Toxicity Effect of Cu 2+ Contaminated Kitchen and Vegetable Wastes on Oxidative Stress Response of Black Soldier Fly Larvae, Hermetia Illucens

Environmental pollution sources including waste or metal accumulation, industrial and agricultural activities can be dangerous. Also, contaminated organic waste (COW) with metals especially, copper ions (Cu 2+ ), can cause toxicity to various ecosystem components, enhance the production of reactive oxygen species (ROS) and consequently cause oxidative stress. The biochemical effect of the COW was monitored by assessing the oxidative stress parameters (OSP) using hydrogen peroxides (H 2 O 2 ), protein carbonyls (PC), lipid peroxides (LP), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), 2,2 diphenyl-1-picrylhydrazyl (DPPH), total antioxidant ability (TAA); β-esterase (β-EST); and total amount of protein (TAP) levels on the organic waste (kitchen and vegetable wastes); Hermetia illucens larvae; and larval excreta collected from 7-day post-treated kitchen and vegetables wastes with (1:10; g:mL) distilled water (DW) or 100 mg/mL Cu 2+ . The OSP levels were significantly higher in the experimental samples from Cu 2+ groups than in the control one. Besides that, the OSP levels of H. illucens larvae feed on vegetable waste was significantly higher than those feed on kitchen waste except for β-EST, PC, and TPA. The best, cheap and easy parameters of antioxidants to ensure the entomoremediation ability are total antioxidant capacity. Possible impacts of accumulated and Cu 2+ contaminated organic waste on H. illucens larvae were discussed. Also, the ability of insects to produce more antioxidants than input or output sources was approved. The potential use of the OSP as a bioindicator method of the bioremediation ability of H. illucens was proposed. The research conducted in this study suggests that significant differences in the oxidative stress parameters, macromolecules concentration, and detoxification enzymes of H. illucens treated with Cu 2+ -contaminated organic waste (KW &VW) were evaluated compared to control value. The elevation levels of antioxidants inside the insect than waste and excreta suggest its role as a source of essential products characterized by economic, environmental and social aspects of sustainability. Besides that, the potential use of oxidative stress parameters as a bioremediation ability of BSFL to various environmental toxins. All these findings are matched by previously accepted knowledge [137, 139-141].


Introduction
The effects of various chemical or environmental pollutants on the insects' physiology and biochemistry have been investigated in various studies [1][2][3][4][5][6][7][8][9]. Environmental pollution sources including waste accumulation, industrial and agricultural activities have a direct effect on the amounts of cupper elevation than normal levels. Excess of

Insect Treatment and Sample Preparation
From the Entomology Department, Faculty of Science, Cairo University, Egypt, the 5 th instar larval of H. illucens was supplied from a colony reared under the rearing conditions (0:24 L:D; 34˚±2; 75% RH). The organic wastes (kitchen and vegetable) were provided routinely from the Giza governorate houses. Each 10 mL of Cu 2+ (100 mg/mL) or 10 mL distilled water was mixed with 1gm organic waste. For each sub-group, different organic waste sources, 50 insects and their feces substances were collected, after 7 days post-application for further analysis and were stored at −20 °C until use. Each experiment was done in three replicates.

