1
UNIVERSITA’ DI PISA
Dipartimento di Scienze Agrarie, Alimentari e Agro-ambientali
Corso di Laurea Magistrale in Biosicurezza e Qualità degli Alimenti
TESI:
Edible insects: food safety aspects related to mycotoxins
Candidato: Relatore:
Elena Favaro Prof.ssa Valentina Meucci
2
Index
Introduction ... 3
Nutritional value of edible insect ... 6
European legislation on food and feed ... 8
Mycotoxins ... 10
Aflatoxins ... 11
Ochratoxin A ... 12
Nivalenol/Deoxynivalenol ... 13
Zearalenone ... 13
Studies about edible insects ... 15
Bosch et al. (2017), Aflatoxin B1 tolerance and accumulation in Black Soldier Fly larvae (Hermetia illucens) and Yellow Mealworms (Tenebrio molitor) ... 21
Van Broekhoven et al. (2014), Exposure of tenebrionid beetle larvae to mycotoxin-contaminated diets and methods to reduce toxin levels ... 23
Niermans et al. (2019), Feeding study for the mycotoxin zearalenone in yellow mealworm (Tenebrio molitor) larvae- investigation of biological impact and metabolic conversion ... 26
Van Broekhoven et al. (2017), Degradation and excretion of the Fusarium toxin deoxynivalenol by an edible insect, the Yellow mealworm (Tenebrio molitor L.) ... 29
Sanabria et al. (2019), Yellow mealworm larvae (Tenebrio molitor) fed mycotoxin-contaminated wheat- a possible safe, sustainable protein source for animal feed? ... 30
Camenzuli et al. (2018), Tolerance and excretion of the mycotoxins Aflatoxin B1, Zearalenone, Deoxynivalenol and Ochratoxin A by Alphitobius diaperinus and Hermetia illucens from contaminated substrates ... 32
Discussion ... 37
3
Introduction
The increasing demand of food is going to be one of the most concerning problems for the next future. There is the need for an alternative protein source to help solve the problem. One of the proposed solutions is the use of edible insects both for food and feed. Edible insects are traditional food in several countries (Murefu et al. 2019) and more than 2000 insect species are known to be edible (Kouřimská and Adámková, 2016). The anthropo-entomophagy is practiced by about 3000 ethnic groups in over 100 countries mainly located in Africa, Asia and Latin America (Baiano, 2020). Around the world, the most frequently consumed species are beetles, caterpillars, bees, wasps and ants. They are followed by grasshoppers, locusts and crickets, cicadas, leafhoppers and bugs, termites, dragonflies, flies and other species (Kouřimská and Adámková, 2016). Regarding animal feed in Europe, insects such as black soldier fly (Hermetia illucens), yellow mealworm (Tenebrio
molitor), and mealworm (Alphitobius diaperinus) constitute the most relevant species used as
derived products for farmed animal feed. Instead, larvae from yellow mealworm and lesser mealworm, black soldier fly, wax moth (Galleria mellonella), grasshoppers, silk moth (Bombix mori), and cricket species are used to produce foods for pets, circus and zoo animals (Baiano, 2020). Although the conventional sources of proteins are affordable to people because of their high productivity, they contribute to enormous environmental costs such as contamination of both surface and ground water with manure, which may can carry pathogenic microorganisms and chemical contaminants like heavy metals, emissions of greenhouse and ammonia gases and possible deforestation as a result of the increasing demand of feed (Imathiu, 2019).
Moreover, the rearing and harvesting of edible insects is more environmentally friendly than production of livestock because of lower greenhouse gas emissions, water pollution and land use (Kouřimská and Adámková, 2016). Currently, the livestock sector uses about 70% of all agricultural
4 land and is responsible for about 15% of the total emission of anthropogenic greenhouse gas (GHG). Expansion of agricultural acreage by land clearing is a major source of GHG emissions and one of the largest contributors to global warming (Oonincx and de Boer, 2012). Moreover, still Oonincx and de Boer (2012), declare that to produce 1 kg of edible protein, mealworms required only 10% of the land needed for beef production. Furthermore, edible insects have the advantage to be farmed vertically (Van Huis et al., 2013). The impact on the environment of products derived from pork, chicken and beef are caused by three main factors: the first one is enteric CH4 production, the second one is
reproduction rate and the third one is feed conversion efficiency (Oonincx and de Boer, 2012). Concerning mealworms, they do not produce CH4, they have a high reproduction rate (females of T.
molitor produce 160 eggs in her life (3 months) and the maturation is short (T. molitor reaches
adulthood in 10 weeks). Feed conversion ratio (FCR) depends on the diet provided and for the mealworms (kg/kg of fresh weight) is similar to chicken but lower than pigs and beef cattle (Oonincx and de Boer, 2012). The proportion of edible weight differs considerably between conventional livestock and insects (van Huis, 2013). In a study by Van Huis, (2013) cricket FCR was analysed. Van Huis in fact, explains that the percentage of edible weight for chicken and pork (both 55% of liveweight) is higher than that for beef (40%). Crickets in the last nymphal stage can be eaten whole, but when eaten as a snack, some prefer that legs (17% of total weight) be removed, and because the chitinous exoskeleton (3%) is indigestible, the percentage edible weight amounts to 80%. Besides that, the FCR of edible weight can be calculated, showing crickets to be twice as efficient as chickens, 4 times more efficient than pigs and 12 times more than cattle (van Huis, 2013). All these findings strengthen the idea that insects can help mankind to solve food/protein shortages (Baiano, 2020). The variability of their nutritional composition must be considered, since it depends on of factors such as: species, development phase, way the insects are killed, and preparation. Subsequently, information on the most suitable rearing condition for growth, development and survivorship for
5 every insect species is a prerequisite for start a mass production (Baiano, 2020). In order to start a mass production, it could be interesting to investigate the possibility to use food wastes, manures or unusual wild resources for rearing edible insects. Since one-third of the food produced annually ends up waste, mass rearing of edible insects on food wastes could combine the need to produce higher amounts of proteins with environmentally friendly procedures (Baiano, 2020).
Another aspect that in a certain way, slow down the production and the consumption of edible insect are sceptical consumers in fact, the consumption of insects as foods is affected by factors such as culture and religion. There are two different psychological reactions to insects as a foods: in countries where populations practice entomophagy, insects are considered as a valued source of nutrients; instead, in western cultures, insects are considered as dirty, disgusting, and dangerous (Baiano, 2020). Other influential factors seem to be familiarity, interest in the environment, interest in sustainable food consumption, convenience, and attachment to meat. As already highlighted, insect acceptance could be improved by transforming them into more conventional forms (hot dogs or fish sticks types) or by adding extracted and purified insect proteins to conventional foods (Baiano, 2020). In this perspective, insects can be peeled, reduced in ground and paste form (drying and grinding). The insect flours can then be used to enrich existing foods such as crisps, bread, pasta, and similar products. Oils, beverages, and confectioneries can also be produced starting from insects (Baiano, 2020). Certainly, a promising strategy to overcome the reluctance to eat insects is to target children for education in entomophagy. In any case, a more acceptable pathway to introduce insects in the human diet could be the use of insects for animal feed (Baiano, 2020).
