does not predict honey toxicity (Fattorini et al. 2023; EFSA 2023).
- In Turkey, honey produced in springtime is generally more toxic, and may contain more grayanotoxin, than honey produced in other seasons (EFSA 2023).
- Nectar is the documented route of GTX into honey. EFSA (2023) found no studies of grayanotoxins in Rhododendron pollen, so pollen’s contribution is unknown. In one multi-species survey, leaves consistently carried more grayanotoxin I than petals or nectar (Fattorini et al. 2023), but leaves are not a honey source.
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Grayanotoxin Content by Species
Rhododendron luteum and Rhododendron ponticum are the most common toxic Rhododendron species in Turkey and the main botanical sources of Turkish mad honey (EFSA 2023; Jansen et al. 2012). Both grow in Turkey’s Black Sea region. R. ponticum also grows naturally in the mountains of Spain and Portugal, and was introduced to Britain and Ireland as an ornamental plant, where it is now invasive (EFSA 2023). Rhododendron ponticum has been analysed by several research groups. Plants grown in German botanical gardens contained 1,300 to 2,300 mg/kg grayanotoxin I by fresh weight (Lechtenberg et al. 2014, as reported by EFSA 2023). Its nectar occurs in at least two chemical types: native Iberian populations are grayanotoxin I dominant, and introduced Irish populations are mostly grayanotoxin III dominant (Egan et al. 2016, as reported by EFSA 2023). Mad honey made from R. ponticum nectar in Turkey’s western Black Sea region, sampled over three years, has been measured at mean grayanotoxin I and III contents of 20.4 and 8.20 mg/kg (Kurtoglu et al. 2014).
Rhododendron arboreum is often linked with Nepali cliff honey, but grayanotoxin levels in the plant itself are not reported in the studies cited here. In a 1981 report of four students poisoned by honey from Eastern Nepal, the habitat of R. arboreum and R. campanulatum, grayanotoxins I, II and III were not detected in the honey but two unidentified grayanotoxin analogues were (Kerkvliet 1981, as reported by EFSA 2023). More recent testing found grayanotoxins I and III in 33 of 60 Nepalese mad honey samples (Ahn et al. 2022). Fattorini et al. (2023) did find grayanotoxin I in the leaves, petals and nectar of R. campanulatum, though their plants were grown in a UK botanic garden rather than sampled in Nepal.
Rhododendron catawbiense and Rhododendron groenlandicum (formerly Ledum groenlandicum) are North American species with documented grayanotoxin content: a 17-species survey found high grayanotoxin I content in R. catawbiense (Lechtenberg et al. 2014), and Labrador tea, made from R. groenlandicum and related species, is reported to contain the toxin (Jansen et al. 2012). Mad honey poisoning was reported in Europe and North America in the nineteenth century, but a documented case from locally produced honey in either region would now be unusual enough to report (Gunduz et al. 2008). One North American honey sample with measured grayanotoxin, from North Carolina, was most likely produced from the nectar of mountain laurel (Kalmia latifolia), another member of the Ericaceae (Jansen et al. 2012).
The same survey found high grayanotoxin I content in six other taxa, among them R. ponticum and the hybrid R. × sochadzeae, while its method did not detect grayanotoxin I in fresh leaves and fruits of R. ferrugineum (Lechtenberg et al. 2014), which grows in the Alps and Pyrenees (EFSA 2023). EFSA (2023) found no reported intoxications from honey of EU origin, and where honey production is scaled up, blending honey from different locations tends to limit severe grayanotoxin contamination by dilution (Jansen et al. 2012).
Geographic and Within-Species Variation
Within a single species, grayanotoxin concentrations are not uniform. Published surveys document substantial variation across geographic populations of the same species. Grayanotoxin I in R. ponticum nectar averaged 1,460 mg/kg dry weight in native Iberian populations against 810 mg/kg in introduced Irish populations (EFSA 2023, citing Egan et al. 2016), and 18% of plants in the introduced range had no grayanotoxin I in their nectar (Fattorini et al. 2023, citing Egan et al. 2016). Within Turkey’s Black Sea region, grayanotoxin III in R. luteum flowers ranged from 1.74 to 760 mg/kg dry weight across sampling locations, and in R. ponticum flowers from below the limit of quantification to 22.9 mg/kg (EFSA 2023, citing Sahin 2014).