Reactive Oxygen Species and Oxidative Damage Concentration
By using a spectrophotometer, the H 2 O 2 concentration of experimental samples determined by Junglee, et al. [54], 150 mg of each sample was directly homogenized with 1 mL of a solution containing 0.25 mL Trichloroacetic acid (TCA) (0.1% (w:v)), 0.5 mL KI (1 M) and 0.25 mL potassium phosphate buffer (10 mM, pH 7.0) at 4˚C for 10 min (one-step buffer: extraction and colorimetric reaction combined). The homogenated sample was centrifuged at 12,000 × g for 15 min at 4˚C. The absorbance was measured at 240 nm. From Levine, et al. [55], we used the procedure for the protein carbonyls assay, with the below-described modifications. In 5 mL ice-cold phosphate buffer (60 mL of 50 mM phosphate buffer, 10 mL of 0.1% Triton X-100, 5 mL of 0.05 mM CaCl 2 ; then completed to 100 mL with distilled water after adjusting pH to 7.0 with 2M HCl or NaOH) samples were homogenized. The samples were centrifuged at 2000 ×g for 10 min at 4 °C after homogenization (mortar, 10 strokes/ 30 seconds), an 800 µl aliquot of the supernatant mixed with 200 µL of 10 mM 2, 4-dinitrophenyl hydrazine (DNPH) prepared in 2 M HCl. The samples were incubated for 30 minutes at room temperature, precipitated with 10% Tricholoroacetic acid (TCA), and left for 10 min at 4°C. At 5000 ×g the samples were centrifuged for 7 min at 4°C. The pellet was washed four times with an ethanol/ethyl acetate (1:1) mixture and redissolved in 1 mL of sodium phosphate buffer (60 mL of 150 mM phosphate buffer, 30 mL of 3% sodium dodecyl sulphate, adjusted to a final volume of 100 mL with distilled water after adjusting the pH to 6.8 with 2M HCl or NaOH). Finally, the absorbance was measured at 366 nm, and the rate of protein carbonyls concentration was expressed as OD/mg protein.
As Hermes-Lima, et al. [56] method, the lipid peroxides concentration was measured. The samples were homogenized in ice-cold methanol (1:5, w/v). At 4 °C , the samples were centrifuged at 2000 g for 10 min after homogenization (mortar, 10 strokes/30 seconds). For the assay, a 5 mL aliquot of the supernatant was used. The following components were consecutively added to the samples (200 µl of supernatant): 400 µl of 1 mM FeSO 4 , 200 µL of 0.25 M H 2 SO 4, and 200 µl of 1 mM xylenol orange. The absorbance was measured at 580 nm. Lipid peroxides concentration was expressed as mM cumene hydroperoxides/µg protein.

Determination of the Enzymatic and Non-Enzymatic Antioxidants Levels
After homogenization using mortar (30 strikes/1 min.), sample preparation of all assessed enzymatic antioxidants was done in 100 mM phosphate buffer saline (PBS) (pH= 7.0). Then centrifugation had done at 4000 ×g for 10 min at 4 C.
The measuring of SOD activity is based on the procedure described by Misra and Fridovich [57]. The reaction mixture was as follows: 0.4 mL of a sodium carbonate buffer (200 mM; pH 10.0), 35 µl of EDTA (10 mM), 87 µl of the supernatant and 0.5 mL of freshly prepared epinephrine (15 mM). The absorbance was measured at 480 nm. SOD activity was expressed as OD/µg protein/min.
The activity of catalase (CAT) was assessed in compliance with Aebi [58] method. The reaction mixture contained 0.9 mL of a potassium phosphate buffer (50 mM, pH 7.0), 60 µL of the supernatant and 40 µL of freshly prepared H 2 O 2 (10 mM). The change in absorbance was measured at 240 nm for 1 min. The CAT activity was expressed as OD/µg protein/min.
According to Mazhoudi, et al. [60] method with slight modifications, the guaiacol peroxidase (GPX) activity was estimated. The reaction mixture containing 0.5 mL of 50 mM potassium phosphate buffer (pH 7.0), 0.2 mL of 1% (m/v) guaiacol, 0.2 mL OF 0.4% (v/v) H 2 O 2 , and 0.1 mL of the enzyme extract from each sample. Changes in the absorbance were measured at 470 nm over 3 min.
The other sensitive, accurate, low sample concentration and low-cost biochemical analysis, (DPPH), was determined by Blois [61]. The reaction mixtures include 1 mL of 0.5M DPPH to different concentrations of sample and incubated for different times before measuring absorbance at 525 nm. DPPH assay is based on scavenging capability measurement. The nitrogen atom contains an old electron which is reduced by delivering a hydrogen atom from antioxidants to hydrazine.
The total antioxidant capacity was measured according to the procedure of Prieto, et al. [62]. The method includes a homogenate sample with the following 0.25 mL 0.6 M sulfuric acid, 0.5 mL 28 mM sodium phosphate, and 0.25 mL 4 mM ammonium molybdate, then incubate at 95˚C for 90 min. The absorbance was measured at 695 nm.

