Food Additives & Contaminants: Part A

ISSN: 1944-0049 (Print) 1944-0057 (Online) Journal homepage: http://www.tandfonline.com/loi/tfac20

Impact of substrate contamination with

mycotoxins, heavy metals and pesticides on
growth performance and composition of black
soldier fly larvae (Hermetia illucens) for use in the
feed and food value chain

Benedict Purschke, Rafaela Scheibelberger, Sonja Axmann, Andreas Adler &

Henry Jäger

To cite this article: Benedict Purschke, Rafaela Scheibelberger, Sonja Axmann, Andreas
Adler & Henry Jäger (2017): Impact of substrate contamination with mycotoxins, heavy metals
and pesticides on growth performance and composition of black soldier fly larvae (Hermetia
illucens) for use in the feed and food value chain, Food Additives & Contaminants: Part A, DOI:
10.1080/19440049.2017.1299946

To link to this article: http://dx.doi.org/10.1080/19440049.2017.1299946

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Download by: [University of Newcastle, Australia] Date: 27 February 2017, At: 11:42
Publisher: Taylor & Francis & Informa UK Limited, trading as Taylor & Francis

Journal: Food Additives & Contaminants: Part A

DOI: 10.1080/19440049.2017.1299946
Impact of substrate contamination with mycotoxins, heavy metals and
pesticides on growth performance and composition of black soldier fly
larvae (Hermetia illucens) for use in the feed and food value chain

Benedict Purschke*,1, Rafaela Scheibelberger1, Sonja Axmann2, Andreas Adler2, Henry Jäger1

1
University of Natural Resources and Life Sciences (BOKU), Department of Food Science
and Technology, Institute of Food Technology, Muthgasse 18, 1190 Vienna, Austria
2
AGES GmbH, Austrian Agency for Health and Food Safety, Institute of Animal Nutrition
and Feed, Wieningerstraße 8, 4020 Linz, Austria

*Corresponding author e-mail address: benedict.purschke@boku.ac.at

Abstract

Edible insects have emerged as an alternative and sustainable source of high-quality, animal-
derived protein and fat for livestock production or direct human nutrition. During production
of insects, substrate quality is a key parameter to assure optimal insect biomass gain as well as
the safety of feed and food derived from commercially reared insects. Therefore, the influence
of a realistic substrate contamination scenario on growth performance and accumulation
behaviour of black soldier fly larvae (BSFL, Hermetia illucens L.) was investigated. Newly
hatched larvae were fed on a corn-based substrate spiked with heavy metals (As, Cd, Cr, Hg,
Ni, Pb), mycotoxins (aflatoxin B1/B2/G2, deoxynivalenol, ochratoxin A, zearalenone) and
pesticides (chlorpyrifos, chlorpyrifos-methyl, pirimiphos-methyl) under defined breeding
conditions (10 d, 28 °C, 67 % RH). The extent of contaminants bio-accumulation in the larval
tissue as well as the effect on growing determinants was examined. The applied heavy metal
substrate contamination was shown to impair larval growing indicated by significantly lower
post-trial larval mass and feed conversion ratio (FCR). Cadmium and lead accumulation
factors of 9 and 2, respectively, were determined, while concentration of other heavy metals
in the larvae remained below the initial substrate concentration. In contrast, mycotoxins and

1
pesticides have neither been accumulated in the larval tissue nor significantly affected the
growing determinants in comparison to the control. The use of BSFL as livestock feed
requires contaminant monitoring - especially for cadmium and lead - in the substrates as well
as in feedstuff containing BSFL to ensure feed and food safety along the value chain.

Keywords: Black soldier fly larvae; rearing; substrate contamination; mycotoxins;

heavy metals; pesticides; contaminants accumulation; feed and food safety;

Introduction

In the last decade, insects have received wide attention as a highly nutritious, efficient and

sustainable source of animal-derived protein and caloric energy for feed and food purposes. In

view of the predicted increase in world population to over 9 billion people in 2050 and the

growing future demand for high-quality protein in human nutrition as well as livestock

husbandry, insects are discussed as a promising alternative to conventional protein sources

such as meat, crops and fishmeal (FAO 2013, van Huis 2013). Compositional data revealed

that edible insects provide high protein and fat contents of up to 77 % on dry base dependent

of the order, species, metamorphic life stage, diet, and habitat (Nowak et al. 2016, Rumpold

and Schlüter 2013b). Beside the promising composition, insects` high fecundity, multivoltine

life cycle, high feed conversion ratio, omnivorous nutrition, low substrate demands, and low

space and water requirements foster the use of insects to contribute to global food security via

feed or directly as food (Rumpold and Schlüter 2013a). However, the risks associated with the

use of insects in the production of feed need to be sufficiently investigated. Furthermore,

insects which are introduced as new feedstuff will impact the whole food value chain as well.

Currently, there is still a lack of scientifically based knowledge on insect rearing and

processing in industrial scale in order to ensure feed and food quality and safety (Schlüter et

al. 2016). Beside safety aspects, environmental impact of insect rearing and processing have

to be taken into account when considering insects as an alternative source of feed and food.

