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Effects of the mycotoxins aflatoxin B1, deoxynivalenol and zearalenone on survival, biomass and toxin accumulation in Musca domestica larvae

In: World Mycotoxin Journal
Authors:
K. Niermans Laboratory of Entomology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, the Netherlands
Wageningen Food Safety Research, Akkermaalsbos 2, 6708 WB, Wageningen, the Netherlands

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E.F. Hoek-van den Hil Wageningen Food Safety Research, Akkermaalsbos 2, 6708 WB, Wageningen, the Netherlands

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R. van Dam Wageningen Food Safety Research, Akkermaalsbos 2, 6708 WB, Wageningen, the Netherlands

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H.J. van der Fels-Klerx Wageningen Food Safety Research, Akkermaalsbos 2, 6708 WB, Wageningen, the Netherlands

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J.J.A. van Loon Laboratory of Entomology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, the Netherlands

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Open Access

Abstract

Insects are receiving increasing attention as a possible ingredient for feed and/or food production. When used efficiently, insects can provide a sustainable and economically favourable contribution to global food security. Housefly larvae (HFL) can grow on a variety of organic side streams and upgrade them by partial conversion into high-quality protein. Organic side streams may be chemically contaminated by naturally occurring toxins, e.g. mycotoxins, therefore, effects on insect survival and biomass as well as other feed and/or food safety issues should be investigated. In this study, the HFL were exposed to a feed substrate spiked with aflatoxin B1 (AFB1), deoxynivalenol (DON) or zearalenone (ZEN) at concentrations of either 1 or 10 times the maximum levels or guidance values set for feed materials by the European Commission. Mortality and biomass of HFL were recorded over five days of exposure. LC-MS/MS analysis was used to determine the concentration of the mycotoxins in the substrate offered, the larvae and the residual feed material. A molar mass balance was calculated to estimate how much of the spiked mycotoxins (and several metabolites), was recovered in the larval body and the residual material. Exposure to either of the three mycotoxins did not affect larval mortality and biomass, and accumulation in the larval body did not take place. Metabolism does seem to occur for AFB1 and ZEN as the molar mass balance revealed an unrecovered fraction of ca. 40-50%. Little DON metabolism occurred as most of the initially present DON was found back unchanged. The results of this study support the potential for safe use of HFL as food- and/or feed when reared on mycotoxin contaminated side-streams, as accumulation of the tested mycotoxins did not take place in HFL. Further research is needed to identify the fate of the unrecovered fractions of AFB1 and ZEN.

1 Introduction

Novel protein sources for food and feed are urgently needed, and insects are considered a valuable source of novel proteins. In order to make insect rearing circular, sustainable and economically feasible, insects could be reared on residual organic streams from agriculture or food production. Fly larvae are receiving increasing attention as they can be reared on a variety of organic residues. Such organic materials potentially contain contaminants which could accumulate in the insect body (Van der Fels-Klerx et al., 2018). Therefore, potential food or feed safety issues should be identified and controlled in advance. A class of contaminants commonly occurring in organic residues are mycotoxins (Van der Fels-Klerx et al., 2018).

Currently, mycotoxin contamination is considered as one of the most important food/feed safety challenges in the food and feed industry (Moretti et al., 2019). Due to climate change, a shift in the mycotoxin contamination pattern as well as geographical distribution pattern is expected (Zingales et al., 2022). Furthermore, modelling of different climate change scenarios predicts an expansion of risk zones leading to an increased exposure to these mycotoxins (Battilani et al., 2016).

Mycotoxins are secondary metabolites produced by fungi and constitute a chemically diverse class of compounds, among which several of the most potent toxic molecules of biological origin. Mycotoxins cause a variety of adverse effects in humans and animals, such as carcinogenicity, hepatoxicity, nephrotoxicity and oestrogenicity among others (Zain, 2011). As mycotoxin exposure causes a variety of detrimental health effects, exposure needs to be kept below safe limits. In order to ensure this, maximum levels (ML) or guidance values (GV) for feed as well as food are set by the European Commission (EC, 2002, 2006). When contamination occurs in levels above these respective levels, contaminated commodities may no longer be used as feed- and/or food. Recycling these contaminated commodities as feed substrate (‘substrate’ in brief) for insect larvae limits feed- and food waste and results in high quality insect proteins.

