Significance New international regulatory guidelines aim to better protect bees from harmful exposures to agrochemicals used in crop protection. Our results provide evidence that the laboratory bioassays in these guidelines can discriminate harmful bioaccumulative insecticides that had formerly been approved for agricultural use. As a test case, we investigated insecticides implicated in the mass mortalities of honey bees in France in the 1990s. We demonstrate that a hitherto overlooked insecticide with a strong capacity to bioaccumulate was very likely responsible.

Abstract Mass mortalities of honey bees occurred in France in the 1990s coincident with the introduction of two agricultural insecticides, imidacloprid and fipronil. Imidacloprid, a neonicotinoid, was widely blamed, but the differential potency of imidacloprid and fipronil has been unclear because of uncertainty over their capacity to bioaccumulate during sustained exposure to trace dietary residues and, thereby, cause time-reinforced toxicity (TRT). We experimentally quantified the toxicity of fipronil and imidacloprid to honey bees and incorporated the observed mortality rates into a demographic simulation of a honey bee colony in an environmentally realistic scenario. Additionally, we evaluated two bioassays from new international guidance for agrochemical regulation, which aim to detect TRT. Finally, we used analytical chemistry (GC-MS) to test for bioaccumulation of fipronil. We found in demographic simulations that only fipronil produced mass mortality in honey bees. In the bioassays, only fipronil caused TRT. GC-MS analysis revealed that virtually all of the fipronil ingested by a honey bee in a single meal was present 6 d later, which suggests that bioaccumulation is the basis of TRT in sustained dietary exposures. We therefore postulate that fipronil, not imidacloprid, caused the mass mortalities of honey bees in France during the 1990s because it is lethal to honey bees in even trace doses due to its capacity to bioaccumulate and generate TRT. Our results provide evidence that recently proposed laboratory bioassays can discriminate harmful bioaccumulative substances and, thereby, address evident shortcomings in a regulatory system that had formerly approved fipronil for agricultural use.

Conspicuous mass mortalities of honey bees were observed in France between 1994 and 1998 (1). Their onset coincided with the introduction of two new-to-market systemic insecticides, imidacloprid (released in 1994) and fipronil (released in 1993), which were used widely on French sunflower (Helianthus annuus) crops (2). Imidacloprid is a neonicotinoid pesticide that disrupts the insect nervous system by acting on nicotinic acetylcholine receptors (nAChRs) (3), and fipronil is a phenylpyrazole insecticide that acts on γ-aminobutyric acid (GABA) receptors (4). Applied as seed dressings, these systemic insecticides are taken up by the growing plant and distributed throughout its tissues, including the flowers (5). Consequently, honey bees are exposed to low-level dietary residues when feeding on nectar and pollen from systemically treated bee-attractive crops (6). Despite being used across similar acreages (7), it was generally believed that the mass mortalities were caused by imidacloprid (1), but the case against imidacloprid is weak for two reasons. First, dietary imidacloprid at environmentally realistic levels does not appear to be able to cause mass mortality in honey bees. Neonicotinoid residues in the nectar and pollen of bee-attractive crops are typically less than 6 ppb (parts per billion) (8), but the consensus dose–response relationship from four previous laboratory studies (9⇓–11) (SI Appendix, section S1) indicates that lethality is infrequent in this range (ca. <5% mortality) even after a 10-d dietary exposure and only dietary concentrations of in excess of 100 times the environmentally realistic level cause substantial mortality (dietary concentration for 50% mortality, LC 50 = 1,750 μg·L−1, or ca. 1,350 ppb). Continuous experimental exposures of honey bee colonies to 5 pbb imidacloprid-laced syrup over 6-wk periods under field conditions (12) found no mass mortality and report only minor sublethal impacts on cardinal indicators of colony performance, such as hive mass and the population size of adult bees.

Second, dietary imidacloprid does not appear to bioaccumulate in individual bees, which decreases the potential harmfulness of the low-level residues that typify its presence in the nectar and pollen of treated crops. Potentially, an insecticide that is present as trace dietary residues eventually may accumulate to a lethal level during a sustained exposure, which is possible in bees because their adult lifespan and the blooming period of mass-flowering crops like sunflower and canola both extend over several weeks. However, honey bees clear ingested imidacloprid rapidly from their bodies (92% within 48 h) (13) and the elimination half-life of imidacloprid and its toxic metabolites is ∼24 h (14). In theory, the bodily concentration of an ingested toxicant reaches steady state in ∼4 elimination half-lives (15), so imidacloprid is unlikely to be bioaccumulative during exposures at an ecologically relevant timescale, which is measured in weeks. Further, it appears that imidacloprid does not accumulate locally at its target sites by binding irreversibly to receptors in the insect nervous system. Instead, rapid postexposure recovery is observed in honey bees (13) and other insects including cockroaches (16), termites (17), and bumble bees (18, 19), which clearly indicates reversible binding. Taken together, this evidence suggests that it is unlikely that even a sustained exposure to dietary imidacloprid at environmentally realistic levels can be the cause of mass fatalities.

