# Chemical contamination of black soldier fly larvae raised on EU-authorized or unauthorized substrate

Marie Papin, Maxime Pattier, Mélanie Davranche, Patrick Borel, Erwan Engel, Christelle Planche

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Marie Papin, Maxime Pattier, Mélanie Davranche, Patrick Borel, Erwan Engel, et al.. Chemical contamination of black soldier fly larvae raised on EU-authorized or unauthorized substrate. Waste Management, 2026, 210, pp.115219. 10.1016/j.wasman.2025.115219. insu-05360216

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Research Paper

Chemical contamination of black soldier fly larvae raised on EU-authorized
or unauthorized substrate

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Marie Papin<sup>a,b,1</sup> <sup>●</sup>, Maxime Pattier<sup>c</sup>, Mélanie Davranche<sup>c,2</sup>, Patrick Borel<sup>b,3</sup> <sup>●</sup>, Erwan Engel<sup>a,4</sup>, Christelle Planche<sup>a,5,*</sup> <sup>●</sup>

ARTICLE INFO

Keywords:

Hermetia illucens

Trace metal elements

Pesticides

Persistent organic pollutants

By-product

School canteen and supermarket biowaste

A B S T R A C T

Black soldier fly larvae (BSFL), *Hermetia illucens*, can efficiently convert biowaste into high-quality protein for the feed industry. However, biowaste can contain several chemical hazards and their fate in BSFL remain largely unexplored, even though they can pose a threat for both the insect sector and the higher levels of the trophic chain. The purpose of this study was to evaluate the chemical contamination of BSFL reared on authorized biowaste (wheat bran, carrots, apricots, salad) or unauthorized biowaste in the EU (school canteen and supermarket biowaste).

PFAS were not quantifiable in the substrates in contrast with persistent organic pollutants, pesticides and trace metal elements. The chlormequat pesticide was the only pesticide quantified in BSFL (15 µg/kg), slightly above the maximum residue limit in the EU for feed (10 µg/kg). Concentrations of PCBs and PCDD/Fs in BSFL were below the maximum limit (ML) but bioaccumulation factors up to 5 were obtained. As and Cd were highly bioaccumulated with concentrations approaching the ML for Cd (0.5 mg/kg) or 8 times below for As (ML = 2 mg/kg). The unauthorized substrate samples tested led to higher chemical safety risks in BSFL, with 10 times higher concentrations of As than in BSFL reared on authorized substrates and the presence of PCDD/Fs and PCBs. The concentrations of chemical contaminants in animal products consumed by humans such as eggs were estimated using transfer models when animals were fed with BSFL reared on unauthorized substrates. The concentrations were below the maximum limits in all cases.

1. Introduction

In 2020, 59 million tons of food waste were produced in the EU (Eurostat, 2023), reinforcing the need for solutions in the management of food waste (European Commission, 2020). One of the promising solutions is the industrial insect-based conversion of biowaste. It has the double advantage of reducing the volume of biowaste, by using them as

growing substrates for insects, while producing larvae, for the feed in-
dustry, and frass, as fertilizer. Black soldier fly larvae (BSFL), *Hermetia
illucens* (Diptera: Stratiomyidae), are particularly suited for the biocon-
version of biowaste since they can grow on a wide range of organic
substrates with feed conversion ratio up to 8 (Scieuzo et al., 2022).
However, for safety reasons (EFSA Scientific Committee, 2015; Lalander
and Vinnerås, 2022), only agricultural by-products and surplus

*Abbreviations:* BSFL, black soldier fly larvae; WB, wheat bran; SC, school canteen; SMKT, supermarket; FW, fresh weight; DW, dry weight; TMEs, trace metal elements; POPs, persistent organic pollutants; ML, maximum limit; MRL, maximum residue limit; BAF, bioaccumulation factor; PCBs, polychlorobiphenyls; DL, dioxin-like; NDL, non dioxin-like; PCDDs, polychlorinated dibenzodioxins; PCDFs, polychlorinated dibenzofurans; PFAS, per and polyfluoroalkyl substances; LOQ, limit of quantification.

* Corresponding author at: INRAE, UR370 QuaPA, MASS group, Saint-Genès-Champanelle 63122, France.

E-mail address: christelle.planche@inrae.fr (C. Planche).

1 https://orcid.org/0009-0008-8162-1330.

2 https://orcid.org/0000-0001-5544-1944.

3 https://orcid.org/0000-0001-9977-3238.

4 https://orcid.org/0000-0001-8550-8046.

5 https://orcid.org/0000-0002-7751-4603.

<footnote><sup>a</sup> INRAE, UR370 Animal Products Quality Unit, MASS Group, Saint-Genès-Champanelle F-63122, France</footnote>

<footnote><sup>b</sup> C2VN, INRAE, Aix-Marseille Univ, INSERM, Marseille F-13005, France</footnote>

<footnote><sup>c</sup> Géosciences, CNRS, Université Rennes 1, UMR 6118, Rennes F-35000, France</footnote>
production of fruits and vegetables, dairy or eggs are currently allowed as insect substrates in the EU (Regulation (EC) No 1069/2009). These authorized substrates can be used to feed rent animals, even humans, and it is counterproductive to use them to feed insects, whether from an economic or environmental point of view (Parodi et al., 2022). Thus, shifting towards more economically and environmentally interesting biowaste, coming from supermarket or school canteen waste for instance, is of great interest. But using these biowaste as substrate for insect larvae will only be authorized in the coming years if the microbiological and chemical safety of these larvae is guaranteed (FAO, 2021). Few studies have been dedicated to the chemical safety of edible insects, especially when reared on unauthorized substrates (Hoek-van den Hil et al., 2023).

TMEs were widely studied in insects (Alagappan et al., 2022; Lievens et al., 2021) and Cd in particular was described as highly bio-accumulated by BSFL (Proc et al., 2020; Purschke et al., 2017). In opposition, mycotoxins and pesticides were not found to be bioaccumulated by BSFL and were not considered to be of concern regarding the legislation for insect used as feed (Meijer et al., 2021, 2023; Spranghers et al., 2025). PCDD/Fs and PCBs were described by Charlton et al. (2015) and Pajurek et al. (2023) as not-threatening because of the low concentrations found in larvae, in regard to the regulation, but further studies are required to confirm these preliminary results and to evaluate any potential bioaccumulation. Per and poly-fluoroalkyl substances (PFAS) are not regulated in feed for now but could be in the future given recent advances in the analysis and toxicological evaluation of these substances. It should be noted that their presence in BSFL was already reported by Li and Bischel (2022) and Wakefield et al. (2024). Previous studies were mostly lab-scale studies where chemical contaminants were spiked at high and sometimes unrealistic concentrations in BSFL substrates. These preliminary data need to be confirmed and extended to more contaminants by implementing industrial rearing conditions.

