3.1. Mineral content changes in Hermetia illucens larvae frass after manure bioconversion
The bioconversion of organic materials was able to cause changes in the mineral content of the substrates. In this study, as shown in Table 2, Cu, Mn, and Na content were significantly increased, and Ca and K content showed a significant reduction in Hermetia illucens frass after cattle manure bioconversion. However, Fe, Mg, P, S, Zn, and the content of the toxic metals showed no marked changes. This finding is similar to the effect of Musca domestica larvae only in Cu, Fe, Na, P, and S content of cattle manure reported by Hussein et al. (2017). These differences could be linked to two factors: 1) the difference between the bioaccumulation factors of Musca domestica and Hermetia illucens, and 2) the higher water and dry matter reduction by Musca domestica in the cited study (37%) than for Hermetia illucens in this study (16.8%). Considering the potential use of larvae frass as biofertilizers, great attention should be given to high rates of reduction of organic matter of waste and low levels of element uptake, which could lead to an accumulation of undesired elements. For example, Zhu et al. (2015) reported a slight increase of 11% and 9% of initial Cd and Cr concentrations, respectively, and a 2.5 fold increase in the Pb concentration after pig manure bioconversion by Musca domestica larvae. The authors also registered a reduction of 74% in water content. On the other hand, Proc et al.(2020a) found a significant increase in the Cd content and a considerable reduction in dry matter (64.7%) of fish feed not spiked by the action of Hermetia illucens larvae. Furthermore, in substrates spiked with cadmium and lead solutions, the concentrations of these elements increased by up to 10%; a 3.7 fold increase on the initial Cd and Pb concentration, respectively, after bioconversion by Hermetia illucens larvae (Fels-Klerx et al. 2016). This effect could be detrimental if the concentrations of the toxic metals in the substrate are very close to the limit values for a particular use. The Hermetia illucens larvae could increase the concentration of these elements, thereby causing these to exceed allowed values. Furthermore, monitoring of toxic elements levels in substrates used as feed for Hermetia illucens larvae is recommended.
Table 2
Mineral content in Hermetia illucens larvae and resídues after cattle manure bioconversion (mean ± 95% confidence interval)
Elements
|
Young larvae
|
Larvae
|
p-value*
|
CM (Residue)
|
CM + HI (frass)
|
p-value*
|
Fish meal**
|
Soybean meal***
|
Micro and macronutrients
|
Ca (g kg− 1)
|
24.62 ± 1.93
|
66.35 ± 1.62
|
0.00
|
7.98 ± 1.74
|
4.95 ± 0.34
|
0.01
|
30.3
|
3
|
Cu (g kg− 1)
|
< 0.006
|
0.02 ± 0
|
-
|
0.04 ± 0.01
|
0.05 ± 0.01
|
0.04
|
0.03
|
0.01
|
Fe (g kg− 1)
|
0.75 ± 0.03
|
1.06 ± 0.12
|
0.00
|
2.06 ± 0.98
|
2.00 ± 0.38
|
0.83
|
0.91
|
0.15
|
K (g kg− 1)
|
17.84 ± 1.11
|
18.69 ± 0.6
|
0.06
|
4.78 ± 0.65
|
8.05 ± 1.89
|
0.01
|
8.5
|
21.5
|
Mg (g kg− 1)
|
4.57 ± 0.29
|
6.65 ± 0.23
|
0.00
|
4.83 ± 0.82
|
5.14 ± 0.83
|
0.31
|
3
|
3.1
|
Mn (g kg− 1)
|
0.10 ± 0.01
|
2.26 ± 0.05
|
0.00
|
0.69 ± 0.16
|
0.34 ± 0.06
|
0.01
|
0.01
|
0.03
|
Na (g kg− 1)
|
2.53 ± 0.11
|
2.63 ± 0.13
|
0.08
|
1.88 ± 0.03
|
2.3 ± 0.16
|
0.01
|
11.1
|
0.01
|
P (g kg− 1)
|
12.68 ± 1.03
|
12.15 ± 0.39
|
0.15
|
3.31 ± 1.44
|
3.67 ± 0.61
|
0.40
|
21.6
|
6.2
|