By contrast, fipronil appears more likely than imidacloprid to have caused the mass mortalities among honey bees observed in France because it is more potent in sustained exposures. After ad libitum feeding on dosed diets, the 10-d LD 50 for fipronil is 3 ng·bee−1 compared with 189 ng·bee−1 for imidacloprid (9). Furthermore, the fact that the LD 50 for exposed honey bees is lower in sustained than acute exposures (10-d LD 50 = 3 ng·bee−1 vs. 48-h LD 50 = 123 ng·bee−1; ref. 9) strongly implicates bioaccumulation because each ingested unit of a bioaccumulative toxicant has greater opportunity to injure the subject in a prolonged exposure than each unit of a nonbioaccumulative toxicant, which spends a much shorter time in the subject’s body. However, it is unclear whether fipronil and imidacloprid are differentially toxic in an environmentally realistic in-hive scenario involving mixed-age groups of honey bees taken directly from outdoor hives rather than in the newly emerged, well nourished, disease-free honey bees conventionally used in the laboratory tests to establish the various LD 50 values for each toxicant. Further, it has been unclear whether the effects measured on individual bees in the laboratory are sufficient to cause mass mortality in a colony. We therefore set out to experimentally quantify the differential toxicity of fipronil and imidacloprid to bees taken from outdoor hives, and we incorporated the emergent dose-appropriate mortality rates into a demographic simulation model (20) to evaluate their potential to cause colony-level impacts under an environmentally realistic scenario.

Our experiments also provided an opportunity to test whether the high toxicity of fipronil is due to bioaccumulation. The mark of a bioaccumulative toxicant is that it is increasingly injurious as the exposure is prolonged because its bodily levels increase, which is termed “time-reinforced toxicity” (TRT) (21). One possible explanation for the historical mass mortalities of honey bees in France is that they were caused by the accumulation of trace residues of a TRT-capable pesticide. We therefore investigated each pesticide’s capacity to generate TRT as suggested in proposed international guidelines for regulatory testing (22) by evaluating Haber’s “constant product” rule. Specifically, we used the results of our experiments to evaluate the exponent, b, in the following constant–product relationship: C t b = k , [1]where C denotes the concentration of the toxicant at its target site and t denotes the duration of the exposure in the dose-duration combinations that produce a specified injury, such as fatality. In theory, the exponent takes the value b = 1 if the toxicant reaches steady state and b = 2 if the toxicant bioaccumulates (SI Appendix, section S2). The evaluation of the exponent in Haber’s constant product rule is a widely recognized approach in toxicology (23) and risk assessment (24) that has been recently recommended for understanding bee–pesticide interactions (21).

We additionally employed a second test for TRT based on recent European Food Safety Authority (EFSA) draft guidance (25). When experimental exposures are conducted at a range of doses and mortality is recorded, toxicokinetic inferences can be made by comparing the ingested mass that precedes fatality across the different doses. A nonbioaccumulative toxicant with a short in-body residence will cause the same injury per unit ingested irrespective of dose (because each unit has approximately the same in-body residence), which means that each bee must ingest the same total mass of toxicant to cause its fatality irrespective of the duration of the exposure. In contrast, a bioaccumulative toxicant retained in the bee’s body has longer to cause injury in the longer-lived bees feeding on lower doses, so these bees need to consume less of the toxicant in total to be killed. Hence, a second signature of TRT is evident when the fatal mass of ingested toxicant declines as the duration of exposure increases, which can be evaluated by testing the ingestion-vs.-longevity relationship for a negative slope.

In addition to investigating the toxicity of the focal historical compounds imidacloprid and fipronil, we also examined two other pesticide compounds in widespread current use, thiamethoxam (a neonicotinoid) and cypermethrin (a pyrethroid). Whereas thiamethoxam has been used worldwide, ongoing concerns over bee health have led the European Union recently to ban the use of neonicotinoids and fipronil on bee-attractive crops (26), but until recently, derogations in the United Kingdom have allowed some farmers to use neonicotinoids (including thiamethoxam) on oilseed rape (Brassica napus) (27). Meanwhile, other farmers are instead using nonsystemic pyrethroid foliar sprays with active ingredients such as cypermethrin, which acts on the sodium channels in the postsynaptic membrane of insect nerve cells (28). Using data from experimental exposures, we estimated demographic mortality rates of all four candidate pesticides and evaluated their potential impact on a computer-simulated colony (SI Appendix, section S3). We investigated whether fipronil’s high potency in sustained exposures (9) emerged from bioaccumulation by testing for the two signatures of time-reinforced toxicity and by using gas chromatography-mass spectrometry (GC-MS) to establish its bioaccumulative potential. Additionally, we used our approach to confirm that neither imidacloprid, thiamethoxam nor cypermethrin generate TRT.