The insect diet has been identified as a core issue determining sustainability of insect based

1
feed. According to Smetana et al. (2016), the use of low value food processing by-products

such as distilled grains and high-impacting waste streams are the most promising strategies

balancing biomass yield and quality as well as environmental performance.

Among other insect species, the black soldier fly (Hermetia illucens, Diptera) is

suggested to have high potential for large scale production and integration in European food

and feed industry (EFSA 2015, Veldkamp et al. 2012). The black soldier fly (BSF) undergoes

a holometabolic life cycle containing the four live stages egg, larva, pupa and imago (Li et al.

2011). Larvae of the black soldier fly (BSFL) are undemanding feeders with no particular

restriction on their diets. In nature they feed on decaying organic matter and animal manure

(Veldkamp, et al. 2012). Originating from humid, warm climates, the conditions tolerated for

breeding range from 24-40 °C while the optimum for larvae was found at 27 °C (Sheppard et

al. 2002). Larval development requires about two weeks depending on breeding temperature,

substrate composition and substrate availability. Latter were reported to be optimal regarding

larval dry weight and development time when feeding 200 mg/day/larvae (Diener et al. 2015).

BSFL are able to convert low value organic waste streams into protein-rich and oil-rich larval

biomass containing between 35-50 % protein and 17-36 % oil on dry base depending on the

diet (Barroso et al. 2014, Kroeckel et al. 2012, Newton et al. 1977, St-Hilaire et al. 2007). In

several feeding studies, larval or pre-pupae meal were found to be a suitable alternative for

the partial substitution of fishmeal or plant protein supplements in livestock husbandry and

aquaculture regarding growing performance (Elwert et al. 2010, Kroeckel, et al. 2012,

Newton, et al. 1977, St-Hilaire, et al. 2007).

The introduction of insect-based feed and food in the European market is regulated by

European legislation. According to Regulation (EC) No 1069/2009 (EC 2009a), insects are

defined as livestock. As a consequence, feed for insects has to meet the same requirements as

feed for livestock farmed for food production given in the Regulations (EC) No 178/2002 and

767/2009 (EC 2002a, EC 2009b). The maximum level of undesired substances such as heavy
1
metals, pesticides, and mycotoxins in feed used for the production of insects as well as

feedstuff containing insects are covered by the Directive 2002/32/EC (EC 2002b). According

to Regulation (EC) No 999/2001, it is currently prohibited to feed processed animal protein

from insects to farmed animals, while insect oil and hydrolyzed proteins are permitted (EC

2001). In addition, at the annual workshop of animal proteins 2016 organized by the European

reference laboratory of animal proteins (EURL-AP), DG SANTE (Directorate General for

Health & Food Safety; European Commission) reported ongoing discussions on a partial lift

or draft amendment to Regulation (EC) No 999/2001 (EC 2001) with a view to permit the

feeding of insect meal to aquaculture animals, and noted that this was expected to enter into

force in 2017. The use of insects and parts thereof in human diet is covered by the revised

Novel Food Regulation (EU) 2015/2283 (EU 2015) which will take effect in 2018.

Accordingly, all insect products have to be subjected to a pre-market authorisation procedure

assessing the food safety in terms of microbiological hazards, environmental contaminants,

veterinary medicines, and allergenic potential.

Feedstuffs contain undesired substances witch can have an adverse effect on the

animal organism and, subsequently, to foodstuffs of animal origin. The presence of these

substances cannot be excluded completely. In this context it is important to determine and

reduce their content in feedstuffs taking into account their acute toxicity, their ability to

bioaccumulation and their degradability to a level where no undesirable or harmful effects can

occur. The content of heavy metals in soil is naturally and geologically limited. The entry via

air as well as fertilizers, plant protection products and waste materials is of crucial importance

for plant feed materials. Assimilation of heavy metals via soil into the plant is the most

frequent entry into the food chain (Wuana and Okieimen 2011). Within the last five years

AGES GmbH (Austrian Agency for Health and Food Safety GmbH) observed in Austria eight

exceedances of the maximum arsenic level in official feed control samples (n = 2005). The

1
maximum cadmium and mercury levels were exceeded once in 2393 and 607 official control

samples, respectively (AGES 2016).

Plant protection products are used because plant diseases, animal pests and the

competition with unwanted frequently toxic plants put crop yield and quality at risk

compromising feed safety and following the supply of food to the population (Al-Saleh 1993).

Maximum values of plant protection product residues are defined within the authorization

process stated in Regulation (EC) No 396/2005 (EC 2005). In 2014 and 2015, the maximum

level of the insecticide chlorpyrifos in soy extraction meal and hexachlorobenzol in fish meal

were exceeded (AGES 2016).

Mycotoxins are metabolic products of fungi with diverse animal and human toxicity.

Particularly monogastrics (e.g. pig, poultry) are endangered when they are fed with

contaminated cereals. Mycotoxin exposure over an extended period of time can cause acute

poisonings or induce mutagenic, carcinogenic or teratogenic effects (Tola et al. 2016).