Leaf age also matters, though not in the same direction in every species: young leaves of R. simsii contained more grayanotoxin I than mature leaves, whereas in wild R. ponticum older leaves contained more (Fattorini et al. 2023, citing Scott-Brown et al. 2016 and Egan et al. 2022). For honey, the documented seasonal pattern is that Turkish honey produced in springtime is generally more toxic than honey from other seasons, and the ratio of grayanotoxins in honey has been linked to night-time temperature and rainfall during flowering (EFSA 2023).
Tissue Distribution Within Plants
Grayanotoxins are not uniformly distributed across plant tissues. Nectar has the highest direct relevance for honey toxicity. In the survey by Fattorini et al. (2023), grayanotoxin I averaged 1,793 mg/kg in extracts of young leaves, 230 mg/kg in petal extracts and 123 mg/l in nectar, and it was detected in the leaves of 60% of plants, the petals of 48% and the nectar of 42%. The authors treat the leaf and nectar comparison as tentative, because fresh and freeze-dried material extract differently, and note that in R. ponticum, nectar concentrations have been reported at a similar order of magnitude to leaf and twig extracts. Leaves themselves are not a honey source. EFSA (2023) found no studies of grayanotoxins in Rhododendron pollen, so how much pollen contributes to honey toxicity is unknown. In one species, R. molle, pollen carried a higher concentration of the grayanane rhodojaponin III than petals or leaves, and none was detected in the nectar (Feng et al. 2024).
Grayanotoxins are also found in twigs (Jansen et al. 2012), which matters for grazing animals, since livestock are poisoned by eating Rhododendron foliage, but is not directly relevant to honey toxicity. The clinical literature on Rhododendron poisoning in livestock (cattle, sheep, and goats grazing on Rhododendron foliage) is distinct from but mechanistically related to the human mad honey poisoning literature.
Sources
- Ahn SY, Kim S, Cho H (2022), Determination of Grayanotoxin I and Grayanotoxin III in mad honey from Nepal using liquid chromatography-tandem mass spectrometry. Analytical Science and Technology 35(2):82-91.
- EFSA CONTAM Panel (2023), Risks for human health related to the presence of grayanotoxins in certain honey. EFSA Journal 21(3):7866.
- Egan PA, et al. (2016), Plant toxin levels in nectar vary spatially across native and introduced populations. Journal of Ecology 104(4):1106-1115.
- Egan PA, Stevenson PC, Stout JC (2022), Pollinator selection against toxic nectar as a key facilitator of a plant invasion. Philosophical Transactions of the Royal Society B: Biological Sciences 377(1853):20210168.
- Fattorini R, et al. (2023), Grayanotoxin I variation across tissues and species of Rhododendron suggests pollinator-herbivore defence trade-offs. Phytochemistry 212:113707.
- Feng HH, et al. (2024), High toxin concentration in pollen may deter collection by bees in butterfly-pollinated Rhododendron molle. Annals of Botany 134(4):551-560.
- Gunduz A, et al. (2008), Clinical review of grayanotoxin/mad honey poisoning past and present. Clinical Toxicology 46(5):437-442.
- Jansen SA, et al. (2012), Grayanotoxin Poisoning: ‘Mad Honey Disease’ and Beyond. Cardiovascular Toxicology 12(3):208-215.
- Kerkvliet JD (1981), Analysis of a Toxic Rhododendron Honey. Journal of Apicultural Research 20(4):249-253.
- Kurtoglu AB, Yavuz R, Evrendilek GA (2014), Characterisation and fate of grayanatoxins in mad honey produced from Rhododendron ponticum nectar. Food Chemistry 161:47-52.
- Lechtenberg M, et al. (2014), Extracts from Rhododendron ferrugineum Do Not Exhibit Grayanotoxin I: An Analytical Survey on Grayanotoxin I within the Genus Rhododendron. Planta Medica 80(15):1321-1328.
- Sahin A (2014), Orman gülü balı ve bitkisindeki grayanotoksin-III (GTX-III) izoformunun LC-MS/MS ile analizi (doctoral thesis). Karadeniz Technical University.
- Scott-Brown AS, et al. (2016), Leaf trichomes and foliar chemistry mediate defence against glasshouse thrips; Heliothrips haemorrhoidalis (Bouché) in Rhododendron simsii. Functional Plant Biology 43(12):1170-1182.