In this study, BSFL contamination and bioaccumulation of various chemical hazards from EU-authorized substrates (wheat bran, salad, carrot and apricot) and EU-unauthorized substrates (supermarket and school canteen biowaste) were assessed after a standard industrial pilot-scale rearing experiment. Three TMEs, 61 persistent organic pollutants (POPs), 18 mycotoxins and 511 residues of pesticides were screened in the substrates. POPs included PFAS, PCDDs, PCDFs and PCBs. When a chemical contaminant was identified in the substrate, its concentration and bioaccumulation in BSFL were evaluated and compared to the current legislation regarding animal feed. Finally, a theoretical transfer model was used to estimate the concentrations of some chemical contaminants in animal products consumed by humans when animals are fed with BSFL.

## 2. Material & methods

### 2.1. BSFL rearing

Preparation of substrates and industrial BSFL rearing conditions were described in detail in Papin et al. (2025). Raw biowaste included EU-authorized ones: wheat bran (WB), surplus waste (carrots, apricots and salad) and EU-unauthorized ones: supermarket (SMKT) and school canteen (SC) biowaste. Unauthorized biowaste were collected on July 1st 2022 among 4 SC and 3 SMKT in France. After industrial packaging removal, biowaste were crushed and mixed. All raw biowaste were fermented for a week and mixed with WB (70–30 % respectively) following conditions previously described. The water content of the mix was adjusted to 70 % and then this mix was used as larvae substrates. After hatching, larvae were fed an organic poultry feed. After 8.5 d of growth, larvae were separated from the frass by sieving (2 × 2 mm) and rinsed using tap water. Larvae were then reared on the aforementioned substrates under industrial conditions for 7 days (28 °C, humidity: 70 %) in darkness, before being frozen to death.

### 2.2. Sample preparation for chemical contaminant analysis

Substrates were analyzed as given to larvae i.e. fermented biowaste mixed with WB. Regarding larvae, between 500 and 900 fresh larvae were pooled before analysis (36–80 g of fresh larvae) in order to obtain mean data without multiplying the number of analysis. Before chemical contaminant analyses, biowaste and larvae were crushed with liquid nitrogen using a homemade stainless-steel ball mill set at 800 tr.min⁻¹ for 1 min and a 2 cm-diameter bead to obtain a homogeneous powder. The powder was freeze-dried (Alpha 1-2 LDplus, Christ, Osterode am Harz Germany) during 24 h and stored at -20 °C before analysis.

### 2.3. Chemical contaminant analysis

The analytical strategy was the following: first of all, a large screening of chemical contaminants was carried out on each replicate of substrate blend. Secondly, if the presence of a contaminant was measured, 2 other replicates of substrates (3 in total) and 2 replicates of the pool of larvae (3 replicates for TMEs) were analyzed. For POPs analysis, only the SMKT and SC groups were screened. Indeed, POPs are mostly present in food from animal origin (Amutova et al., 2021; Arrebola et al., 2018) which were only present in these two biowaste.

#### 2.3.1. Trace metal elements (TMEs)

All glassware and materials were decontaminated beforehand by successive bath of 4 N HNO₃ and MilliQ water at 40 °C. A high purity acid used for digestion and analysis is produced from NORMAPUR 69 % grade in nitric acid (VWR Chemicals, Rosny-sous-Bois, France), distilled at low temperature (sub-boiling) in PFA containers. Fifty mg of samples were weighted in Teflon vessels with 5 mL of 69 % HNO₃ and 1 mL of H₂O₂ 30 % (Suprapur, Supelco, Bellefonte, Pennsylvania, USA), and mineralized in a microwave digestion system (Multiwave 7000, Anton Paar, Graz, Austria). The following parameters were used: temperature ramp from ambient to 250 °C in 35 min, temperature maintained at 250 °C for an additional 25 min. Digestion was carried out under magnetic agitation and a maximum of 140 bar. Samples were diluted to match 0.37 mol/L HNO₃ and trace metal element concentrations were measured using a quadrupole ICP-MS (Agilent technologies 7700, Santa Clara, California, USA). The instrument was tuned to account for oxide and double-charged ion, in Helium (collision cell) and nitric oxide gas mode. The concentrations were determined using a 7 points external calibration procedure using multi-elemental solutions (Analytichem, 0.37 mol/L HNO₃) with online Rh internal standard introduction. Rh is used to correct instrumental drift and possible matrix effects. The international geostandard SLRS-6 was used to check the validity and reproducibility of the results. The detection limits were calculated following AFNOR protocol and are inferior to 4 ppt for As, Cd and Pb before sample dilution. Substrates and larvae were analyzed in triplicate. TMEs were not analyzed in the salad substrate and corresponding BSFL due to analytical issues. In this study, the concentrations of 3 of the 4 TMEs regulated in feed are presented: As, Cd and Pb.

#### 2.3.2. PCDD/Fs and PCBs

The PCDDs, PCDFs and PCBs analyzed are listed in Table S1. These analyses were performed by the French National Reference Laboratory (Laberca, Nantes, France). Samples (3 g of biowaste and 2 g of larvae) were extracted using a pressurized liquid extraction system (SpeedEx-tractor E-914; BÜCHI Sarl, France) and three extraction cycles were performed at 120 °C using a mixture of toluene and acetone (70/30, v/v). The ¹³C-labeled internal standards (Wellington Laboratories, Guelph, Canada) were added to the samples before the extraction step. The extracts were concentrated under dryness for gravimetric determination of the lipid content. After redissolution in n-hexane, the extracts were transferred to the top of a set of four columns filled with different sorbents (i.e. silica gel impregnated with 10 % (w/w) silver nitrate, silica gel impregnated with 44 % (w/w) sulfuric acid, active carbon and
activated alumina), mounted into an automated purification device (Autosym GO-4-HT; Miura-Shimadzu, Japan). Elution with n-hexane was performed for clean-up purpose, followed by back-flush injection of toluene to collect the mono and di-ortho PCBs fraction from the alumina column, then PCDD/Fs and non ortho PCBs from the carbon column. PCDD/Fs and PCBs were separated and detected by gas chromatography with electron ionization coupled with high resolution mass spectrometry GC-EI(+)-HRMS (Agilent Technologies 6890 gas chromatograph coupled with a JEOL 800D mass spectrometer) using a HT8-PCB capillary column (60 m × 0.25 mm, 0.25 µm, Trajan Scientific and Medical, Ringwood, Victoria, Australia) for PCBs and a DB-5MS column for PCDD/Fs analysis (60 m × 0.25 mm, 0.25 µm, Agilent Technologies, Santa Clara, California, USA).