Detoxification Enzymes Activity
By using the method of Gomori [63], the activity of β-esterase was determined. The reaction mixture contained 18 mg of fast blue B salt dissolved in 10 mM potassium phosphate buffer (pH 7.0), 600 µL of 0.113 M βnaphthyl acetate which was dissolved in 50 % acetone. A volume of 240 µL of this solution was mixed with 10 µL of each sample, and the absorbance was measured at 570 nm over 3 min.

Biomolecules Concentration
Spectrophotometrically, the total protein concentration of samples was determined by Bradford [64] method. Briefly, 0.9 mL of the dye reagent (10 mg COBB + 5 mL methanol + 10 mL 85% O-phosphoric acid, completed to 100 mL with distilled water) were added to 0.1 mL of each sample in a separate test tube. The contents of the tube were mixed by gentle shaking and left to stand for 2 min. The OD of the protein sample was measured at 595 nm. 2% albumin concentration was used as a standard.

Statistical Analysis
The effect of both control treatment and contaminated Cu 2+ treated samples on the oxidative stress parameters levels of organic waste, 5 th instars larvae of H. illucens, and excreta were assessed by performing Kruskal-Wallis H (P<0.05). All statistical analyses were performed using IBM SPSS Statistics for Windows (Version 17.0. Armonk, NY: IBM Corp.).

Reactive Oxygen Species and Oxidative Damage
The levels of H 2 O 2 , PC and LP were generally lower in control than Cu 2+ -treated KW or VW waste samples (Table 1 and Fig. 1). In control samples, elevated levels of H 2 O 2 were observed in the larval excreta than larvae and input organic waste materials. The H 2 O 2 increased folds were 10.4 and 17.5-x in the larval excreta; 5.4 and 2.9-x in insect larvae for kitchen and vegetable waste, respectively (P value < 0.05) ( Table 1). While, the H 2 O 2 levels in the Cu 2+ -treated samples showed an increased level within the larval with 6.5, and 6-x fold than VW and KW, respectively. The concentration of H 2 O 2 was increased in the kitchen waste than vegetable input waste. However, the insect and excreta output samples showed an elevation level of ROS in vegetable treated samples, compared with samples collected from the kitchen waste pool. The protein carbonyls (PC) amount in the control samples showed a uniform decrease pattern along with input (KW), process (insect larvae), and output (larval EX), while the VWtreatment showed an accumulation pattern of PC inside insect larvae than VW and EX. In Cu 2+ -treated samples, there was a significant decrease of PC levels in the EX from the KW treatment samples (Fig. 1C), however, the VW treatment samples showed no significance in the PC amount among VW, BSFL, and EX (P>0.05) (Fig. 1D). However, the Cu 2+ -treated VW samples showed a slightly significant increase inside BSFL than VW and EX (Fig.  1F). While in the KW treatment, the results showed uniform stability between KW, BSFL, and EX (Fig. 1E). The protein carbonyls amount in 5 th instar larvae of H. illucens feed on kitchen waste were significantly higher in relation to those feed on vegetable waste, however, lipid peroxide BSF feed on vegetable waste showed a higher concentration than those feed on kitchen waste ( Fig. 1). In the control treatment, the significant decrease level of lipid peroxides (LP) levels within the insect larvae were shown in both KW&VW than the waste or EX (Table 1).

