Abstract
Yellow mealworm (Tenebrio molitor, YMW) is a potential new source of protein for food and feed. YMW farmers are looking for more sustainable, circular, and economical substrates than those currently allowed for all livestock; but safety concerns are cause for maintaining legal barriers. YMW were reared on broiler manure; category 2 meat-and-bone meal from animal rendering; biodegradable municipal waste; former foodstuffs sourced from supermarkets, of both vegetable and animal origin; and a standard wheat-based chicken diet. Performance in terms of survival and larval biomass yield were determined, and larvae and frass samples were analysed to determine nutrient composition and assess chemical and microbiological safety. Highest mean larval biomass yields were observed for chicken feed and category 2 meat-and-bone meal. Substrate-specific results showed accumulation of Cd, Se, Cu, and Zn; and dioxins; but larval concentrations did not exceed legal maximum levels. The biodegradable municipal waste, supermarket mix, and chicken feed contained residues of pesticides; some were transferred to the larvae. Supermarket mix contained residues of nicotine, assumed to originate from improper handling, suggesting a need for continuous control of the material as being crucial for safety. Since a municipal-waste stream implicitly offers less opportunities for control of the material composition and associated safety, in current collection systems, this limits its suitability for use at this time. Broiler manure was high in several elements and contained residues of coccidiostats; these were carried-over to the larvae and resulted in non-compliance with applicable limits. High microbiological contamination for this stream also raises concerns on its safety although post-harvest processing is anticipated to be capable of reducing contamination to acceptable levels. Larval performance on the category 2 meat-and-bone meal was equivalent to the wheat-based chicken feed and food safety parameters were all acceptable, presumably due to extensive processing. We conclude that when appropriate control measures are applied and substrate materials are mixed for optimized composition and growth; tested residual streams could be a nutritious, safe and sustainable substrate for insect rearing.
1 Introduction
Reared insects are increasingly seen as an alternative source of protein for food and feed due to their ability to grow on a variety of residual streams. One species of insect that has received such interest is the yellow mealworm (YMW, Tenebrio molitor L. 1758; Coleoptera: Tenebrionidae) (Costa et al., 2020; Deruytter and Coudron, 2022; Gkinali et al., 2022; Grau et al., 2017; Hong et al., 2020; Moruzzo et al., 2021). In the European Union (EU), YMW is at this time permitted to be processed into animal feed for pets (Regulation (EC) No 142/2011), aquaculture (Regulation (EU) 2017/893), and pigs and poultry (Regulation (EU) 2021/1925). The processing requirements for petfood differ from those for livestock: insect-based feed products for livestock may only consist of ‘processed animal proteins’ (PAPs), which must have undergone more rigorous processing than petfood (Regulation (EC) No 142/2011). Furthermore, the European Food Safety Authority (EFSA) has thus far evaluated four ‘novel food’ applications for YMW: dried (EFSA, 2021a), two separate entries of frozen and dried formulations (EFSA, 2021b; 2025), as UV-treated powder (EFSA, 2023). All of these novel food products are now legally allowed to be marketed as novel food for human consumption – except for the frozen and dried formulations that had been submitted by the Belgian find and Insect Industry Federation (BiiF), but with EFSA’s positive evaluation, approval is expected soon at time of writing (Regulation (EU) 2017/2470). A major side-stream from commercial insect farming called ‘frass’, primarily consisting of excrements and up to 5% dead insects in volume/3% in weight, may also be processed to be turned into fertiliser (Regulation (EU) 2021/1925).
Historically, YMW are a common pest that infested stored grains or grain products (Kavallieratos et al., 2019; Walkowiak-Nowicka et al., 2023). This makes the use of former foodstuffs (FFS) from the bakery industry as rearing substrate self-evident (Mancini et al., 2019). Use of FFS as insect rearing substrate is allowed, as long as they do not contain meat or fish (Regulation (EC) No 142/2011). Indeed, various vegetable-based waste streams have thus far been investigated and showed great potential for rearing this insect species (Kotsou et al., 2024). Certain other residual streams that contain animal by-products, that are at this time not yet permitted as rearing substrate in the EU for conventional livestock animals such as poultry and pigs, could also prove to be suitable and more sustainable for insect rearing (Rumbos et al., 2020). However, before the authorization of such substrates can be considered, the safety of the insect biomass to be used for food or feed purposes must be assured.
For this study, a variety of residual streams were selected to be used as substrate, these were streams with diverse origin and all widely available. The selected streams were: broiler manure, category 2 meat-and-bone meal from animal rendering, biodegradable municipal waste, and former foodstuffs sourced from supermarkets containing materials of both vegetable and animal origin. These streams all have in common that they are currently not permitted to be used in the EU for applications in feed for any livestock animal, including reared insects.
Many of the restrictions on the types of feed materials that are permitted to be fed to farmed animals in the EU can be traced back to the recycling of animal proteins resulting in outbreaks of zoonotic and animal diseases such as bovine spongiform encephalopathy (BSE), African and classical swine fever, foot and mouth disease, and others (Shurson et al., 2023). Various relaxations of these bans have been implemented in recent years, but certain barriers still persist (Meijer et al., 2023; Van Raamsdonk et al., 2023). In addition to the potential transfer of diseases, alternative insect-rearing substrates warrant consideration of potential hazards that are not, or less commonly, associated with conventional feed materials that are already safely being used for the rearing of YMW. Potential hazards are largely specific to the material in question (Meyer et al., 2021). For instance, in the case of broiler manure, micro-organisms, residues of veterinary pharmaceuticals such as antibiotics and coccidiostats or antimicrobial resistance (AMR) genes are a potential concern (Martins et al., 2022), but little is known on effects and substance transfer of such substances in relation to reared YMW. For most considered substrates with materials of vegetable origin, residues of pesticides could be present and have adverse effects on larval performance, and substance transfer from substrate to insect biomass could result in non-compliance (Houbraken et al., 2016; Meijer et al., 2022, 2024). Certain (heavy) metals and metalloids such as arsenic (As, Van der Fels-Klerx et al. (2016)) and mercury (Hg, Truzzi et al. (2019)) have been shown to accumulate in YMW, so verification of safe levels when reared on residual streams is paramount. Although environmental contaminants such as dioxins and polychlorinated biphenyls (PCBs) do not appear to bioaccumulate in YMW, high substrate concentrations (such as those potentially found in residual streams) could be transferred and concentrate in the insect product upon drying (Pajurek et al., 2023; Ratel et al., 2023). Ensuring microbiological safety of any food or feed product is of course critical: common processing steps will likely reduce contamination to safe levels, but this may not suffice in the case of heat-resistant bacterial endospores (Kooh et al., 2020; Pöllinger-Zierler et al., 2023; Yan et al., 2023). In addition, little is known on YMW facilitating horizontal transfer of AMR genes (Andrade-Oliveira et al., 2023).