6
Nutritional value of edible insect
Edible insects are excellent source of energy, proteins as well as amino acids, they have also a high content of mono- and polyunsaturated fatty acids, they are rich in trace elements like copper, iron, magnesium, manganese, phosphorus, selenium and zinc. Edible insects are also a source of vitamins such as riboflavin, pantothenic acid, biotin and in some cases, folic acid (Kouřimská and Adámková, 2016). According to Kouřimská and Adámková (2016), the energy value depends on their composition, especially on the fat content, therefore high protein insect species have lower energy content. Moreover, larvae or pupae are usually richer in energy compared to adults. Concerning proteins, a study revealed that in 100 different species, the protein content was in a range of 13% to 77% by dry matter (Xiaoming et al., 2010). The amount of nitrogenous substances in adult insects may be higher since some nitrogen is also bound in the exoskeleton and not included in the measurements (Kouřimská and Adámková, 2016). Referring to amino acids, they contain a high number of them, including high levels of phenylalanine, tyrosine, lysine, tryptophan and threonine (Kouřimská and Adámková, 2016). Some analysis done in insects by Xiaoming et al. (2010), showed that the content of essential amino acids represents the 46–96% of the total amount of amino acids. Lipids represents on average 10% to 60% of fat in dry matter and this level is higher in the larval stages than adult forms (Kouřimská and Adámková, 2016). Fat is found in different forms in the insects: triacylglycerols constitute about 80% of the total fat and they serve as an energy reserve for periods of high energy intensity, such as long flights. Then, phospholipids which are the second most important group and usually represent less than 20% but it depends on the life stage and insect’s species (Kouřimská and Adámková, 2016). In edible insects can be found also C18 fatty acids such as oleic, linoleic and linolenic acids (Kouřimská and Adámková, 2016). Although the nutritional importance of linoleic and α-linolenic acid and essential amino acids is well recognized, the presence
7 of high amounts of unsaturated fatty acids will also give rise to rapid oxidation of insect food products during processing, causing them to go rancid quickly (EFSA, 2015).
Concerning fiber, edible insects contain a significant amount of fiber and insoluble chitin is the most common form of fiber in the body of insects. Chitin is contained mainly in their exoskeleton (Van Huis et al. 2013) and it acts like cellulose in the human body and because of this effect is often called “animal fiber” (Kouřimská and Adámková, 2016).
Edible insects are also rich in minerals, such as iron, zinc, potassium, sodium, calcium, phosphorus, magnesium, manganese and copper (Kouřimská and Adámková, 2016). There is also a good source of vitamins: they contain water soluble and lipophilic vitamins (Kouřimská and Adámková, 2016). It is important to underline that the level of vitamins and minerals in wild edible insects is seasonal, but in case of farmed species it can be controlled with the administration of feed (Kouřimská and Adámková, 2016).
8
European legislation on food and feed
Insects can be used as a source of food and feed due to their nutritional value. It is also important to underline that edible insects should be non-toxigenic and non-pathogenic towards humans and animals (Schrögel and Wätjen, 2019).
Insects that are harvested by humans and used to produce food or any other product obtained from animals, are considered as livestock in the European Union according to Article 3 (6) of the EC Regulation 1069/2009 (EC 2009a). Therefore, the regulations regarding animal feed apply to insect farming as well (Schrögel and Wätjen, 2019). While some insect species are capable of converting waste into valuable biomass, under EC Regulation 1069/2009 (EC 2009a) and EC Regulation 767/2009 (EC 2009b) it is not allowed to feed animals on various waste stream materials, such as household waste or manure (Schrögel and Wätjen, 2019).
Currently, it is also not allowed in the EU to feed processed animal protein to farmed animals, except insect oil and hydrolysed proteins according to EC Regulation 999/2001 (EC 2001) on the prevention of transmissible spongiform encephalopathies (Schrögel and Wätjen, 2019).
In the feed production, maximum levels are set by the European Commission for contaminants such as heavy metals (e.g., cadmium), pesticides, and mycotoxins (e.g., aflatoxin B1) (EC 2002). For the mycotoxins deoxynivalenol, zearalenone, fumonisins, ochratoxin A and T-2 toxin and HT-2 toxin reference values are set by the European Commission (EU 2016). These specifications apply to both the farming of the insects as well as to using insects as animal feedstock (Schrögel and Wätjen, 2019). To ensure consumer safety, the basic principles of UE legislation on contaminants in food are described in Regulation 315/93/EEC which explains that every food containing an amount
9 unacceptable for public health of contaminants shouldn’t be placed on the market (EU 1993). In addition, EC Regulation 1881/2006 (EU 2006) sets maximum levels of certain contaminants such as mycotoxins and metals in specific
foodstuffs. With respect to human consumption in the EU, farmed insects and products thereof are covered by the novel food regulation EU 2015/2283 (EU 2015). Under this regulation, food or food ingredients are defined as novel which had not been used for human consumption in the EU before 15 May 1997. Before
placing them on the market in the EU, novel food materials either need to undergo an authorization procedure in accordance with Article 10, which includes a safety assessment regarding human health (Schrögel and Wätjen, 2019). Nevertheless, the Regulation 2015/2283 offers a simplified authorization procedure for novel foods that are new for the European Union markets but have been traditionally used in third countries. In such situation, the food can be commercialised on the basis of a simple notification of the food business operator, provided the possibility to demonstrate that the traditional food is safe (consumption has continued for longer than 25 years in the customary diet of a significant number of people in at least one third country) and that there are no safety concerns raised by EU Member States or EFSA. This notification procedure requires only 5 months (Baiano, 2020).
The legislative framework specifically related to insects used as food and feed, however, is still under development (EFSA, 2015).
Figure 1: overview of regulatory framework regarding food and feed in the European Union (Schrögel and Wätjen, 2019).
10 On the 13th January 2021, EFSA concluded that is possible to consume dried larvae of Yellow
mealworm (Tenebrio molitor), whole or in the form of powder. The production of this novel food does not raise concern. The Panel underlines that the levels of contaminants depend on the levels of these substances in the insect feed, so if the EU legislation regarding feed is applied, the composition of the larvae does not raise safety concern (EFSA, 2021).
Mycotoxins
Mycotoxins are low molecular weight secondary metabolites produced by molds (Schrogel and Watjen, 2019). These compounds are toxic, and they are naturally produced by certain types of moulds. These moulds (fungi) can produce mycotoxins and grow on numerous foodstuffs such as cereals, dried fruits, nuts and spices. Mould growth can occur both before harvest or after harvest, during storage, on/in the food itself often under warm, damp and humid conditions. Most mycotoxins are chemically stable and survive to food processing. (WHO, 2018)
A lot of mycotoxins have been identified, but the most observed mycotoxins that represent a concern to human and animal health include aflatoxins, ochratoxin A, patulin, fumonisins, zearalenone and nivalenol/deoxynivalenol (WHO, 2018).
The effects of mycotoxins can be acute with severe illness and may appear quickly after the consumption of foods that are contaminated. Other mycotoxins have chronic toxicity effects on health, including cancer and immune deficiency (WHO, 2018).
11
Aflatoxins
Aflatoxins are mycotoxins produced by Aspergillus flavus and Aspergillus parasiticus which grow in soil, decaying vegetation, hay, and grains (WHO, 2018). Different kind of crops can be affected by
Aspergillus spp. such as cereals (corn, sorghum, wheat and rice), oilseeds (soybean, peanut,
sunflower and cotton seeds), spices (chili peppers, black pepper, coriander, turmeric and ginger) and tree nuts (pistachio, almond, walnut, coconut and Brazil nut) (WHO, 2018).
Chemically, aflatoxins are a group of polyketide-derived mycotoxins. They are secondary metabolites and their biosynthesis start during idiophase when the mould’s growth slowed or stopped and the chemical differentiation ensue (Dutton, 1998). There are four major aflatoxins B1, B2, G1 and G2, with the letters referring to the colour of their fluorescence under ultraviolet light, blue or green. The most toxic aflatoxin is Aflatoxin B1 (AFB1), which has a carcinogen effect (Klich, 2007). IARC in 1987, included aflatoxins in Group 1 (Carcinogenic to humans). This conclusion was re-affirmed in 1993 and 2002. An IARC working group in 1993 concluded that in experimental animals, there was enough evidence for the carcinogenicity of naturally mixture of aflatoxins and aflatoxins B1, G1 and M1 (Ostry et al., 2016).
