Using fly larvae to convert food waste for growing Oujiang color common carps: health risk assessment of polycyclic aromatic hydrocarbons

The present study used Chrysomya megacephala larvae (CML) to transform food waste into safe and high-quality fish feed to substitute fish meal as a source of protein for growing Oujiang color common carps followed by a human health risk assessment of polycyclic aromatic hydrocarbons (PAHs). The results showed the ∑PAH concentration in the CML fed with food waste ranged from 50 to 370 μg kg−1, and the most abundant PAH compound in the CML was BaP, contributing 59–84% of ∑PAHs. The Pearson correlation analysis results indicated no correlation between the ∑PAH concentrations and the culture substrate ratio (p > 0.05). Concentrations of BaP in the CML decreased with the increase of breeding density (p < 0.01). The residues as organic fertilizers have no potential ecological risk for PAHs. The biotransformed larva meal was used to partially or completely replace the fish meal as supplementary protein in the experimental feeds (T0, 0%; T50, 50%; T100, 100%). No significant difference (p < 0.05) of survival rate, lipid, and protein content in Oujiang color common carp was noted among T0, T50, and T100 fish feeds. Concentrations of ∑PAHs in Oujiang color common carp fed with the CML fish feeds all met the food safety standards in the European Union (EU). Furthermore, the consumption of Oujiang color common carps fed with the CML feed does not pose any health risks of PAHs for humans.


Introduction
Polycyclic aromatic hydrocarbons (PAHs) are highly hydrophobic and organic lipophilic compounds with a fused ring structure comprising two or more aromatic rings (Domingo and Nadal 2015;Singh et al. 2016). The PAHs are mainly formed by the incomplete combustion or pyrolysis of organic materials and natural diagenesis, such as forest fires, fossil fuels and wood combustion, volcanic eruptions, industrial processes, and cooking (Singh et al. 2016;Sun et al. 2018). PAHs have Technical specification for the disposal of food waste.
received extensive attention as a persistent organic pollutant in the environment due to their high toxicity, carcinogenicity, teratogenicity, and mutagenicity to humans and animals. Previous studies demonstrated that several PAHs such as benzo(a)pyrene (BaP), chrysene (Chry), indeno [1,2,3-cd] pyrene (IP), and benzo(b)fluoranthene (BbF) had carcinogenic, mutagenic, and genotoxic effects on animals (Rundle et al. 2000;USEPA 2019). In addition, the breast, lung, bladder, and skin cancers of humans are also related to exposure to PAHs (USEPA 2017). The PAH-DNA adducts formed by the carcinogen benzo(a)pyrene diol epoxide may increase the risk of lung cancer (Li et al. 2001). Different cooking methods (e.g., frying, smoking, roasting, and grilling) significantly affect the formation of PAHs in food (Ledesma et al. 2016;Li et al. 2015;Lin et al. 2011). Furthermore, it was also found that the concentrations of PAHs produced by cooking Chinese food were 29.5-130 ng m −3 , which were about twice those of cooking Japanese food and fast food (Li et al. 2003).
Food waste can be defined as the end products of various food processing industries, retailers, restaurants, and consumers (Lin et al. 2013). There has been a growing concern over the yield and treatment of food waste in both developed and developing countries (Grizzetti et al. 2013). Most food wastes are mainly disposed of in landfills or by incineration (Mo et al. 2017;Zhou et al. 2014). However, these traditional disposal methods have the disadvantages of occupying the land, producing greenhouse gases, and consuming social resources (USEPA 2018). According to the "Technical specification for the disposal of food waste" issued by the Ministry of Housing and Urban-Rural Development of the People's Republic of China (MOHURD), food waste can be used as insect breeding feed, and the types of insects to be fed should be determined according to the nutritional characteristics of food waste, the climatic adaptability of insects, and the market demand for insect feed (CJJ184 2021). The high organic content of food waste makes it a recyclable resource, and many studies have confirmed that fly maggots can efficiently biotransform food waste into animal feeds, fertilizer, and biodiesel (Wen et al. 2016). Niu et al. (2017) demonstrated that using housefly larvae to convert food waste can obtain value-added larva protein (57.06 ± 2.19%), maggot oil (15.07 ± 2.03%), and organic fertilizers. Li et al. (2012) showed that the maximum yield of fatty acid methyl esters (FAME) obtained by feeding Chrysomya megacephala larvae (CML) with food waste was 87.7%, and its properties were within the specifications of the USA and European biodiesel fuel blend stock standards. Tenebrio molitor, Musca domestica, and Chrysomya megacephala larvae raised with food waste meet the national hygienical standard for feeds and food safety standards and are used as raw materials for animal feed production (Gao et al. 2019;GB2762 2017GB13078 2017;GB19164 2003;Huang et al. 2022). In addition, FAO also believes that insects as feed have potential in ensuring food security and should be vigorously promoted (Vantomme et al. 2012).
Chrysomya megacephala (CM) belongs to the Chrysomya genus (Calliphoridae family, order Diptera), which undergoes complete metamorphosis with four distinct stages (egg, larva or maggot, pupa, and adult) (Gabre et al. 2005). The CML is rich in proteins, essential amino acids, fat, vitamins, trace elements, and active substances such as lectin, lysozyme, antimicrobial peptide, and chitin (Badenhorst and Villet 2018, Huda et al. 2015. The maggot grows fast and can consume a large amount of organic waste (e.g., livestock and poultry manure and food waste) such that the material and energy in the organic waste can be transformed into insect nutrients (such as protein and fat) (Cickova et al. 2015). The maggot products after bioconversion can be used as animal feed protein, and the remaining residue can be reused as organic fertilizer , Sing et al. 2014, Yang and Liu 2014. Sing et al. (2014) reported that the CML as a protein source in fish feeds for culturing tilapia could improve the growth, feed efficiency, and survival of tilapia fry. However, the food waste (30-40%) and maggot products (25-32%) contain high lipid content Niu et al. 2017), which may lead to PAH enrichment in feeds, which accumulate in poultry and aquatic products, and eventually reach humans. Therefore, it is necessary to ensure that aquatic products and poultry are raised by the feeds of larva products that are relatively free of PAHs. The major objectives of this study were to (1) investigate concentrations of PAHs in the CML fed with food waste; (2) investigate the bioaccumulation of PAHs in Oujiang color common carp fed with the CML fish feed; and (3) assess potential health risks based on PAH concentration in the Oujiang color common carp.