The main objective of this study was to assess the safety of the reared YMW by analysing substrate, larvae, and frass samples for the presence and levels of prioritised hazards for each type of tested substrate. The second underlying objective was to determine the performance of larvae (in terms of growth, survival, and nutritional values) when reared on such materials.
2 Materials and methods
Substrates
Yellow mealworms were reared on six primary substrates consisting of different types of feed/ingredients. These were: broiler manure (BM), category 2 meat-and-bonemeal (Ct2), biodegradable municipal waste (MW), supermarket mix containing meat (SM), wheat bran (W), and a standard chicken feed (CF). The rationale for this selection of substrates as well as a more in-depth justification of certain experimental design choices (e.g. concerning pre-treatment) are provided in Hoek-van den Hil et al. (2022). In summary, these experimental substrates represented four distinct residual streams that are, at this time, not (yet) permitted for insect farming in the EU – each with its own set of potential hazards associated with it.
The used substrate-materials were procured and processed as follows. The broiler manure (BM) was acquired from AVINED (Nieuwegein, The Netherlands). This was dried at 50 °C in an oven and milled using a cutting mill (shredder) and sieve (3 mm). The meat-and-bonemeal was sourced exclusively from Category 2 materials (Ct2, in the context of Regulation (EC) No 1069/2009) from poultry, pigs, and cattle. This has been processed in accordance with ‘method 1’ (Regulation (EC No 142/2011), by Darling Ingredients, Sumar, The Netherlands. The biodegradable municipal waste (MW) was collected from the residential neighbourhood of IJburg in Amsterdam (AMS Institute, Amsterdam, The Netherlands). In principle, this stream may contain for instance: peels and remains of vegetables, fruit and potatoes; remains of cooked food (including meat/fish); vegetable oil. Although the pilot-stream from this particular municipality should be limited to kitchen waste; it may also contain weeds and other fine garden waste such as twigs and leaves, as well as a variety of other materials in case of erroneous disposal. After collection, this material was processed by removal of large pieces of contaminants such as plastic cups and textile, size reduction using a rotating grater with holes of 3 mm, and removing large pieces of fibres, such as flower stems, that could not pass the grater. The supermarket mix (SM) contained former foodstuffs, including meat (Attero, Tilburg, The Netherlands). This was processed by mechanical unpacking, and ground and mixed afterwards. In addition to these primary residual streams, a standard wheat-based chicken feed (CF, De Valk, Wekerom, Lunteren, The Netherlands); wheat bran (W; Bonda, Den Bosch, The Netherlands), and organic carrots (C; sourced via local retailer) were used. CF and W were stored in paper bags at room temperature and fresh organic carrots were stored at 7 °C for a maximum of one week prior to the start of the experiment. Dry residual streams (BM and Ct2) were stored in closed containers at room temperature, whereas wet residual streams (MW and SM) were stored in closed containers at −18 °C prior to the experiment, and at 7 °C during the experiment.
Results of compositional analysis for each of the used materials are shown in Table 1 below. The materials and methods for proximate analyses are presented in Appendix A. The highest value both for crude protein and total fat was found in the Ct2 substrate component, while sugar and starch were absent there. Due to the comparatively high inherent moisture content of the MW and SM materials, these were mixed with W in order to achieve a total moisture of the substrate that was anticipated to support YMW growth. Substrate combinations were assembled according to their dry matter content (DM), as shown in Table 2. While total feed amounts (in fresh weight) varied across treatments, the input of dry matter was controlled and harmonised within treatment types. This hybrid approach allowed to simulate production conditions realistically while keeping nutrient delivery balanced.
Results of proximate analysis for residual stream components
Citation: Journal of Insects as Food and Feed 2026; 10.1163/23524588-bja10373
Substrate compositions
Citation: Journal of Insects as Food and Feed 2026; 10.1163/23524588-bja10373
Animal procedures
The YMW used in this study were bred by Wadudu (Hoogersmilde, The Netherlands): they originated from beetles that were placed on egg-laying substrate for one week. Indicated age of YMW was an approximation, as the exact age may vary depending on the duration adults remained in the breeding crates, which influences the timing of egg deposition and hatching. YMW were hatched and reared according to the company’s standardised rearing methods, shown in Table S1 in the Supplementary material. Mealworms (aged approx. 36 days; i.e. in the 5th rearing week) were harvested, packed, and transported from Wadudu to HAS Green Academy (’s Hertogenbosch, The Netherlands); 7 days before the start of the experiment. After transportation, the mealworms were separated from the substrate and frass with a 1 mm sift and shed skins were removed. During the acclimatization period, one week prior to the experiment, mealworms were housed in 600 × 400 × 165 mm (l × d × h) euro norm crates and fed with the chicken feed (CF) (De Valk, Wekerom, The Netherlands) and carrots (Table S1 in the Supplementary material). Crate densities were not experimentally adjusted but reflected natural variation in larval densities provided by the breeder. Based on typical supplier estimates and back-calculations from individual mealworm weight, crate populations were estimated at 16 000-20 000 larvae, though actual values may vary beyond this range.