### 2.3.3. PFAS

The rearing substrates were analyzed for PFAS by the French National Reference Laboratory (Laberca, Nantes, France). PFAS were extracted from 1 g of sample by solid/liquid extraction using a mixture of methanol and potassium hydroxide. ¹³C-labelled internal standards (Wellington Laboratories, Ontario, Canada) were added to the sample before the extraction step. After one night of contact, water was added to the sample extract and then purification was carried out on two solid phase extraction cartridges (polystyrene divinyl benzene (Chromabond PFAS, Macherey-Nagel, Dueren, Germany) and carbon stationary phases (ENVI Carb, Supelco, Bellefonte, Pennsylvania, USA)). Extraction yields ranged from 43.9 to 132.7 %. Final purified extracts were analyzed by LC-ESI(-)-MS/MS (Agilent Technologies 6495C, Santa Clara, California, USA). The explored analytes are listed in supplementary material, along with the limit of quantifications (LOQ) (Table S2).

### 2.3.4. Pesticides

Five hundred eleven residues of pesticides (Table S3) were screened in all substrates by Inovalys lab (Tours, France). The sample preparation and extraction were made following a QuEChERS (Quick, easy, cheap, effective, rugged and safe) method (Anastassiades et al., 2003), where 5–10 g of samples were used. Samples were extracted using acetonitrile with 4 g of MgSO₄, 1 g of NaCl and citrate buffer. Purification salts were used: 0.9 g of MgSO₄ and 0.15 g of PSA. Samples were centrifuged for 20 min. For pesticides analyzed using liquid chromatography, 5 µL of sample was injected in a LC-MS/MS system using a Thermo Scientific Accucore C18 (150 mm × 2.1 mm, 2.6 µm) column. The mobile phase was a mixture of 5 mM ammonium formate, ultrapure water and methanol. For pesticides analyzed using gas chromatography, 2 µL of sample was injected in splitless mode in a GC–MS/MS system with a RTX-5MS (30 × 0.25 × 0.25) column. Calibration curves of the pesticides were prepared in each matrix. Blanks in solvents and in matrices were made.

### 2.3.5. Mycotoxins

The following mycotoxins regulated in the EU were analyzed in every substrate blend (Table S4): aflatoxins, trichotecenes A and B, fumonisins, ochratoxin, zearalenone and ergot alkaloids. These EU-regulated mycotoxins were analyzed by a certified French lab (Inovalys, Tours, France). Samples were crushed, if necessary, to a size below 0.5 µm. Two g of sample was used. The sample preparation and extraction were made following a QuEChERS method, using water and acetonitrile as extraction solvents. MgSO₄ and NaCl (4:1) were used as extraction salts. Samples were centrifuged for 5–10 min. MgSO₄ (0.15 g) and 0.05 g of C18 were used as purification salts. The analysis was realized on a LC-MS/MS system coupled with a UPLC chain Infinity II 1290 II AGILENT 6495C using a Zorbax Eclipse Plus column C18 Agilent (Santa Clara, California, USA). The mobile phase was a mixture of water/methanol, 5 mM of ammonium formate and 0.1 % of formic acid. Three µL of samples were injected in the system. A calibration in matrix was made. Blanks in solvents and matrices were made. The quality of the extraction and analysis was verified using extraction and injection

markers.

## 2.4. Estimation of the fate of chemical contaminants in laying hens and pigs fed with BSFL raised on supermarket waste

The purpose of this part was to evaluate the potential risk of bio-magnification in the food chain (Ali and Khan, 2019; Tison et al., 2024; Wu et al., 2022) of chemical contaminants when BSFL are reared on unauthorized biowaste and then used as laying hen or pig feed. Transfer models available at www feedfoodtransfer nl were used. Worst-case scenarios were used to evaluate these transfers: laying hens were fed entirely with dried BSFL raised on supermarket waste and pigs with 10 % of these BSFL. According to Hong and Kim (2022), up to 10 % of BSFL meal were incorporated in pig diets in most scientific studies. The concentrations of NDL-PCBs and total PCDD/Fs/PCBs in egg yolk fat and body fat from laying hens were estimated using the ‘Transfer model NDL-PCBs (congener-specific) – laying hen, version 1.0’ and the ‘Transfer model dioxins (total TEQ) – laying hen, version1.1’ respectively. The concentrations of total PCDD/Fs/PCBs in the body fat of pigs and of Cd in the liver and kidney of pigs fed with 10 % of BSFL raised on supermarket waste were evaluated using the following models: ‘Transfer model dioxins (total TEQ) – pig, version 2.0’ and ‘Transfer model cadmium – pig, version 2.0’. The parameters used such as the feeding regime, feed intake and duration of exposure are described in Supplementary Information (Table S6).

## 2.5. Calculations

The bioaccumulation factor (BAF) of each chemical contaminant was calculated according to Diener et al. (2015):

$$BAF = \frac{\text{Concentration in BSFL (dry weight)}}{\text{Concentration in the substrate (dry weight)}}$$

## 2.6. Statistical analysis

Results are expressed as means ± SEM in dry weight. Normality of residues was tested using Shapiro-Wilk test. Differences between groups regarding the chlormequat concentration in BSFL and the TMEs concentrations in substrates and BSFL were tested using one-way ANOVA. Homogeneity of variances was tested using a Brown-Forsythe test and in case of inhomogeneity a Welch's correction was applied. When a significant effect was detected, a Tukey post-hoc test was performed. Two-ways ANOVA were performed to test differences between groups regarding PCDD/Fs, PCBs and TMEs concentrations in substrates and BSFL, using a full model with the group and the matrix (substrate or BSFL) as the two variables. Heteroscedasticity was tested using the Spearman's rank correlation test. When a significant effect was detected, a Bonferroni post-hoc test was performed to compare the mean concentration of the substrate to the corresponding BSFL concentration in each group. Values of p < 0.05 were considered significant. All statistical analyses were performed using GraphPad Prism version 10.0.3 for Windows.