Food waste
In the present study, there were two major kinds of food waste, dish waste and staple food waste, which were mainly cooked food collected from local restaurants and hotels. After picking out the bones and other debris, the dish waste was pretreated and washed 3-5 times with tap water to reduce the excess amounts of salt and oil, and a handcranked meat grinder was used to crush and homogenize the waste. The treated dish waste was strained and preserved at 25 °C. Each batch of food waste samples was randomly sampled and stored at − 18 °C for subsequent analysis.

CM rearing experiment
The CM eggs used in this research were obtained from the ecotoxicology Laboratory of Sichuan Agricultural University, China. Under standard laboratory conditions, it takes 8-12 h for CM eggs to develop into larvae and pupate after 5-6 days, and then, the CM adults appear after 5-7 days. The CM larva rearing experiment kept the temperature at 28 ± 3 °C humidity at 75 ± 5%.
The culture medium of food waste used in this experiment was a mixture of dish waste and staple food waste, which was divided into five types based on the mass ratio of dish waste (100%, 90%, 80%, 70%, and 60%). The culture medium (1000 g) was mixed uniformly in an enamel pot (350 × 250 × 150 cm 3 ), and five replicates of each treatment were inoculated with 0.5-g, 1.0-g, 1.5-g, 2.0-g, and 2.5-g CM eggs, making up 25 group treatments (Table S1). In this study, the hatching percentage and surviving rate of all groups of larvae ranged from 98 to 99% and 93-99%, respectively. The culture medium was stirred evenly twice a day to ventilate the whole culture medium system from the 2-day age of larvae. After 5 days of rearing, the fourth instar larvae were collected with a sieve (20 mesh, 0.85 mm of aperture).
Oujiang color common carp (Cyprinus carpio var. color) is a local breeding species in the Oujiang River basin of China, with a traditional farming model of farming fish in paddy fields. Recently, the Oujiang color common carp has been widely cultured in China due to its ease of breeding, fast growth, and fewer diseases (Lu et al. 2011). Oujiang color common carp fry (initial length, 8.62 ± 0.88 cm; initial weight, 10.5 ± 2.58 g) was provided by the Chinese Academy of Fishery Sciences, Guangzhou, China. The experimental fish feeds were prepared according to the methods described by Wen et al. (2013) with slight modifications, and the biotransformed larva meal was used to partially or completely replace the fish meal as supplementary protein in the experimental feeds (T 0 , 0%; T 50 , 50%; T 100 , 100%). The formulation and chemical composition of the fish feeds are shown in Table S2. The twelve fiberglass tanks (1.15 × 0.84 × 0.60 m 3 ) were filled up with aerated tap water, each with an approximate capacity of 150 L. Water quality parameters were maintained in the tanks at 6.5-7.2 mg L −1 dissolved oxygen, 6.9-7.5 pH, and 26 ± 3 °C temperature. There were four replicates of each group rearing 60 fish per tank with a photoperiod of 12:12 (light:dark) hour cycle. The fishes were fed two times daily at 4% of body weight for 60 days. During the experiment, fish were weighed every 2 weeks and the quantity of feed was adjusted accordingly. All fish samples were collected after fasting for 24 h, and the lengths and weights of the fish were recorded.