Maximum value of aflatoxin B1 in feed is regulated in the Directive 32/2002/EC (EC 2002b).

Guideline values of deoxynivalenol (DON), zearalenone (ZON), ochratoxin A and fumonisins

are defined in Regulation (EC) No 476/2006 (EC 2006). Exceeding of maximum values will

result in a ban on mixing, while exceeding of guideline values will allow a dilution with less

contaminated feed. Within the last five years AGES GmbH detected three times increased

DON in feed for pig and one times increased aflatoxin B1 and ZON values in grain out of

1764 feed control samples (AGES 2016).

Several studies already indicated that insects - either wild harvested or farm-raised -

are potentially vulnerable to accumulation of chemical contaminants ingested via

contaminated feed or water (Marone 2016). Accumulation of mercury, arsenic, and lead were

reported for wild harvested insects (Green et al. 2001, Handley et al. 2007, Zhang et al. 2012).

Vijver et al. (2003) showed that lead and cadmium concentration of mealworm larvae

(Tenebrio molitor) reared on spiked soils linearly increase with exposure time. Diener, et al.
1
(2015) investigated the bioaccumulation of heavy metals in BSFL reared on spiked chicken

feed containing cadmium, lead and zinc, respectively. Level of accumulated cadmium in

larvae and prepupae was higher than the original cadmium concentration in the substrate

while lead and zinc were not accumulated. Interestingly, heavy metal concentration in adult

BSF significantly decreased. This phenomenon was ascribed to defecation prior pupation.

Tschirner and Simon (2015) showed accumulation of lead and cadmium in BSFL reared on

plant based substrates. van der Fels-Klerx et al. (2016) reported slower development and

lower live weight of BSFL reared on cadmium-spiked substrate. Charlton et al. (2015)

assessed the chemical safety of cultivated larvae of different fly species reared using different

methods and waste substrates in four geographically dispersed locations. Contaminants

belonging to the group of heavy metals, pesticides, mycotoxins, and veterinary medicines

were detected, whereas only cadmium level in three house fly samples exceeded the EU limit

for animal feed (500 µg/ kg). High levels of phosphorous-containing pesticides around 750

µg/kg and medium levels of chlorinated pesticides were found in locusts captured in Kuwait

(Saeed et al. 1993). Furthermore, studies showed that mealworm larvae (Tenebrio molitor)

accumulate, degrade, and enantiomerize several types of pesticides such as benalaxyl (Gao et

al. 2013), epoxiconazole (Lv et al. 2014), and metalaxyl (Gao et al. 2014) as well as the

mycotoxin zearalenone (Hornung 1991).

Consequently, it is of crucial importance to evaluate whether bioaccumulation or up-

take of contaminants occurs during insect rearing on contaminated substrate. Hence, this

study aims to investigate the growth performance and the accumulation of relevant feedstuff

contaminants by black soldier fly larvae (BSFL) reared on spiked substrates containing

defined amounts of heavy metals, pesticides, or mycotoxins. Thereby, the work contributes to

assure appropriate rearing conditions for insects considering substrate quality and to assure

the safety of insect derived feed and food used for animal and human nutrition.

1
Material and methods

Raw material
Newly hatched, seven days old larvae were purchased from a commercial breeder (Bioflytech,

Alicante, Spain). During the first week after hatching, young larvae were fed on wheat flour.

In order to select larvae of homogeneous size, they were sieved using 1.2 mm mesh size

according to the procedure of Tschirner and Simon (2015). The larvae which did not pass the

mesh were excluded from the trials. Mean individual larvae mass were determined by

weighing 20 larvae in a ten-fold replication. The larvae used had a mean individual weight of

5.8 ± 0.1 mg. Initial dry matter content (23.4 ± 0.1 %) of young larvae was determined

according to method 950.46 (AOAC 2002).

The rearing substrates for the experimental part were based on corn semolina (Strobl

Naturmühle GesmbH, Linz-Ebelsberg, Austria). According to the manufacturer`s

specification corn semolina was composed as follows: 76 % carbohydrates, 10 % fibre, 9 %

protein, 4 % fat, and 1 % minerals on dry base. The moisture content was found to be

9.12 ± 0.04 %. For the introduction of mycotoxins, ground naturally DON and ZON

contaminated corn grains from an official control sample were used while the corn semolina

was used as a carrier for pesticides. Particle size of both corn products was fairly equalized by

grinding the corn semolina using a pin mill (Pallmann, Zweibrücken, Germany) to obtain a

homogeneous particle spectrum of the substrate. Determination of particle size was done via

dynamic image analysis (Camsizer 4, Retsch Technology, Haan, Germany). Results are based

on the minimal Martin diameter and summarized in Table 1.

Substrate preparation
Composition of control substrate and contaminated substrates are presented in Table 2.