Several feeding studies on a variety of insects examined the effects of mycotoxins on insect biomass and survival as well as the possible transfer of mycotoxins from the substrate into the insect body (Bosch et al., 2017; Camenzuli et al., 2018; Meijer et al., 2019; Piacenza et al., 2020). These studies showed that biomass and survival of the insects studied is not at all affected by the presence of mycotoxins in their substrate. A recent systematic literature review concluded that the use of mycotoxin-contaminated waste streams as substrate for insect rearing seems to provide a promising approach for the future of mycotoxin remediation and a circular economy (Niermans et al., 2021). Most available studies focussed on Hermetia illucens L., Alphitobius diaperinus Panzer and Tenebrio molitor L. (the latter two are beetles (Coleoptera: Tenebrionidae). The larvae of the house fly (HFL) Musca domestica L. (Diptera: Muscidae) are considered as an additional promising source of proteins for feed. However, data for a safety assessment for HFL fed on mycotoxin contaminated substrates are lacking. Hermetia illucens and the HFL both belong to the order Diptera, but to different families and have a considerably different ecology (Kortsmit et al., 2023; Van Huis et al., 2020). Data gathered on H. illucens may therefore not be generally valid for Diptera.

Only few studies on HFL, dating back to the 1970s, have been published (Al-Adil et al., 1972; Nevins and Grant, 1971). These indicate that oral uptake of aflatoxin B1 (AFB1) and G1 (AFG1) increases insect mortality, act as a temporary chemo-sterilant (Al-Adil et al., 1972) and resulted in a ten-fold higher concentration of AFB1 in the HFL relative to the substrate (Nevins and Grant, 1971). Studies performed on other insect species reared for food and feed, including H. illucens, A. diaperinus and T. molitor concluded that accumulation and transfer of mycotoxins has, until now, not been observed, although a portion of the ingested mycotoxins could not be recovered (Bosch et al., 2017; Camenzuli et al., 2018; Charlton et al., 2015; Leni et al., 2019; Meijer et al., 2019; Niermans et al., 2019; Piacenza et al., 2020; Schrögel and Wätjen, 2019). These unrecovered mycotoxin fractions could indicate the formation of unknown metabolites or masked forms caused by metabolic processes and interactions with other substances in the substrate (Gützkow et al., 2021; Meijer et al., 2022).

The aims of the present work were (1) to determine the effect of AFB1, deoxynivalenol (DON) and zearalenone (ZEN) exposure on HFL survival and biomass, (2) to determine whether mycotoxin accumulation takes place in the HFL body, and (3) to get a better insight into mycotoxin metabolism by determining how much of the initially fed mycotoxins can be recovered in the larval body and the residual materials (mycotoxin recovery).

2 Materials and methods

Chemicals and standards

Mycotoxin standards were purchased from Romer Labs (Getzersdorf, Austria): AFB1, aflatoxin B2 (AFB2), AFG1, aflatoxin G2 (AFG2), aflatoxin M1 (AFM1), ZEN, α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), DON, 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), DON-3-glucoside (DON-3G); from Enzo Life Sciences (Brussels, Belgium): aflatoxicol (AFL) and from TRC (Toronto, ON, Canada): aflatoxin P1 (AFP1) and aflatoxin Q1 (AFQ1).