# 3. Results and discussion

## 3.1. Impact of the different substrates on the growth and composition of BSFL

General data of the larvae reared on the different substrates such as the weight, protein and lipid content were described in detail by Papin et al. (2025). BSFL fresh weights were similar to the reference group (120.5 ± 7.3 mg), except for those reared on apricot (84.9 ± 3.4 mg) and SMKT (90.3 ± 4.2 mg) substrates. However, no significant differences in terms of dry weight (270 ± 23 mg FW), protein or lipid content (48.3 ± 2.6 % and 22.2 ± 2.6 % DW on average) were noticed.
## 3.2. Trace metal elements (TMEs)

The concentrations of As, Cd, and Pb in the different substrates and corresponding BSFL groups are summarized in Table 1. Arsenic levels in authorized substrates were below the LOQ (<10 µg/kg DW), whereas in unauthorized substrates As concentrations were approximately ten times higher, reaching 88.2 ± 5.5 µg/kg for SMKT and 110.1 ± 11.7 µg/kg for SC. It was shown that animal products and market foods, present in these unauthorized substrates, contain significant concentrations of As (Upadhyay et al., 2019), which could explain the differences observed in this study. This indicates that these specific unauthorized substrates raise greater safety concerns compared to the authorized substrates of this study. Regarding Cd, the highest concentration was measured in WB at 81.8 ± 1.0 µg/kg while the lowest was in SMKT at 69.7 ± 0.7 µg/kg. The Pb concentrations were comparable across substrates, ranging from 22.7 ± 2.1 µg/kg for WB to 42.7 ± 2.4 µg/kg for SC. Despite these differences, all substrate concentrations remained well below the maximum limits (ML) for complete feed (Directive 2002/32/EC) being 20–543 times lower for As, 6.8–8 times lower for Cd and 133–250 times lower for Pb (Table 1). Consequently, although TME concentrations differ between authorized and unauthorized substrates of this study, these differences alone do not preclude the use of these

<table><caption>**Table 1**
Concentrations (mean ± SEM, µg/kg in dry weight) in Arsenic (As), Cadmium (Cd) and Lead (Pb) in rearing substrates and black soldier fly larvae (BSFL, n = 3), corresponding bioaccumulation factors (BAF) and maximum limits (ML) authorized for feed (Directive 2002/32/EC).</caption><thead><tr><th></th><th></th><th>As</th><th>Cd</th><th>Pb</th></tr></thead><tbody><tr><td rowspan="3">Wheat bran</td><td>Substrate</td><td>&lt;10</td><td>81.8 ± 1.0<sup>a</sup></td><td>22.7 ±<br>2.1<sup>a</sup></td></tr><tr><td>BSFL</td><td>23.6 ± 2.7<sup>c</sup></td><td>544.5 ± 23.6<br>******</td><td>23.3 ±<br>1.6<sup>a</sup></td></tr><tr><td>BAF</td><td>2.4</td><td>6.7</td><td>1.0</td></tr><tr><td rowspan="3">Carrot</td><td>Substrate</td><td>&lt;10</td><td>74.1 ± 1.6<sup>ab</sup></td><td>39.2 ±<br>11.5<sup>a</sup></td></tr><tr><td>BSFL</td><td>32.7 ± 1.0<sup>c</sup></td><td>471.9 ± 22.7<br>b****</td><td>39.6 ±<br>6.9<sup>a</sup></td></tr><tr><td>BAF</td><td>3.3</td><td>6.4</td><td>1.0</td></tr><tr><td rowspan="3">Apricot</td><td>Substrate</td><td>&lt;10</td><td>74.2 ± 1.6<sup>ab</sup></td><td>24.3 ±<br>3.1<sup>a</sup></td></tr><tr><td>BSFL</td><td>27.0 ± 2.9<sup>c</sup></td><td>388.0 ± 8.6<br>c****</td><td>32.7 ±<br>9.3<sup>a</sup></td></tr><tr><td>BAF</td><td>2.7</td><td>5.2</td><td>1.4</td></tr><tr><td rowspan="3">Supermarket</td><td>Substrate</td><td>88.2 ± 5.5<sup>a</sup></td><td>69.7 ± 0.7<sup>b</sup></td><td>28.1 ±<br>2.9<sup>a</sup></td></tr><tr><td>BSFL</td><td>296.2 ± 14.7<br>a***</td><td>403.9 ± 2.2<br>bc****</td><td>27.2 ±<br>1.9<sup>a</sup></td></tr><tr><td>BAF</td><td>3.4</td><td>5.8</td><td>1.0</td></tr><tr><td rowspan="3">School canteen</td><td>Substrate</td><td>110.1 ± 11.7<br>a</td><td>76.7 ± 3.4<sup>ab</sup></td><td>42.6 ±<br>2.4<sup>a</sup></td></tr><tr><td>BSFL</td><td>202.6 ± 6.6<sup>b</sup></td><td>374.3 ± 8.0<br>c****</td><td>21.3 ±<br>2.3<sup>a*</sup></td></tr><tr><td>BAF</td><td>1.8</td><td>4.9</td><td>0.5</td></tr><tr><td>ML (µg/kg DW)</td><td></td><td>2273</td><td>568</td><td>5682</td></tr><tr><td>ML (µg/kg 12 %<br>moisture)</td><td></td><td>2000</td><td>500</td><td>5000</td></tr></tbody></table>

BAF = bioaccumulation factors (BSFL concentration of a TME divided by substrate concentration of this TME). Differences between the mean concentrations in the rearing substrate and BSFL were assessed with Two-ways ANOVA followed by a Bonferroni post-hoc test, *: p < 0.05; **: p < 0.0005; ***: p < 0.0001. Different letters in the same column between the groups of substrates or BSFL indicate significant differences between the means (One-way ANOVA with Tukey post-hoc test or unpaired t-test for As in substrates, p-value < 0.05).

unauthorized substrates as feed materials regarding the regulation. Interestingly, Hoek-van den Hil et al. (2023) measured As and Pb concentrations above the ML (2.7 and 116 mg/kg 12 % moisture, respectively) in the organic fraction of municipal household waste. The As, Pb and Cd concentrations measured in this substrate were between 10 (As) to 3000 (Pb) times higher than the concentrations of the SC or SMKT substrate of this study. Similarly, concentrations of As, Cd, and Pb in carrots and apricots (24–200 µg/kg) from the Second French Total Diet Study (EAT 2, ANSES, 2011) and processed food waste (1–146 mg/kg) reported by Alagappan et al. (2022) are higher than those measured here (10–74 µg/kg). These variations underline the strong influence of sampling site, seasonality, and substrate variability on TME content.

In BSFL, As concentrations ranged from 23.6 ± 2.7 for WB to 296.2 ± 14.7 µg/kg for SMKT. Larvae reared on authorized substrates showed similar As levels while those fed on unauthorized substrates accumulated higher concentrations. Cd levels in BSFL ranged from 374.3 ± 8.0 (SC) to 544.5 ± 23.6 µg/kg (WB). Finally, Pb concentrations in BSFL did not significantly differ among groups, ranging from 21.3 ± 2.3 (SC) to 39.6 ± 6.9 µg/kg (C).