Aflatoxins can also be found in milk of animals that are fed with contaminated feed, in the form of aflatoxin M1 (WHO, 2018).
There are two forms of the disease, called aflatoxicosis, caused by exposure to aflatoxin: acute aflatoxicosis results in death and chronic aflatoxicosis which causes cancer, with the liver as the primary target organ, immunosuppression,
Figure 2: in the image the oxidative metabolism products of AFB1 (Guengerich et al., 1997).
12 teratogenicity and other symptoms (Klich, 2007). Aflatoxins have been shown to be genotoxic, meaning they can damage DNA and cause cancer in animal species. There is also evidence that they can cause liver cancer in humans (WHO, 2018).
In the image the oxidative metabolism products of AFB1 (Guengerich et al., 1997).
Ochratoxin A
Ochratoxin A (OTA) is produced by several species of Aspergillus and Penicillium. It can contaminate cereal and cereal products, coffee beans, dry vine fruits, wine and grape juice, spices and liquorice and the contamination occur worldwide (WHO, 2018). Traces of ochratoxin A have been found in fermentation products like beer and coffee. OTA occurs in animal-derived food products such as pork and poultry meat by carry-over as a result of feeding animal with contaminated feed (Petzinger and Ziegler, 1999).
When the mycotoxin is ingested is very persistent in blood (half-life 35 days) and it renders the toxin among the most frequent mycotoxin
contaminants in human blood. Ochratoxin A is neither stored or deposited in the body, but evenly distributed in the body and it impose serious damage to the kidneys. It is indeed
classified a 2B cancer compound, being possibly carcinogenic for humans. Referring to the toxicological profile, it includes teratogenesis, nephrotoxicity and immunotoxicity (Petzinger and Ziegler, 1999).
13
Nivalenol/Deoxynivalenol
Deoxynivalenol (DON) and Nivalenol (NIV) belong to the group B of trichothecenes toxins, produced by Fusarium fungi (Pestka, 2010). In the A group can be found T-2 and HT-2 toxins (Pestka, 2010). The formation of the moulds and toxins occur on a variety of
different cereal crops (WHO, 2018). DON is also called vomitoxin because high doses of it induce vomiting (Rotter, 2010). This mycotoxin has the ability of induce toxic and immunosuppressive effects in several animal species; at cellular level, the main effects is inhibition of
protein synthesis (Rotter, 2010). Symptoms of this disease included nausea, emesis, diarrhoea, leukopenia and haemorrhage sometimes culminating in shock-mediated death (Pestka, 2010). In animals, moderate to low ingestion of toxin can cause poorly defined effects associated with reduced performance and immune function. The main effect at low dietary concentrations seems to be a reduction in food consumption (anorexia), while higher doses induce vomiting (emesis) (Rotter, 2010).
Zearalenone
Zearalenone (ZEN) is a resorcyclic acid lactone produced by the fungi Fusarium graminearum (Gibberella zeae), F. culmorum, F. cerealis, F. equiseti and F. semitectum. Zearalenone can be produced on numerous substrates, including wheat, barley, corn, corn silage, rice, rye, sorghum, and occasionally in forages. Production in soybeans is uncommon (Gupta et al., 2018). The ZEN concentration in maize and other grains depends on water activity, temperature conditions, duration of the growth period, and fungal species (Santos Alexandre et al., 2019). ZEN is majorly formed in Figure 4: In the image, structure of DON (Rotter, 2010)
14 pre-harvest period, but its production can continue even under poor storage conditions (Santos Alexandre et al., 2019).
ZEN has been shown to have hormonal, oestrogenic effects and can cause infertility at high intake levels (WHO, 2018).
In animals, the metabolic effects of ZEN in males mainly affect the reproductive system, testosterone synthesis, spermatogenesis, sex organ weight, and testicular histology. In females, the reported adverse effects are on the reproductive tract, animal fertility, and embryo survival, as ZEN mimics the effects of the oestrogen hormone estradiol (Santos Alexandre et al., 2019).
ZEN undergoes reduction and two different compounds are formed: α and β-zearalenol. The α-zearalenol metabolite is three times more oestrogenic than zearalenone and it seems an activation process of the toxin, unlike the production of β-zearalenol which seems a deactivation process (Gupta et al., 2018).
15
Studies about edible insects
Several studies have been carried out on edible insects and their risk of consumption. In these investigations have been analysed risks connected to the rearing, harvesting and consumption of these insects.
It is recommended to consider the potential hazards in terms of microbial hazards, chemical hazards, allergenic potential, environmental hazards, and impact of processing and storage (Schrögel and Wätjen, 2019). Foodstuff may contain undesired substances which can be harmful for animals and subsequently for foodstuff of animal origin (Purschke et al., 2017).
Focusing on mycotoxins, there are different studies concerning their toxicity, metabolism and excretion in edible insects (Table1).
Larvae species Rearing substrates Duration of feeding period Analytes Treatment prior to analysis Reference Hermetia illucens, Tenebrio molitor Poultry feed spiked with AFB1 at levels of 0.01, 0.025, 0.05, 0.10, 0.25, and 0.5 mg/kg dry feed Hermetia illucens: 10 days Tenebrio molitor:
Until first pupa was observed AFB1 AFM1 analysed directly (T. molitor) vs. 2 days on non-contaminated feed (H. illucens) Bosch et al., 2017
16 Alphitobius diaperinus, Tenebrio molitor, Zophobas atratus Tenebrio molitor and Zophobas atratus: Wheat bran spiked with ZEN, OtA, T-2 toxin at 500 µg/kg Alphitobius diaperinus: Diet containing maize, wheat, soy, limestone, palm-, sunflower- and soybean oil, spiked with ZEN, OTA, T-2 toxin at 500 µg/kg Alphitobius diaperinus: 15 days Tenebrio molitor: 28 days Zophobas atratus: 40 days (depending on species-specific developmental time) ZEN OTA T-2 toxin analysed directly vs. 24 h/48 h/72 h fasting Van Broekhoven et al., 2014
17 Tenebrio molitor Wheat flour spiked with toxins at levels of approx. 500 µg/kg ZEN and approx. 2000 µg/kg ZEN blended with artificially contaminated wheat flour at levels of approx. 500 µg/kg ZEN and approx. 2000 µg/kg ZEN blended with naturally contaminated wheat flour 4 weeks (short-term trial) and 8 weeks (long-term trial) ZEN ZEN-14-O-glucuronide ZEN14Sulf ZEN-14-O-glucoside ZEN-16-O-glucoside Hydrolyzed ZEN decarboxylated hydrolyzed ZEN α-ZEL α-ZEL-14-O-glucuronide ZELSulf β-ZEL β-ZEL-14-O-glucuronide ZAL ZAN DON 24 h fasting Niermans et al., 2019
18 at levels of approx. 600 µg/kg ZEN and approx. 900 µg/kg ZEN Tenebrio molitor wheat flour Naturally contaminated with mycotoxins at levels of 4.9 mg/kg DON, 86 µg/kg 15-ADON, 300 µg/kg DON-3-glucoside and wheat flour spiked with 8 mg/kg DON 14 days DON DON-3G 15-ADON analysed directly vs. 24 h fasting Van Broekhoven et al., 2017
19 Tenebrio molitor Naturally contaminated grain at levels of 0.2 ppm, 2 ppm, 10 ppm, and 12 ppm DON 32.8 ± 3.2 days (until 2 pupae were observed) DON 3-ADON NIV Directly analysed Sanabria et al., 2019 Alphitobius diaperinus, Hermetia illucens Commercial wheat-based rearing substrate spiked with AFB1, DON, OTA and ZEN in concentrations of 1, 10, and 25 times the maximum EC limits or guidance values Alphitobius diaperinus: 14 days Hermetia illucens: 10 days AFB1 aflatoxicol AFP1 AFQ1 AFM1 DON 3-ADON 15-ADON DON-3G ZEN α-ZEL β-ZEL 2 days on non-contaminated feed Camenzuli et al., 2018
20 for specific complete feed spiked with mixtures of mycotoxins (with an average of 8- to 20-fold increase of the EU limits) Legenda
AFB1: Aflatoxin B1, AFM1: Aflatoxin M1, AFP1: Aflatoxin P1, AFQ1: Aflatoxin Q1, OTA: Ochratoxin A, ZEN: Zearalenone, ZEN-14-Sulf: Zearalenone-14-sulfate, α-ZEL: α- zearalenol, β-ZEL: β-zearalenol, ZEL-Sulf: α-zearalenol-sulfate, α-ZAL: α- zearalanol, β-ZAL: β- zearalanol, ZAN: zearalanone, DON: deoxynivalenol, DON-3G: deoxynivaleol-3-glycoside, 15-ADON: 15-acetyl-deoxynivalenol, 3-ADON: 3-acetyl-deoxynivalenol, NIV: nivalenol.