S (g kg− 1)
|
4.35 ± 0.44
|
4.88 ± 0.15
|
0.02
|
2.58 ± 0.61
|
2.78 ± 0.23
|
0.29
|
4.00a
|
4.2
|
Zn (g kg− 1)
|
0.43 ± 0.03
|
0.54 ± 0.05
|
0.00
|
0.41 ± 0.15
|
0.50 ± 0.17
|
0.17
|
0.05
|
0.05
|
Toxic elements
|
Cd (mg kg− 1)
|
0.02 ± 0.04
|
0.34 ± 0.03
|
0.00
|
0.45 ± 0.04
|
0.42 ± 0.05
|
0.08
|
-
|
-
|
Cr (mg kg− 1)
|
1.09 ± 0.13
|
2.46 ± 0.11
|
0.00
|
2.89 ± 2.03
|
5.76 ± 5.51
|
0.14
|
-
|
-
|
Pb (mg kg− 1)
|
< 0.002
|
0.19 ± 0.36
|
-
|
< 0.002
|
< 0.002
|
-
|
-
|
-
|
The mineral content of Hermetia illucens larvae frass obtained in this study was compared with European, Canadian, American, and Brazilian maximum limits of contaminants in organic fertilizers to assess its suitability for agricultural use. As shown in Tables 2 and 3, Cu, Zn, Cd, and Pb content met the maximum limits established by cited legislations; however, Cr content only met the Canadian maximum limits. A reason for this finding could be linked to Cr supplementation in cattle feeds and subsequent transfer to excreta. Usually, diverse forms of Cr are used in feed to treat mental, physical, or metabolic stress in cattle They sometimes can exceed the maximum limits for animal feed by six fold, as reported by Li et al.(2019). Therefore, in this case, it would be recommendable that a subsequent treatment process can be used to improve the suitability of larvae frass, for example, for co-composting with other residual materials that have low Cr content.
Table 3
Maximum limits of mineral content in animal feed and organic fertilizer
Elements
|
Maximum Limits for feed
|
Maximum Limits for organic fertilizer
|
EU1
|
USA2
|
Canada3
|
EU4
|
USA5
|
Canada6
|
Brazil7
|
Ca (g kg− 1)
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
Cu (g kg− 1)
|
-
|
0.25p, s; 0.1f
|
-
|
0.3
|
1.5
|
0.4
|
-
|
Fe (g kg− 1)
|
-
|
0.5p; 3.0s
|
-
|
-
|
-
|
-
|
-
|
K (g kg− 1)
|
-
|
10.0a
|
-
|
-
|
-
|
-
|
-
|
Mg (g kg− 1)
|
-
|
5.0p; 2.4s; 3f
|
-
|
-
|
-
|
-
|
-
|
Mn (g kg− 1)
|
-
|
2.0p; 1.0s
|
-
|
-
|
-
|
-
|
-
|
Na (g kg− 1)
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
P (g kg− 1)
|
-
|
10.0a
|
-
|
-
|
-
|
-
|
-
|
S (g kg− 1)
|
-
|
4.0a
|
-
|
-
|
-
|
-
|
-
|
Zn (g kg− 1)
|
-
|
0.5p; 1s; 0.25f
|
-
|
0.8
|
2.8
|
0.7
|
|
Cd (mg kg− 1)
|
2.0
|
10a
|
0.4
|
1.5
|
39
|
3
|
3.0
|
Cr (mg kg− 1)
|
-
|
100s; 500p
|
-
|
2.0
|
-
|
210
|
2.0
|
Pb (mg kg− 1)
|
10.0
|
10.0a
|
8.0
|
120.0
|
300
|
150
|
150.0
|
1Source: Directive 2002/32/EC of the European Parliament and of the Council of 7 May 2002 on undesirable substances in animal feed. 2Source: National Research Council (2005). Mineral tolerance of animals. 3Source: Canadian Food Inspection Agency (2015). RG-8 Regulatory Guidance: Contaminants in Feed. 4Source: Regulation (EU) 2019/1009 of the European Parliament and of the Council of 5 June 2019 laying down rules on the making available on the market of EU fertilising products.5Source: Code of Federal Regulations Part 503-Standards for the Use or Disposal of Sewage Sludge.6Source: Canadian Council of the Ministers of the Environment (2005). Guidelines for compost quality.7Source: Instrução Normativa SDA N°27 do 05 de Junho de 2006, Anexo V: Limites máximos de contaminantes admitidos em fertilizantes orgânicos e condicionadores de solo. a: concentration value for poultry, swine and fish feed. f: concentration value for fish feed. p: concentration value for poultry feed. s: concentration value for swine feed.