Spiked substrate preparation

Spiking solutions of AFB1, DON and ZEN were dissolved in methanol (MeOH). Table 1 presents the intended and the final (analytically determined) concentrations in the substrate. Four ml of the spiking solution was mixed with 216 ml water, 20 ml nipagin solution in water (final concentration: 0.9 mg/l; Merck, Darmstadt, Germany) and 160 g dry food mix (37% wheat bran, 56% wheat flour, 4% full fat milk powder (28.2 g fat/100 g) and 2% dry instant baker’s yeast) to obtain four separate batches of wet substrate of in total 400 ml (60% moisture; 1% MeOH). In the solvent control- and the control substrate the four ml spiking solution was either replaced by MeOH or water. The wet substrate was mixed manually for 5 min, and subsequently for 30 min in a head-over-head shaker (Reax 2, Heidolph Instruments GmbH & Co, Schwabach, Germany). The control substrates were prepared in the same way as the spiked substrates, with an equal amount of MeOH except for the blank.

Table 1
Table 1

Overview of experimental substrates

Citation: World Mycotoxin Journal 16, 4 (2023) ; 10.1163/18750796-20222826

In order to determine whether the spiked substrates could be considered homogeneous, 10 replicates from the AFB1 1× ML substrate were analysed for the presence of AFB1, and four replicates of each of the other treatment substrates were analysed. When the measured concentrations (relative standard deviation of the replicates) in the samples differed ≤20% from each other, the samples (and substrates) were considered homogeneous. The control substrates were also analysed in order to verify that these were free of mycotoxins.

Housefly rearing

The HF eggs used in this study were taken from the HF colony reared at the Laboratory of Entomology (Wageningen University, the Netherlands), originally obtained from dr. Leo Beukeboom, Faculty of Science and Engineering, University of Groningen. Adult HF were kept in mesh BugDorm insect rearing cages (W24.5 × D24.5 × H24.5 cm, MegaView Science Co., Ltd.„ Taichung, Taiwan) in a climate cell with the following settings: 25 °C, relative humidity of 65% and a day/night rhythm of 12/12 h. The adult flies had access to tap water, a 20% sucrose solution, full fat milk powder (28.2 g fat/100 g) and dry instant baker’s yeast at all times. HF eggs were collected in a nylon sock which was hung into a paper cup above approx. five cm of full fat cow milk and covered by an aluminium lid with holes, providing an optimum environment for egg laying. The necessary number of eggs was collected from the nylon sock, and used in the current study. For colony maintenance purposes, the rest of the eggs were weighed, and placed in a 480 ml rearing cup (BugDorm insect pots; MegaView Science Co., Ltd.) on top of the wet substrate (ca. 0.012 g of HF eggs per 62.5 g wet substrate). The wet substrate consisted of a dry food mix containing 37% wheat bran, 56% wheat flour, 4% full fat milk powder (28.2 g fat/100 g) and 2% dry instant baker’s yeast. Water was added to the dry substrate mix to obtain the wet substrate (water:dry substrate mix; 60:40). Mature larvae were left in the substrate to develop into pupae and two days before the expected emergence the rearing cups were placed in new clean BugDorm insect rearing cages.

Experimental set-up

HFL were exposed to a control substrate (with or without solvent), or a substrate spiked with AFB1, DON or ZEN. Mycotoxins were spiked to the substrate in a concentration of either 1× or 10× the ML or GV allowed by the European Commission (EC). The concentration chosen for AFB1 was based on the ML for all feed materials, as set in Directive 2002/32/EC (EC, 2002). The chosen concentrations for DON and ZEN were based on the lowest GV for feed materials as mentioned in EC Recommendation 2006/576/EC (EC, 2006). Table 1 presents an overview of the substrates tested. Per substrate treatment, four replicates were performed leading to a total of 32 treatment groups.

Two-hundred eggs were collected from the HFL culture and placed on top of 62.5 g mycotoxin spiked wet substrate (preparation described below) in each of the 480 ml rearing cups (BugDorm insect pots; MegaView Science Co., Ltd.) and placed in a climate cell at 25 °C, relative humidity of 65% and a day/night rhythm of 12/12 h. On the fifth day of exposure, larvae were separated from the residual material (a mixture of left-over substrate and frass), transferred to a clean (non-spiked) substrate for ±4 h and collected afterwards. The residual material of the spiked substrates and the control substrates was weighed, collected and stored for further analyses. After each step, larvae were washed and their body surface dried using a paper towel. Per rearing cup both the total fresh larval biomass and the number of larvae were quantified by either weighing or counting, respectively. Larval- and residual material were stored at −20 °C until further analyses.