Contaminants were introduced either via spiked corn flour/semolina (mycotoxins, pesticides)

or water (heavy metals). Contaminant concentrations of the substrates were chosen according

1
to common contamination levels found in animal feed. Final moisture content of all substrates

was adjusted to 45 %. Heavy metal substrate (HM) was spiked with an aqueous solution of

heavy metal ions resulting in the following final concentration (values on wet base):

2 mg/kg As, 1 mg/kg Cd, 10 mg/kg Pb, 0.1 mg/kg Hg, 10 mg/kg Cr, and 10 mg/kg Ni.

Mycotoxin substrate (MT) was inoculated with a spiked corn standard. Final mycotoxin

concentrations in the substrate on wet base were as follows: 4600 ± 1400 µg/kg DON

(naturally contaminated), 88 µg/kg Aflatoxin B1, 17 µg/ kg Aflatoxin B2, 46 µg/kg Aflatoxin

G2, 260 µg/kg Ochratoxin A, and 860 ± 260 µg/kg ZON (naturally contaminated). For the

pesticide substrate (PC), 100 g of spiked corn flour was used to adjust the final substrate

concentrations of chlorpyrifos, chlorpyrifos methyl, and pirimiphos methyl to 2.5 mg/kg on

wet base.

Feeding trial
The feeding trials were conducted with two replicates per substrate in a climate chamber at

28 °C and 67 % RH under exclusion of light. Control trial was done in quadruplicate. Plastic

boxes (39 x 28 x 14 cm) sealed with plastic tulle (pores size < 1 mm) allowing air circulation

were used as rearing containers. The substrate supply rate was set to 200 mg/day/larvae

according to feeding trials conducted by Diener, et al. (2015). The total substrate amount of

1 kg (wet weight, 45 % moisture) was evenly distributed in each container forming a substrate

layer of about 4 cm. According to preliminary trials, approximately 700 larvae based on mean

individual weight were placed in each container. Along with the moisture content of the

substrate increasing bacterial and mold growth in the preliminary trial proved to be a limiting

factor with regard to the possible duration of the incubation. High germ contents of Bacillus

species, enterobacteria and mainly lactic acid bacteria were detected in the substrate by

microbiological analysis according to VDLUFA (2012) after 13 days of rearing. The mold

growth in this time increased visibly. Predominantly Mucorales and Penicillium species were

1
detected but Aspergillus species as possible aflatoxin formers were not found. To achieve

sufficient biomass gain, the larvae were incubated for ten days under above mentioned

conditions without further addition of feed or water. Under these conditions the substrate was

allowed to dry slowly to facilitate the harvest.

At the end of the incubation time, larvae were collected from the containers using a

sieve (mesh size 1.2 mm) and forceps. The collected larvae were weighed and counted,

followed by a washing step to remove residual substrate from the surface. Postharvest dry

matter content of larvae were analysed in duplicate according to method 950.46 (AOAC

2002). Larvae were frozen at -30 °C and subsequently freeze dried at -49 °C and 0.2 mbar

(FreeZone 6, Labconco, Kansas City, USA). After four days of freeze-drying, the larvae were

ground using a kitchen mixer (Mini Multi Deluxe, Moulinex, Alencon, France), freeze-dried

for three more days and stored until further analysis.

The following parameters were determined according to Tschirner and Simon (2015)

to describe the rearing performance of the larvae: initial total larvae mass, initial larvae count,

post-trial total larvae mass, post-trial larvae count, post-trial individual larvae mass, post-trial

larvae dry matter, and substrate consumption which is defined as mass difference of initial

and residual substrate on dry weight. Furthermore, the feed conversion ratio on dry base

(FCRd) as well as on wet base (FCRw) was calculated according to Eq. (1) and Eq. (2).

substrate consumption [g dry weight] (1)

=
post trial total larvae mass [g dry weight] − initial total larvae mass [g dry weight]

substrate consumption [g wet weight] (2)

=
post trial total larvae mass [g wet weight] − initial total larvae mass [g wet weight]

Analysis of contaminants

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Mycotoxins
Ground larvae or residual substrate were weighed and mixed with an extraction solution

containing of 79 % acetonitrile, 20 % deionized water and 1 % pure acetic acid to obtain a

ratio of sample and solution of 0.25 g/ml. The mixture was stirred on a magnetic stirrer for

2 hours. The resulting extract was then centrifuged for 2 minutes at 3500 rpm. Following, the

supernatant was tenfold diluted with 1 % pure acetic acid in deionized water and filtered with

a 0.2 µm sterile filter. The diluted extract was directly measured after HPLC Agilent 1290

Infinity- separation system (Agilent Technologies, Santa Clara, CA, USA)) with the liquid

column Phenomenex C18 “Gemini” 150 4.6 mm 5 µm-particle (Phenomenex Inc., Torrance,

CA, USA) using the triple-quadrupole mass spectrometer AB Sciex 5500 QTRAP with

“Turbo ion spray”; ESI source (SCIEX, Framingham, MA, USA)). The column is operated at

a flow rate of 1 ml per minute. Determination of mycotoxins was done in duplicate.