Overview of weight (g) of feed constituents (dry and wet ingredient) provided to yellow mealworm, for each of the
Citation: Journal of Insects as Food and Feed 2026; 10.1163/23524588-bja10373
The experiment was conducted during the late larval exponential growth phase of Tenebrio molitor. Larvae were reared during 21 days from approx. 44 to 65 days of age, after which the experiment was terminated just before the onset of pupation. This timing was selected based on commercial practice (Wadudu) and supported by literature describing typical larval development under comparable conditions (e.g. Deruytter and Coudron, 2022), which identify this period as optimal for measuring feed conversion efficiency prior to growth plateau and metamorphosis. After the 7-day acclimatization period, the mealworms (aged approx. 44 days) from the stock colony were sieved and separated from the frass with 1 mm sieve. To estimate individual mealworm weight and the proportion of residual non-mealworm mass, five replicate subsamples ranging from 5.00 to 5.80 g were collected. After manual separation of larvae from remaining particles (e.g. feed), the mealworm-only mass ranged from 4.44 to 5.47 g. The estimated proportion of non-mealworm material in these sieved samples ranged from 1.0-11.3%. Based on these data, the average individual mealworm weight was 29.6 ± 1.2 mg, while the apparent weight including residual substrate was 31.8 ± 0.9 mg. From this sieved stock, 500 ± 0.5 g of material – containing an estimated 7% residual non-mealworm mass – was added to each experimental crate (600 × 400 × 165 mm). This quantity was chosen to reflect standard commercial conditions and corresponded to approximately 15 000-16 000 larvae per crate, based on earlier weight-based sampling. Given the internal dimensions of the crates (55.5 × 35.5 cm), this equates to a larval density of approx. 8-10 larvae/cm2, which is within the range reported as suitable for high-yield rearing (Deruytter and Coudron, 2022). A randomised block design was applied, with crates assigned to treatment groups at the start of the experiment. Each treatment was applied to
At the end of the experiment, live samples of the YMW (aged approx. 65 days) were directly transported to the WUR analytical laboratories in The Netherlands for microbiological analysis on the following day (see below). For the remaining YMW biomass, intended for proximate and chemical analysis: the mealworms were harvested, using a 2 mm sieve, separating the mealworms from the frass. The total rearing weight of the mealworms and frass were then determined separately (accuracy ± 0.1 g). Finally, the YMW were euthanised by freezing at −18 °C for at least 24 hours.
Chemical and microbiological analyses
A detailed overview of materials and methods for proximate, chemical and microbiological analyses are presented in Appendix A. These methods included: proximate analysis for composition, metals and trace elements, pesticides (including analytical scope of the multi-residue method (Table S2 in the Supplementary material)); environmental contaminants: PFAS, dioxins, PCBs; antibiotics and coccidiostats; bacteria; and antimicrobial resistance (AMR) genes.
Statistics and data analysis
The measures and calculations used for data analysis are as follows. The mean individual larval weight (mg) was calculated based on the weight of 100 larvae divided by that number. Survival (%) was calculated by dividing the number (N) of mealworms at the end of the experiment by the number at the start (N). The waste reduction index (WRI) was calculated by subtracting the weight of the frass (F) from the weight of the substrate at T = 0 (
Significant differences in the effect of substrates on mean larval biomass yield (g), survival (%), mean individual larval weight (mg), WRI, ECI, and analysed concentrations for chemical hazards in larval and frass samples were tested using SPSS Statistics for Microsoft Windows 6 (version 25.0.0.2, IBM, Armonk, NY, USA). This was done using a non-parametric independent samples Kruskal–Wallis test with
Total larval biomass yield (mg) of yellow mealworm per container after rearing (YMW age 44-65 days) on a variety of residual streams. Arithmetic mean and standard deviation for
Citation: Journal of Insects as Food and Feed 2026; 10.1163/23524588-bja10373
Most analytical results for chemical hazards were reported in separate tables for each of the respective contaminant groups, in Tables S5-S7 in the Supplementary material. Microbiological results are shown directly alongside the core text in this manuscript. The results reported in theses tables are the values as analysed. For some contaminant groups such as heavy metals and dioxins and PCBs, legal limits are relative to feed with a standardised moisture content of 12%. For these contaminant groups, the analysed values were corrected using the moisture content of the sample before assessment of this value against the respective legal limit in the core text of this manuscript.
For the comparison of contaminant levels in the larvae against levels initially present in the rearing substrate, the bioaccumulation factor (BAF) was calculated. A BAF > 1 signifies that accumulation of the contaminant has occurred to some extent. Calculation was done by dividing the concentration in the larvae by the concentration in the substrate. Each substrate as provided to the insects, consisted of a wet and dry component which were analysed separately. The total substrate concentration of each respective analyte was calculated based on the inclusion level as reported in Table 1.
3 Results
Performance and composition
Larval performance: All results in terms of larval performance for each of the provided treatments (mean larval biomass yield, survival, mean individual larval weight) are shown in Supplementary Table S3. Figure 1 shows the results for total larval biomass yield (mg) upon harvest of the mealworms on day 22 of the experiment. Highest larval biomass yields were observed for the CF, Ct2, W and BM treatments. The two treatments containing MW or SM performed significantly less in terms of larval biomass yield. No significant differences between treatments were observed for the mean survival rate.