Extraction

Before extraction, the frozen five-day-old HFL were ground under liquid nitrogen to obtain a fine powder, the sample was then stored at −80 °C. Sample extraction of the substrate and residual material was performed in accordance with an in-house validated method based on the QuEChERS (quick, easy, cheap, effective, rugged, and safe) method, with a slightly adjusted protocol for the larval samples. A quantity of 2.5 g of the substrate or residual material was weighed in a 50 ml tube, 7.5 ml water was added and shaken. After 15 min, 10 ml extraction solvent (acetonitrile, 1% acetic acid) was added and mixed for 30 min in a head-over-head shaker (Reax 2; Heidolph Instruments GmbH & Co, Schwabach, Germany). Afterwards, 4 g of magnesium sulphate was added, mixed manually and vortexed for 1 min. Samples were centrifuged (MSE Falcon 6-30) for 10 min at 3,000 rpm. The same procedure was used for the HFL analyses, however, in order to prevent use of excessive amounts of larval sample material, extractions were done with 200 mg of larval sample. Amounts of solvents and sample material used in the extraction procedure were adjusted accordingly. Finally, 200 μl of sample extract was added together with 200 μl water in a syringeless PTFE filter file (Mini-UniPrep; Whatman, Marlborough, MA, USA), capped, vortexed and placed in the refrigerator for 30 min. Afterwards, the vials were closed and stored at 4 °C until LC-MS/MS analyses. In order to calculate the mycotoxin concentrations in the samples, a matrix-matched calibration was prepared in blank extract of each of the matrices (initial substrate, larvae and residual material).

Chemical analyses

The LC-MS/MS system consisted of a Waters Acquity injection and pump system (Waters, Milford, MA, USA) and an Waters Micromass Ultima triple quad system equipped with an electrospray ionization (ESI) source which was operated in positive- and negative mode (instrumental MS/MS parameters of the mycotoxins analysed are shown in Supplementary Tables S2 and S3). LC separation was performed by an Acquity HSS T3 1.8 μm 100 × 2.1 mm column (Waters). Eluent A consisted of H2O and eluent B was composed of MeOH:H2O 95/5 (v/v); for the positive ionization mode both eluents contained 1 mM ammonium formate and 1% formic acid, while for the negative ionization mode both eluents contained 5 mM ammonium acetate and 0.1% acetic acid. The LC eluent gradient for both ionisation modes was similar and started with an initial period of 2 min at 100% A. The proportion of B was linearly increased to 50% at 3 min and followed by a linear gradient of 100% B at 8 min (5 min for the negative mode) and was kept for 2 min. In the positive mode, the initial conditions were restored at 10.5 min and the elution ended at 15 min, while for the negative ionisation mode this happened after 8.5 min and 11 min, respectively. The flow rate was 0.4 ml/min, the column temperature was 35°C, and the injection volume 5 μl. The conditions set for electrospray ionization were as follows: spray voltage 2.5 kV/2.0 kV, desolvation temperature 350 °C, desolvation gas flow 565 L/h. MassLynx v4.2 software (Waters) was used to analyse the LC-MS/MS data obtained.

Identification of peaks was performed according to the criteria of an in-house validated method. The limit of detection (LOD) was determined as the lowest concentration included in the calibration curve with a signal to noise ratio ≥3, and the limit of quantification (LOQ) was determined by following the criteria for identification and recovery mentioned in SANTE/12682/2019 (EC, 2019) and can be found in Supplementary Table S4. To obtain the final mycotoxin concentration in the samples the calculated mycotoxin concentrations were corrected for the determined average recovery of the mycotoxins in the different matrices (Supplementary Table S5).