|
3.2. Mineral content changes in Hermetia illucens larval biomass
Fly larvae can degrade different types of substrates, assimilating the minerals therein for growth and development. Table 2 shows the mineral concentration of initial larvae (young larvae) and larvae (first prepupae appeared) of Hermetia illucens. Some macronutrients (K, Na, and P) did not have marked changes at the end of the bioconversion process. On the other hand, other micro and macronutrients, such as Ca, Cu, Fe, Mg, Mn, S, and Zn, presented significant increases. Proc et al.(2020b) also found increases in Ca, Cu, Fe, Mg, and Mn concentrations; however, K, Na, P, S, and Zn content decreased in larvae at the end of fish feed bioconversion. The reason for the differences between these studies could be linked to the nutritional composition of substrates. In this regard, Tschirner and Simon (2015) revealed that mineral content changes in biomass of Hermetia illucens larvae depend on the type of substrate. For example, the authors found that K, Na, P, and Mg content decreased in larvae fed with a mixture of middlings from a feed mill. When larvae were fed with dried distillers’ grains with solubles made from barley, corn, wheat, and sugar syrups (protein group), the Cu, Fe, K, Mg, Mn, Na, P, and Zn content decreased. However, in larvae fed with dried sugar beet pulp, only Cu, Fe, P and Cu decreased, and the remaining elements increased.
Among all the evaluated elements, calcium was the most increased in larval biomass at the end of the experiment, with a 2.6 fold increase in the initial concentration. This result is consistent with the increase in the calcium content in Hermetia illucens larvae fed with fish feed, up to 2.3 times the initial concentration of young larvae, reported by Proc et al.(2020b), and it is also similar to the results obtained in larvae fed with co-products (dried sugar beet pulp), up to 2.7 times the initial calcium concentration, highlighted by Tschirner and Simon (2015). Previous studies with other fly larvae (Musca autumnalis) have revealed that large amounts of Ca are ingested and stored in the Malpighian tubules during the larval stage, to subsequently be used during the pupariation process (Darlington et al. 1983; Grodowitz and Broce 1983). This calcium uptake by fly larvae depends on the concentration of this element in the diet (Dube et al. 2000).
Regarding the toxic elements, the Cd, Cr, and Pb concentrations in larvae were significantly increased (Table 2). The Cd content increase could be linked with the high Ca content in Hermetia illucens larvae, inherent to insects. Cd uptake is through Ca2+ channels in the intestinal cells (Braeckman et al. 1999; Craig and Hare 1999; Buchwalter and Luoma 2005) and this element is mainly accumulated in the larvae body (47%-93%) and less amount is excreted in the feces (Wu et al. 2020). However, the Pb content increase would be linked with the Pb content in the substrate as related by Tschirner and Simon (2015) and Diener et al.(2015b), both for spiked and not spiked substrates with Pb solutions, respectively.
On the other hand, a comparison of the mineral content in larval biomass was carried out in other studies to identify if the concentrations of minerals depend on the type of substrate (not spiked with mineral solutions) when it is feed co-product or waste. Table S1 (in supplementary material) shows a wide variation in the mineral content of larval biomass with apparently no pattern according to the type of substrate used for rearing. For instance, calcium content obtained in larvae was higher than the values obtained in larvae fed with feeds and co-products, and only similar with larvae reared in dried sugar beet pulp. However, this does not mean that all larvae reared in residues will have more calcium than those obtained from co-products or animal feed. For example, in the case of larvae fed with restaurant waste (Spranghers et al. 2017), kitchen waste and chicken manure (Shumo et al. 2019) had lower amounts of calcium than those from the other substrates. In this study, the Cu content in larvae was higher than that fed with feeds and in line with larvae fed with chicken manure and kitchen waste (Shumo et al. 2019) but lower than the Cu content in commercial larvae (Irungu et al. 2018). Furthermore, regarding K, Mg, and P content, these were similar to the content of larvae fed with chicken feed (Dierenfeld and King 2008) and Na content was in line with the content in larvae grown in poultry and pig manure (Newton et al. 2005). Zn and Fe content in the present study were higher in larvae fed with feeds or co-products and only lower than larvae reared in chicken manure (Shumo et al. 2019), and the Mn content was higher than all the Mn concentrations in larvae from the cited studies.