The waste reduction index (WRI) and efficiency of food conversion (ECI) indexes are shown in Table S3 in the Supplementary material. Figure 2 shows the calculated WRI indexes. The WRI was highest in the CF and W treatments followed by Ct2, MW, and SM. The WRI was lowest at BM. The ECI for the SM (1.23) treatment was significantly lower than in all other treatments (arithmetic mean 0.54 for
Waste reduction index (WRI) on a dry-matter basis. Arithmetic mean for
Citation: Journal of Insects as Food and Feed 2026; 10.1163/23524588-bja10373
Sample composition: Table S4.1 in the Supplementary material shows the results of the proximate analysis of the larval samples. These results are summarised for moisture content, ash, crude protein, crude, fibre, and N-free substances in Figure 3. Significant differences between treatments were identified for all measured parameters. Most importantly, the moisture content of the larvae reared on the slurry-type substrates (MW and SM) was relatively low compared to the substrates for which carrot was provided as source of moisture. Despite the large range of sugar and starch levels in the substrate components (as shown in Table 1), neither were detected in the larvae. Significant lowest crude protein content has been found in BM (107 g/kg) and the significant highest crude protein content in SM (183 g/kg). Crude fat was significant highest in YMW grown on MW (137 g/kg) and significant lowest when YMW were grown on BM (31 g/kg). Finally, YMW grown on Ct2 has a significantly higher Ca content (0.20) than YMW grown on other residual streams.
Results of proximate analysis of larval samples for moisture/water content, crude ash, crude protein (with conversion factor nitrogen → protein of 4.76), crude fat, crude fibre, and N-free substances. CF, chicken feed (with carrot, +C); BM, broiler manure (+C); W, wheat (+C); Ct2, category 2 meat-and-bone meal (+C); MW; biodegradable municipal waste (with wheat, +W); SM, supermarket mix containing former foodstuffs with meat (+W). Arithmetic mean and standard deviation for
Citation: Journal of Insects as Food and Feed 2026; 10.1163/23524588-bja10373
Table S4.2 in the Supplementary material and Figure 4 show the results of the proximate analysis for frass. With respect to nitrogen, the Ct2 residual stream resulted in the frass with the highest concentration (8.0 g/kg) while the other treatments had more similar results between 2.3 and 3.6 g/kg. Phosphorus in frass was lowest in BM compared to the other residual streams varying from 1.09 g/kg in CF to 3.06 g/kg in Ct2. Potassium was highest in frass from BM compared to the other residual streams varying from 0.89 g/kg in Ct2 to 2.37 g/kg in CF.
Results of proximate analysis of larval samples for nitrogen (N), phosphorus (P), and potassium (K). CF, chicken feed (with carrot, +C); BM, broiler manure (+C); W, wheat (+C); Ct2, category 2 meat-and-bone meal (+C); MW; biodegradable municipal waste (with wheat, +W); SM, supermarket mix containing former foodstuffs with meat (+W). Arithmetic mean and standard deviation for
Citation: Journal of Insects as Food and Feed 2026; 10.1163/23524588-bja10373
Chemical safety
Metals and trace elements: Table S5 in the Supplementary material shows the concentrations (mg/kg) of a variety of metals and metalloids, as analysed in samples of substrate, larvae, and frass. The elements As, Hg, Tl, Pb, and U were all not detected in the larvae samples and were mostly absent in the substrate and frass samples. Shown in Figure 5 are the results for Cd, Se, Cu, and Zn. These elements accumulated from substrate to larval biomass in some of the tested treatments, with the highest mean BAF being 4.1 ± 0.2 for Se observed in the SM treatment. For the only heavy metal Cd that accumulated, BAFs from 0.93 ± 0.07 (in the CF; i.e. no accumulation) to 1.52 ± 0.10 (MW) were observed. Conversely, larval concentrations of Mn and Fe were less than 20% and 50%, respectively, than found in corresponding substrates. In all cases except for Cd, the mean larval concentration of each element was highest in the larvae corresponding to the BM treatment, which is largely owed to the concentrations of elements in the corresponding BM substrate also being comparatively high.
Concentrations (mg/kg) of cadmium (A), selenium (B), zinc (C), and copper (D Cu) in substrate, larvae, and frass samples. Arithmetic mean and standard deviation for
Citation: Journal of Insects as Food and Feed 2026; 10.1163/23524588-bja10373
Pesticides: Analytical results for pesticide residues in all analysed samples are shown in Table S6 in the Supplementary material. Pesticide residues were not detected in samples of the CF, Ct2, BM, and C substrate components. Traces of hydroxy-tebuconazole were found in the BM substrate (0.009 mg/kg), but not in the associated larvae and frass samples for this treatment. A variety of pesticides was detected in the MW, SM, and W substrate components, and most of those substances were also recovered in the frass samples for the treatments containing those components. While residues of some pesticides were detected in the SM, concentrations were generally low: ≤0.02 mg/kg. Figure 6 shows the analytical results for substrate, larvae, and frass samples of the MW treatment for those pesticides for which concentrations ≤0.05 mg/kg were found in at least one tested matrix. Pyrimethanil was detected in the larvae reared on the MW and SM, as was the case for the larvae of the W treatment; but it was only detected in the MW and SM substrate components, not the W or C. Since pyrimethanil appears to have also accumulated in the larvae reared on the SM treatment, it is possible that this substance also accumulated in the W treatment from a substrate concentration that was <LOQ. Finally, nicotine was detected in the SM substrate at disproportionately high concentrations (approx. 2 mg/kg) and comparatively high concentrations of this substance were also detected in the larvae (approx. 0.47 mg/kg) and frass (1 mg/kg) samples of the SM treatment. For this reason, nicotine was omitted from Figure 6.
