Multi-Elemental Analysis of Edible Insects, Scorpions, and Tarantulas from French (Online) Market and Human Health Risk Assessment Due to Their Consumption: A Pilot Study

Edible insects are becoming increasingly popular as protein alternatives to traditional animal-based products. As such, information on their elemental composition is important to ensure they are safe for human consumption. This article describes the development and validation of a rapid, reliable method for the simultaneous determination of 19 elements (Al, As, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, Pb, Se, Sr, and Zn) in edible insects by inductively coupled plasma mass spectrometry (ICP-MS) following closed vessel microwave digestion. The method was validated using three insect certified reference materials, namely black soldier fly larvae meal (BFLY-1), cricket flour (KRIK-1), and mealworm powder (VORM-1). The method was applied to analyze twelve different (whole) insect species. The maximum amount of each sample was calculated for As, Cd, and Pb with respect to their provisional tolerable daily intake values established by the Food and Agricultural Organization/World Health Organization. Most of the samples, except for scorpions and tarantulas, were safe to consume at large doses (1000–10,000 insects per day). Furthermore, most of the samples contained high levels of Fe, K, Na, and Zn, providing a preliminary overview of the nutritional profile of these novel protein alternatives.


Introduction
Entomophagy is a term used to describe the practice of eating insects [1].While common in some countries, Western regions are still overcoming psychological and cultural barriers [2].Recent efforts have been made to eliminate these barriers and promote the consumption of insects as an alternative protein source, given that the world's population is estimated to reach approximately 9.7 billion by 2050 and a shortage of current animal-based proteins is expected [3][4][5].An increasing number of edible insect products are emerging on the market, providing consumers with environmentally friendly and nutritious options, taking into account that insects consume less water and land, emit less greenhouse gases compared to livestock, and they contain proteins, essential vitamins, and minerals [6][7][8][9][10].However, information on the total elemental composition of these commercially available edible insects is required to ensure their safety for human consumption.
Trace elements in the human body, which are generally present at levels < 250 µg g −1 are classified as essential, nonessential, and potentially toxic, depending on the dose and duration of intake [11][12][13].Essential trace elements play a vital role in human health and functions.They include Cu and Fe, which participate in energy metabolism via oxidationreduction reactions, and Fe enables transport of oxygen throughout the body via the formation of hemoglobin [11,13].Meanwhile, potentially toxic elements (PTEs) include As, Cd, Hg, and Pb, which can lead to cancer and damage to the nervous system and organs Foods 2024, 13, 2353 2 of 13 when consumed at high doses for long periods of time [11,[13][14][15][16].There is a possibility that insects, such as the black soldier fly larvae (BSFL, Hermetia illucens) that convert organic matter into biomass, are bioaccumulating these PTEs from their feed, posing health risks for humans if ingested [17,18].To minimize these risks, trace element levels in edible insects must be monitored.
The aim of this study was to develop and validate a rapid and reliable method for the determination of minor, major, potentially toxic, and essential elements in a selection of commercially available edible insects using ICP-MS.For this purpose, nineteen trace elements were studied: Al, As, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, Pb, Se, Sr, and Zn.As the proposed method has not previously been applied to such matrices, it was validated through the analysis of several insect certified reference materials (CRMs) before being applied to the analysis of real-life insect samples.
An Agilent 8900 ICP tandem mass spectrometry (ICP-MS/MS) instrument (Agilent Technologies, Courtaboeuf, France) equipped with a MicroMist concentric nebulizer and Scott-type double-pass quartz spray chamber was used for analysis.Sample digests were introduced directly into the instrument via a peristaltic pump from tubes connected to an SPS 4 autosampler (Agilent Technologies, Courtaboeuf, France).The operating conditions are detailed in Table 1.These parameters were optimized daily by performing short-term stability tests with the tuning solution (1 mg L −1 ) in both detection modes (standard and collision mode) to ensure maximum sensitivity and minimal interference from oxide (CeO + /Ce + < 1.2%) and doubly charged ions (Ce 2+ /Ce + < 2%).The signals were obtained using Scientific MassHunter software (Agilent Technologies) and the raw data were processed using Excel.were used throughout the study.High purity argon (99.996%,Linde Gas, Montereau-Fault-Yonne, France) was used for the plasma, auxiliary, and nebulizer gases.
Stock solutions containing 1000 mg L −1 of each analyte (Al, As, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, Pb, Se, Sr, and Zn) were purchased from LGC Standards (Molsheim, France) and were used to prepare calibration standard solutions daily in 6% (v/v) HNO 3 .
Internal standard solutions were prepared using 1000 mg L −1 standard stock solutions of Y, Re, and Bi that were purchased from LGC Standards.These solutions were added to all samples, calibration standards, and blanks to compensate for drift.
A tuning solution and a Factor P/A solution were prepared separately using multielement solutions (Agilent Technologies) to ensure optimal instrument sensitivity over a wide range of masses and linear response of the detector between pulse and analog detection modes, respectively.
Solutions of HNO 3 at 6% (v/v) and 10% (v/v) were used for rinsing the ICP-MS/MS system between analyses.