All samples (substrate, larvae and residue) were analysed for the concentration of the mycotoxin (AFB1, DON, ZEN) spiked to the substrate, and its respective metabolites (mentioned above).

Data analysis

Data on the number of larvae and larval biomass at harvest were used to determine the effect of mycotoxin exposure on HFL survival and biomass. A Kruskal-Wallis test and a Dunn’s Multiple Comparison Test (comparison of all treatments with the solvent (1% MeOH) control treatment) was performed in GraphPad Prism v4 (La Jolla, CA, USA) to determine whether survival and individual larval biomass measured in the treatment groups differed significantly ( P < 0.05). A Mann-Whitney test U was performed to determine whether the absolute amount (μg) of mycotoxins differed significantly ( P < 0.05; IBM SPSS statistics, Armonk, NY, USA) between the substrate and residue samples. Molar mass balance calculations were performed for all substrates other than the control substrates. Detected concentrations from the LC-MS/MS analyses were adjusted for the wet weight measured for each of the treatments. Mycotoxin concentrations below the LOQ were set to zero. The concentrations (μg/kg) of the mycotoxins determined in larval biomass and mass of residual substrate by LC-MS/MS analysis were multiplied by the total wet weight of larval biomass or residual substrate mass respectively to yield the total amount of mycotoxin recovered, including the amounts of metabolites analysed. The molar mass balance was calculated by dividing the sum of the mycotoxin mass detected in the larvae and residual material by the mycotoxin molar mass, including that of metabolites quantified, present in the initial substrate on a wet weight basis. All figures were made in GraphPad Prism v4.

3 Results

Control substrates and sample homogeneity

No concentrations above the LOQ for any of the three mycotoxins and their metabolites included in the analyses were detected in the control substrate. For all spiked substrates, the relative standard deviation of the mycotoxin concentration between the replicates was ≤20% and the mycotoxin concentrations in the substrates were therefore considered as homogeneous (Supplementary Table S1).

Larval survival and biomass

The average survival of the HFL was 66-83% and was not affected by exposure to AFB1, DON and ZEN for 5 days (Figure 1A). Exposure to the mycotoxins did also not result in an effect on insect biomass when comparing the individual treatments with the solvent control substrate (Figure 1B).

Figure 1
Figure 1

(A) Larval survival and (B) individual larval weight recorded after five days feeding on the included substrates.

Citation: World Mycotoxin Journal 16, 4 (2023) ; 10.1163/18750796-20222826

Mycotoxin accumulation and metabolism

Chemical analytical results showed that parent compounds and metabolites were detected in the substrate and residual materials (in concentrations above the LOQ), but that these were not present in concentrations above the LOQ in any of the larval samples.

A molar mass balance was calculated in order to express which fraction of the initially present parent compounds (or metabolites) in the substrate was recovered in the larvae and the residual material. The molar mass-balances of the AFB1 treatments – as based on averages – were 50% (1× ML) and 63% (10× ML). Even though seven known aflatoxins (AFB2, AFG1, AFG2, AFM1, AFL, AFP1 and AFQ1) were analysed, neither the substrate, the larvae nor the collected residual material contained AFB1 metabolites in concentrations above their LOQ. The absolute amount of AFB1 (μg) in the substrate was significantly lower than in the residual material for both the 1× ML and the 10× ML substrates ( P < 0.05).

The calculated molar mass balance of DON in both treatments was 112% (1× GV) and 95% (10× GV) (Figure 2). This indicates that a complete mycotoxin recovery was obtained by the DON metabolites analysed. DON itself and the metabolites DON-3G, 15-ADON and 3-ADON were present in the residual materials of the DON-spiked substrates. For both 1× and 10× GV DON-spiked substrates, DON was responsible for the majority of the recovered fraction, and the absolute amount of DON (μg) in the substrate did not significantly differ from the concentration in the residue in both the 1× GV ( P = 0.886) and the 10× GV ( P = 0.886) substrates (Supplementary Table S7). Contribution of the metabolites; DON-3G, 15-ADON and 3-ADON to the total amount of DON-related compounds ranged between 0.01% and 1.3%, indicating that only little metabolism occurred (Supplementary Table S6).