Heavy metals
The samples were digested using a pressurized microwave assisted acid digestion. Therefore,

0.3 g sample was weighed into a quartz vessel and 3 ml of 65 % nitric acid as well as 0.5 ml

30 % hydrochloric acid were added. The vessels were closed with caps and placed in the

UltraCLAVE (MLS GmbH, Leutkirch, Germany), which was pressurized with nitrogen (40

bars). The samples were digested according the following program: ramp in 5 min to 80 °C /

in 15 min up to 140 °C / in 15 min up to 230 °C / 20 min at 230 °C. After digestion, all quartz

vessels were filled to the 15 ml mark with ultrapure water and transferred into 16.5 ml PP

vials. Before the analysis an aliquot of this is diluted tenfold with ultrapure water and directly

used for multi-element measurement with an Agilent 7500 ce single quadrupole ICPMS

(Agilent Technologies, Santa Clara, CA, USA). The instrument was equipped with a micro

mist nebulizer, a Scott double pass spray chamber, a 2.5 mm ID quartz torch, a sampler cone

made from copper with nickel tip and a skimmer cone made from nickel. The instrument was

1
tuned for suitable sensitivity and robustness with oxide ratios < 2 % (156CeO+/140Ce+) in

nogas mode and < 3.0 % doubly charged ions (70Ce+/140Ce++) in no gas mode. Oxide ratios

and doubly charged ratios were lower in collision mode respectively. Determination of heavy

metals was done in duplicate.

Pesticides
2,5 g ground larvae or residual substrate were weighed into a 50 ml PP reaction tube, mixed

with 20 ml extraction solution composed of acetonitrile/deionized water (1:1, v/v) and mixed

for 10 min on a mechanical shaker (400 swings/min). Afterwards 6.5 g salt mixture (ratio

8:2:2:1: magnesium sulfate water free, sodium chloride, tri-sodium citrate di-hydrate, di-

sodium hydrogen citrate sesqui-hydrate) were added, immediately shaken thoroughly for

1 min by hand and centrifuged for 5 min at 4000 rpm. The organic phase was further cleaned-

up with dispersive SPE (solid phase extraction: 100 mg magnesium sulfate water free (Merck

Chemicals and Life Science GesmbH, Darmstadt, Germany), 75 mg Bulk Sorbent Bondesil

PSA 40 µm (Agilent Technologies, Santa Clara, CA, USA) and 50 mg Discovery® DSC-18

SPE (Sulpeco, Bellefonte, PA, USA) per ml acetonitrile extract). After mixing thoroughly for

1 min by hand to get sufficient homogeneity an additional clean-up step was performed (LLE;

liquid-liquid extraction). Therefore, 5 ml sodium chloride solution (20 % sodium chloride in

water) were mixed with 1 ml SPE cleaned extract and 1 ml n-hexane, shaken thoroughly for

1 min by hand and further 10 min with a mechanical shaker. After centrifugation for 5 min at

4000 rpm/min, 1 µl of the hexane phase (upper phase) was injected via splitless injector into

HP7890 GC-system with multimode inlet injector (Agilent Technologies, Santa Clara, CA,

USA) and detected with 7010 triple quadrupole GC/MS (Agilent Technologies, Santa Clara,

CA, USA). Determination of pesticides was done in duplicate.

1
Statistical analysis
Statistical analyses were carried out using Statgraphics Centurion XVI (Statpoint

Technologies Inc., Warrenton/USA). Data are expressed as mean ± standard deviation (SD) of

the performed replications. Data were subjected to one-way analysis of variances (ANOVA),

and means were generated and adjusted using Bonferroni post-hoc test. Unless otherwise

stated, a 95 % confidence level (statistical significance at p ≤ 0.05) was considered.

Results and Discussion

Rearing parameter

1
Table 3 presents the growth performance parameters of BSFL reared on control and

chemically contaminated corn substrates. Independent of the feeding substrate, BSFL

consumed around 100 g substrate on dry base which equals to about 20 % of the total

available substrate dry matter. Substrate consumption between 38-66 % (dry matter) within

15 days were reported for BSFL reared on different vegetable substrates (Tschirner and

Simon 2015). Diener, et al. (2015) found overall reduction of organic waste substrate (dry

matter) during the development of BSF from egg to prepupae (16-42 days) between 26-43 %

depending on the food supply. Newton et al. (2005) and Sheppard et al. (1994) reported 39 %

and 50 % reduction of pig manure (dry matter) and chicken manure (wet matter) by BSFL.

Due to the shorter rearing period used in the present study, a lower substrate consumption was

found.

Survival rate as well did not significantly differ between the tested substrates. Post-

trial larvae count was found to be in the range of 65-70 % of the initial larvae amount. Within

their study, Tschirner and Simon (2015) reported survival rates between 22-78 % of BSFL

reared on different plant based substrates. van der Fels-Klerx, et al. (2016) reported survival

rates of BSFL in the range of 81-97 %. Differences between larvae reared on control and

contaminated substrate spiked with cadmium, lead, and arsenic were not found.