Enzymatic and Non-Enzymatic Antioxidants Response
The enzymatic and non-enzymatic antioxidants response showed a significant decrease inside the control samples than Cu 2+ -treated samples in both KW and VW (Table 1, Figs. 2, 3). In control samples, unlike Cu 2+ -treated samples, each antioxidant showed a unique pattern among wastes, insects, and excreta. For instance, the constitutive CAT activity of KW and VW were significantly decreased than BSFL and EX, while the constitutive activity of SOD and GPx of KW were significantly increased than insect larvae and excreta. However, the non-enzymatic DPPH levels of BSFL showed significantly increased levels than waste and excreta in both KW&VW. In kitchen waste treatments, the non-enzymatic TAA showed a significant decrease inside BSFL than waste and excreta, while the excreta from VW treatments showed a significant increase than other samples (P<0.05) ( Table 1). The enzymatic activity and non-enzymatic levels of Cu 2+ -treated samples showed a uniform trend pattern in both KW and VW samples (Figs. 2, 3). As the antioxidant's levels inside BSF were significant increase than waste and excreta. This trend occurred in CAT, SOD, GPx, DPPH, and TAA in both KW and VW ( Fig. 1A-D, F and Fig. 2A-D), except the case of GPx activity in the KW, the GPx activity in the KW was significantly increased than the other samples (P<0.05) (Fig. 2E). The activity of the three principal antioxidant enzymes (CAT, GPx, and SOD) and nonenzymatic levels showed that 5 th instar H. illucens feed on vegetable waste higher than those feed on kitchen waste (Figs. 2, 3).

Detoxification Enzyme Activity and Biomolecules Concentration
The constitutive detoxification enzyme activity (β-esterase) showed a different pattern in VW compared to KW treated samples. The β-esterase activity of BSFL was significantly increased than KW and EX; however, the depletion levels of the detoxification enzymes inside the insect larvae occurred compared to VW and excreta (Table  1). Yet, the concentration of biomolecules, protein concentration, has a uniform pattern in the KW and VW treatment samples. The results showed a significant increase in the excreta than BSFL and waste (Table 1). In Cu 2+treated samples, both activities of detoxification enzymes and biomolecules concentration showed a significant increase in the excreta than input waste and insect for VW and KW (Figs. 4, 5). Yet there was no significant difference among VW and BSF in the activities of β-esterase (Fig. 2B). Also, the non-significance difference among BSF and KW or VW has occurred in the concentration of total protein (Fig. 5A, B). The level of β-esterase activity in BSF feed on kitchen waste had higher activity than those feed on vegetable waste (Fig. 4). Yet, the total protein amount didn`t reveal significant differences in BSF feed on kitchen waste or vegetable waste (Fig. 5).

