Evaluation of the risks for animal health related to the presence of hydroxymethylfurfural (HMF) in feed for honey bees

Abstract The European Commission has asked the EFSA to evaluate the risk for animal health related to the presence of hydroxymethylfurfural (HMF) in honey bee feed. HMF is a degradation product of particular sugars and can be present in bee feed. HMF is of low acute toxicity in bees but causes increased mortality upon chronic exposure. A benchmark dose lower limit 10% (BMDL10) of 1.16 μg HMF per bee per day has been calculated from mortalities observed in a 20‐day study and established as a Reference Point covering also mortality in larvae, drones and queens for which no or insufficient toxicity data were available. Winter bees have a much longer lifespan than summer bees and HMF shows clear time reinforced toxicity (TRT) characteristics. Therefore, additional Reference Point intervals of 0.21–3.1, 0.091–1.1 and 0.019–0.35 µg HMF/bee per day were calculated based on extrapolation to exposure durations of 50, 90 and 180 days, respectively. A total of 219 analytical data of HMF concentrations in bee feed from EU Member States and 88 from Industry were available. Exposure estimates of worker bees and larvae ranged between 0.1 and 0.48, and between 0.1 and 0.51 μg HMF/per day, respectively. They were well below the BMDL10 of 1.16 μg HMF/bee per day, and thus, no concern was identified. However, when accounting for TRT, the probability that exposures were below established reference point intervals was assessed to be extremely unlikely to almost certain depending on exposure duration. A concern for bee health was identified when bees are exposed to HMF contaminated bee feed for several months.


Introduction
In line with EFSA's policy on openness and transparency, and in order for EFSA to receive comments on its work from the scientific community and stakeholders, EFSA engages in public consultations on key issues. Accordingly, the draft Opinion on the opinion on the evaluation on the risks to animal health related to the presence of hydroxymethylfurfural (HMF) in feed for honey bees was released for public consultation from 3 December 2021 to 10 February 2022 by means of an electronic comment submission tool together with explanatory text on the scope of the public consultation on the EFSA website (See Appendix 1).

Comments received and responses
Comments were received from two interested parties (Table 1).

Stakeholder Country
Verein der Zuckerindustrie e.V. Germany

JKI -Julius Kühn Institut -Federal Research Centre for Cultivated Plants Germany
The comments received were duly evaluated by the WG on HMF in bee feed and the CONTAM Panel and wherever appropriate taken into account for finalisation of the draft Opinion.

