1575 Journal of Food Protection, Vol. 84, No. 9, 2021, Pages 1575–1581 https://doi.org/10.4315/JFP-21-099 Published 2021 by the International Association for Food Protection Copyright Ó, Her Majesty the Queen in Right of Canada, as represented by the Canadian Food Inspection Agency. This is an open access article. Research Paper Analysis of Microbiological and Chemical Hazards in Edible Insects Available to Canadian Consumers BEATA M. KOLAKOWSKI https://orcid.org/0000-0002-5604-6978,* KRYSTYNA JOHANIUK, HELEN ZHANG, AND ETSUKO YAMAMOTO Canadian Food Inspection Agency, Food Safety Science Services Division, 1400 Merivale Road, Ottawa, Ontario, Canada K1A 0Y9 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/84/9/1575/2893105/i0362-028x-84-9-1575.pdf by guest on 26 September 2021 (ORCID: https://orcid.org/0000-0003-4786-3535 [H.Z.]; https://orcid.org/0000-0001-5533-4540 [E.Y.]) MS 21-099: Received 10 March 2021/Accepted 5 May 2021/Published Online 6 May 2021 ABSTRACT Edible insects are a novel food in most countries; their popularity is growing because of their high-protein and low-fat content, ease of cultivation, and small environmental impact. To our knowledge, this is the first report that addresses both microbiological and chemical hazards in edible insects. Samples were collected from retail stores or purchase through e- commerce. A total of 51 samples of dried whole insects or insect powder were tested for Escherichia coli, which serves as an indicator of the overall sanitation conditions throughout the food production chain, and the bacterial pathogen Salmonella spp. Neither Salmonella spp. nor E. coli (.100 CFU/g) was found in the samples analyzed. A total of 43 samples of crickets (protein bars, powders, flour, and whole insects) and 4 samples of silkworm (whole insects) were analyzed for up to 511 pesticides. Of these, 39 samples contained up to four pesticides; 34 samples were compliant and 5 samples were noncompliant with Canadian regulations. Seven pesticide residues were detected, with glyphosate and its metabolite, aminomethylphosphonic acid, as the predominant residues. Nineteen of the samples tested for pesticides were also analyzed for arsenic, cadmium, mercury, and lead; there was insufficient material remaining to allow testing of pesticides and metals. The positive rates for arsenic, cadmium, lead, and mercury were 100, 79, 58, and 74%, respectively. The detected concentrations ranged from 0.030 to 0.34 mg/kg for arsenic, from 0.031 to 0.23 mg/kg for cadmium, 0.019 to 0.059 mg/kg for lead, and from 0.94 to 28 μg/kg for mercury. Based on the lack of detection of microbiological contamination, and the positive rates and levels of pesticides and metals observed in the products, Health Canada determined that all insect products analyzed were safe for human consumption. This is a limited study; the Canadian Food Inspection Agency will continue to monitor this novel food. HIGHLIGHTS Edible insects were tested for potential microbiological and chemical health hazards. No Salmonella spp. or E. coli (.100 CFU/g) was detected. 89% compliance was found with Canadian pesticide regulations. Glyphosate and its metabolite, AMPA, were the most commonly detected pesticides. All insect products tested were deemed safe for human consumption. Key words: Edible insects; Escherichia coli; Glyphosate; Pesticides; Salmonella; Toxic metals Edible insects and food products containing insect healthier and more nutritious food. The environmental ingredients are not commonly consumed by humans in footprint of insect production is minimal compared with the North America and Europe. By contrast, people in Africa, production of beef, pork, and chicken (31). Asia, and Latin America have been consuming insects for In response, a growing number of edible insect centuries. There are 1,000 to 2,000 species of insects products have appeared on the Canadian market in recent consumed globally (22). The Food and Agriculture years, such as whole and powdered insects and processed Organization of the United Nations (10) and other insect products. Dried whole insects (roasted, smoked, and organizations have been promoting the cultivation and flavored) are intended to be consumed as is, whereas consumption of edible insects as alternative sources of powdered insects are used as ingredients in other foods. protein (food security) and as sustainable forms of Processed products, such as protein bars, chips, crackers, agriculture. Edible insects have high protein-to-fat ratios and cookies, are manufactured with insects as the principal relative to plants or traditional meats, making insects a source of protein. Edible insect products can be purchased online, at specialty stores, and at mainstream grocery stores. * Author for correspondence. Tel: 613-773-6313; Fax: 613-773-5959; To date, there are no specific regulations, standards, or E-mail: [email protected]. guidelines regarding potential microbiological or chemicals 1576 KOLAKOWSKI ET AL. J. Food Prot., Vol. 84, No. 9 hazards established in Canada for edible insects or insect- multiresidue