Pesticide concentrations (mg/kg) in samples (substrate components, larvae, frass) associated with the wheat (W)+biodegradable municipal waste (MW) treatment. Arithmetic mean and standard error for
Citation: Journal of Insects as Food and Feed 2026; 10.1163/23524588-bja10373
Environmental contaminants: PFAS, dioxins, PCBs: PFAS was not detected in any of the analysed substrate samples (<LOQ) and therefore assumed to be absent in larvae and frass as well. Analytical results for dioxins, dioxins and dioxin-like PCBs (dl-PCBs) and non-dl-PCBs (ndl-PCBs) are shown in Table S7 in the Supplementary material. In Figure 7, the upper bound values are shown which have been corrected for 12% moisture content, which is the value to which EU legal limits apply in case of feed materials. For all tested treatments, bioaccumulation (on 12% wet weight basis) from substrate to larval biomass was observed for the ub values of ndl-PCBs. This was also the case for all TEQ values for the SM treatment; and with the exception of the ub dioxin TEQ value, for the MW treatment. The ub ndl-PCBs values were comparatively higher in the larvae than in the substrate, i.e. these bioaccumulated. Further, bioaccumulation of all TEQ values was observed for the MW and SM treatments with BAF values between 1.0 (ub, dioxins in MW) and 3.3 (ub, ndl-PCBs in MW).
Analytical results for dioxins (expressed as WHO2005-PCDD/F-TEQ), dioxins and dioxin-like PCBs (expressed as WHO2005-PCDD/F-PCB-TEQ), and non-dioxin-like PCB (ICES-6). Bars signify upper bound (ub). Analysed values corrected for 12% moisture content, which is the value to which EU legal limits apply (Directive 2002/32/EC). Arithmetic mean and standard deviation for
Citation: Journal of Insects as Food and Feed 2026; 10.1163/23524588-bja10373
Antibiotics and coccidiostats: No antibiotics were found in any of the substrates, and coccidiostats only in the BM. Results for coccidiostats in this substrate material, and related larvae and frass samples, are shown in Table 4. Residues of monensin, narasin, and 4,4′-dinitrocarbanilide (DNC) – which is a marker for nicarbazin – were found in all analysed samples. Concentrations in larval samples were all lower than in the substrate, <1% for monensin and narasin and < 2% for DNC; indicating that bioaccumulation did not occur.
Analytical results of coccidiostats monensin and narasin, and the nicarbazin-marker 4,4′-dinitrocarbanilide (DNC)
Citation: Journal of Insects as Food and Feed 2026; 10.1163/23524588-bja10373
Microbiological safety
Bacteria: Analytical results for bacteria in all analyzed samples, i.e. substrate, larvae and residue, or a mixture of larvae and residue are shown in Table 5 (substrate) and Table 6 (larvae and frass). Listeria monocytogenes, Campylobacter, MRSA, and Salmonella were not detected in any of the tested substrates, mixture of larvae, or residue at the end of the rearing period. Spore forming bacteria were present in most substrates but not detected in the C and W, nor in the larvae or residue from the CF, W, and Ct2 treatments. In addition, anaerobic spore forming bacteria were not detected in the Ct2 and in the larvae of the BM and SM treatments.
Overview of results for microbiological safety in substrate components before the start of the experiment (presence of bacteria in log cfu/g)
Citation: Journal of Insects as Food and Feed 2026; 10.1163/23524588-bja10373
Overview of results for microbiological safety in larvae, frass and mixture larvae/frass samples at the end of the experiment (presence of bacteria in log cfu/g)
Citation: Journal of Insects as Food and Feed 2026; 10.1163/23524588-bja10373
In the substrates at start of the rearing, Bacillus cereus was present at detectable levels (
Antimicrobial resistance (AMR) genes: Results for AMR genes are shown in Table 7. All four antimicrobial resistance genes ermB, tetW, aph(3′)-III, sul2 were found in all substrates, except or only low values of aph(3′)-III and sul2 (<1.0 E2 copies/μl) in W and aph(3′)-III in MW. The tested antimicrobial resistance genes were also found in most of the larvae and residues of the different treatment groups at the end of the rearing period, with the highest counts in BM and W. However, for some treatment groups, the resistance genes were found in the larvae but not in the corresponding residue – or the other way around.
Overview of results for antimicrobial resistance (AMR) genes in substrate (pre-experiment) and larvae and frass samples (post-experiment) (DNA copies per μl)
Citation: Journal of Insects as Food and Feed 2026; 10.1163/23524588-bja10373
4 Discussion
This is, to our knowledge, the first experimental study to assess the comprehensive safety of Tenebrio molitor reared on a diverse set of residual streams. Results showed that the overall larval performance, in terms of mean larval biomass yield, was highest with the two substrates consisting of the chicken feed and category 2 meat meal. The mean larval biomass yield was only slightly lower in the wheat treatment. These substrates have in common that they were all with carrot as a moisture source. All treatments containing broiler manure (BM), supermarket mix containing former foodstuffs with meat (SM), or household municipal waste (MW) performed significantly worse in that regard. However, the comparatively reduced mean larval biomass yields for BM, SM, and KW do not necessarily lead to the conclusion that these residual streams are not (optimally) suitable for YMW rearing. Rather, it is recommended that future research with these materials should consider some type of pre-processing, different ratios of wet and dry components, and/or combining these materials with other ingredients to optimise nutritional value and particle structure.
Results of proximate analyses showed significant differences in the composition of both larval and frass biomass, depending on the substrate on which the respective larvae were reared. For instance, moisture content of the BM larvae was significantly higher than that of the MW and SM treatments, at the expense of both crude protein and crude fat contents, which were significantly lower. Although the fat contents of these three substrate constituents was not meaningfully different, the crude protein content of the BM treatment (25.9%) was substantially higher than for the MW and SM treatments (approx. 3.3%). For the frass, the most important difference was that nitrogen and phosphorus for the Ct2 treatment were significantly elevated, whereas potassium was significantly lower than most other treatments. For BM, nitrogen and potassium were also relatively high, but phosphorus was lowest. For use as a fertiliser, ideal NPK ratios differ depending on the plant it is being applied to. Furthermore, since insect frass is being used as a constituent of multi-component fertilisers, final NPK ratios will shift depending on those of other (mineral-based) constituents (Nogalska et al., 2024). These differences, both for larvae and frass, require more research to determine trends and detailed analysis of micronutrients.