In all the studies, the larvae are fed with feed spiked with different mycotoxins at different concentrations. They are reared for different time, depending on the species-specific developmental time. When the time is over, the larvae are analysed. The larvae are directly analysed immediately after the treatments or in some studies, the larvae undergo fasting for 24h, 48h or 72h or they are fed on uncontaminated substrates for a short amount of time. Then, the results are compared.
21
Bosch et al. (2017), Aflatoxin B1 tolerance and accumulation in Black Soldier Fly larvae (Hermetia illucens) and Yellow Mealworms (Tenebrio molitor)
In the study of Bosch et al. (2017), 100 larvae of each specie (Hermetia illucens and Tenebrio molitor) are fed with poultry feed spiked with AFB1 at the following concentrations: 0,01, 0,025, 0,05, 0,10, 0,25 and 0,5 mg/kg of dry feed. They used a solvent (methanol and chloroform) as a vehicle to mix the mycotoxin in the feed. Bosch et al. (2017) fed insects with and without solvent, but since the methanol and the chloroform are extremely volatile at ambient temperature, it is likely evaporated during the mixing of the feed. Hermetia illucens (BSF) larvae were reared in a climate cabinet (27°C, 88% relative humidity) for 10 days, then the larvae and the boxes (containing remaining feed, exuviae, excreta) were collected and weighted. The residues of the boxes were collected and stored at -18°C until further analyses. The larvae were transferred to a new container with 10g of spiked poultry feed (with no solvent) and collected after 2 days. BSF larvae were weighted, cleaned and then stored al -18°C until further analyses. Tenebrio molitor (YMW) larvae were reared in containers in climate chambers at 28°C and 70% relative humidity. When the first pupa was observed, the larvae were counted, the residual material was weighted and then this material and the YMW larvae were stored at -18°C until further analyses. To distinguish the amount of AFB1 in the gut from the larvae as a whole, three additional containers with 50 first instar YMW each were prepared and provided with the 0.5 mg AFB1/kg feed. These larvae were also removed from the container when the first pupa was observed, and then placed in a new container with 4g of poultry feed (no solvent and AFB1). After 2 days were collected, weighed, cleaned and then stored at -18°C until further analyses. The mycotoxin in the feed did not affect survival or body weight of the BSF larvae and it indicates the high tolerance to AFB1, in fact, they were not affected by the varying levels of AFB1 compared to those which were fed with only the solvent. YMW had similar survival rate in all the dietary groups and the
22 same findings were obtained for the YMW, except for the larvae fed the feed containing 0.204 mg/kg AFB1, which were heavier than those fed the feed without AFB1.
The amount of AFB1 and AFM1, in BSF was below the detection limit of the applied analytical method (LOQ = 0,10 µg/kg). In the larvae of YMW, the AFB1 was detected in the larvae fed with 0,025 mg/kg and in the higher concentrations and part of AFB1 might have been bound to proteins and left undetected, instead the AFM1 was below the detection limit. However, the mass balance suggest that a considerable amount of AFB1 was lost in both insect species ( 83.0%- 95,1% in BSF and 89.0%- 95,6 in YMW). The residues of YMW contained AFM1 when the larvae were fed with 0,023, 0,084mg/kg AFB1 or more and this suggest that this mycotoxin is at least partially trasformed by YMW in AFM1, which excreted later on. These findings suggest that BSF and YMW seem to metabolise AFB1 in a duferent way.
The authors colncluded that further studies are needed to evaluate the presence of other AFB1 metabolites and the tolerance of these insects to other mycotoxins. The lack of accumulation of AFB1 in the larvae need to be investigate before using BSF and YMW as a strategy to use condemned mycotoxins-contaminated crops as substrate in the food chain.
23
Van Broekhoven et al. (2014), Exposure of tenebrionid beetle larvae to mycotoxin-contaminated diets and methods to reduce toxin levels
In the study of Van Broekhoven et al. (2014), the larvae of YMW, Zophobas atratus and Alphitobius
diaperinus (LMW) were reared on contaminated substrated (ZEN, OTA and T-2 toxin). A control diet
was provided and it consisted in pure wheat bran for YMW and Z. atratus and maize, wheat, wheat bran, soy, limestone and palm-, sunflower- and soybean oil for LMW. Then, toxic powder containing mycotoxins was added to the feed in a concentration of 500 µg/kg. Larvae were allowed to grow in a climate chamber at 28°C, 65% RH until the start of the experiment (21 days for LMW, 26 days for YMW and 33 days for Z. atratus). Larvae were weighed and then transferred to either a control diet or a diet containing one of the three mycotoxins. They used 5 replicates per diet and each replicate contained 70 larvae for LMW, 50 larvae for YMW and 35 larvae for Z. atratus. Differences in larval density were based on the different sizes of the species. Larvae were allowed to feed on toxic diet for 15 days for LMW, 28 days for YMW and 40 days for Z. atratus. These differences were based on the different development times of the species. Then, the larvae were collected (about 1g dry weight) when they stopped growing and went into pupation. Larval samples were then stored at -20°C until further analysis.
After the feeding experiment, larvae of each specie (1g dry weight) were cleaned and transferred to uncontaminated control diet for 24, 48 and 72 hours before harvest, and after that, frozen at -20°C until further analyses. Another sample (about 1g) of each specie, after the feeding experiment, submitted fasting for 24, 48 and 72 hours and subsequently weighted and stored at -20°C until further analyses.
The survival and larval growth was determined after feeding the larvae with control diets and spiked diets. Survival exceded 80% for LMW, 90% for Z. atratus and YMW. No difference on survival rate
24 between the larvae fed on control or spiked diets was observed for LMW and Z. atratus, higher survival was detected for YMW when reared on OTA-spiked diet and T-2 toxin compared to the ones reared on control diet and this could have been due to the experimental setup. YMW larvae that fed on control diet were smaller at the beginning of the feeding experiment than the larvae that fed on toxin-containing diets. Smaller larvae are more vulnerable to handling and this might also explain why the survival percentage of this larvae was lower overall than for the two larger species.
No difference in weight gain for LMW between control or spiked diet. For YMW, larvae fed with OTA and T-2 toxin gained more weight and for Z. atratus larvae fed with T-2 toxin gained less weight than larvae fed with control diet.