pesticide method (505 pesticides), a phenoxy containing foods. Internationally, the European Food Safety herbicide method (2 pesticides: 2,4-dichlorophenoxyacetic acid Authority requires premarket approval of individual edible [2,4-D] and 2-methyl-4-chlorophenoxyacetic acid [MCPA]), a insect species (8). A few countries in the European Union quat screen (diquat/paraquat) method, and a glyphosate method permit the marketing and sale of certain edible insect (parent compound plus metabolite) for a total of 511 pesticides. species (25). Edible insects produced for human consump- Metal method analyses were used for up to 18 metals, but only the tion and available to Canadian consumers must meet the toxic metals (arsenic, cadmium, lead, and mercury) are discussed in this article. same safety and hygiene standards as other foods available in Canada (6). All pesticide residues in insect products are Chemical analysis: multiresidue pesticide method. The subject to the general maximum residue limit (MRL) of 0.1 multiresidue pesticide method began with soaking in water for 2 h mg/kg (7). Each pesticide is assessed separately against the (1). Then, the sample was extracted in 1% acetic acid in MRL, including cases with multiple pesticides per sample. acetonitrile in the presence of isotopically labeled internal Notable exceptions are a pesticide and its metabolite or standards. Cleanup was performed using dispersive solid-phase Downloaded from http://meridian.allenpress.com/jfp/article-pdf/84/9/1575/2893105/i0362-028x-84-9-1575.pdf by guest on 26 September 2021 metabolites or multiple forms of a pesticide (e.g., spinosyn extraction with primary-secondary amine and C18. The extract A and spinosyn D, assessed as spinosad), which are was then analyzed by gas chromatography–tandem mass spec- summed and then compared against the MRL. There are trometry (GC-MS/MS) and liquid chromatography–tandem mass no regulations regarding permissible levels of arsenic, spectrometry (LC-MS/MS). cadmium, lead, or mercury in insect products in Canada (7). GC-MS/MS was performed using electron impact ionization, In this context, this article presents baseline surveillance with helium as the carrier gas (1). The samples were also analyzed data on the prevalence and levels of chemical and using reverse-phase liquid chromatography with gradient condi- microbiological hazards of edible insects. These data are tions. Detection was performed by tandem mass spectrometry with electrospray ionization in positive or negative mode with multiple important in the assessment of potential human health risks. reaction monitoring (1). A total of 505 pesticides were analyzed MATERIALS AND METHODS by these two methods. Two transitions were monitored per pesticide residue, regardless of detection method. Supplemental Insect samples. Each sample consisted of a single or Table S1 contains a list of all pesticides screened as part of this multiple unit or units (e.g., individual consumer-sized packages) method. from a single lot with a total weight of at least 50 g for microbiological analysis or at least 200 g for chemical analysis. Chemical analysis: phenoxy herbicide method. With the All samples were acquired from online retailors or collected at phenoxy herbicide method (2), the sample was extracted in 0.007 retail establishments located in Ottawa, Canada, with the intent of M HCl, pH was adjusted to 2, and ethyl ether was added. After capturing as wide a range of retail forms (i.e., brand and product centrifugation, the ethyl ether layer was evaporated to near type) and insect types as possible. The samples included dried dryness, diluted to volume with methanol, centrifuged again, and whole insects, insects in powdered form, or insects incorporated then analyzed with LC-MS/MS. The samples were analyzed using into finished products (e.g., protein bars). The products included reverse-phase liquid chromatography with isocratic conditions. domestically cultivated and processed products, domestically Detection was performed by tandem mass spectrometry with processed products, and imported products. Some products were electrospray ionization in negative mode with single reaction labeled organic. To be included in the study, the products had to be monitoring. For MCPA, m/z 199 and 201 were monitored, whereas intended for human consumption. Therefore, insects available for for 2,4-D, m/z 219.2 and 221.2 were monitored. Table S2 contains purchase as animal or pet feed were excluded. A total of 51 a list of all pesticides screened as part of this method and their samples of dried whole insects or insect powder were collected for limits of detection (LODs). microbiological hazard testing. A total of 43 samples of crickets (protein bars, powders, flour, and whole insects) and 4 samples of Chemical analysis: quat method. With the quat method (9), silkworm (whole insects) were collected for chemical hazard samples were extracted in acidic methanol (2% HCl). After testing. incubation, two rounds of centrifugation, and filtration, the samples were ready for analysis by LC-MS/MS.