These concentrations were compared to the ML for insect used as complete feed (Directive 2002/32/EC) (Table 1). Both insects and substrates are considered feedstuff in the legislation, so the ML is the same for both categories. While As and Pb concentrations in BSFL were lower than the ML (8–100 times and 143–267 times lower, respectively), Cd concentrations were at or just below the ML. These Cd concentrations, irrespective of substrate authorization, represent a potential risk for the insect industry. This phenomenon was already observed by Hoek-van den Hil et al. (2023) where larvae reared on either the chicken feed substrate or municipal household waste exhibited Cd concentrations above the ML.

Finally, bioaccumulation was assessed by comparing TME concentrations in BSFL to those in their respective rearing substrates (Table 1 and Fig. S1). For As, bioaccumulation factors (BAF) ranged from 1.8 (SMKT) to 3.4 (SMKT), with substrate values below the LOQ replaced by the LOQ for BAF calculation. Notably, Grosso et al. (2024) also observed As bioaccumulation in BSFL reared on school canteen substrates. These findings contrast with most prior studies (van der Fels-Klerx et al., 2020; Proc et al., 2020; Purschke et al., 2017), which reported no or limited As bioaccumulation (BAF: 0.08–0.9) likely due to differences in substrate concentration or substrate type. The present study performed in industrial conditions involved lower substrate As concentrations (max 0.1 mg/kg in SC) compared to spiked laboratory studies with concentrations between 0.32 mg/kg and 3.52 mg/kg (van der Fels-Klerx et al., 2020; Proc et al., 2020; Purschke et al., 2017), suggesting that As bioaccumulation may occur preferentially at low environmental concentrations. At high concentrations, As could be toxic for BSFL, thus triggering detoxification mechanisms (Arnone et al., 2022; Deng et al., 2024). Cadmium exhibited strong bioaccumulation in BSFL, with BAF ranging from 4.9 (SC) to 6.7 (WB), consistent with literature (Biancarosa et al., 2018; Diener et al., 2015; van der Fels-Klerx et al., 2020; Hoek-van den Hil et al., 2023; Proc et al., 2020; Purschke et al., 2017; Schmitt et al., 2019; Spranghers et al., 2025). Cd is thought to cross the intestinal barrier through the calcium channels (Braeckman et al., 1999; Diener et al., 2015). However, huge disparities in BAF values were reported, ranging from 1 to 20, either due the type of substrate, the initial Cd concentration in the rearing substrate (Bulak et al., 2018) or its contamination mode, natural or spiked, which could impact its bioaccessibility (Poland et al., 2021). In contrary to the other TMEs, Pb was not bioaccumulated; its concentrations in BSFL were equal or lower than the substrate concentrations. BAF values were close to 1 for WB and carrot and slightly above 1.4 for apricot, and 0.5 for SC, meaning that Pb is only transferred to the larvae without bioaccumulation. Pb bioaccumulation appeared substrate-dependent in line with previous observations (Diener et al., 2015; van der Fels-Klerx et al., 2020; Proc et al., 2020), although some studies report BAF > 2 (Purschke et al., 2017; Tschirner and Simon, 2015).
### 3.3. POPs: PFAS, PCDD/Fs, PCBs

POPs include PFAS, PCDDs, PCDFs, non dioxin-like PCBs (NDL-PCBs) and dioxin-like PCBs (DL-PCBs). PCDD/Fs and PCBs were measured only in SC and SMKT substrates because these molecules are mostly found in food from animal origin, so potentially only present in these two bio-waste. All regulated PCDD/Fs and PCBs were found in the substrates and BSFL (Table S1). The concentrations were compared to the regulation levels in feed in the EU (Directive 2002/32/EC) in Table 2. For all categories the levels found in SMKT and SC substrates and BSFL are at least ten times lower than the action threshold or ML. Charlton et al. (2015) and Belghit et al. (2024) also obtained NDL-PCB values in BSFL below the EU regulatory limit, ranging from 0.05 to 4.28 µg/kg 12 % moisture, comparable to BSFL of this study (0.270–1.07 µg/kg 12 % moisture). Concentrations in BSFL were higher than in their respective substrates, with a significant difference in all cases except for NDL-PCBs of the SC group (Fig. 1D).

PCDD/Fs and PCBs (NDL and DL-PCBs) were bioaccumulated in BSFL with BAF between 1.25 and 2.86, in agreement with Belghit et al. (2024) (BAF: 1.2–2.4). However, the BAF range was wider when looking at individual congeners of PCBs and PCDD/Fs (Table S1): from 0.37 (HpCDF) to 4.37 (PCB 52) for SMKT BSFL and from 0.73 (PeCDD) to 5.44 (PCB 81) for SC BSFL. Independently of the type of substrate, there was systematically more than 50 % of PCDD/Fs and PCBs that had a BAF above 1 and even 100 % of PCBs for the SMKT BSFL. TCDD, which is the reference value for PCDD in toxic equivalency factor (TEF) terms (TEF = 1), had a BAF of 2.0 for SMKT BSFL and 2.1 for SC BSFL. Overall, the most toxic molecules; with the highest TEF values; were bioaccumulated by BSFL (Table S1). Bioaccumulation was also substrate-dependent, for example the BAF for PCDDs in SMKT was 1.4 while for SC it was 2.9, as observed by van der Fels-Klerx et al. (2020) on various former food-stuffs. Pajurek et al. (2023) concluded that PCDD/Fs and PCBs will not pose a risk for insect to be used as feed because of the low concentrations in BSFL compared to the legislation. However, Belghit et al. (2024) showed that BSFL reared on aquaculture sludge exceeded the ML for PCDD/Fs and PCDD/Fs + DL-PCBs because of the bioaccumulation and the initial concentrations in the sludge 20 times higher than in the present SMKT and SC substrates. These biowaste are highly heterogeneous and the POPs concentrations in animal products contained in these biowastes are dependent of multiple variables. Therefore, if the proportion of animal products in biowaste used as substrate was higher than in the present study, PCDD/Fs and PCBs concentrations in the substrate could also be higher and consequently higher PCDD/Fs and PCBs concentrations in BSFL could be expected. Therefore, the fact that BSFL bioaccumulate these molecules including the most toxic ones is quite concerning, despite concentrations being 10–20 times lower than recommended by European legislation for feedstuffs (Table 2).

PFAS were detected in all rearing substrates but none of them were quantifiable (results < LOQ) (Table S2). Although these results were obtained by a certified laboratory, they should be treated with caution as Wakefield et al. (2024) identified br-PFHxS in supermarket waste and PFHpA in catering waste. Indeed, the intra substrate variability is high, which could explain these results. Wakefield et al. (2024) also reported

low and inconsistent recoveries and chromatographic retention shifts when analyzing the same type of samples, suggesting potential underestimations of the real PFAS concentrations.