Discussion
The problem of dealing with estimation the oxidative stress factors, or more specifically, the mechanisms of using oxidative stress, are considered as the main point of interest to scientists dealing with phenomena of bioremediation, toxicology, and adaptation. In recent studies, these researches topics have gained significant value, mainly due to the increased stress levels as a result of the human and environments-related activities pressure [21,65]. These abnormal changes require organisms' adaptation and mitigation. These responses can be vital as they can allow the studying of specific defense mechanisms against various and different stress factors [2,5,7,8,14].
Organic waste accumulation and contamination have a serious problem with ecosystem function and structure [65][66][67][68]. Accumulation of organic waste is dealing with the quantity of organic waste production. As, within the early decades of this century, annual organic waste production was over 600 million tones, and by 2025, it is expected to reach around one billion tones [69]. The Landfill is currently the most common method for disposing of organic waste, which has the potential to contribute to global warming as well as soil and water pollution, making it an increasing problem for society Christensen, et al. [70]. Otherwise, organic waste contamination, especially heavy metals, can be a source of environmental pollution as a result of oxidative stress magnification inside the various and surrounding living organisms [71][72][73].
Exposure to environmental pollutants, including organic waste, chemical pollutants, and heavy metals, is linked with the elevated levels of oxidative stress through increasing the production of ROS; which cause deleterious effects on the macromolecules of the living organisms [2,3,5,7,8,14,65]. Besides that, heavy metals, especially Cu 2+ , can increase ROS production by disturbing the respiratory metabolism rate [74]. The imbalance between oxidants and antioxidants can lead to oxidative damage products [75][76][77]. So, the use of insect to convert organic waste into biomass, which can then be used for an innovative purpose, were studied in various studies [65,[78][79][80][81][82][83][84][85], however, the ability to use insect as a bioremediation agent of pollutants contamination is still needed more studies and clarifications mechanisms [86,87].
In the present study, the oxidative stress parameters, detoxification enzymes activity and biomolecules concentration of the control and Cu 2+ contaminated organic waste (kitchen waste and vegetable waste) on the input organic waste, insect 5 th instar larvae, Hermetia illucens, and insect excreta were studied. Although the insect larvae fed on the control organic waste, the levels of H 2 O 2 don't tend to zero value (Table 1). This may be due to the necessity of maintaining the optimal concentration of H 2 O 2 in cells as it can be used in intracellular signalling and regulation [88]. However, the interactions between the organic waste and heavy metals especially Cu 2+ was not clear, some differences might come from the different chemical compositions of the organic waste, such as organic matter, which can affect the ability to uptake the other chemical pollutants [89][90][91].
The organic waste type includes vegetable waste, food waste, plant tissues waste, animal offal, vegetable waste, restaurant waste and animal manure [65,92,93] can lead to oxidative stress especially when contained chemical pollutants, as arsenic [94] as a result of ROS production [95,96]. In the present work, the levels of oxidative stress were evaluated directly and indirectly to evaluate the ability of BSFL to bioremediate the Cu 2+ -organic waste contamination. These oxidative stress parameters were evaluated, also, for control organic waste through assessment of the H 2 O 2 levels, macromolecules damaged products (protein carbonyls and lipid peroxides), as well as measuring the enzymatic and non-enzymatic antioxidants levels (SOD, CAT, GPx, DPPH or TAA), besides that the biomolecules concentration (proteins) and related detoxification enzymes in the KW or VW, 5 th instar larvae of BSF and larval excreta. These were done according to the generally accepted previous studies [7,65,97,98].
The results showed a significant decrease in the H 2 O 2 levels, as well as macromolecules oxidatively damaged products, protein carbonyls and lipid peroxides levels, in the control samples than Cu 2+ -treated samples in both kitchens and vegetables waste treatment samples (Table 1 & Fig. 1). Our results are similar to other studies which showed that copper exposure leads to a stress-dependent production of ROS which leads to protein carbonylation and lipid peroxidation elevation [99,100]. The studies of Baryla, et al. [101] and Lushchak [102] emphasized the use of lipid peroxides levels as an indicator of the oxidative stress parameters, moreover, the study of Ahmed [32] & Yousef, et al. [96] revealed that protein carbonyls amount, as well as lipid peroxides levels, can be used as an indicator of oxidative stress in Aedes caspius and Aiolopus thalasinus, respectively. Also, Zaman, et al. [94] showed a significant increase in the lipid peroxides and protein carbonyls amount as a result of treatment of adult female house flies, Musca domestica, and 4 th -instar cabbage loopers, Trichoplusia ni with As 3+ , (NaAsO 2 ) and As 5+ , (Na 2 HAsO 4 ).