No toxin was detected in samples of larvae which had only fed on control diets. ZEN was found in LMW immediately after the consumption of the contaminated diet. OTA and T-2 toxin were found after 48 hours of fasting and OTA and ZEN after 72 hours of fasting. T-2 toxin was also found after 24 hours of uncontaminated control diet. Unexpected results were obtained after LMW had been fasting or transferred to a control diet.
Refferring to YMW all the mycotoxins were found immediately after the consumption of contaminated diet. T-2 toxin was also found after 24 hours of fasting. After 48 hours of fasting were found OTA and T-2 toxin. T-2 toxin was also detected after 24 hours of uncontaminated control diet along with OTA. After 48 hours of uncontaminated diet were still found T-2 toxin and OTA but after 72 hours of control diet only OTA was found. Toxin concentration in larvae which fasted or fed on control diet decreased in general, which was according to expectation. OTA was only detected in a very low level in one sample and conclusions cannot be made on decrease of toxin concentration with time.
25 In Z. atratus after the contaminated diets only OTA was revealed. After 24 hours of fasting were found OTA and T-2 toxin. All the mycotoxins were below the detection limit in the uncontaminated control diet.
In this study, YMW did not retain mycotoxins in unmetabolised form in their body, despite the high concentration present in the diets. Furthermore, mycotoxin concentration decreased rapidly during fasting and feeding on control diets. The authors recommended to allow insects to fast at least 24h before harvest. Mycotoxins are expected to be largely
excreted by the insects through the faeces and this would be a plausible explanation for the rapid decline in toxin concentration detected in YMW when larvae fasted or fed on control diet for longer periods of time, but in this study residues were not analysed.
Further research is needed to obtain better understanding on the ingestion, excretion and possible metabolism of mycotoxins by edible insect species
26
Niermans et al. (2019), Feeding study for the mycotoxin zearalenone in yellow mealworm (Tenebrio molitor) larvae- investigation of biological impact and metabolic conversion
In the study of Niermans et al. (2019), the larvae of YMW were analysed. Larvae were fed with 7 different diets spiked with ZEN and DON in different concentrations: toxin-free control, spiked and artificially contaminated and two naturally contaminated diets.
The exposure of the larvae to the diets was 4 weeks (short-term) or 8 weeks (long-term). YMW larvae were kept on wheat bran and subsequently selected at an age of 42 days (circa 1cm long). Feeding groups, each containing 100 larvae were incubated at 25°C with a humidity of 75%. Biological triplicates were obtained for the seven diets prepared. Biological triplicates of the short-term exposed samples consisted of 200 individuals to obtain sufficient sample amount for further mycotoxin analysis and biological triplicates of the long-term exposure included 100 individuals. Three replicates of larvae and residues sample (combination of faeces and residual feed) were obtained for analyses for every group, short-term and long-term exposure for each of the seven diets. Then, the larvae were harvested and submitted fasting for 24h and subsequently freezed at -18°C. The residue was kept separately and analysed.
In this study was analysed the presence of ZEN and its reductive metabolites and phase II metabolites. The presence of this mycotoxin did not affect the survival rate of the larvae neither in the treatment
DON (µg/kg) ZEN (µg/kg) α- ZEL(µg/kg) β- ZEL(µg/kg)
Blank control (C) 572,0 <LOQ ND ND
Blank spiked (low conc.) (S1) 568,4 588,5 ND ND
Blank spiked (high conc.) (S2) 576,5 2254 ND <LOQ
Blank+ artif. contam. (low conc.) (A1) 939,0 427,0 ND 11,3 Blank+ artif. contam (high conc.) (A2) 2101 2283 ND 76,2 Blank + naturally contam. (low conc.) (N1) 2854 602,3 ND <LOQ Naturally contam. (high conc.) (N2) 4588 919,3 ND <LOQ
27 nor in the control groups (no dead animals were found). The larvae reared on N1 and N2 diet gained more weight than larvae fed with other diets after 8 weeks of exposure. Initially, these substrates contained an higher level of proteins (N1 16% and N2 25%), so the relative increase of weight was corrected for the higher amount of protein available, but still clear difference between the N1 and N2 and the other groups after normalisation for the protein content is visible.
The calculation of the total recovery of ZEN is based on measured contents, the total amount of residue (containing faeces and not consumed feed) collected and final weight of the larvae. Recovery rates were calculated for the amount of ZEN present in the feed in relation to the amount of ZEN, α- and β-ZEL present in the residue. No ZEN or metabolites were found in larvae neither in the short-term exposure, nor in the long-short-term exposure, but detectable levels were found in the residues (ZEN, α- ZEL and β- ZEL) in all samples except for the control sample. The detected amount of unmetabolized ZEN in the residues accounted for 15% up to 63% of the total ZEN amount that was fed to the respective insect groups. However, the reductive ZEN metabolites (α- and β-ZEL) together account for up to another 30% of the overall ZEN intake that is recovered in the residual samples. It is clear that S1 and S2 feed showed a higher recovery rate than artificially and naturally contaminated samples (based on the mass balance data of ZEN). In the 4-week feeding trial, the amount of ZEN that was recovered on the residues either as unchanged ZEN or in its reduced form in lower than in the 8-week experiment. Moreover, α- and β-ZEL were detected in all residues samples but they were not present in all feed samples. The detected levels for α- and β-ZEL were higher in the long-term exposure samples compared to the short-term exposure.
Some phase II metabolites (ZEN14Sulf) were found in diets N1, N2, A1 and A2 but weren’t found in larvae. In the residues were found peaks related to ZEN14Sulf and ZELSulf. In none of the sample of YMW were detected glucuronide or sulphate. In no residue sample
28 collected for the spiked feed groups (S1 or S2) or the control, any phase II metabolite peak of ZEN was detectable, indicating that in YMW larvae, no sulfation occurs.
DON was present in the naturally contaminated flour and in the control flour, measurements have been carried out in all feed, YMW and residue samples for this toxin as well. No detectable amounts of DON were present in the larvae, but it was detected and quantified in all residue analyses. Based on the mass balance, the recovery of DON was around 35% after 4 weeks of exposure and 56% after 8 weeks of exposure and is found to be the highest mycotoxin in the control and spiked samples. DON was not the original focus of this study thus no metabolites were investigated.
The present experiment shows that in some feeding groups, more than 50% of the presumably ingested ZEN remained undetected after analysis of the insects, the unconsumed feed as well as the larval faeces.
29
Van Broekhoven et al. (2017), Degradation and excretion of the Fusarium toxin deoxynivalenol by an edible insect, the Yellow mealworm (Tenebrio molitor L.)
In another study of Van Broekhoven et al. (2017), larvae of YMW were fed with DON -contaminated diets: one of them was naturally contaminated (4,9 mg/kg) and it contained among DON, also different mycotoxins (15-acetyldeoxynivalenol, deoxynivalenol-3-glucoside, beauvericin enniatin A1, enniatin B, enniatin B1, NIV, zearalenone ergotamine and ergotaminine) and the other one was artificially contaminated (8 mg/kg). A wholegrain wheat control diet was also used. Five weeks old larvae were used to either of the three diets. Every diet included six replicates and every sample counted 50 larvae. Once larvae had consumed all diet, they were placed in a new container with fresh diet, and faeces were stored at -20 °C until analysis.
The experimental feeding lasted 2 weeks until they were at harvest weight (>100mg). Larvae were cleaned of faeces manually, counted and subsequently every larvae sample and faeces were weighed per replicate. For each replicate, half of surviving larvae were killed immediately by freezing at -20 °C, and half of them were allowed to fast for 24 h in empty containers. Larvae were subsequently killed by freezing at -20 °C.