It must be noted that the results of this study are inherently limited to the state of the tested substrates and the presence of contaminants (as highlighted previously in Hoek-van den Hil et al. (2023)). Different batches of residual streams that were tested in this experiment may contain hazards at different levels, or of a different nature.
Heavy metals
Maximum levels for undesirable substances – including the heavy metals As, Pb, Cd, and Hg – in animal feed have been established in Directive 2002/32/EC. Limits established in this Directive are applicable relative to a feed with a moisture content of 12%; concentrations of undesirable substances in feed with a different moisture content should therefore be normalized for this percentage. Limits in this Directive differ depending on the nature of the feed material, e.g. if it is of vegetable or animal origin, or if it is a complementary or complete feed. The limits most applicable to freshly harvested YMW larvae would be: 2 mg/kg for Cd in feed materials of animal origin, and; 2 for As, 10 for Pb, and 0.1 mg/kg for Hg in feed materials in general. For As, Pb, and Hg, larval concentrations were all <LOQ and between 0.02 and 0.06 mg/kg for Cd: thus none of these limits were at risk of being exceeded in any of the analysed larval biomass samples. These findings are largely in line with those of Van der Fels-Klerx et al. (2016) and Truzzi et al. (2019). The effects of dietary exposure to heavy metals and other elements on YMW were assessed in both studies. Van der Fels-Klerx et al. (2016) used grain that had been artificially contaminated (‘spiked’) with Cd, Pb, and As; whereas Truzzi et al. (2019) used increasing levels of olive pomace added to wheat to determine transfer of Cd, Pb, Ni, As, Hg. Both studies found that, respectively, As and Hg bioaccumulated in YMW; but these metals were <LOQ in the larval samples in this study.
Accumulation from some substrates to larvae was observed for the elements Cd, Se, Cu, and Zn. Although bioaccumulation factors (BAF) were not uniform for these elements, this is primarily a function of substantial differences in concentrations in the substrates rather than any indication of differences in bioaccumulation patterns. Keil et al. (2020) found that Zn supplementation in the diet of YMW (via ZnSO4-spiked wheat bran) caused a strong reduction in Cd. This appears to correspond with some of the findings in this study: Zn was relatively elevated in the BM and Ct2 substrates and Cd was indeed <LOQ in the related larval samples, but more research would be needed to further substantiate this correlation. Overall, highest larval concentrations of Mn, Fe, Co, Ni, Cu, Zn, and Mo were found in the larval samples associated with the BM treatment, corresponding to that substrate also containing the highest concentrations of those elements compared to the other tested substrates. High concentrations of such elements is not necessarily a safety concern – any may even be a benefit if marketed as a complementary feed product.
Pesticides
For all identified pesticides, the MRLs as laid down in Regulation (EC) No 396/2005 for the product type insects are set at the substance-specific lower limit of analytical determination, as indicated by an asterisk (approx. 0.01*). The MRLs in this Regulation are applicable to both food and feed products. For most substances, including pyrimethanil due to its higher MRL of 0.05 mg/kg, the limits in larval biomass were not exceeded. However, for thiabendazole (MRL: 0.01 mg/kg) and nicotine (MRL: 0.01 mg/kg) the limits were exceed in the larvae. This does not necessarily imply an immediate safety concern, but rather points to a legal issue of default MRLs being set for reared insects despite the potential for carry-over.
Overall, a comparatively large variety of pesticides was identified in the MW, which was anticipated to be due to their presence on fruit and vegetable outer peels that form the bulk of that substrate (Nguyen et al., 2020). It cannot be excluded that the presence of multiple pesticide residues in the MW, SM, and W substrates had an adverse effect on larval performance in terms of, e.g. larval biomass yield and survival: such ‘cocktail’ effects had previously been observed for reared black soldier fly larvae (Hermetia illucens) (Meijer et al., 2024). Since the objective of this study was to determine the larval performance and safety of YMW reared on actual residual streams, such potential effects were not controlled for. As indicated above, the nature and levels of specific hazards are substrate- and batch-specific: this highlights the need for adequate traceability and monitoring of these types of residual streams.
The detection of nicotine in the SM substate and subsequently in the larvae and frass samples was unexpected. Although nicotine has historically seen use as an insecticide, its use as the active substance in a plant protection product has not been allowed since 2009 in the EU (Authority, 2023). Considering the relatively high concentrations found, we speculate that the presence of nicotine in former foodstuffs originating from a supermarket was more likely to be attributed to contamination from cigarettes (e.g. ash, butts) from personnel involved with the handling of this material. Although nicotine was not detected in the MW in this study, the apparent lack of possibilities for control of this stream in conventional collection systems imply a reduced suitability for large-scale utilization. Novel, specifically designed collection systems, for instance in the case of centralised collection in high-rise buildings, could offer higher levels of control.
Pyrimethanil was detected in the larvae reared on the W treatment, but not the W or C substrate components: suggesting that a trace-amount was present in the W substrate, which bioaccumulated; or that distribution of this substance in the substrate was not homogenous. Higher concentrations of pyrimethanil were also observed in the larvae than in the MW and SM substrates, which substantiates the hypothesis that this substance accumulates in YMW. More research is thus recommended to assess the effects of pyrimethanil exposure to YMW. For conventional livestock animals such as ruminants, swine, and poultry; this involves a metabolic study to determine the distribution of the pesticide residue between muscle and fat tissue (Liu et al., 2020). The octanol-water coefficient (log Kow) of a substance is generally seen a main predictor for concentration in either tissue and some studies have also suggested this correlation between fat content and log Kow value of a pesticide for Tenebrio molitor (Dreassi et al., 2020; Houbraken et al., 2016). Hill et al. (2024) further found that exposure to some pesticides (they studied the insect growth regulator pyriproxyfen) resulted in altered fat/protein ratios compared to a control. It is unclear how processing by fractionation could affect distribution of pesticide residues in the fat or protein fraction, which should receive follow-up study. This recommendation can be extended to all analytes in this study, due to a general lack of data on effects of processing on chemical safety parameters.