Figure 2
Figure 2

Molar mass balance of AFB1, DON and ZEN for each of the spiked substrates: (A) 1× maximum level (ML) or guidance value (GV) and (B) 10× maximum level (ML) or guidance value (GV).

Citation: World Mycotoxin Journal 16, 4 (2023) ; 10.1163/18750796-20222826

Figure 3
Figure 3

Overview of the absolute amount of ZEN, α- and β-ZOL (μg per kg total material) in the substrate, larvae and residues for each of the two substrates: (A) ZEN 1× GV and (B) ZEN 10× GV.

Citation: World Mycotoxin Journal 16, 4 (2023) ; 10.1163/18750796-20222826

The molar mass balance of ZEN was 54% (1× GV) and 63% (10× GV) (Figure 2). In the mass balance of both ZEN treatments, ZEN was the main compound recovered. α- and β-ZOL contributed – on average – 4.1 and 4.7% to the mass balance of the ZEN 1× GV substrate. In the ZEN 10× GV substrate, α- and β-ZOL contributed around 2.3 and 8.3% to the mass balance, respectively (Supplementary Table S6). As mentioned above, no metabolites were detected in the substrate and residual material of the AFB1 treatments and for DON no significant difference in absolute concentration between the substrate and residual material was found (Supplementary Table S7). In the case of ZEN and its metabolites the absolute amount and their contribution – though low – in the substrate and residual material was calculated (Figure 3A and 3B). The metabolites α- and β-ZOL were already present in the ZEN-spiked substrates. Interestingly, the concentration of ZEN is significantly ( P < 0.05) decreased in the residual material of both the 1× and 10× GV ZEN-spiked substrates as compared to the initial substrate (Figure 3A and 3B). Also, the absolute concentration (μg) of α-ZOL, in the 10× GV substrate, and β-ZOL, in both ZEN substrates, changed significantly ( P < 0.05).

4 Discussion

Our study is the first to evaluate the effects of mycotoxin exposure of HFL using LC-MS/MS and to present molar mass balances quantifying compound recovery. Survival of HFL was not significantly affected by mycotoxin exposure. Exposure started in the egg stage and consequently survival reflects effects on hatchability and exposure of neonates to the mycotoxins. Furthermore, the presence of unfertilised eggs (Gadallah and Marei, 1973) or damage caused while handling the eggs could have contributed to the mortality that occurred in this study. No other comparable studies on HFL have been performed. Studies on H. illucens larvae (also order Diptera, class Brachycera, but family Stratiomyidae instead of Muscidae) did not start in the egg phase, but in a later larval stage (five or seven days since hatching, when the larvae were switched from a starter substrate to the organic residue substrate) and therefore likely resulted in a higher survival (Bosch et al., 2017; Camenzuli et al., 2018; Meijer et al., 2019). HFL develop into pupae already on day six or seven and no switch from starter to organic residue substrate is applied in practice. We therefore started exposure in the egg stage to match the practice of commercial HFL mass rearing. A study on Drosophila melanogaster Meigen strain A11 showed that eggs exposed to AFB1 were less likely to develop into adults than when two-day-old larvae were exposed to the same concentration of AFB1 (Chinnici et al., 1979). No significant effect on HFL body mass gain was found when fed mycotoxins in concentrations corresponding to 1× and 10× the ML or GV of AFB1, DON and ZEN. Studies on H. illucens showed that exposure of larvae to concentrations of AFB1, DON and ZEN in lower (Purschke et al., 2017) and in even higher concentrations (Camenzuli et al., 2018; Meijer et al., 2019) than tested in this study also showed no effect on body mass.