Survival and larval growth were not affected by the presence of the mycotoxin in every diet. No DON or metabolites were found in the larvae immediately after the consumption of the spiked diets or after 24 hours of fasting. Some residues of DON were found in the faeces of both the contaminated diets: the percentage of excreted DON in faeces from larvae on DON-spiked diet was higher (ca. 41%) than in faeces from larvae on naturally contaminated diet (ca. 14%).
Mycotoxins that were detected in larvae were present in low concentrations compared to the levels found in the infected wheat, with the exception of enniatin A (30μg/kg in the diet vs 10μg/kg in the larvae).
30 In this study, the authors suggest that possibly, the presence of other fungal metabolites in the naturally contaminated flour interfered with excretion of DON in YMW larvae. Another possible explanation is that mycotoxins in naturally contaminated substrates may differ in stability or bioavailability due to matrix effects. Moreover, it’s
also known that enzymes involved in detoxification of xenobiotics (es P450 and glutathione-S-transferases) are present in YMW, but which enzymes are responsible for DON metabolism in YMW and what kind of metabolites are produced, remains a topic of investigation.
Sanabria et al. (2019), Yellow mealworm larvae (Tenebrio molitor) fed mycotoxin-contaminated wheat- a possible safe, sustainable protein source for animal feed?
In another study of Sanabria et al. (2019), the effects and excretion of DON in YMW larvae was observed. Two types of wheat were mixed and sorted into six fractions to obtain low (2000 µg/kg), medium (10,000 µg/kg) and high (12,000 µg/kg) levels of spiked-DON diets. Wheat containing 200 µg/kg DON was used as the control diet. Five hundred larvae and 50 beetles of YMW were reared in plastic containers with ground wheat. Pupae were moved into separated container twice weekly. Once beetles emerged, they were placed into a new container. 6000 larvae were selected from the previous colony and three hundred of them were allocated per replicate (five replicates for every diet) and placed in plastic drawers. Larvae were reared until the first pupae was observed in a replicate (32-34 days). After collecting the final weights, larvae were placed in an empty container to
31 fast for 24 h and then washed with water. The frass was collected and refrigerated at 4°C and the larvae were euthanized by freezing at -20°C.
The survival rate and weight gain of YMW reared on DON-contaminated wheat for 32–34 days was comparable to those reared on the control wheat, indicating that they could tolerate up to 12,000 µg/kg DON.
Mycotoxin concentration was tested in wheat, freeze dried larvae and frass and DON, 3-ADON and NIV were searched. DON was found in the larvae (but did not exceed 131 µg/kg) and in the frass in all the diets as well as 3-ADON (but only in one sample out of three). NIV was below the detection limit in the larvae. In the frass, DON was found in all the samples as well as 3-ADON. NIV was found in the frass related to the medium concentration and in the one related to the control diet.
32
Camenzuli et al. (2018), Tolerance and excretion of the mycotoxins Aflatoxin B1, Zearalenone, Deoxynivalenol and Ochratoxin A by Alphitobius diaperinus and Hermetia illucens from contaminated substrates
In the study of Camenzuli et al. (2018), larvae of LMW and BSF were fed with 15 different spiked diets and two control diets (one specifically unspiked and the other one spiked with solvent, methanol). These two insect species were exposed to individual mycotoxins (AFB1, ZEN, DON and OTA) as well as to a mixture of the four mycotoxins via feed contaminated at different concentrations (3 mix at different concentration). Each treatment was performed in triplicate.
Every replicate included 100 one-week-old BSF and the larvae were placed in a Petri dish and fed with spiked feed. After 10 days, the larvae were separated from the residual material (remaining feed, frass and exuviae), cleaned, dried and put in another container with unspiked feed. After two days, the larvae were separated again from the residual material, cleaned and then harvested. Per replicate, 200 two-week-old LMW larvae were placed in a plastic container and fed with spiked feed. After 14 days, the larvae were separated from the residual material, rinsed, dried and transferred to another container and fed with control diet. Two days after, the larvae were separated again from the residual material, cleaned and the harvested.
After every step, the larvae were weighted as well as the residual material.
Both insects were reared under standard condition in a climate chamber: LMW larvae at 28°-29°C and 50-60% relative humidity, and BSF larvae at 26°C and 80% relative humidity.
33 Insects and residual material samples were freeze-dried and subsequently stored at -20°C until further analysis. The research lasted 12 days for BSF and 16 days for LMW.
No difference in the survival rate was observed between the larvae of BSF reared on control feed and the larvae reared on mycotoxin contaminated feed. Additionally, survival rate was not different between the different treatments (single and mix). The weight gain in BSF had no significant difference between all the treatment. This underline that individual mycotoxins or mixture of them, do not impact the development of the larvae. The survival rate of LMW was not affected by the presence of mycotoxins or the mixture of them, in fact there was no significant differences amongst the various treatments. Moreover, there was no significant difference in the weights between the various treatments compared to the controls. Regarding LMW, the larvae reared on OTA-L3 had an average weight significantly lower than the larvae reared on the control feed, as well as significantly lower weight than larvae from treatment OTA-L2. Larvae from treatment M2 had an average live weight significantly higher than the larvae from treatments DON-L1, ZEN-L2 and OTA-L3.
The mycotoxin concentration was measured in the feed, larvae and residual material from the period when the insects were reared on spiked diets (RM spiked feed) and also the residual material from the period when the insects were put on clean feed (RM gut clean) for both BSF and LMW larvae species.
No accumulation of AFB1 was observed in the BSF larvae even when reared on feed spiked with mycotoxin mix. DON, ZEN and OTA were detected but their concentration were several orders of magnitude lower than the concentration in the feed.
Referring to LMW larvae, the concentration of all mycotoxins and mix were below the detection limit in all the treatments.
The increasing mycotoxin concentration in the spiked feed (from M1 to M3) is consistent with the increasing concentration in the RM (spiked feed) of both insects for the respective treatment,
34 however AFB1 concentration in the RM (spiked feed) of both insects is lower than the AFB1 concentration in the feed. The highest concentration of AFB1 was measured in the RM (spiked feed) related to the M3 diet for both insects. No clear difference could be observed amongst the treatments on the ratio of AFB1 in feed and in the RM (spiked feed) for BSF and LMW larvae. The DON concentration in the RM (spiked feed) of the BSF larvae was on average higher than the respective spiked concentration in feed for the individual and mixture treatments. Similar results were observed for ZEN and OTA treatments of the BSF larvae reared on feed spiked with individual and mixture mycotoxins. On the other way around, the DON, ZEN, and OTA concentrations in the RM (spiked feed) of the LMW larvae was generally not different from the concentration in feed.
Referring to RM (gut clean), AFB1, DON, ZEN, and OTA were detected in the BSF larvae in every treatment except for AFB1 and ZEN in the L1 treatments in both the individual and mixture treatments. The mycotoxin concentrations were several orders of magnitude lower compared to the concentration in the RM (spiked feed). The highest AFB1 concentration in the RM (gut clean) of the BSF was 50 times lower than the AFB1 concentration in the RM (spiked feed) and 60 times lower than the AFB1 concentration in the feed. The highest DON concentration in the RM (gut clean) of the BSF was in the L3 treatment, which is 17 times lower than the concentration in the spiked feed. For the LMW larvae, concentrations of AFB1 and ZEN in the RM (gut clean) were all below the respective LOQ. DON was detected in the RM (gut clean) of the L3 treatment with but it was 430 times lower than the concentration in the feed. OTA was detected in the RM (gut clean) M3 and in one treatment of the M2. The measured concentrations of DON and OTA were slightly above the LOQ.
In this study the metabolites (aflatoxicol, AFP1, AFQ1, AFM1, 15- and 3- ADON and DON-3G, α and β-ZEL) were analysed. In BSF larvae all the aflatoxin metabolites and all DON metabolites were below the LOQ in all treatments. ZEN metabolites were found in the L2, L3, M2 and M3 treatments. In LMW larvae, all the metabolites analysed were below the detection limit.