Reference Materials and Samples
Three insect-based CRMs provided by the National Research Council Canada (Ottawa, ON, Canada) were used throughout the study to validate the method, namely: BFLY-1 (black soldier fly larvae meal), KRIK-1 (cricket flour), and VORM-1 (mealworm powder).The mussel tissue ERM-CE278k CRM from the Institute for Reference Materials and Measurements (LGC Standards) was also used throughout the study as an internal quality control sample.
Fourteen dry samples including authorized edible crickets (Acheta domesticus), worms (Tenebrio molitor), ants (Atta laevigata), water bugs (Nepidae), locusts (Locusta migratoria), scorpions (Heterometrus longimanus), and tarantulas (Haplopeima albostriatum) were purchased from the French market and online.To ensure sufficient quantities for analysis, multiple packages of some samples were purchased.Although these packages originated from the same batch, they were each treated as a single sample to distinguish any variations between packages, which led to the analysis of 27 samples (see Table 2 for details).Information on the life stage and origin of the samples is not available.Each sample was removed from its original packaging and stored at room temperature in a closed polyethylene tube while awaiting preparation for analysis.As most packages contained multiple insects, the total weight (in g) was measured.From this, the weight per insect (in g) was approximated by weighing 20 whole insects.
Each sample was then homogenized using a BM500 benchtop laboratory ball mill (Anton Paar, Courtaboeuf, France) and three 10 mm agate beads.The grinding frequency and time varied depending on the type of insect.Those with hard exoskeletons (scorpions, water bugs, and tarantulas) were ground at 30 Hz for 2 min, whereas others were ground at 25 Hz for 45 s.

Multi-Elemental Analysis by ICP-MS/MS
Approximately 0.3 g of homogenous sample was weighed in a quartz digestion vial, to which 3 mL of HNO 3 (67%, v/v) and 3 mL of ultrapure water were added.The samples were then completely digested using a closed microwave digestion system for 30 min at 250 • C and a pressure of 140 bar.Once cooled to room temperature, the sample solutions were transferred into 50 mL polyethylene flasks and 100 µL of a mixture of internal standard solution (Y, Re, and Bi) were added before final dilution to 50 mL with ultrapure water and subsequent analysis by ICP-MS.

Data Processing
Concentrations were expressed in mg kg −1 .Calibration curves were produced for each analysis series to verify linearity (R 2 ≥ 0.995).Humidity was measured in all three insect CRMs to correct the result.A Grubbs test at the 95% confidence level was performed to identify and remove any outliers in the experimentally obtained data.
To determine the method's accuracy when analyzing the three insect CRMs, the difference between the certified and measured values (∆ m ) were compared to their combined uncertainties (U ∆ ) using the Equations ( 1)-(3) [32,33]: where c m is average measured concentration, c CRM is certified concentration, s m is standard deviation of the measured value, and s CRM is uncertainty of the certified value.Note, a coverage factor (k) of 2 was used, corresponding to 95% confidence level.

Method Validation
To ensure that the proposed method is applicable to insect matrices, three CRMs (BFLY-1, KRIK-1, and VORM-1) were analyzed in triplicates on three different days (n = 9, unless otherwise specified) over a period of seven weeks.The comparison of the average total concentration and standard deviation determined for each of the 19 elements in the three CRMs and the corresponding certified value is provided in Figure 1.
Foods 2024, 13, x FOR PEER REVIEW 5 of 14 insect CRMs to correct the result.A Grubbs test at the 95% confidence level was performed to identify and remove any outliers in the experimentally obtained data.
To determine the method's accuracy when analyzing the three insect CRMs, the difference between the certified and measured values (Δm) were compared to their combined uncertainties (UΔ) using the Equations ( 1)-(3) [32,33]: where cm is average measured concentration, cCRM is certified concentration, sm is standard deviation of the measured value, and sCRM is uncertainty of the certified value.Note, a coverage factor (k) of 2 was used, corresponding to 95% confidence level.

Method Validation
To ensure that the proposed method is applicable to insect matrices, three CRMs (BFLY-1, KRIK-1, and VORM-1) were analyzed in triplicates on three different days (n = 9, unless otherwise specified) over a period of seven weeks.The comparison of the average total concentration and standard deviation determined for each of the 19 elements in the three CRMs and the corresponding certified value is provided in Figure 1.Overall, most of the elements had average percent recoveries ranging from 90 to 110% for all three CRMs, hence confirming the method's fit for the analysis of insects.Furthermore, upon comparing Δm with UΔ (Table S1), all the elements except for Co, K, and Se in BFLY-1, were found to have Δm values lesser than UΔ, indicating that there were no statistically significant differences between the measured and certified concentrations.Overall, most of the elements had average percent recoveries ranging from 90 to 110% for all three CRMs, hence confirming the method's fit for the analysis of insects.Furthermore, upon comparing ∆ m with U ∆ (Table S1), all the elements except for Co, K, and Se in BFLY-1, were found to have ∆ m values lesser than U ∆ , indicating that there were no statistically significant differences between the measured and certified concentrations.