None of the mycotoxins and metabolites included in the analyses were detected in the larval samples and therefore accumulation in the larvae did not take place. This is in contrast with a previous study in which a ten-fold higher concentration of AFB1 in 2nd instar HF larvae was found after two days of exposure to 20 μg/kg AFB1 (Nevins and Grant, 1971). The results from the present study are in accordance with previous studies on H. illucens, A. diaperinus and T. molitor, showing that AFB1, DON and ZEN did not accumulate in the insect body (Bosch et al., 2017; Camenzuli et al., 2018; Niermans et al., 2019).

The results of this study lead us to infer that mycotoxin metabolism occurs in HFL. Additionally, substrate-specific enzymes and microorganisms or photochemical degradation over time could be responsible for mycotoxin metabolism or breakdown leading to an incomplete mass balance. These events were not examined in the current study and therefore no statements can be made about this.

For AFB1 ca. 40-50% is not accounted for in the molar mass balance, even though seven aflatoxins were included in the analyses. This finding suggests that as yet unknown metabolites are formed. It is important to trace detoxification pathways and resulting metabolites for AFB1 in view of the future use of HFL larvae as feed/food. The percentage of AFB1 recovered in this study seems comparable to what was found for A. diaperinus, while AFB1 recovery in H. illucens seems lower (Camenzuli et al., 2018). This suggests that aflatoxin metabolism and the possible formation of unknown metabolites might also be relevant for these species. Though no aflatoxin metabolites were detected in the residual materials of the HFL, some residual materials of A. diaperinus contained AFL and AFM1, and AFL was also detected in residual materials of H. illucens (Camenzuli et al., 2018). The formation of novel AFB1 metabolites was confirmed for T. molitor (Gützkow et al., 2021).This suggests differences in metabolism routes between insect species. However, these insect species were reared on distinct substrates which might have been responsible for metabolite formation. Other studies observed low aflatoxin recovery rates after spiking a food/feed matrix (Sulyok et al., 2020; Warth et al., 2012). This suggests that non-enzymatic conjugation of AFB1 to, for example, proteins in the matrix could be an additional reason for the incomplete mass balance.

The molar mass balance of DON showed that DON was completely recovered in this study. Most of the DON was found back unchanged, however, DON-3G, 3-ADON and 15-ADON were found in low concentrations in the residual material. These three DON metabolites seem to be the main metabolites of DON formed. Also, for DON, the percentage recovered in this study was comparable to what was found for A. diaperinus, but it was lower in H. illucens (Camenzuli et al., 2018). The residual materials of A. diaperinus and H. illucens were also analysed for the presence of 15-ADON and DON-3G, but neither of these metabolites were detected (Camenzuli et al., 2018), suggesting a difference in metabolism routes occurs in a HFL and BSFL study.

The molar mass balance for ZEN varied between 54 and 63%, depending on the initial concentration, indicating that 37-46% had been converted. Although the metabolites α- and β-ZOL were already present in the ZEN-spiked substrates, changes in their concentrations were observed, indicating a possible metabolism of ZEN into metabolites. The concentration of ZEN decreased in the residual material as compared to the concentration in the initial substrate. The concentration of β-ZOL in the residue was significantly higher in both ZEN substrates as compared to that in the spiked substrate. A preferred conversion of ZEN into β-ZOL rather than into α-ZOL has also been observed in bovine liver preparations and liver subfractions from laying hens, but seemed to depend on the species as in pigs mostly α-ZOL is formed (EFSA, 2011, 2017). The relative potency factor (RPF) for oestrogenicity determined for α-ZOL is 60.0, (RPF ZEN is 1.0), while the RPF for β-ZOL is 0.2 (EFSA, 2016). Therefore, it seems promising that HFL may convert some of the more toxic ZEN into the less toxic metabolite β-ZOL.