35 In the RM (spiked feed), aflatoxicol was only detected in the highest treatment levels (L3 and/or M3) for both insects, while AFM1 was only detected at the two highest treatment levels (L2, L3, M2, and M3) for the LMW but not for the BSF. One signal for AFP1 was detected in the L2, L3, M2 and M3 treatments for the BSF larvae but the concentration could not be determined because of matrix interferences. All DON metabolites were below their LOQ in the RM (spiked feed), as well as in the RM (gut clean) of all treatments of both insects. Both α- and β-ZEL were detected in the RM (spiked feed) of all treatments for both insects, except for the β-metabolite in the individual L1 treatment of the LMW. Even if the BSF and LMW were reared on the same spiked feed, the concentration of ZEN metabolites was up to 50 and 40 times higher in the RM (spiked feed) of the BSF compared to LMW. The maximum mean concentration of the α-metabolite in the RM (spiked feed) of the BSF was four times lower than the mean concentration of ZEN in the same treatment.
36 The authors in this study relying on the results of the mass balance for AFB1 and DON in BSF, suggest that this insect species may form other metabolites, such as de-epoxynivalenol (DOM), a metabolite of DON which can be produced by bacteria in the gut, or conjugated forms of mycotoxins. Further analytical and toxicological research is needed to obtain certainties into mycotoxin metabolism of BSF and LMW and to fully understand the safe limits of mycotoxins in insect feed and thus the safety of the insects.
37
Discussion
Edible insects are considered the future of the animal and human nutrition due to their high nutritive value and to their easy breeding. An aspect that must be taken into account though, it is the safety, especially the chemical safety of this animals.
Several studies have been carried out to investigate the presence of mycotoxins in insects. The evaluation of the absorption and excretion of these toxins are extremely important for human and animal safety. Numerous insects are used as food, such as LMW, YMW, Zophobas atratus and BSF, these species were used to evaluate the possible use as food and feed.
The most common evaluated mycotoxins in the studies are ZEN, OTA, T-2 toxin, DON, NIV and AFB1. Some of their metabolites are also investigated to understand the biotransformation of the mycotoxins inside the insect’s body. Unfortunately, just few studies took into account the metabolites, so this topic must be deepened and for example, seeking for different metabolites or different kind of elimination of the toxin. Few studies reported the accumulation and excretion of combination of mycotoxins in different diets and different concentration (Carmenzuli et al., 2018). It is important to better understand the effects and fate not just of a single mycotoxin, but also the combination of them because the action of a single mycotoxin can be enforced by the presence of other kind of mycotoxins. The co-occurrence of mycotoxins in food and feed has led to several studies on possible interactions. Some studies indicated that the toxic potential may vary with the combinations and/or proportions of individual mycotoxin. The data on combined toxic effects of mycotoxins are limited, thus the health risk from this multi-exposure is not well-known.
The tolerance and efficient excretion of mycotoxins by insects might add a further aspect to the debate on pros and cons of insect use in food and feed. In addition to the biological value of insect
38 protein and ecological benefits, certain insects might be capable of ‘detoxifying’ grain lots with AFB1 contents that would be extremely harmful to humans and livestock.
The majority of the studies evaluated in this thesis indicates that the presence of mycotoxins or combination of mycotoxins did not significantly affect the larval growth or the survival rate compared to the control group and shows the high tolerance of these larvae towards the presence of mycotoxins. Just in one study of van Broekhoven et al. (2014), the survival rate was higher in the larvae fed with contaminated-diets instead of the control and this could be due to the experimental setup: the larvae reared on the controlled diets were smaller than the ones reared on the spiked diets (van Broekhoven et al., 2014).
Weight gain in YMW during the experiment of Niermans et al. (2019) and van Broekhoven et al. (2017) showed no significant difference between spiked diets and control diets but underlined an enhanced growth of YMW larvae reared on naturally DON-contaminated diet compared to spiked or DON- uncontaminated diets. Same result is showed in the experiment led by van Broekhoven et al. (2014), where YMW larvae fed with OTA and T-2 toxin gained more weight compared to control diets. Therefore, the presence mycotoxins seem to be responsible for an enhanced growth (Niermans et al., 2019). Moreover, in the study of Bosch et al. (2017), the presence of AFB1 in YMW and BSF did not significantly affect the weight gain in larvae fed with spiked diets compared to the ones fed with solvent, but the larvae of YMW fed with diet containing 0.204 mg/kg AFB1, were heavier than those fed with the feed without AFB1.
The mycotoxins have a different fate in each larvae species and in some studies they are not found in the larvae or residues immediately after the spiked feed was given, even if the concentration of the mycotoxin exceed the UE limit in the feed. In other studies, instead, mycotoxins were found in the larvae immediately after the consumption of the spiked diets. Same as well happens with
39 mycotoxins’ metabolites. These differences are due to different times of exposure or different concentration of the mycotoxins in the diets.
In the study of Bosch et al. (2017), the accumulation and excretion of AFB1 in BSF and YMW was investigated using 6 different spiked diets and it was detected in YMW when the larvae were fed with the highest concentrations of mycotoxin in the diet. In BSF larvae was below the detection limit. This is in line with another study (Camenzuli et al., 2018), in which the AFB1 was not detected in BSF larvae. The mass balance for AFB1 resulted in less than 20% of the parental compound spiked in the diets for BSF in the study made by Camenzuli et al. (2018). This is the same result obtained in the previous study of Bosch et al. (2017). Moreover, in the Bosch’s et al. study (2017) also the presence of AFM1 was evaluated and it showed that YMW seem to partially transform AFB1 into AFM1 (when fed with the diets with the highest concentrations of mycotoxin). The author concludes that YMW and BSF seem to metabolize AFB1 differently. In the study of Camenzuli et al. (2018), beside AFM1 three additional aflatoxin metabolites were analysed (aflatoxicol, AFP1 and AFQ1). However, the detected concentration of these additional metabolites could not explain the mass unaccounted for. In the study of Camenzuli et al. (2018) the mass balance for AFB1 in LMW was high (56%-80%) and it indicates that these larvae have a lower capacity to metabolize AFB1 instead of BSF and YMW. In all the reported study, appears that insects metabolise AFB1 in various ways, so additional researches have been recommended.
In the study by Sanabria et al. (2019) the DON detected from YMW larvae fed with low and high concentration was 15% and 6% of the DON concentration in the diet, respectively. This is in line with the DON detected in the study of van Broekhoven et al. (2017) where YMW larvae were fed with naturally contaminated diet and the DON detected in the excreta was 14% of the DON provided in the diet. The DON detected in excreta of larvae grown on artificially DON-spiked feed was 41%. The
40 reason for the great difference in excretion levels of DON after feeding on naturally contaminated wheat flour compared to artificially spiked wheat flour could not be clarified. On the contrary Sanabria et al. (2019) showed that DON was measurable in all insects’ larvae even after 24h fasting prior to analysis. A major difference in the experimental setup between the two reported studies was the much longer feeding period, which was nearly doubled, and the age of the larvae. Very low levels of DON were detected in the larvae of the BSF (Camenzuli et al., 2018) compared to the levels available in the feed. The remaining DON fraction could not be found in the larvae and in the excreta, so it remains unclear where the remaining DON may be. Still in Camenzuli’s et al. study (2018), no DON was detected for the LMW and the 3-ADON metabolite was undetectable in larvae of LMW and BSF fed with spiked diets. However, due to matrix interference, the LOQ in this study was several times higher for the metabolites compared to the other studies. So, a clear difference can be observed between the two species and their way to metabolize DON. The authors showed that while the concentration of DON in the residual material (spiked feed) was higher than the feed, the overall mass balance was less than 100% indicating that DON was in fact metabolized by the BFS larvae. Similarly, van Broekhoven et al. (2017), didn’t detected any DON metabolites (15-ADON and 3G-DON) in YMW fed with spiked diets containing DON. On the other hand, in the study made by Sanabria et al. (2019), DON and 3-ADON was detected in YMW larvae, maybe because of the high dose of this mycotoxin in the diets and its conversion in 3-ADON. Moreover, Sanabria et al. (2019) exposed the larvae to higher levels of DON and it led to a longer exposure of the larvae to the mycotoxin (32-34 days) instead of the trial made by van Broekhoven et al. (2017) of 15 days.
