Environmental concentrations of TCEP and TDCIPP induce dysbiosis of gut microbiotal and metabolism in the honeybee (Apis mellifera L.)

Organophosphorus flame retardants (OPFRs) have emerged as significant global pollutants, yet their harmful effects on pollinating insects remain largely unexplored. This study explored the toxicological effects of tri(2-chloroethyl) phosphate (TCEP) and tri(1,3-dichloro-2-propyl) phosphate (TDCIPP) on the gut microbiota and metabolic pathways of honeybees (Apis mellifera L.). Exposure to TCEP led to a 35 % reduction in intestinal wall thickness and significantly suppressed the expression of pyrimidine metabolism-associated enzymes, including CAD, DHODH, and ODCase (p < 0.05). In contrast, TDCIPP exposure increased the relative abundance of Snodgrassella and Lactobacillus by 40 % and 25 %, respectively, while exerting more extensive toxicity by disrupting nucleotide metabolism, oxidative stress responses, and microbial diversity. Histological assessments revealed that both chemicals compromised intestinal wall integrity and induced crypt loss in the midgut epithelium. Multi-omics analyses underscored distinct toxicity mechanisms: TCEP primarily inhibited pyrimidine biosynthesis, impairing nucleotide synthesis and DNA repair processes, whereas TDCIPP caused broader metabolic disturbances, likely attributed to its greater hydrophobicity. Notably, the enhanced prevalence of certain microbial taxa suggests potential microbial adaptations to TDCIPP-induced stress. This comparative analysis highlighted the detrimental effects of TCEP and TDCIPP on gut health and metabolism, critical factors for honeybee survival and ecological function. These findings underscored the urgent need for further investigation into the ecological hazards posed by OPFRs and provided a basis for developing mitigation strategies to address the impacts of persistent organic pollutants (POPs) on pollinators.

Pollinators play a critical role in the reproduction of wild plants, agricultural productivity, food security, and overall ecosystem health [33]. Among them, honeybees, the most extensively managed pollinators, are indispensable for both enhancing crop yields and producing valuable consumables [31]. However, the alarming decline in global honeybee populations poses a severe threat to the long-term viability of these essential ecosystem services [36]. This decline is driven by multiple factors, including habitat destruction, monocultural farming, pathogens, parasites, pesticide exposure, and, importantly, environmental pollution [35]. Persistent organic pollutants (POPs), characterized by their longevity, bioaccumulative nature, and inherent toxicity, present profound risks to ecosystems, particularly to vital pollinators such as honeybees.

With the increasing restrictions on certain brominated flame retardants, organophosphorus flame retardants (OPFRs) have garnered significant attention as alternative chemical additives [30], [39]. Among them, tri(2-chloroethyl) phosphate (TCEP) and tri(1,3-dichloro-2-propyl) phosphate (TDCIPP) are frequently detected in diverse environmental matrices and have been identified in honey at concentrations ranging from 0.25 to 2.5 μg/kg [4], [24]. Studies indicate that these OPFRs are not confined to honey but are also present in airborne particulate matter, agricultural soils, and surface waters, raising concerns regarding their environmental persistence and potential for long-range transport [21], [34], [46]. Despite differences in hydrophobicity, TCEP being moderately hydrophilic (log Kow ≈ 1.44) and TDCIPP exhibiting greater lipophilicity (log Kow ≈ 3.65–3.77), both compounds can readily adsorb onto particulate matter and infiltrate nectar and pollen, the primary food sources for bees [2], [27], [41]. Consequently, honeybees are at risk of exposure to these pollutants through multiple pathways, including ingestion of contaminated nectar and pollen or inhalation of airborne particulates during foraging activities [27].

While the toxic effects of TCEP and TDCIPP on aquatic organisms have been extensively documented [1], their sub-lethal impacts on pollinators remain largely unexplored. This knowledge gap is particularly concerning, as environmentally relevant exposures may impair critical physiological functions in honeybees, including immune defense, detoxification mechanisms, and nutrient assimilation [19]. The potential ramifications extend beyond individual bees, posing threats to colony stability and overall pollinator health [10], [18]. Although previous research has primarily focused on the acute lethality of POPs, emerging evidence suggests that prolonged sub-lethal toxicity may induce irreversible physiological alterations, ultimately compromising colony sustainability [28]. Given these risks, it is imperative to investigate the mechanistic pathways through which OPFRs disrupt vital biological processes in honeybees. Advancing our understanding of these interactions is essential for safeguarding pollinator populations and mitigating the broader ecological consequences of OPFR contamination.

Honeybees depend on a well-balanced gut microbiota, a highly specialized microbial consortium that plays a crucial role in digestion, immune regulation, and pathogen defense [17], [43]. However, exposure to OPFRs, which are not chemically bound to substrates and are prone to volatilization and leaching, may disrupt this delicate microbial equilibrium, potentially impairing metabolic efficiency and immune resilience [26]. Similar disturbances in gut microbiota composition have been documented in fish and turtles following OPFR exposure, underscoring the capacity of these contaminants to alter host-microbe interactions across taxa [29], [47]. Despite these findings, the long-term consequences of such dysbiosis in honeybees remain poorly understood, particularly under chronic, low-dose exposure conditions that closely reflect real-world contamination levels [44].

To bridge this critical knowledge gap, we conducted an oral exposure study in which worker honeybees were subjected to TCEP and TDCIPP at environmentally relevant concentrations over a 14-day period. We systematically assessed biochemical enzyme activity, histopathological alterations, gut microbiota composition, and metabolic disruptions to construct a comprehensive profile of OPFR-induced physiological changes. By examining TCEP and TDCIPP, two widely detected OPFRs with distinct physicochemical properties, this study elucidated the underlying mechanisms of sub-lethal toxicity in pollinators. Our findings not only advanced understanding of the ecological risks associated with OPFR contamination but also emphasized the urgent need for targeted conservation strategies to safeguard honeybee health and maintain ecosystem stability.