Dioxins and PCBs
The legal maximum levels for dioxins and PCBs in feed materials of animal origin are: 0.75 ng WHO-PCDD/F-TEQ/kg for dioxins; 1.25 ng WHO-PCDD/F-PCB-TEQ/kg for dioxins and PCBs; and 10 μg/kg for non-dioxin-like PCBs (all upper bound concentrations). As with heavy metals, legal limits for dioxins and PCBs are set for a standardised feed with a moisture content of 12%. When this correction factor is applied to analysed values, using the analysed moisture contents of each sample as described in section 3.2, all values for the substrate component and larval samples were far below (<10%) the legal limits for feed materials. An additional limit for dioxins and dioxin-like PCBs is laid down for T. molitor as a novel food, but only for the frozen, dried and powder forms (≤ 0.75 pg/g fat; applicant Fair Insects BV), and not the dried form (applicant SAS EAP Group). Regardless, even if the YMW from this experiment were corrected for the measured water content, analysed concentrations would by far not exceed this limit.
Results from this study showed that some accumulation had occurred, but that final concentrations of dioxins and PCBs in reared YMW were (far) below legal limits. The Ct2 substrate showed a slight trend of comparatively elevated concentrations of each of the TEQ values, but this did not translate to similarly elevated concentrations in the respective larval samples. The observation that dioxin and PCB concentrations were low is in line with previous experimental research, in which artificially contaminated substrates were provided to YMW (Pajurek et al., 2023). Ratel et al. (2023) suggested that a drying step, which is commonly used when producing insect-based food or feed, could concentrate the dioxins in the insect biomass, thereby elevating the relative concentration. Given the low larval concentrations found in this study (<10% of MLs), a drying step is still unlikely to raise a concern for concentrations of dioxins or PCBs. However, a substrate that is higher in dioxin levels than those tested in this study, may still result in concentrations in insect-based products that could be of some concern: for instance, in the case of compost- or biosolid/sludge-based substrates (Focker et al., 2022).
Antibiotics and coccidiostats
No residues of antibiotic substances were detected in any of the analysed samples. This absence was somewhat surprising: although application of antibiotics in poultry farming for routine prophylactic and metaphylactic use has been prohibited in the EU since 2022 (Regulation (EU) 2019/6), therapeutic use can still result in residues remaining manure (Muhammad et al., 2020). A total of three coccidiostats or their markers (monensin, narasin, nicarbazin) were detected in the BM substrate and related larvae and frass samples. This is not especially remarkable, since these substances are allowed and often used as poultry feed additives to control for Coccidia parasites (Martins et al., 2022). Insects reared on the tested residual streams (including and especially BM) are not anticipated to be sold as food, but rather as a feed product. As with heavy metals, maximum levels for coccidiostats in animal feed have been established in Directive 2002/32/EC; for monensin sodium (1.25 mg/kg), narasin (0.7 mg/kg), and nicarbazin (1.25 mg/kg) – and these limits are also relative to a feed with a moisture content of 12%. The Directive specifies that these limits only apply to non-target feed following unavoidable carry-over. In this study, the coccidiostats in the larvae resulted from substance transfer from the substrate, rather than carry-over, which means that such limits are technically not applicable – although most probably the most relevant in case of BM being allowed as as substrate. Nonetheless, the mean analysed concentrations in larval samples from the BM treatment would exceed these limits. This means that the use of YMW reared on BM-containing coccidiostats results in a non-compliant feed product. More research is therefore recommended to determine whether, e.g. a fasting treatment could reduce coccidiostat concentrations in exposed larvae. Conversely, this provides some perspectives for the potential application of manure from organically reared broilers, for which preventive use of coccidiostats is not permitted.
Micro-organisms
Due to the limited sample set, the absence of certain (pathogenic) micro-organisms in analyzed samples is not conclusive, but the presence of micro-organisms such as spore forming bacteria in most substrates, and more specifically Bacillus cereus, sulphite reducing Clostridia and Clostridium perfringens in e.g. Ct2, MW, and SM are indicative for the type of hazards that can be present in these residual streams. Also, the absence of Campylobacter in BM might be affected by the limited number of samples and sampling moment, taking into account that the presence of Campylobacter in broiler flocks is highly seasonally dependent (Pacholewicz et al., 2024). High total bacterial counts e.g. in BM are not unexpected, as this substrate will contain faecal bacteria from broiler gut microbiome. The low bacterial count in Ct2 is related to the processing from meat-and-bone-meal to a dry powder. This shows that processing the substrates before use as rearing material can affect the presence of micro-organisms in reared insects at the moment of harvest. Nonetheless, the high total aerobic counts – even in the larvae reared on Ct2 – indicate that processing of the substrate prior to rearing cannot fully substitute processing of larvae post-rearing.
For insect-based processed animal proteins (PAPs) intended for feeding to other animals, limits have been established in Regulation (EC) No 142/2011 – but these limits only apply to processed products, rather than the unprocessed insects that have been analyzed in this study. Early in 2024, the European Commission’s Standing Committee on Plants, Animals, Food and Feed (PAFF) clarified that live insects may be fed to animals other than ruminants (SANTE, 2024), but it is unclear which microbiological criteria these insects must comply with. Insects for food are subject to novel food legislation: product-specific microbiological criteria have been laid down in the respective approvals (see Regulation (EU) 2017/2470)), but these also only apply to fully processed products rather than the unprocessed larvae that have been analysed in this study. Further research should thus evaluate different processing methods and parameters for their effects on microbiological safety.