Niermans et al. (2019) investigated the presence of DON in YMW, in feed, larvae and excreta. No detectable amounts of DON were present in the larvae, but it was detected and quantified in all the excreta. Based on the mass balance, the recovery of DON was around 35% after 4 weeks of exposure
41 and 56% after 8 weeks of exposure. DON was not the original focus of this study thus no metabolites were investigated. Since not all the DON ingested from the diets was sequestered by larvae or excreted, it is possible that the remaining portion was metabolized into uncommon or unknown metabolites and were so undetected in the samples. Processes other than acetylation convert DON into uncommon derivatives. Further studies into the enzymatic processes could provide insight into DON metabolism in insects and help guarantee the safety of insect-derived protein for use in animal feed and human food.
Van Broekhoven et al. (2014) assessed the tolerance and accumulation of ZEN, OTA and T-2 toxin in YMW, LMW and Z. atratus where they showed that although these mycotoxins were initially retained in the larvae, the concentrations dropped rapidly when the larvae fasted before harvesting. In Camenzuli’s et al. (2018) study, more than 88% was accounted for in both ZEN and OTA treatments for the LMW and this underline that the larvae of the LMW excreted the majority of ZEN and OTA taken up from their feed and metabolize these mycotoxins only to a small portion. Still in the study by Camenzuli et al. (2018) were administered mixes of mycotoxins (AFB1, ZEN, DON, and OTA) and the percentage of the metabolites in the ZEN mass balance between the mixture and the single mycotoxin is comparable, which indicates that the insect larvae (LMW and BSF) metabolisms are independent of the presence of other mycotoxins. In the Camenzuli’s et al. study (2018) mass balance was performed for OTA too and it showed that roughly half of the OTA was detected in the BSF, which is in strong contrast with the LMW, where more than 97% of the OTA was accounted for.
In the study of van Broekhoven et al. (2014) OTA was instead detected in little levels above the LOQ in YMW and Z. atratus but was not detected in LMW, but possibly it could have been found in LMW too, but the level was too low to be detected. In the case of YMW the concentrations of OTA decreased faster on fasting compared to feeding on control feed. ZEN and T-2 toxin were found in
42 relatively high concentrations in YMW directly after feeding but were not detected in Z. atratus or LMW. ZEN concentrations in YMW seemed to drop below LOD already after 24 h of fasting or feeding on control diet. For T-2 toxin, the concentration decreased more gradually. Unexpected results were obtained after LMW had been fasting or transferred to a control diet. For ZEN and T-2 toxin, higher concentrations of toxin were detected in samples where lower concentrations were expected because larvae had been fasting or feeding on control diet for longer periods of time. Mycotoxins are expected to be excreted through faeces and this could be an explanation for the rapid decline showed for YMW. However, accumulation in faeces were not determined in this study. Niermans et al. (2019) investigated the presence of ZEN in YMW larvae too, carrying out two different time exposure of mycotoxins: short-term (4 weeks) and long-term (8 weeks). The larvae were exposed to ZEN in three different scenarios and larvae were kept fasting for 24h prior to analyses. In some feeding groups, more than 50% of the presumably consumed ZEN remained undetected in the larvae, in unconsumed feed and in larval excretion which confirms findings from van Broekhoven et al. (2014). Niermans et al. (2019) investigated also reductive metabolites (α-ZEL and β-ZEL) and they accounted for a fairly proportion up to 30% on the total ZEN intake in the larvae of YMW. This metabolic pathway was also observed by Camenzuli et al. (2018) for larvae of BSF. No phase II metabolites were detected in the larvae of all feeding groups in the study of Niermas et al. (2019), but ZEN14Sulf and ZELSulf were found in the residues of the groups reared on naturally and artificially contaminated feed. The fact that no phase II metabolites were found in residues of the feeding group with pure ZEN-spiked substrate suggests that no sulfation occurs in the larvae of YMW. This suggested that both free ZEN and ZEN14Sulf were converted either by the larvae or due to contact of unconsumed feed with intestinal bacteria originating from larval faeces. Furthermore, it was concluded by authors, that no ZEN-related substances were detectable in the larvae after 24 h of fasting. Thus, they concluded that
43 regarding feed and food safety that the majority of the ingested toxin was excreted quickly and efficiently and should be irrelevant after a fasting period of 24 h.
Since not all the mycotoxins or metabolites are found in the residues or in the larvae themselves it is suggested in most of the study to investigate the presence of other types of metabolites which can be produced by the insect or bacteria in the gut, or conjugated forms of mycotoxins (bound to protein, sulphates or glucuronide). In just one study led by Janković-Tomanić (2019) the antioxidant defense enzyme such as superoxide dismutase (SOD), catalase (CAT) and phase II biotransformation enzyme, glutathione-S-transferase (GST) were investigated in YMW after the exposure of this larvae to DON-contaminated diets. Larval antioxidant enzyme activities of SOD and GST were altered by the presence of DON in the diet. SOD and GST activity increased in comparison to the control diets. Referring to CAT, no significant differences were found for its activity in the experimental groups when compared to the control. This is an important result because shows that a sort of detoxification occurs in the larvae, but it is still unclear why CAT activity was not altered and what other kind of detoxification occurs in YMW larvae. Moreover, antioxidant enzymes in LMW, BSF or other insects are not being investigated yet, so their data are lacking.
Another important aspect is the well-being of the insect: all the studies consider just the larval growth and the survival rate, but no other parameter is taken into account. Mycotoxins have different toxic action on the body, even long-term damages and problems in the subsequent generations. An example could be ZEN and its metabolites because of their oestrogenic effect (first place α-ZEL) in animals. These metabolites are detected in YMW in some studies but are unknown their effects on reproduction or pupation of the larvae. On the other hand, we must consider that in all studies, the concentration of the mycotoxins or mixes of them, were way higher than the ones set by the EC for food and feed. It is so reasonable to think that these insects could be used as food or/and feed in the
44 next future if the substrate where they are reared on do not exceed the EU limit. It is essential though, to first understand what kind of metabolite or compound is naturally formed once the insect ingest the toxic diet. Most of the authors also recommend a period of fasting (24h or even more) or rearing at least for period of 24h (or more) on uncontaminated diet so that the major part of the mycotoxin can be excreted through faeces. This could be an important practice because the levels of this contaminants seem to drop after these methods in all the studies present in this thesis.
In conclusion, just few studies have been performed regarding the fate and effects of mycotoxins in edible insects. Furthermore the available studies regarded only few types of mycotoxins and didn’t report data regarding the exposure to combinations of mycotoxins. It could be interesting to know how these different mycotoxins interact with insects’ body and how these mycotoxins are metabolized or excreted by larvae. Moreover, it could be important to know how the emerging mycotoxins (such as beauvericin and enniatin) could interact with other mycotoxins in different mixture and at different concentration. Finally it should be evaluated the safety of the animal in first place, how these toxins affect the larvae development, their survival and what kind of damages could affect the future generations.