Also, Copper can catalyze the production of ROS, such as superoxide anion radical (O 2 •-), hydrogen peroxide (H 2 O 2 ) and hydroxyl radical ( • OH), through Fenton and Haber-Weiss reactions. ROS can react with lipids, proteins, or DNA molecules and can initiate chain reactions that engage lipid peroxidation, protein oxidation and fragmentation, and DNA strand breaks [103]. The obtained results approved that the proteins are the most important targets of free radical attack in the cells [102]. This phenomenon occurred as ROS can convert amino groups of proteins and cause changes in the protein structure and function. Therefore, oxidative stress correlates with several modified carbonyls groups of protein damage [103]. The proteins damage leads to conformation disruption of protein molecules, including enzymes [104,105]. Besides that, the significant elevation of lipid peroxides occurred when the antioxidants are depleted by the actions of ROS [5,7,8,96,106].
The present work showed the elevation production of ROS in the larval excreta than larval tissues (Table 1) which confirm the criteria of the ability of the insect to detoxify toxicants through the action of antioxidants, excretion [9,107]. However, in Cu 2+ -treatment samples, the elevated levels of H 2 O 2 inside the BSF 5 th instar larvae than the other samples in both kitchen and vegetables waste treatment, indicate the ability of the insect to store the toxicants and ROS [47]. Additionally, the present study showed the depletion levels of protein carbonyls amount in the kW treatment samples than VW, however, the elevated levels of both enzymatic and non-enzymatic antioxidants in the VW than KW in both control and Cu 2+ -treated samples ( Table 1; Figs. 1, 2, 3). These results are following previously accepted knowledge that deals with the use of vegetables as input waste to produce BSFL and frass. These BSFL products are comparable to other products and are considered valuable and sustainable resources for the agricultural industry [108].
The action of toxicants and oxidants may lead to an increase in the levels of non-enzymatic antioxidants, such as DPPH or TAA as well as the activity of antioxidants and detoxification enzymes such as SOD, CAT, GPx, and βesterase, respectively. Moreover, SOD and CAT are considered as key antioxidant enzymes responsible for ROS scavenging [106], while, GPx can overcome the low concentration of ROS as, H 2 O 2 [96]. Also, esterases are considered as a vital detoxification enzyme in various insects' species such as Hermetia illucens (unpublished data), Blattella germanica [109], Culex quinquefasciatus [110], and Trichoplusia ni [111]. However, DPPH radical scavenging can practice for the antiradical properties assessment of multiple compounds [112]; as well as it can be occurred by the action of antioxidants through donating a hydrogen atom to terminate the free radical chain. Also, Aromatic amino acids like phenylalanine and tyrosine can stabilize DPPH free radicals by donating a proton to electron-deficient free radicals [113]. Additionally, β-esterase is an important component of insect defense against xenobiotic chemicals, such as pesticides [114]. The role of the insect's detoxification system is one of the mechanisms behind the effects of secondary metabolites on pesticide susceptibility [115]. Yet, the total antioxidant capacity can assess the ability of all antioxidants in the living organisms to neutralize the oxidation effects of oxidants [116], so it can be used as a tool for evaluating the redox status [117].
The results of the present study emphasized that the redox-active metals could increase the levels of antioxidants and detoxification enzymes, as SOD, CAT, GPx, β-esterase, DPPH radical scavenging, and TAA compared with control samples value (Table 1; Figs. 2, 3, 4). The same observation was showed in the Zaman, et al. [94] study which approved that SOD, CAT, glutathione-s-transferase (GST), the peroxidase activity, glutathione reductase (GR), lipid peroxidation and protein oxidation levels were induced by As 3+ in M. domestica, however, CAT and GSTPX were not affected. Additionally, As 5+ did not affect M. domestica. So, the suggestion of using Cu 2+ and As 3+ as a pro-oxidant was proposed especially for H. illucens and M. domestica, respectively. Additionally, the study of Tuncsoy, et al. [100] showed a significant elevation of SOD, CAT, GPX, GST, acetylcholine esterase after treatment with copper oxide nanoparticles with Galleria mellonella. Also, the elevation of total antioxidant capacity, protein carbonyl content and activities of SOD, CAT, POX or GST of the Helicoverpa armigera adults after UV light exposure for 30 min were tested and approved the increased oxidative stress as a result of exogenous factors. Meng, et al. [118], suggested that the elevation activity of SOD and CAT in the cells after detoxification may appear due to increased ROS generation. Briefly, SOD can dismutase the superoxide anion radical and CAT [119] or GPx [120] can decompose H2O2 at high or low levels, respectively [5,96]. All these findings can answer the 3rd question of our hypothesis which is related to the ability of BSFL to produce more antioxidants, especially within contaminated or polluted zones.