Human health risk assessment of PAHs
The non-cancer and cancer risk of human caused by PAH exposure via fish consumption was evaluated according to the USEPA standard models (USEPA 1989) (detailed cal. parameters in supplementary data). The hazard quotients (HQ) of non-carcinogenic PAHs was calculated using the following equations: where i is individual PAHs; CDI is chronic daily intake; C is the PAH concentrations in fish; IR is the fish ingested rate; (1) (4) CR = CDI × OSF EF is the exposure frequency; ED is the exposure duration; BW is the body weight; AT is the averaging exposure time; RfD is the reference dose; CR is cancer risk; and OSF is oral slop factor.
Details of the PAH analyses, QA/QC, and calculations are described in the Supplementary material.

Statistical analyses
The data obtained were submitted to analysis of variance, using the general linear model procedure of SPSS 19.0 software (IBM, Chicago, USA). The significance of differences among treatments was tested by the Duncan multiple range test, and a level of p < 0.05 was used as the criterion for statistical significance. Pearson correlation analysis was used to analyze the relations between the PAH concentrations and the inoculum density, proportion of dish waste, and lipid content.

CML
Under optimal conditions (2.0 g kg −1 , breeding density; 80-100%, the proportion of dish waste), 1-kg food waste could produce approximately 172-253 g of CML, which contains 41.6-51.8% protein and 36.7-48.5% lipid, while the food waste mass was reduced by 63.5-79.4% in 5 days (Fig. 1). Figure 2 shows that the ∑PAH concentration in the CML ranged from 50 to 370 μg kg −1 . The Pearson correlation analysis results indicated no correlation between the ∑PAH concentrations and the culture substrate ratio (p > 0.05). Concentrations of ∑PAHs and 5,6-ring PAHs in the CML were the highest when the breeding density was 1.0 g kg −1 . The most abundant PAH compound in the CML was BaP, contributing 59-84% of ∑PAH. The results were similar to those reported in earthworms used for remediating sewage sludge contaminated with PAHs (Contreras-Ramos et al. 2009). Like earthworms, CML's PAH accumulation pathway may involve the passive diffusion-mediated concentration of PAHs in aqueous phases by the epidermis and ingestion of PAHs through food (Contreras-Ramos et al. 2009;Krauss et al. 2000). In addition, previous studies on earthworms and black soldier flies showed that the amount of PAHs accumulated by the larvae via the ingestion pathway was much more significant than that via the epidermis (Fan et al. 2020;Ma et al. 2012).
In the present study, the bioconcentration factors (BCF) describing the ability of PAHs to concentrate in the CML is shown in Table S4. The results show that the CML has a strong bioaccumulation ability for 5,6-ring PAHs in the food waste (BCF > 5). The CML's median concentration of carcinogenic PAHs (BaP, DahA, IP, BbK, BfK, BaA, and Chry) was 148 μg kg −1 , accounting for 81.3% of the ∑PAHs; especially, the ECF value of BaP reached 19.8. However, when the breeding density was higher than 1.5 g kg −1 , the BCF value of the CML was less than 1, and concentrations of ∑PAHs and 5,6-ring PAHs were also decreased (p < 0.05) (Fig. S2). In addition, concentrations of BaP in the CML decreased with the increase of breeding density (p < 0.01), and the proportion of BaP in ∑PAHs decreased by 62.8-93.7%. Therefore, appropriately improving the breeding density can effectively reduce the bioconcentration effect of the CML on the PAHs in food waste. When the breeding density was between 0.5 and 1.5 g kg −1 , the weight and length of the CML reached a peak, indicating that the CML can get enough food to meet its growth and development needs of nutrition, but PAHs could also be absorbed with the diet and enriched in the larvae (Green et al. 2003). As the breeding density increased, the amount of food waste converted by the CML had also increased, resulting in a relative shortage of CML food and reducing PAH intake. When the breeding density continued to increase, the yield tended to be stable. At relatively high breeding density, the larvae cannot take enough nutrients for growth and development, Fig. 1 The length (A), weight (B), food waste mass reduction rate (C), and yield (D) of Chrysomya megacephala larvae and the CML was forced to ingest low nutritional value staple food waste such as cereals to maintain their survival (Cickova et al. 2015). The proportion of 2,3-ring PAHs in staple food waste was 70%, which may be one of the reasons why the proportion of 5,6-ring PAHs in the CML decreased with the increase in breeding density (Fig. 2).