PFAS were not quantifiable in the rearing substrate so they were not analyzed in BSFL since this study aims at assessing the potential bio-accumulation of chemical contaminants in larvae. Moreover, Li and Bischel (2022) reported no bioaccumulation of PFBA, PFOA, PFBS and PFOS in BSFL. Wakefield et al. (2024) found the highest PFAS level in larvae for PFOA in BSFL reared on poultry manure, with a mean of 7.4 µg/kg. However, for two PFAS compounds, the levels were higher in BSFL than in the substrate, suggesting a potential bioaccumulation: HFPO.DA in larvae from manufacturing waste (substrate 0.14 µg/kg; larvae 0.20 µg/kg) and BrPHFX in larvae from supermarket surplus (substrate 0.49 µg/kg; larvae 0.68 µg/kg). Further studies are thus required to clarify the bioaccumulation phenomenon in BSFL.

### 3.4. Pesticides

Among the 511 pesticides investigated (Table S3), 4 were identified in the substrates: chlormequat (in carrot, apricot and salad substrates), phosmet (in apricot substrate), spirotetramat-BYI08330-enol (in salad substrate) and propamocarb (in SMKT substrate). The authorized substrates came from fruits and vegetables grown following sustainable agricultural practices, using the lowest amount of pesticide possible. It is also possible that the fermentation step during the preparation of the substrates has influenced their pesticide concentration (Regueiro et al., 2015). The chlormequat pesticide is used as a plant growth regulator and is widely used in the cereal industry under the name chlormequat chloride. For example, Reynolds et al. (2004) observed concentrations of chlormequat in the range of 0.2–1.5 mg/kg in WB. Therefore, its presence in the substrate can be traced back to the WB used to produce the different substrate blends. The phosmet was the only pesticide quantified, above the LOQ, in the apricot substrate (35.3 ± 4.6 µg/kg) (Table 3). Its presence in apricot, however, was not reported in the second French Total Diet Study (ANSES, 2011), in which apricot blends were analyzed, which may have overwrite extreme values and thus contamination. According to Regulation (EC) No 396/2005, the maximum residue limit (MRL) for phosmet in apricot used as feed is 10 µg/kg, therefore this feed does not comply with EU regulation. Hoek-van den Hil et al. (2023) detected more than 15 pesticides from the organic fraction of municipal household waste substrate, different from the ones detected in this study, despite being part of the pesticide screened list. Again, considering the heterogeneity of the composition of this type of substrate, depending of the sampling site, eating habits, season and agricultural practices, it is not surprising to obtain different results.

With the results obtained on the substrates, only BSFL raised on apricots, salad and SMKT substrates were investigated focusing on the 4 pesticides detected previously in these substrates. Chlormequat was the only pesticide quantified in all BSFL groups (Table 3). Although Spranghers et al. (2025) observed an impact of the nutritional composition of the substrate on the pesticide transfer in BSFL, our results indicate that there are no significant differences in chlormequat concentrations between BSFL reared on the different substrates considered

<caption>**Table 2**</caption>

PCDD/F and PCB concentrations (mean ± SEM) found in supermarket and school canteen substrates and corresponding black soldier fly larvae (BSFL) and comparison with regulatory limits in feed in the European Union (Directive 2002/32/EC).

<table><thead><tr><th rowspan="2"></th><th colspan="2">Regulatory limits</th><th colspan="2">Supermarket</th><th colspan="2">School canteen</th></tr><tr><th>Maximum level</th><th>Action threshold</th><th>Substrate</th><th>BSFL</th><th>Substrate</th><th>BSFL</th></tr></thead><tbody><tr><td>PCDDs + PCDFs (TEQ ng/kg 12 % moisture)</td><td>0.75–1.75</td><td>0.5–1.25</td><td>0.025 ± 0.005</td><td>0.034 ± 0.000</td><td>0.019 ± 0.004</td><td>0.028 ± 0.001</td></tr><tr><td>PCDDs + PCDFs + DL-PCBs (TEQ ng/kg 12 % moisture)</td><td>1.5–5.5</td><td></td><td>0.043 ± 0.007</td><td>0.082 ± 0.004</td><td>0.029 ± 0.004</td><td>0.055 ± 0.002</td></tr><tr><td>DL-PCBs (TEQ ng/kg 12 % moisture)</td><td></td><td>0.35–2.5</td><td>0.018 ± 0.002</td><td>0.048 ± 0.004</td><td>0.010 ± 0.001</td><td>0.026 ± 0.001</td></tr><tr><td>NDL-PCBs (µg/kg 12 % moisture)</td><td>10</td><td></td><td>0.45 ± 0.04</td><td>1.07 ± 0.06</td><td>0.22 ± 0.02</td><td>0.27 ± 0.00</td></tr></tbody></table>

In the regulation, the ML is given in toxic equivalency (TEQ) values. TEQ concentrations were calculated using the individual toxic equivalency factor (TEF) from 2022 of each congener (Devito et al., 2024). TEF values represents the degree of toxicity of a congener; the higher the TEF value, the higher the toxicity of the congener.
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Sum of PCDDs (A), PCDFs (B), DL-PCBs (C) and NDL-PCBs (D) (mean ± SEM) in supermarket waste, school canteen waste (n = 3) and BSFL reared on these substrates (n = 2). Differences between the mean concentrations in the rearing substrate and BSFL were assessed with two-ways ANOVA followed by a Bonferroni post-hoc test, *: p < 0.05; **: p < 0.005; ***: p < 0.0005; ****: p < 0.0001. The framed numbers are the bioaccumulation factors (mean BSFL concentration in each family of contaminant divided by the mean substrate concentration of the same family of contaminant).</figcaption></figure>

<caption>**Table 3**</caption>

Concentrations of pesticides (µg/kg DW) detected above the LOQ in apricot, salad and supermarket substrates (n = 3) and the corresponding pools of BSFL (n = 2) and comparison with regulatory limits in feed in the European Union (Regulation (EC) No 396/2005).

<table><thead><tr><th rowspan="2">Pesticides</th><th colspan="2">Apricots</th><th colspan="2">Salad</th><th colspan="2">Supermarket</th></tr><tr><th>Substrate</th><th>BSFL</th><th>Substrate</th><th>BSFL</th><th>Substrate</th><th>BSFL</th></tr></thead><tbody><tr><td>Chlormequat<br>(+ salts)</td><td>d &lt; LOQ</td><td>15.5<br>± 0.5</td><td>d &lt; LOQ</td><td>14.0<br>± 0.0</td><td>d &lt; LOQ</td><td>14.5<br>± 1.5</td></tr><tr><td>Phosmet</td><td>35.3 ±<br>4.7</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>Regulatory<br>limits*</td><td>10</td><td>10</td><td>10</td><td>10</td><td>NA</td><td>10</td></tr></tbody></table>

LOQ = 10 µg/kg DW; ND = Not detected; NA = Not Applicable because supermarket substrate is not allowed for animal feed; * applicable for both pesticides.