Such follow-up research should take into account a variety of factors that may complicate the efficacy of the method to reduce microbiological contamination to safe levels. For instance, whether the matrix is comparatively wet or dry can play a large role in the log reductions observed for thermoresistant fractions of avian influenza virus and Salmonella; D values for dry matrices could be up to 100 fold higher (Hayrapetyan et al., 2019). Similarly, combinations of processing methods and the use of ‘Tyndallisation’ (involving several heating/resting cycles) could be effective in reducing spore counts, but appropriate storage conditions (pH, water activity, temperature) remain crucial to prevent outgrowth of spores (Keratimanoch et al., 2022; Osimani and Aquilanti, 2021; Vandeweyer et al., 2020).
Antimicrobial genes
The detection of AMR is based here on a set of previously described PCRs used for comparison of AMR-levels in livestock between EU countries and other environmental studies (Van Gompel et al., 2020; Yang et al., 2022a,b). These genes were chosen as ubiquitously present genes in multiple bacterial species, not because of their specific importance to human health. Relative quantities of these genes can be compared between samples and were previously linked to increased usage of antimicrobials in livestock. The presence and variation that is measured in these samples of diet, larvae and residues, could be expected as AMR genes are widely spread and these variations in matrix can affect the growth of the specific bacteria in which the genes were detected.
5 Conclusion and recommendations
Yellow mealworm were reared on different substrates, including not yet legally authorised residual streams: broiler manure, category 2 meat-and-bone meal from animal rendering, biodegradable municipal waste (fruits, vegetables and other foods), and former foodstuffs containing meat from supermarkets; to determine larval performance and safety. Highest larval biomass yields were observed for a standardised CF and the Ct2 meat meal, followed by W. Performance on slurried MW and SM substrates was less successful and future studies should adapt diet formulations accordingly. For all substrates, some accumulation of chemical hazards such as heavy metals and dioxins was observed, but generally not exceeding legal limits for food or feed (if applicable). The number of microorganisms present appears to be comparatively high, but presence of potential pathogens can be reduced by selection and possible pre-treatment of substrates. Furthermore, pathogens are likely neutralised by processing and adequate storage – as is common and required for both food and feed applications. It is recommended that the efficacy of specific processing methods to reduce microbiological contamination to safe levels should be validated in follow-up studies.
For the BM substrate; the presence (and transfer) of drug residues like coccidiostats, and potentially antibiotics; and high levels of metals as well as the presence of fecal microorganisms might raise some concerns for the suitability of this substrate. Thus BM should ideally originate from poultry farms free of specific pathogens and from untreated flocks. Both MW and SW each contained certain contaminants at levels that may make larvae reared on them non-compliant, and the potential for pathogenic contamination is estimated to be higher than other tested substrates. Control mechanisms to prevent some of these issues should be considered and validated. Experimental results from this study suggest the Ct2 meat meal to be a homogenous substrate that can provide larval biomass yields that are equivalent to a vegetable-based CF substrate. Based on the results of this study, rearing YMW on Ct2 does not raise safety concerns. Overall, the results of this study suggest that the evaluated substrates each have strong potential to be used for rearing insects that are safe for food and/or feed. Certain safety issues are substrate-specific and may currently still result in non-compliance, but these could be addressed by technological solutions; which require further experimental verification.
Corresponding author; e-mail: Nathan.meijer@wur.nl
Acknowledgement
We wish to thank Marc Groenen (WFSR) for assistance with statistical analysis.
Author contributions
N. Meijer: Writing (original draft), data curation, visualization. K. van Zadelhoff: Formal analysis, investigation, writing (review and editing), project administration. M.A. Dame-Korevaar: Writing (review and editing). A. Borghuis: writing (review and editing), supervision, project administration, conceptualization. J.W. van Groenestijn: Writing on substrate origin, collection and processing, reviewing part of the manuscript. J. Boonstra: Methodology and formal analyses (of viruses). M. Brouwer: Methodology and formal analyses (of AMR). H. Brust: Investigation, writing (review and editing). E. de Lange: Investigation, formal analysis, writing (review and editing). L. Leenders: Writing (review and editing), investigation. N. te Loeke: Methodology and formal analyses (of pathogenic bacteria). X. Luinenburg: Methodology and formal analyses (of pathogenic bacteria). M. Tienstra: Investigation writing (review and editing). A.F.G. Antonis: writing (review and editing), supervision, project administration, conceptualization. M. Appel: Writing (review and editing). M.E. Bruins: Conceptualization, Writing (review and editing). T. Veldkamp: Conceptualization, writing (review and editing). S. Naser El Deen: Writing (review and editing). E.F. Hoek-van den Hil: Conceptualization, writing (review and editing), supervision, project administration, funding acquisition.
Disclosure and funding
The project “Safe insect rearing on yet to be legally authorised residual streams” (LWV20102, BO-64-001-025) was co-financed by the Top Consortium for Knowledge and Innovation’Agri & Food’ by the Dutch Ministry of Agriculture, Fisheries, Food Security and Nature. Commercial partners in the project were: Darling Ingredients International Rendering & Specialties B.V.; Dorset; HAS; Ingredient Odyssey SA (Portugal; Meatco. B.V.; Nederlandse Vereniging Diervoederindustrie NEVEDI; Nijsen company; Stichting Amsterdam Institute for Advanced Metropolitan Solutions (AMS)/Stad Amsterdam; Stichting AVINED; The Insectory; Venik (Verenigde Nederlandse Insectenkwekers); Wadudu Insectencentrum.
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