Analysis of Commercially Available Edible Insects
The levels of 19 elements measured in commercially available edible insects are presented as the following: PTEs, major elements, essential elements, and others (see Tables 3-6, respectively).It is worth to note that amongst the species analyzed in this study, only four are currently authorized as novel food in the European Union (EU), namely the meal-worm (Tenebrio molitor), the lesser mealworm (Alphitobius diaperionus), the locust (Locusta migratoria), and the cricket (Acheta domesticus) [34].
The Food and Agricultural Organization/World Health Organization established provisional tolerable daily intake (PTDI) values for As, Cd, and Pb of 2.10 µg kg −1 , 0.82 µg kg −1 , and 3.57 µg kg −1 bodyweight per day, respectively, in this type of foodstuffs [35,36].The maximum amount of whole edible insects that can be safely consumed daily for a 60 kg adult and an 18 kg child was calculated using the approximated weight per insect (in g) and is summarized in Table 3.
Based on the measured concentrations, it appears that most of the samples, notably the crickets, locusts, and worms, can be safely consumed at rather large quantities (from 1000 to 100,000 individuals) by both children and adults.However, the maximum number of scorpions that can be consumed in terms of Cd were significantly lower than the other samples, with 12-32 recommended for adults and 4-10 for children.
Moreover, in the case of tarantula samples, the consumption of only 1 individual per day is recommended for children and 2-3 for adults.However, this finding should not pose too much concern considering the context in which insects are being considered as a protein alternative.For instance, in Europe, insects are marketed as a food ingredient, to be added in crackers, pasta, etc. [34].
With regard to the samples originating from the same lot, similar contamination patterns were observed for the ants, crickets 1-3 and 7-8, and worms 4-6 samples.Contrarily, measured results varied significantly between the following samples (see Tables 3-5 and 7): crickets 5-6, locusts 1-2, scorpions 3-4, tarantulas 1-3, water bug 1-2, and worms 1-3.Larger variances observed in scorpions, tarantulas, and water bugs can be explained by the number of individuals in each lot.In fact, especially for these species, one lot was composed of only one or two individuals.
Table 4 summarizes the levels of major (naturally abundant) elements (Na, K, Ca, and Mg) in each sample.Overall, high levels of K were observed for all the samples (from 4-to 10-fold compared to Ca, Mg, and Na), suggesting that insects are rich in this element.This is beneficial as K plays a role in proper kidney and heart regulation, muscle contraction, and nerve transmission [37].
Table 3. Concentrations ± standard deviation (n = 2; µg kg −1 ) of potentially toxic elements in edible insects, scorpions, and tarantulas (with corresponding sample name) by ICP-MS as well as the number of individual of each type that can be safely consumed (per day) in relation to provisional tolerable daily intake (PTDI) a for human adults b and children c .Although natural samples (free from additives or other ingredients besides the insect) were purchased for this study, some were found to have salt added to them (see ingredients listed in Table 2).This was reflected by high levels of Na being reported in the scorpion and tarantula samples (up to 10,597 and 40,454 mg kg −1 , respectively).Sodium, being an essential nutrient, helps maintain plasma volume, acid-base balance, nerve transmissions, and healthy cell functions [38].Table 7 compares the results obtained in previous studies with those obtained in this work for potential toxic and major elements.Many results are comparable despite the samples being from different origins, prepared differently, etc.For example, two out of three samples of cricket in the previous study had up to 10-38% of seasonings, whereas only whole dehydrated crickets were analyzed in this work.Yet, except for As, the levels of Cd, Pb, Ca, Mg, K, and Na are fairly similar.The largest differences were observed for water bugs, which were from India in the previous study.
As can be seen in Table 5, high Cu levels were measured in the scorpion (81-130 mg kg −1 ) and tarantula samples (80-121 mg kg −1 ).Copper is involved in energy production, iron metabolism, brain development, and immune system functioning [39].Most of the samples (crickets, locusts, worms, scorpions, ants, tarantulas, and water bugs) also contained high levels of Fe, ranging from 33 to 474 mg kg −1 .This element is necessary for human growth and development, with it circulating oxygen throughout the body [40].Lastly, high levels of Zn were observed for all the samples (from 100 mg kg −1 to 1000 mg kg −1 ).This is noteworthy, as zinc is involved in cellular metabolism (catalytic activities of enzymes, protein and DNA synthesis), immune health, and growth and development [41].Table 8 shows that the levels of several essential elements are consistent with those previously obtained in similar samples.Differences may be attributed to different sample origin, sample preparation, etc.The systematically lower concentrations in water bugs reported previously might be a result of the legs and antennas having been removed prior to analysis, whereas the whole insect was analyzed in this work.

Table 2 .
List of edible insects, scorpions, and tarantulas samples used in the study with corresponding code number, sample name, ingredients, and origin (if available).
a c weight of 18 kg.

Table 4 .
Concentrations ± standard deviation (n = 2; mg kg −1 ) of major elements in edible insects, scorpions, and tarantulas (with corresponding sample name) measured by ICP-MS.

Table 6 .
Concentrations ± standard deviation (n = 2; mg kg −1 ) of remaining elements in edible insects, scorpions, and tarantulas (with corresponding sample name) measured by ICP-MS.