ZEN recovery in HFL was similar to that observed in T. molitor (Niermans et al., 2019), while ZEN recovery in H. illucens and A. diaperinus seemed to be complete (Camenzuli et al., 2018). Also in these insects species, α- and β-ZOL were, as in this study, present in the residual materials (Camenzuli et al., 2018; Niermans et al., 2019). α-ZOL even contributed to around 50% of the total ZEN recovery in H. illucens (Camenzuli et al., 2018). Further studies are needed to clarify the contribution of the HFL and the substrate in this.

In conclusion, data collected in this study contribute to understanding whether HFL reared on mycotoxin contaminated side streams could be used safely as feed and- or food ingredients. This study suggests that mycotoxin metabolism occurs in HFL, as for AFB1 and ZEN, which were two of the three investigated mycotoxins, recovery of the initial amount of mycotoxin present in the substrates was partial (ca. 50-60%). Most of the initially present DON was found back unchanged, suggesting that little DON metabolism occurred. Therefore, further studies are needed to examine the role of the HFL as well as its substrate in mycotoxin metabolism.

Contamination with unknown metabolites can lead to an underestimation of toxicity and exposure. Therefore, further research is needed to identify the currently missing fraction of AFB1 and ZEN. For future research it would also be of interest to include other known mammalian phase I metabolites as zearalanone (ZAN) and phase II glucosides, glutathione conjugates and sulphate esters commonly found in insects (Wilkinson, 1986). Additionally, analysis of conjugates such as sulphates or glucosides which are known to be formed by plant enzymes (Brodehl et al., 2014), might help in determining the role of the substrate in mycotoxin metabolism.

Tracking of conversion or metabolic pathways through e.g. isotopic labelling is required to determine the presence of unknown metabolites to map AFB1 metabolism in HFL. Such metabolites do not seem to be toxic to HFL, however, they might confer toxicity to consumers of HFL. Overall, this study showed that HFL mortality and biomass were not affected after exposure to AFB1, DON and ZEN. Furthermore, HFL do not accumulate the tested mycotoxins indicating their possible safe use as food- and/or feed when reared on mycotoxin contaminated side-streams. Mycotoxin metabolism seems to occur for AFB1 and ZEN, while metabolism of DON seems limited.

*

Corresponding author; e-mail: elise.hoek@wur.nl

Supplementary material

Supplementary material is available online at: https://doi.org/10.6084/m9.figshare.24071325

Table S1. Concentration (μg/kg) of the mycotoxin measured in the different replicates of the initial substrates.

Table S2. Instrumental MS/MS parameters of mycotoxins analysed in positive ionisation mode.

Table S3. Instrumental MS/MS parameters of mycotoxins analysed in negative ionisation mode.

Table S4. LOD and LOQ (ng/ml) per mycotoxin in the different matrices.

Table S5. Average recovery of the mycotoxins in the different matrices analysed.

Table S6. Overview of the average contribution of AFB1, DON and ZEN and their metabolites to the overall molar mass balance (%).

Table S7. Overview of the absolute amount of mycotoxin (metabolite) recovered (μg) in the substrate, larvae and residues calculated per kilogram of material.

Acknowledgements

This study was financially supported by the Dutch Research Council (NWO; NWA programme, InsectFeed project, NWA.1160.18.144). Additional financing from the Knowledge Base program of the Ministry of LNV via project KB-33-001-045 is acknowledged. We would like to thank Anna Dörper, Parth Shah, Sevasti Maistrou and Yvonne Kortsmit for their assistance during sample collection, Geert Stoopen for his advice and assistance, and Marcel Dicke and Anna Voulgari Kokota for constructive comments to the final manuscript.

Authors’ contribution

Conceptualisation: KN, EH, HF, JL, RD; investigation: KN; formal analysis: KN, RD; writing-original draft preparation: KN; writing-review and editing: KN, EH, HF, JL, RD; visualisation: KN; supervision: EH, HF, JL; funding acquisition: HF, JL. All authors have red and agreed to the published version of the manuscript.

Conflict of interest

The authors declare no conflict of interest.

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