Significant differences could be observed for the post-trial larvae mass. Total as well

as individual mass of larvae harvested from HM substrate were significant lower in

comparison to the control, MT, and PC substrate, respectively. Thus, growth performance of

BSFL was negatively influenced by the applied heavy metal concentrations in the substrate,

whereas the presence of mycotoxins or pesticides did not alter the generation of larval

biomass compared to the control. This result is in consistency with Diener, et al. (2015) who

showed slightly increasing development time of BSFL with increasing substrate concentration

of lead and cadmium. Similar results were reported by van der Fels-Klerx, et al. (2016). BSFL

reared on cadmium-spiked substrate (0.25 mg/kg) showed significant lower total larval live
13
weight and longer development time in comparison to larvae fed with control, lead-, and

arsenic-spiked substrate, respectively. Individual larvae wet weight reached a minimum of

45 mg (HM) and a maximum of 84 mg (PC). In several feeding studies, post-trial BSFL mass

on wet base were reported to range between 88-175 mg (Diener, et al. 2015), 34-290 mg

(Tschirner and Simon 2015), 110-220 mg (Sheppard, et al. 1994), 100-160 mg (St-Hilaire, et

al. 2007), 194-315 mg (Banks et al. 2014), and 138-220 mg (Diener et al. 2011), respectively.

The generally low individual mass gain in comparison to the values reported in literature may

be explained by different feeding trial design and parameters such as rearing period, feed

composition, feed supply, and breeding conditions.

Determination of post-trial larvae dry matter revealed a significant higher dry matter

content of larvae fed on HM substrate. Similar trend were reported by Diener, et al. (2015). In

their study, larvae fed on substrate spiked with cadmium exhibited significantly increased dry

matter contents in comparison to the control. This was traced back to the accumulation of

heavy metal ions in the larval tissue resulting in an increased proportion of dry matter.

According to the lower biomass gain of larvae reared on HM substrate, feed

conversion ratio (FCRd and FCRw) as well significantly differs among the tested substrates.

Larvae reared on control, PC and MT substrate achieved a dry based FCRd of around 10.

These findings are in accordance to results reported for BSFL reared on pig manure and

organic waste by Newton, et al. (2005) and Diener, et al. (2011) who found FCRs of 9.6 and

14.5, respectively, based on dry weight. In contrast, feeding of BSFL on HM substrate

resulted in a significant higher FCRd of 18. This may be ascribed to the negative influence of

heavy metal accumulation on growth performance and development velocity. Wet base FCRw

were found to range between 13 and 19 for control, MT and PC substrates while rearing on

HM substrate led to a significant higher FCRw of about 31. Feeding trials of BSFL on

vegetable products, human faeces, chicken manure, and chicken/cow manure resulted in wet

weight based FCR of 4.6-29.4 (Tschirner and Simon 2015), 2.0-33.9 (Banks, et al. 2014),
14
13.4 (Sheppard, et al. 1994), and 10.0 (Morgan and Eby 1975) depending on the substrate

composition and feeding regime.

Contaminants

Error! Reference source not found. shows the analytical results of the harvested larvae and

the corresponding residual substrate after rearing with mycotoxins, heavy metals or pesticides

contaminated substrates. The values are all calculated on dry weight. In both independent

feeding trials with MT substrate no toxin could be found in the larvae emerged from it.

Whereas the residual substrate provides toxin values comparable with the spiked amounts of

the feed. Only DON as an exception showed significant higher values in the residual substrate

than in the starting material. The reason for that could be masked mycotoxins which are for

example bound to carbohydrate or protein matrix what is catalyzed by plant enzymes mostly

involved in detoxification processes (Berthiller et al. 2013). These mycotoxin derivatives are

undetectable by conventional techniques what may have led to an underestimation of the

DON content in the corn substrate. During digestion process of the insects, the masked

mycotoxins could have been reverted to their original chemical structure by hydrolysis and

thus can be detected in the residual substrate. Berthiller et al. (2011) showed in their in vitro

study that several lactic acid bacteria, normally found in the intestinal flora, have a high

capability to hydrolyze the derivative deoxynivalenol-3-β-d-glucoside (D3G) back to its toxic

form DON. The already proven high number of lactic acid bacteria in the substrate after

rearing might be responsible for the chemical conversion of masked mycotoxins and explain

the increased mycotoxin concentration in the residual substrate.

Heavy metals act different during the rearing process. In the present study significant

bioaccumulation of cadmium and lead was observed in the larvae. The bioaccumulation factor

(BAF, i.e. concentration in organism divided by the concentration in substrate) was calculated

according to Walker (1990). BAF of 9.1 ± 1.4 for cadmium and 2.3 ± 0.3 for lead were

determined. In their study, Tschirner and Simon (2015) achieved comparable cadmium and
15
lead BAFs of 7.4 ± 2 and 2.6 ± 0.6 depending on the substrate used. Similar BAFs were

reported by van der Fels-Klerx, et al. (2016) for cadmium (6.1-9.5) and lead (1.2-1.4)

depending on the initial concentration in the contaminated substrates. In the residual substrate,

cadmium and lead concentrations fairly corresponding to the initial quantity were detected. In

contrast, chrome and nickel were present in the larvae almost four times less than in the initial

quantity, whereas the residual substrate showed slightly higher values. Arsenic was found in

the larvae and the residual substrate in equal amounts to the starting material. Mercury was

not accumulated in the larvae, and the residual substrate yielded slightly higher mercury

content in comparison to the initial substrate.