Discussion Based on our findings, we postulate that fipronil is a credible cause of the mass mortalities of honey bees that were associated with agricultural sunflower in France during the 1990s. Fipronil can be lethal to honey bees in dietary exposures to the trace residues that typify those in nectar and pollen from treated crops, due in part to its capacity to generate TRT. We estimate that the resulting increase in the demographic mortality rate is capable of causing mass mortality among adult bees. Our present study has examined in detail only a lethal endpoint, but fipronil may also have various sublethal impacts, such as detrimental effects on foraging intensity and homing success (31), which could further accelerate colony failure. Our hypothesis that fipronil is capable of causing major impacts on honey bees is supported by the occurrence of occasional mass mortalities of honey bees, such as the 2014 event that involved 172 hives across 23 apiaries in the Canton of Bern, Switzerland (32). Despite the ongoing ban on the use of fipronil in European agriculture, the accident in Bern arose from fipronil residues present as an accidental contaminant in a batch of fungicide that had been used to treat nearby fruit trees, which were foraged by honey bees. It is not yet possible to identify that fipronil definitively was a culprit in the French incidents because there is a lack of data to prove the historical levels and prevalence of its residues in nectar and pollen, but the findings of our laboratory experiments provide strong evidence that justifies a future program of field experimentation to further test the hypothesis. Imidacloprid and Mass Mortalities of Honey Bees. Our results suggest that dietary exposure to imidacloprid in nectar and pollen is an unlikely cause of mass mortality in adult honey bees because trace dietary residues at environmentally realistic levels cause only low levels of mortality, even in sustained exposures. This finding is consistent with the levels of mortality that have been reported by previous researchers (SI Appendix, section S1). Of course, some recent mass mortalities of honey bees were instead caused by the release of insecticidal neonicotinoid dust created when treated maize seeds were planted by pneumatic drilling machinery, such as the 2008 incident in Baden-Würtemberg in Germany (33), but dust emission cannot account for the mass mortalities that coincided with the midsummer bloom of French sunflower crops, however, because agricultural sowing (including maize) occurs earlier in the year. Imidacloprid’s low toxicity in sustained dietary exposures to trace residues (12) appears to be due in part to its rapid elimination by bees, which makes it unable to bioaccumulate and thereby generate TRT. Bees can eliminate ingested imidacloprid in part because it binds reversibly to its target receptor, which is indicated by two lines of evidence. First, in vitro experiments using radio-labeled ligands show that imidacloprid can be displaced from the neuroreceptors of stable flies, Stomoxys calcitrans (34) (SI Appendix, section S7), and that this process can be rapid (dissociation half-life of ∼10 min) in house flies (Musca domestica) (35) and aphids (Myzus persicae) (36). In principle, similar reversible ligand-receptor binding therefore appears likely in bees. Second, the timescale of postexposure recovery (24–48 h) in honey bees (13) and bumble bees (18, 19) coincides with the timescale of metabolic elimination (elimination half-life ∼ 24 h) (13, 14, 19), which logically suggests that imidacloprid increasingly dissociates from its receptors as detoxification reduces the concentration of its unbound form. Taken together, this collection of evidence indicates that imidacloprid binds reversibly to its receptors in bees and, if so, toxicodynamic-kinetic theory relating to toxicants with a short elimination half-life (15) predicts the absence of TRT in experimental exposures to imidacloprid, just as we observed in the present study. Our conclusion that imidacloprid fails to cause TRT is further supported by the results of a previous laboratory exposure (9), which also produced results that indicate the absence of TRT (C-vs.-t relationship, Haber exponent b = 1.1; SI Appendix, section S8). While our results demonstrate that dietary imidacloprid is not lethal to honey bees at environmentally relevant levels, we emphasize that this should not be taken to mean that it is harmless to bees. We found that dietary imidacloprid produced a hormesis in longevity (days of exposure survived) in honey bees, which is not unexpected; various chemical stressors produce hormesis in insects (37) and imidacloprid itself causes hormetic responses in stink bugs (38), aphids (39) and, notably, in the longevity of spider mites (40). However, the increased longevity that we observed at low doses should be viewed as an intoxication symptom, which is likely to displace affected honey bees from their normal physiological equilibria (37, 39). Finally, we note that although our present study did not consider the potentially detrimental impact of imidacloprid on other demographic variables that are relevant to colony health, such as queen fecundity and larval performance, the lack of mass mortalities in exposed colonies under field