No significant differences in the mean chlormequat concentrations between groups of BSFL were found (one-way ANOVA, p < 0.05).

in this study. This pesticide was transferred to the larvae from the substrate, more specifically from the WB used to produce the different substrate blends. As the same amount of WB was present in every substrate (30 %, w/w), this could explain why BSFL reared on different substrates contain the same amount of chlormequat. However, as the

chlormequat concentrations in larvae (14.0–15.5 µg/kg) were close to the LOQ (10 µg/kg) and the concentrations in the substrates were below the LOQ, it is not possible to conclude precisely on the bioaccumulation of chlormequat in BSFL. Charlton et al. (2015) did not detect any pesticide in BSFL reared on a mixture of brewery solid waste, dry fish feed, factory waste and yeast. Similarly, Hoek-van den Hil et al. (2023) did not measure any pesticide above the LOQ among the 268 pesticides screened in BSFL when reared on an organic household fraction substrate. Regarding other pesticides, most of the previous studies showed no bioaccumulation, even suggesting degradation in some cases, of 24 pesticides, fungicides, herbicides or insecticides in BSFL reared on spiked substrates (Lalander et al., 2016; Meijer et al., 2021, 2023; Purschke et al., 2017; Spranghers et al., 2025). Nevertheless, bifenthrin was bioaccumulated by BSFL (BAF = 2.2) according to Li and Bischel (2022). Therefore, as pesticide bioaccumulation seems molecule-dependent, further studies including a step of spiking into the rearing substrate would be necessary to thoroughly evaluate the phenomenon of chlormequat bioaccumulation. The EU legislation has set a limit of 10 µg/kg for chlormequat in feedstuff (Regulation (EC) No 396/2005). The concentrations measured in the larvae were thus above but very close to the authorized limit, even for BSFL reared on authorized substrates such as apricots and salad. Regarding chlormequat chloride, only one study on *Tenebrio molitor* is available among all edible insect species (Dreassi et al., 2020). Unstarved larvae presented relatively low concentrations
of chlormequat chloride compared to the substrate. Moreover after 24 h of fasting, a complete elimination of the pesticide was reported and after 18 h of fasting, BSFL gut is totally emptied (Gold et al., 2025), suggesting that chlormequat was naturally excreted with the gut content and was not transferred to other tissues. Besides, Houbraken et al. (2016) observed in *Tenebrio molitor* that pesticides with lower log K<sub>OW</sub> are excreted more easily suggesting that chlormequat, as an hydrophilic pesticide (log K<sub>OW</sub> = -1.58), could be easily excreted. In our study, larvae were not starved but it could be a strategy to mitigate the chlormequat content in BSFL in the future.

### 3.5. Mycotoxins

Among the mycotoxins analyzed in the substrates (Table S4), only zearalenone and ergot alkaloids were detected at concentrations above the LOQ. Zearalenone was quantified in WB (0.02 mg/kg FW), carrots, apricots, salad and SMKT substrates (0.01 mg/kg FW for each substrate). Zearalenone was already measured in WB from Greece, with concentrations ranging from 0.021 to 0.023 mg/kg (Irakli et al., 2017), in agreement with our results. The sum of ergot alkaloids was 46.0 ± 0.0 and 65.7 ± 14.0 µg/kg FW in WB and SMKT substrates, respectively. These concentrations are much lower than the ML (1000 mg/kg 12 % moisture, Directive 2002/32/EC) and those reported by Mavungu et al. (2011) at 174 µg/kg among 18 WB samples from Belgian shops. In the present study, the substrates were prepared according to standard industrial rearing conditions. They were fermented and mixed with WB as explained in the Material and Methods section. Previous studies have shown that food fermentation could be a strategy for mycotoxin degradation (Adebiyi et al., 2019; Gavahian et al., 2021). Therefore, the concentrations in mycotoxins presented here might have been impacted by the industrial preparation steps. It is noteworthy that Wakefield et al. (2024) discovered other mycotoxins, currently not regulated in feed, in supermarket surplus (Enniatin B1, moniliformin, penicillic acid) and in catering substrate (Roquefortune C). Therefore, it would be interesting to expand the list of analyzed mycotoxins to deepen the future risk assessment.

Ergot alkaloids, although found in the different substrates, were below the LOQ (0.04 mg/kg DW) in all larvae (Table S5). Therefore, these molecules are not bioaccumulated by BSFL and are not of concern for the assessment of the chemical safety of BSFL. These results on mycotoxins agree with previous studies (Charlton et al., 2015; Purschke et al., 2017). Charlton et al. (2015) did not find any mycotoxin above the LOQ in BSFL reared on a mixture of brewery waste, dry fish feed factory waste and yeast. However, deoxynivalenol (DON) was detected by Purschke et al. (2017) in the residual substrate obtained after BSFL rearing on corn semolina spiked with a mix of mycotoxins (DON, aflatoxin B1, aflatoxin B2, aflatoxin G2 and ochratoxin A). They hypothesized that there were, in the rearing substrate, DON derivatives, bound to carbohydrates or proteins, not extracted by their extraction method. Nevertheless, they can be extracted in the frass after digestion of macronutrients by BSFL enzymes. Therefore, an underestimation of mycotoxins in the different substrates and BSFL cannot be excluded, and further studies are needed to verify this hypothesis. Mycotoxins did not affect the larval growth, survival rate or body mass of BSFL, suggesting a high tolerance to these contaminants (Bosch et al., 2017; Camenzuli et al., 2018; Meijer et al., 2019). Camenzuli et al. (2018) and Meijer et al. (2019) even suggested that mycotoxins could be degraded by BSFL, which means that potential degradation products and their toxicity will need to be studied in the future.