Bioaccumulation could not be observed in the larvae cultured on PT substrate.

Pesticide concentrations below those of the starting material were detected. Beyond that,

lower amounts of pesticides in the residual substrate were detected as well. During breeding a

large and diverse number of microorganisms proliferated, which in turn can be responsible for

a variety of processes. The degradation of pesticides by microorganisms might be a decisive

factor for the decrease of pesticides in the residual substrates. Singh, et al. verified in their

work that a consortium of bacteria such as Pseudomonas, Klebsiella, Stenotrophomonas,

Ochrobactrum and Bacillus species can efficiently degrade chlorpyrifos in a soil-water system

(Singh et al. 2016). Additional, chlorpyrifos-methyl was reported to be hydrolyzed by water,

the rate being dependent on temperature and pH value (JMPR 1975). The analytical detection

of the metabolites was not included.

During control experiments, insects were fed on non-contaminated substrate. The

determination of the contaminant concentration in larvae and residual substrate revealed the

expected results which are presented in Table 5. Mycotoxins and heavy metals were not

detected in relevant quantities in larvae as well as in the residual substrates. Pesticides were

not found at all.

16
 The analysis results of the contaminants correlate with the observations from the

rearing experiments. Growth on HM substrate significantly reduced biomass gain of the

larvae associated with a significant bioaccumulation of the heavy metals cadmium and lead.

Furthermore, MT and PT substrate did not affect the growth of the larvae and mycotoxins and

pesticides were not absorbed in relevant amounts or accumulated in the larval tissue.

Conclusions
Insect production for animal feed and human food is currently evolving in Western countries.

Beside the lack in agricultural and technical know-how regarding the industrial-scale

production of insect biomass and appropriate post-harvest processing concepts, there are

major concerns connected with feed and food safety issues such as microbiological status,

allergenicity, and toxic environmental contaminants.

The present study aimed to investigate the impact of plausible substrate contamination

levels on the mass gain performance of BSFL and their bio-accumulation of relevant feedstuff

contaminants. Larval development was found to be negatively influenced by the applied

heavy metal substrate contamination level indicated by the low post-trial larvae mass and feed

conversion ratio. This phenomenon was shown to be associated with a considerable

accumulation of cadmium (BAF of 9) and lead (BAF of 2) in the larval tissue exceeding the

statutory thresholds for animal feed. Hence, production of BSFL as a livestock feed requires a

strict monitoring of heavy metal concentration – especially cadmium and lead - in the

substrates as well as in feedstuff containing BSFL in order to secure safe (accumulation) as

well as economically reasonable (growth performance) products.

In contrast, tested mycotoxins and pesticides were not accumulated by the larvae.

Also, growth performance of BSFL was not compromised by the presence of mycotoxins and

pesticides in the rearing substrate. According to these findings, BSFL may be used to convert

contaminated substrate exceeding the allowed level of mycotoxins and/or pesticides into

17
insect biomass for feed or non-food application without the transmission of those

contaminants in the feed chain.

In order to generate a deeper knowledge about the interaction between edible insects,

substrate contaminants and feed/food safety, studies are needed regarding the accumulation

behaviour depending on species, metamorphic development stage, kinetics, and other relevant

contaminants or harmful substances (e.g. dioxins or biogenic amines). Considering that

sustainability of insect production is decisively determined by the feedstuff used for rearing,

alternative substrates such as food by-products or organic waste streams have to be taken into

account. Beside the possible safety risks accompanied with the use of those side streams,

important issues such as substrate homogeneity, standardisation and availability have to be

tackled.

Acknowledgements
The authors would like to thank Franz Mlynek, Maximilian Rührlinger, Andreas Della-Rosa,

Wolfgang Brodacz, Karolina Lichtmannegger and Hermann Unterluggauer for providing and

spiking the corn flour with contaminants as well as for performing heavy metal, mycotoxin

and pesticide analysis.

Conflict of interest
The authors have declared no conflicts of interest.

18
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24
List of Tables

Table 1 - Particle size characteristics in terms of D values of the substrate base (pin-milled
corn semolina) and the ground corn used for the inoculation of the substrate with pesticide
and mycotoxin standards. Values are based on the minimal Martin diameter.

D values Corn semolina Solid corn inoculum

D10 [µm] 90.6 23.5
D50 [µm] 366.3 238.9
D90 [µm] 620.5 573.6

Table 2 - Composition of the control substrate as well as the substrates spiked with heavy
metals (HM), mycotoxins (MT), and pesticides (PC).

Substrate Corn Solid corn Aqueous Water Total

semolina inoculum inoculum addition
[g] [g] [g] [g] [g]
Control 605.20 - - 394.80 1000
HM 605.20 - 37.13 357.67 1000
MT 505.20 100.00 - 394.80 1000
PC 505.20 100.00 - 394.80 1000

25
Table 3 - Initial and post-trial parameters for feeding trials of Black soldier fly larvae reared on control and chemically contaminated substrates
containing defined amounts of heavy metals (HM), mycotoxins (MT) and pesticides (PC).