Residue
Concentrations of PAHs in the food waste residues after bioconversion by the CML are shown in Fig. S3. The median and mean concentrations of ∑PAHs in the residues were 87.1 μg kg −1 and 102 μg kg −1 , respectively, which were significantly lower than the concentrations of ∑PAHs in food waste feeds (staple food waste, 186 μg kg −1 ; dish waste, 118 μg kg −1 ) (p < 0.05). The concentration of ∑PAHs in the residue at the breeding density was 2.0 g kg −1 which was higher than that at the breeding density of 0.5 g kg −1 (78.0 μg kg −1 ) and 1.5 g kg −1 (82.7 μg kg −1 ) (p < 0.05). The proportion of 2,3-ring PAHs in food waste feeds was 70.5% (Fig. S1), but 5,6ring PAHs were the most abundant component of PAHs in the residue (p < 0.05), accounting for 91.3%. The BaP was the most abundant congener in the residues (p < 0.05), accounting for 76.2%, ranging from 49.6 to 90.5%. The concentrations of BaP in food waste residues increased by 13.7 and 4.34 times compared to the dish food waste and staple food waste (p < 0.05), respectively. In addition, there was no correlation between the total and congener concentrations of PAHs and the culture substrate ratio or breeding density (p > 0.05). Previous studies showed that resistance of PAHs to degradation increases with an increase in molecular weight. (Baldantoni et al. 2017). Therefore, the CML could excrete excessive 5,6-ring PAHs by its immune system and detoxification function during the bioconversion of food waste (Fan et al. 2020). In addition, the 2,3-ring PAHs in the food waste might be degraded during bioconversion. Wong et al. (2002) found that pig manure as a co-composting material could effectively increase the amount of soluble organic carbon, ammoniacal nitrogen, and soluble phosphorus in the compost and increase the population of PAH-degrading bacteria (e.g., thermophilic and mesophilic bacteria). Subsequently, it improved the degradation rate of PAHs in the soil; the maximum degradation rate of 3, 4-ring PAHs was up to 90% during the first 20 days of composting. In the present study, the phosphorus and potassium contents increased by 94-630% and 38-119%, respectively, during CML's bioconversion of food waste. Furthermore, PAH degradation involves the microorganism breaking organic compounds down into less complex metabolites and mineralizing into inorganic minerals, H 2 O, CO 2 , or CH 4 (Haritash and Kaushik 2009). Previous studies have observed that the earthworms have little effect on the degradation of PAHs in the soil; however, they can improve soil aeration, promote soil microbial activity, and then increase Fig. 2 Concentrations of PAHs in Chrysomya megacephala larvae (CML). Note: 100%, 90%, 80%, 70%, and 60% are the proportion of dish waste (%); 0.5 g kg −1 , 1.0 g kg −1 , 1.5 g kg −1 , 2.0 g kg −1 , and 2.5 g kg −1 are the breeding density the degradation rate of PAHs (Yuan 2009). The CML degraded food waste mainly by drilling and peristalsis in the culture medium, which increased the temperature and pH of the culture medium. The loose porous culture medium promoted the release of ammonia and moisture and stimulated the growth of aerobic microorganisms (Niu et al. 2017;Zhu et al. 2012).
The residues could be used as organic fertilizers for agricultural production because of their rich nutrient compositions, such as nitrogen (1.2-2.11%), phosphorus (0.33-4.01%), and potassium (0.38-0.78%). However, the potential ecological risk of PAHs in residue cannot be neglected when applied to the soil. There is no clear limit for PAH content in soil environment in China. BaP equivalent concentration (TEQ BaP ) and toxicity equivalency factors (TEFs) are used to evaluate the potential ecological risk of PAHs in the residues (Nisbet &Lagoy 1992). Fig. S4 depicts the TEQ BaP concentrations of 10 PAHs in the residues. The TEQ BaP of 10 PAHs in the residues ranged from 44.1 to 160 μg kg −1 , with a median of 70.5 μg kg −1 . According to the previous studies that reported the amount of organic fertilizer (Zhu et al. 2012), assuming that the amount of residue used accounts for 10% of the soil mass, none of the TEQ BaP values in soil exceeded the limit value of Dutch (30.3 μg kg −1 ) and Canada (100 μg kg −1 ) agricultural soil standard (Wang et al. 2009). The results indicated that there was no potential ecological risk for PAHs when the food waste residues after bioconversion by CML were used as organic fertilizers for agricultural production.