General Comments
The EFSA-Panel comes to the conclusion, that "it is regarded appropriate to mainly rely on the data from Jashimowicz and El Sherbiny (1975) and Gregorc et al. (2020), considering the lowest BMDL to be 1.16 -18 μg/bee per day". The study of Gregorc et al., who elaborated the higher BMDL of 18 μg/bee per day, has not been taken into account within the further considerations. Instead, EFSA solely based the main reference point for bee health at 1.16 μg/bee per day. This approach is not comprehensible. It is true that the HMF concentrations used in Gregorc et al. are about twice those used in Jashimowicz and El Sherbiny, and that the number of bees in the feeding studies is lower. However, these cannot be reasons to completely disregard a study.
There are some aspects to be considered regarding the study from 1975: • Inverted sugar syrups from the 1970s are not comparable with today's products. In nowadays, the inversion is carried out in a more gentle and controlled manner.
In section 3.1.6.1 (Derivation of a reference point based on benchmark analyses) the studies relevant for more detailed dose-response analysis are discussed and contrasted. As noted in the last paragraph of this section, it is concluded that based on consideration of the differences between studies, including the quantitative analyses of the dose-response data, the CONTAM Panel did not consider it reasonable to disregard the lower end of the BMDL interval across studies in favour of more recent investigations. However, selecting 1.16 µg/bee per day as the "main Reference Point" did not imply that the possibility for higher values was disregarded, as suggested by this comment. In fact, the quantitative uncertainty analysis (section 3.7.5, Table 9) considers the full BMDL interval across studies, assuming that any value in this range is equally probable. The overall conclusions given in the opinion related to the risk characterization, i.e., the likelihood of exceeding the reference point for different exposure durations (20 days to 180 days), noted in the abstract, summary and conclusion, are based on consideration of the full BMDL interval across the two studies, with no preference for any study.
The Jachimowicz and El Sherbiny (J&E) study used mostly "synthetic" test solutions obtained by mixing different sugars to imitate inverted sugar syrups. These artificial inverted sugars were adjusted to pH 3.9 and spiked with commercial HMF to obtain the feeding solutions. Only one real inverted sugar solution with pH 3.9, in which the HMF content was determined analytically, was included for comparison. It is certainly an advantage, if today's sugar syrups are less acidic. However, the J&R study has clearly shown that it is not the pH of 3.9 but the HMF content that accounts for the toxicity (see response to next bullet point). • The pH-value of the feeding solution was adjusted with citric acid.
It´s well known and also demonstrated by Jashimowicz and El Sherbiny that acids and storage time have a big impact on the formation of HMF.
Since the HMF content of the different feeding solutions has not been analytically verified, it can be assumed that the feeding solutions contained higher HMF levels than originally calculated.
• There is a lack of information on the purity of the used HMF. Research has shown that the purity of HMF available for purchase in 1970s was about 97 -98 %. Therefore, the kind and level of by-products included in the used HMF preparation is unknown. A negative influence on bee health by possible toxic by-products cannot be excluded.
• The Winkler method used to determine the HMF content in the feed solutions underestimates the real HMF contents. The Winkler photometric method is less selective and susceptible to interferences, so that the use of the current HPLC method would probably have analyzed significantly higher HMF contents in the feed solutions used, from which higher BMDLs would have been derived.
It is clearly a deficiency of the J&E study that the actual HMF concentration in the feeding solutions (prepared from synthetic inverted sugar by spiking with HMF) were not verified analytically. However, the J&E study shows in Table 1 that the pH-dependent formation of HMF decreases very markedly from pH 2.88 (112 mg HMF in 100 mL solution) to pH 3.30 (6 mg). It can be assumed by extrapolation that HMF formation was negligible at pH 3.9 (pH of the feeding solutions). Consistent with this notion is the finding that the "synthetic" inverted sugar solution adjusted to pH 3.9 with citric acid was as non-toxic as the non-acidified solution; in contrast, inverted sugar solution with pH 3.9 containing HMF was highly toxic (Fig. 2 of the J&E study). Based on the above we do not think that the feeding solutions contained higher HMF levels than originally calculated.
The CONTAM Panel is not aware of research showing the purity of HMF available for purchase in the 1970s. 97-98% purity is quite high and about the same as todays HMF, leaving little room for impurities. Presumably, the unknown impurities in commercially available HMF are, at least in part, formic acid and levulinic acid, which originate from degradation of HMF (see 1.1.4.2) and which were shown by Bailey (1966) to have about the same toxicity as HMF.
In general, an analytical method that is less selective and prone to interferences gives higher values than a selective one. Therefore, HPLC analysis should give lower values than the Winkler method. This has indeed been found for HMF in the study by Zappala et al. (2005), as reported in section 1. The leaflet may be of interest for EFSA, as it covers the topic of HMF. As a general conclusion based on our findings and knowledge, we support the conclusions of the draft scientific opinion of EFSA.
We support the recommendations (Chapter 5, p. 68): • Further chronic toxicity tests of HMF on honey bees are necessary for the more detailed risk assessment of HMF • For estimating the worst-case scenario of HMF uptake during overwintering it is important to generate more consumption data • A maximum level of HMF for bee feed at the time of delivery should be established • Bee feed should be labelled with use-by date and storage conditions The following documents (Lüken DJ and von der Ohe W, 2016; Lüken DJ and von der Ohe, no date) were uploaded by JKI. The documents and statement were provided by LAVES Bee Institute Celle. The content is fully shared by JKI, and therefore represent our joint view.
Thank you. Note that ML setting and product labelling are not within the remit of EFSA and thus such recommendations cannot be made.

4.
Appendix 1 -Scope of the consultation EFSA's Panel on Contaminants in the Food Chain (CONTAM) has launched an open consultation on the draft scientific opinion on the risks for animal health related to the presence of hydroxymethylfurfural (HMF) in feed for honey bees. This document presents estimations of exposure to HMF in honey bees via uptake of bee feed and an assessment of health risks for bees related to the exposure to HMF. Interested parties are invited to submit their comments by the indicated deadline. When submitting specific comments to certain lines or paragraphs, reference to the line and page numbers to which the comments relate must be made. Additional data or files to support the comments may be submitted using the relevant function in the digital form. If data on chemical contaminants are submitted to support a comment, these must be submitted in SSD format to the EFSA Data Collection Framework (DCF) via the call for collection of chemical contaminants occurrence data in food and feed. Please contact data.collection@efsa.europa.eu for further information and to receive the access credentials for the DCF web interface. Comments will not be considered if they: • are submitted in other languages than English; • are submitted after the closing date of the consultation; • are still in 'draft' status on the closing date of the consultation; • are presented in any form other than what is provided for in the instructions and the relevant function in the tool (e.g. comments made by email will not be considered); • are made outside the corresponding fields of the form, for instance as part of supporting files uploaded in the tool; • are not related to the contents of the document or scope of the consultation; • contain complaints against institutions, personal accusations, irrelevant or offensive statements or material; • are related to policy or risk management aspects, which are out of the scope of EFSA's activity.
Comments will be assessed in line with the criteria above and taken into consideration if found to be relevant.
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