Yet, the SOD antioxidant enzyme activities were suppressed in Trichoplusi ni, by 29.4% as a result of treatment with As 5+ [94], as well as SOD depletion occurred in Galleria mellonella after treatment with 10 µg Cu/L of CuO [100]. Also, the reduction of glutathione reductase activity and total thiol (SH) content of Scenedesmus sp after Cu 2+ and Zn 2+ treatment [121]. These findings may link with the present results related to depletion of total protein content after treatment of Cu 2+ (Fig. 5) as the enzymes are protein in nature.
Additionally, free radical scavenging is generally done by the action of redox status through Fenton reaction [5]. As well, the study of Kaur, et al. [122] showed that plant gall extract has free radical scavenging to inhibit oxidative damage to biomolecules, briefly, Fe-ascorbate can inhibit lipids peroxidation through termination of the lipid peroxidation chain reaction. Moreover, the study of Oghenesuvwe and Paul [123] mentioned the DPPH antioxidant properties as an insect's isolated compounds product as, Reticulitermes speratus, Tenodera aridifolia, Camponotus obscuripes [124], Allomyrina dichotoma [125], and Protaetia brevitarsis [126]. Besides that, the elevated levels of total protein amount in the insect larvae than input waste emphasized the ability of BSFL to convert the organic waste into bio-based valued products ( Table 1). Besides that, the availability of bioactive compounds inside BSF biomass to enhance their nutritive value which can use as animal feed [65,127,128]. Many types of organic waste, such as food waste, plant tissues waste, animal offal, and animal manure, have been recommended as effective organisms for turning them into insect biomass [129]. The resultant larval (H. illucens) biomass has proved to be a beneficial feed component for pigs, chickens, and fish farmed animals [130]. In addition, BSFs are considered a great source of energy and easily digested amino acids [131]. As the BSFs' primary source of amino acids, the protein composition of the substrate has an impact on their amino acid availability [132]. Overall, BSFs have a higher concentration of essential amino acids [133][134][135] and a better amino acid profile than soya bean meal and most traditional protein sources [136]. The amount of total protein found in the 5 th instar H. illucens feed on Cu 2+ contaminated kitchen waste or vegetable waste revealed no significant differences (Fig. 5).
The previous study investigated the ability of BSF to be used in entomoremediation of heavy metal pollution as it has a strong tolerance to cadmium stress, this was done through identifying and characterizing three MTs genes in BSF [137]. This finding supports our results with measuring oxidative stress parameters (Table 1; Figs. 1, 2, 3, 4, 5). These investigations have the answer to one of our questions related to the ability to use the oxidative stress parameters as an indicator of bioremediation ability. Besides that, the present results can emphasize the 1 st question in our hypothesis which is related to the capability of management of Cu 2+ -contaminated organic waste using BSFL, as well as, the study of Diener, et al. [138] confirmed the great potential of BSFL as a waste manager in low and middle-income countries, yet, the high percentage of larval mortality due to elevated zinc concentrations in the waste material and anaerobic conditions in the experimental trays may occur.
The research conducted in this study suggests that significant differences in the oxidative stress parameters, macromolecules concentration, and detoxification enzymes of H. illucens treated with Cu 2+ -contaminated organic waste (KW &VW) were evaluated compared to control value. The elevation levels of antioxidants inside the insect than waste and excreta suggest its role as a source of essential products characterized by economic, environmental and social aspects of sustainability. Besides that, the potential use of oxidative stress parameters as a bioremediation ability of BSFL to various environmental toxins. All these findings are matched by previously accepted knowledge [137,[139][140][141].

Declarations Ethical Approval and Consent to Participate
This article does not contain any studies with human participants or animals that require ethical approval.