Substrate Initial total Initial Substrate Post-trial Post-trial Post-trial Post-trial Feed Feed
larvae mass larvae count consumption total larvae larvae count individual larvae dry conversion conversion
mass [-] larvae mass matter ratio FCRd ratio FCRw
[g WW] [-] [g DW] [g WW] [mg WW] [%] [-] [-]
Control 4.10 ± 0.12 707 ± 21 106 ± 10 34.76 ± 5.27b 464 ± 63 74.8 ± 2.9b 34.8 ± 0.8a 9.6 ± 0.6a 17.7 ± 3.3a
HM 4.06 ± 0.37 700 ± 6 117 ± 4 20.34 ± 0.04a 452 ± 0 45.0 ± 0.1a 36.8 ± 1.0 17.9 ± 0.2b
b
31.5 ± 0.3b
MT 4.08 ± 0.01 703 ± 1 102 ± 15 31.66 ± 2.98ab 492 ± 47 65.0 ± 12.3ab 34.9 ± 0.5a 10.1 ± 0.3a 19.3 ± 2.7a
PC 4.07 ± 0.04 701 ± 6 121 ± 6 41.21 ± 1.09b 489 ± 10 84.3 ± 0.5b 33.8 ± 0.7a 9.4 ± 0.1a 13.7 ± 0.6a

Values are means ± standard deviation. In the same column, means followed by different superscripts are significant different among samples
(p ≤ 0.05) according to one-way ANOVA and Bonferroni post hoc test.
DW = dry weight; WW = wet weight

26
Table 4 - Analytical results of initial substrate, ground larvae and residual substrate after
rearing on mycotoxin (MT), heavy metal (HM) and pesticide (PC) contaminated substrates.
Values are calculated on dry weight and specified with measurement uncertainty.

Feeding trials
Residual
Initial substrate Larvae
Mycotoxins substrate
[µg/kg] [µg/kg]
[µg/kg]
Deoxynivalenol* 697.7 ± 212.3 n.d. 1135.7 ± 345.7
Aflatoxin B1 13.3 n.d. 10.9 ± 5.5
Aflatoxin B2 2.6 n.d. < LOQ 4
Aflatoxin G2 7 n.d. < LOQ 16
Ochratoxin A 39.4 n.d. < LOQ 20
Zearalenone* 130.4 ± 39.4 n.d. 103.7 ± 31.6
Residual
Initial substrate Larvae
Heavy metals substrate
[mg/kg] [mg/kg]
[mg/kg]
Chrome 15.2 3.4 ± 0.5 19.9 ± 3.0
Nickel 15.2 4.2 ± 0.6 19.7 ± 3.0
Arsenic 3.0 2.8 ± 0.4 3.8 ± 0.6
Cadmium 1.5 13.7 ± 2.1 1.8 ± 0.3
Mercury 0.2 0.1 ± 0.03 0.3 ± 0.08
Lead 15.2 35.6 ± 5.3 19.8 ± 3.0
Residual
Initial substrate Larvae
Pesticides substrate
[mg/kg] [mg/kg]
[mg/kg]
Chlorpyrifos 0.4 0.006 ± 0.003 0.228 ± 0.114
Chlorpyrifos-methyl 0.4 < LOQ 0,001 0.068 ± 0.034
Pirimiphos-methyl 0.4 0.001 ± 0.0005 0.172 ± 0.086
n.d. = not detected; LOQ = limit of quantification;
*detected values of naturally contaminated DON and ZON

27
Table 5 - Analytical results of ground larvae and residual substrate after rearing with non-
contaminated control substrate. Values are calculated on dry weight and specified with
measurement uncertainty.

Control trials
Residual
Larvae
Mycotoxins substrate
[µg/kg]
[µg/kg]
Deoxynivalenol n.d. < LOQ 80
Aflatoxin B1 n.d. n.d.
Aflatoxin B2 n.d. n.d.
Aflatoxin G2 n.d. n.d.
Ochratoxin A n.d. n.d.
Zearalenone n.d. n.d.
Residual
Larvae
Heavy metals substrate
[mg/kg]
[mg/kg]
Chrome 0.064 ± 0.01 0.045 ± 0.007
Nickel 0.048 ± 0.007 0.189 ± 0.028
Arsenic < LOQ 0.024 < LOQ 0.024
Cadmium 0.048 ± 0.007 < LOQ 0.005
Mercury < LOQ 0.012 < LOQ 0.012
Lead 0.032 ± 0.005 0.024 ± 0.004
Residual
Larvae
Pesticides substrate
[mg/kg]
[mg/kg]
Chlorpyrifos n.d. n.d.
Chlorpyrifos-methyl n.d. n.d.
Pirimiphos-methyl n.d. n.d.
n.d. = not detected; LOQ = limit of quantification;

28
Graphicaal Abstract

29