From Hormonal Pathways to Colony Development: Multiscale Investigation of Diethylhexyl and Di-n-butyl Phthalate Impact on Bombus terrestris (Hymenoptera: Apidae)

Phthalates are widely used as plasticisers to increase the flexibility of plastic materials or as solvents in detergents, paints, and pesticides. As they are not chemically bound to polymer matrices, they leach easily into the environment and have become ubiquitous pollutants, detected both in highly anthropogenic environments and remote locations. Identified as endocrine disruptors in vertebrates, phthalates also affect fertility, immunity, and development in aquatic invertebrates. Yet, their impact on terrestrial invertebrates, and especially insects, is understudied. Due to their lipophilic nature, phthalates are actively trapped by the lipid-rich cuticle of insects, exposing bees through flight and contact with contaminated surfaces. In Europe, two of the most common phthalates, di-(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DnBP), have been detected on honeybee and bumblebee cuticles, as well as in hive materials such as honey and wax. Bees in urban and agricultural environments, already under multiple anthropogenic pressures, are therefore additionally exposed to phthalates, which may disrupt conserved hormonal and metabolic pathways with cascading effects on health and development. During this project, we investigated for the first time the impact of DnBP and DEHP on a bee species, Bombus terrestris, a key European pollinator and established surrogate species in wild bee ecotoxicology. B. terrestris workers were exposed to environmental concentrations of phthalates, to assess multi-level impact from the molecular to the colonial scale. First, short-term exposure assays revealed a non-monotonic dose-response relationship for mortality following DnBP exposure, alongside significant antagonistic effects of DEHP combined exposure and of social contacts. At the molecular level, low-dose DEHP altered gene expression related to ecdysteroid signalling, a key developmental hormonal pathway, while surviving DnBP-exposed workers showed dysregulated expression of metabolism-related genes. Secondly, long-term repeated exposure of workers in microcolonies further highlighted transgenerational obesogenic effects. The DnBP-exposed workers displayed increased lipid storage and produced drone offspring with increased body mass. These results were associated with a dysregulation of the expression of genes involved in the insulin-signalling pathway. DEHP, alone or in combination with DnBP, caused elevated worker mortality after 35 days of repeated exposure and impacted ecdysteroid-related gene expression. These novel findings provide the first evidence of phthalate impact on bees and towards risk assessment of these pollutants in pollinators. Despite growing evidence of their disruption of conserved hormonal and metabolic pathways across taxa, existing EU legislation addresses phthalates exclusively through direct human exposure. This work underscores the need for broader environmental regulation and further research to evaluate the role of phthalates in pollinator decline.