Regarding the content of the toxic element, the Cd concentration in larvae fed with cattle manure of this study was lower than all the Cd content in larvae fed with other types of waste and co-products, and only in line with larvae fed with chicken feed (Fels-Klerx et al. 2016). However, Cr content was only lower than the content of this metal in larvae fed with municipal sewage sludge and Pb concentrations were higher than for all the Pb contents in larvae grown in feeds, some co-products, and especially municipal sewage sludge.
The high variations among the mineral contents in larvae fed both feeds, co-products or waste, could be linked to different factors, such as different experimental setups (Bosch et al. 2019a, b), different harvest times (Liu et al. 2017), different fly strains (Zhou et al. 2013) and the different nutritional contents of substrates (Tschirner and Simon 2015).
The micro and macronutrients in larvae were compared with the mineral content of fish and soybean meals, as shown in Table 2. The larvae's Ca, Fe, Mg, Mn, and S contents were higher than in fish and soybean meal. The Cu, Na, and P contents were higher in the fish meal than in the others; however, they were higher in larvae than in the soybean meal. The K content in larvae was lower than in soybean meal but higher than in fish meal. The Zn concentration was similar to that in larvae, fish meal, and soybean meal. Therefore, the mineral content of Hermetia illucens larvae is comparable with fish meal and possibly better than soybean meal.
The mineral content in larvae was also compared with the maximum limits for feeds established by different countries. As shown in Tables 2 and 3, the toxic elements in larvae met the EU, USA (United States of America), and Canada concentration limits for Cd, Cr, and Pb. For micro and macronutrients, EU and Canadian legislation has not established maximum limits; therefore, the values of these elements in larvae were compared with the limits established by the USA. The Cu level in larval biomass met the maximum limit for pig, poultry, and fish feed. Furthermore, Fe and Zn content only met the maximum limit for pig feed. However, K, Mg, Mn, P, and S concentrations were slightly higher than the maximum limits for poultry, pig, and fish feed. These results are promising for two reasons: 1) the safe levels of toxic elements and 2) despite the slightly higher mineral levels compared with maximum levels for feed, the larval biomass could be used partially in feeds by adjusting the mineral content according to the animal's nutritional requirements.
3.3. Bioaccumulation of toxic elements, micro and macronutrients in Hermetia illucens larvae
The bioaccumulation factors (BAF) of micro and macronutrients and toxic elements in larval biomass increased from young to adult larvae (Fig. 1). The BAF of elements in larvae that were higher than 1 showed the following trend in this study: Ca > K > Mn > P > S > Mg > Na > Zn > Cd.
These results were compared with the BAF of different elements in Hermetia illucens larvae fed with artificially uncontaminated substrates of previous studies (Tschirner and Simon 2015; Biancarosa et al. 2017; Schmitt et al. 2019; Proc et al. 2020a). As was previously observed in the larvae concentrations, there is a wide variation range in the BAF of elements of different studies; however, the BAFs of Ca, Mg, Mn, and Cd in all studies were > 1, thereby revealing bioaccumulation. Ca, Mg, and Mn are essential elements for insects' metabolic processing and development (Clark 1958; Ben-Shahar 2018); however, Cd is considered a toxic element and, as mentioned, is linked with the calcium content in fly larvae. For instance, in Musca domestica larvae that generally contain tenfold less calcium than Hermetia illucens larvae (Gold et al. 2018), the BAF of cadmium is < 1(Wang et al. 2017; Negi et al. 2020), on the other hand, in Hermetia illucens larvae, this factor often exceeds the value of 1. This Cd and Ca relationship is also in line with the BAF obtained when Hermetia illucens larvae were fed with substrates spiked with cadmium solutions at different concentrations (Diener et al. 2015b; Fels-Klerx et al. 2016; Purschke et al. 2017; Bulak et al. 2018; Wang et al. 2021). Consequently, great care should be taken regarding the cadmium content in substrates used as feed for Hermetia illucens larvae since this can accumulate up to almost tenfold the cadmium concentration of substrate in larval biomass (Tschirner and Simon 2015).