conditions (12) suggests that we have not overlooked a crucial factor. We add two further notes about the imidacloprid hormesis. First, a hormetic increase in mean longevity in the low-dose range is not incompatible with a small increase in daily mortality rate in the same range (such as we observed), because the increased lifespan of the surviving majority more than offsets the days lost by infrequent deaths. Second, the increased longevity observed in our lowest doses confirms the presence of the active substance and, in conjunction with dose-dependent mortality that conforms closely to previous studies (SI Appendix, section S9), validates our dose preparations. Inferences About Cypermethrin and Thiamethoxam. Based on our findings, cypermethrin has the lowest potential for impact on colony performance by directly causing adult mortality. In individual honey bees, detoxicative enzyme systems can reduce harm from ingested pyrethroids (41) and preclude bioaccumulative toxicity, or TRT, which may explain the low mortality rate due to cypermethrin in our present study. In contrast, thiamethoxam appears capable of causing a more substantive impact on colony performance by elevating the mortality rate among adult workers. Nevertheless, thiamethoxam produced neither signature of TRT. It appears probable that thiamethoxam does not bioaccumulate in honey bees because it is subject to detoxicative metabolism, like the closely similar neonicotinoid clothianidin (42). Performing the analytical chemistry to clarify the toxicokinetics of thiamethoxam in honey bees should be a target for future research. The relatively low levels of mortality in adult honey bee workers caused by dietary exposures to thiamethoxam and cypermethrin do not preclude harmful sublethal effects on colony performance (43). Future Research and Regulatory Implications. We used laboratory toxicology and demographic simulation to predict the potential of four dietary pesticides to produce mass mortality of adult honey bees. The postulated absence of mass mortality in environmentally realistic exposures to 5 ppb imidacloprid is already supported by the outcome of in-hive exposures under field conditions (12), but further research is necessary to provide a similar experimental evaluation of the other predictions, especially the proposition that fipronil has the capacity to cause mass mortality in honey bees. Various insects besides honey bees forage on the flowers of pesticide-treated crops, including other kinds of bees that may be affected more strongly (44). Consequently, other species should be tested to assess more broadly the impacts of pesticides on farmland insect faunas. When Haber’s Rule successfully describes the manifestation of toxic injuries, it suggests underlying proportionalities between exposure concentration, bodily concentration, and the accrual rate of injury. We speculate that these proportionalities arise when physical processes (e.g., diffusion, concentration-dependent association-dissociation of ligand–receptor complexes) fundamentally determine the levels of toxic effects. In itself, however, Haber’s Rule cannot elucidate the details of pharmacological mechanisms and a comprehensive theory of bee-pesticide toxicology, e.g., pharmacokinetic/pharmacodynamic models, should be a goal for future research. The potentially severe impact of dietary fipronil highlights the need to identify agrochemicals that cause TRT before they are used widely in agriculture because TRT enables trace contaminants to become disproportionately harmful by sustained exposure. Formerly, international regulatory procedures for the risk assessment of plant protection products have relied on short-term laboratory exposures of honey bees (so-called “first tier” tests), which do not take account of the possible harm that results from TRT during realistically sustained exposures. Our findings illustrate the potential value of a bioassay aimed at revealing TRT. Specifically, we show that TRT can be detected both by evaluating the exponent of Haber’s Rule [i.e., evaluation of b in the log(t)-vs.-log(C) relationship] and by testing for exposure dependence of the lethal dose (i.e., evaluation of the ingestion-vs.-longevity relationship), which endorses the value of these analyses as specified in the newly formulated draft guidelines issued by both the EFSA for risk assessment in bees (25) and the Organisation for Economic Co-operation (OECD) (22). By explicitly including an evaluation of TRT due to dietary exposure, future risk assessments will enable regulators to better protect farmland bees and the valuable ecosystem services that they provide in pollinating crops and wild flowers.

Acknowledgments P.J.H. was supported by a doctoral studentship (Grant 1200662) from the Natural Environment Research Council.

Footnotes Author contributions: P.J.H., C.R.T., and J.E.C. designed research; P.J.H. and A.J. performed research; P.J.H., A.J., and J.E.C. analyzed data; and P.J.H., C.R.T., and J.E.C. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The datasets generated and analyzed during the current study are available in the University of Exeter ORE repository, https://ore.exeter.ac.uk/repository/ (doi: 10.24378/exe.943).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1804934115/-/DCSupplemental.