### 3.6. Estimation of the fate of chemical contaminants in laying hens and pigs fed with BSFL raised on supermarket substrate

In order to assess the chemical risk in human food resulting from livestock fed with BSFL reared on these different substrates, transfer models of chemical contaminants were used. A worst-case scenario was

chosen by using the highest concentrations in chemical hazards, found in BSFL reared on SMKT substrate (Tables 1 and 2). Laying hens and pigs figure among the authorized animals to be fed with BSFL (Commission Regulation (EU) 2021/1372). Using transfer models (www feedfoodtr ansfer.nl), it was possible to estimate the concentration of NDL-PCBs and the sum of PCDD/Fs + DL-PCBs in egg yolk fat and body fat from laying hens fed exclusively with SMKT BSFL. Similarly, the concentrations in total PCDD/Fs and DL-PCBs in pigs body fat and Cd concentrations in liver and kidney of pigs fed with 10 % of SMKT BSFL were calculated over time. The obtained data were then compared to the regulatory limits (Table 4). According to the model, none of the chemical contaminants followed would have been transferred at concentrations above the maximum regulatory limits for these animal products. For example, the total NDL-PCBs concentration was estimated at 0.009 ng/g egg yolk fat, which is 4440 times lower than the ML. The sum of DL-PCBs and PCDD/Fs in laying hen body fat (estimated at 0.6 pg TEQ/g body fat) was also 5 times lower than the ML. For these chemical contaminants, and based on the model used in the present study, raising BSFL on an unauthorized substrate such as SMKT waste and their subsequent use as feed does not seem to lead to a biomagnification phenomenon along the food chain. Moreover, this was a worst-case scenario and the proportion of BSFL incorporated in animal feed is generally lower than 100 % for laying hen. Indeed 25 % of BSFL meal replacement for laying hens is recommended (Bovera et al., 2018; Schiavone et al., 2017) while pig experiments incorporated maximum 10 % of BSFL meal (Hong and Kim, 2022). Furthermore, this meal could be at least partially defatted according to the literature, meaning that lipophilic contaminants such as PCDD/Fs and PCBs might be partially eliminated during the process. Nonetheless, this model was not developed for insects as feed, and since the transfer level of chemical contaminants from the diet is matrix-dependent (Rose et al., 2012), these theoretical estimates must be validated *in vivo*. Besides, the chemical contaminant concentrations

<caption>**Table 4**</caption>

Concentrations and regulatory limits (Commission Regulation (EU) 2023/915) of NDL-PCBs, PCDD/Fs + DL-PCBs and Cd in egg yolk fat and body fat of laying hens and kidney and liver of pigs estimated using www feedfoodtransfer.nl.

<table><thead><tr><th>Animal</th><th>Contaminant</th><th>Egg yolk fat</th><th>Body fat</th><th>Kidney</th><th>Liver</th></tr></thead><tbody><tr><td rowspan="3">Laying hen</td><td>NDL-PCB (ng/g tissue)</td><td>0.009</td><td>0.009</td><td></td><td></td></tr><tr><td>PCDD/Fs +<br>DL-PCBs (total<br>TEQ) (pg TEQ/<br>g tissue)</td><td>40</td><td>40</td><td></td><td></td></tr><tr><td>Pig</td><td>PCDD/Fs +<br>DL-PCBs (total<br>TEQ) (pg TEQ/<br>g tissue)</td><td>5</td><td>3</td><td></td><td></td></tr><tr><td></td><td>Cadmium<br>(mg/kg tissue)</td><td>0.04</td><td>0.04</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td>1.25</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>0.06</td><td>0.015</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>0.5</td></tr></tbody></table>

Some cells are left empty because the regulation only applies to one of the two species, depending on the animal product concerned.
in BSFL used for these estimations were obtained with only one type of SMKT waste, which are highly variable and thus these results are not transferable to other biowaste. For example, the sum of DL-PCBs and PCDD/Fs in laying hen body fat, 5 times lower than the ML, could exceed this limit with higher concentrations of these contaminants in other biowaste, particularly in view of the high BAF observed in the present study. A study from Heuel et al. (2023) has already investigated the transfer of Cd, Pb and Aflatoxin B1 from BSFL substrate to eggs and meat of laying hens fed with BSFL in vivo. They did not find any Pb in the meat and eggs and no Cd in the eggs, Cd and Pb concentrations in the tissues were also lower than the ML, except for the kidneys. Biancarosa et al. (2019) also demonstrated that Atlantic salmon fed with BSFL meal did not accumulate As. Moreover, Pajurek et al. (2023) have shown that PCBs and PCDD/Fs intake related to consumption of poultry meat and livers were not different when poultry were fed insect protein compared to traditional feed. These preliminary results, which are only valid for the specific SMKT substrate tested, seem to indicate that recycling yet-unauthorized biowaste with BSFL do not entail safety issues when introduced in the food chain.

European legislation regarding chemical contaminants in feed and substrate authorizations is among the strictest in the world. While the current study primarily targets the EU insect sector for feed, the results could also be applied to the US or Singaporean insect rearing systems, as the SMKT and SC biowaste are also unauthorized in these countries (Lähteenmäki-Uutela et al. 2021; Singapore Food Agency 2024). On the contrary, countries like Thailand which have more permissive legislation regarding substrate authorization and feed safety, could use these results to refine the risk assessment related to the insect-based bioconversion of SC and SMKT biowaste (Krongdang et al. 2025).

## 4. Conclusion

PCDD/Fs, PCBs, As and Cd were bioaccumulated in BSFL, with bioaccumulation factors up to 5.4 for PCDD/Fs and PCBs and 6.7 for Cd. Concentrations of Cd in all BSFL were just below or at the ML (0.5 mg/kg 12 % moisture) and the chlormequat pesticide was quantified above the MRL (15 µg/kg) in BSFL reared on different substrates. The unauthorized biowaste of SMKT or SC used in this study overexposed BSFL to concerning chemical contaminants such as As, PCDD/Fs and PCBs. Nevertheless, BSFL concentrations were below the ML and theoretical calculations do not suggest safety issues related to animal products from animal fed with these larvae. For a more comprehensive risk assessment, other chemical contaminants should also be included such as veterinary and drug residues, polycyclic aromatic hydrocarbons, microplastics, and other toxic TME such as mercury. Further research with other unauthorized substrates is also necessary to validate that these biowaste are safe for insect rearing and the higher levels of the trophic chain.

### CRediT authorship contribution statement

**Marie Papin:** Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization. **Maxime Pattier:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Mélanie Davranche:** Writing – review & editing, Resources, Methodology. **Patrick Borel:** Writing – review & editing, Supervision, Conceptualization. **Erwan Engel:** Writing – review & editing, Supervision, Resources, Funding acquisition, Conceptualization. **Christelle Planche:** Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

This work was supported by INRAE Metaprogramme Bioeconomy for urban areas (BETTER). The authors thank Inovalys for the pesticides and mycotoxins analysis and Laberca for the PFAS, PCBs and PCDD/Fs analysis. The authors also thank the company BioMiMetiC for having performed larval rearing.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.wasman.2025.115219.

### Data availability

Data will be made available on request.

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