Fish
The composition and nutrient contents of experimental feeds are shown in Table S2. T 0 contained the highest proteinenergy ratio, cellulose, lipid, and ash contents (p < 0.05) and the lowest gross energy and phosphorus contents (p < 0.05). No significant differences in crude protein were found among the three fish feeds (p > 0.05). According to Table S5, the growth performance in Oujiang color common carp decreased in the order fed with T 50 > T 100 > T 0 (p < 0.05). In addition, no significant difference (p < 0.05) of survival rate, lipid, and protein content in Oujiang color common carp was noted among T 0 , T 50 , and T 100 fish feeds (p > 0.05). Therefore, using CML protein to replace fish meal partially or wholly as a feed protein source can achieve better yield and similar product quality. In the present study, concentrations of ∑PAHs (78.6 ± 6.81 μg kg −1 DW) and 5,6-ring PAHs (49.3 ± 4.73 μg kg −1 DW) in T 100 feeds were higher than other experiment feeds (p > 0.05); especially, the content of BaP accounts for 32% of ∑PAHs (Table 1). However, after 60 days of the experiment period, concentrations of the total PAHs (25.7 ± 6.62 μg kg −1 wet weight (WW)) and 2,3-ring PAHs (18.1 ± 6.80 μg kg −1 WW) in the fish fed with T 50 were 2 and 4 times higher than those in the fish fed with T 100 and T 0 , respectively (Table 1). In addition, there was no significant difference in the concentrations of 5,6-ring PAHs in the fish fed the three experimental feeds (p > 0.05), and the proportion of 5,6-ring PAHs (mean 32.9%) was significantly lower than that in the fish feeds (mean 56.0%) (p < 0.05). The results showed that Oujiang color common carp has low bioavailability of 5,6-ring PAHs in the CML fish feeds. The PAH concentration of the fish species does not exceed the European Union maximum levels of contaminants in food (50 μg kg −1 WW)(EU 2008). The better body weight growth rate and specific growth rate would make it possible for the fish to improve the accumulation of PAHs, especially 2-3-ring PAHs (Cheung et al. 2007). Concentrations of ∑PAHs (0.54-0.61 μg kg −1 , WW) in the fish fed with CML fish feeds were similar to those of grass carp (12.6-18.8 μg kg −1 , WW), bighead carp (5.90-15.7 μg kg −1 , WW), and mud carp (6.58-11.7 μg kg −1 , WW) fed with food waste fish feeds (Cheng et al. 2015) and those collected from the markets in Pearl River Delta (1.57-54.4 μg kg −1 , WW) (Cheung et al. 2007;Wang et al. 2010). Figure 3 shows cancer and non-cancer risks of PAHs in adults and children through the consumption of Oujiang color common carp fed with experiment fish feeds. In this study, the hazard index values of the fish fed with three experiment fish feeds were below 1, suggesting that the estimated daily intake below the recommended reference doses for the PAHs and the level of non-cancer risk is considered acceptable (USEPA 2019). A lifetime cancer risk below the 10 −4 value is regarded as the upper limit of the acceptable risk levels (NYSDOH 2007). The cancer risk values for fishes range from 2.86 × 10 −6 to 7.65 × 10 −6 , indicating that the consumption of Oujiang color common carps fed with the CML fish feed had a low cancer risk for PAHs and the fish is safe for human consumption. Man et al. (2020) also demonstrated that it is safe to use food waste to grow freshwater fish (Nile tilapia (Oreochromis niloticus) and jade perch (Scortum barcoo) based on the PAHs in fish. This study shows it is a great potential to use CML to transform food waste into safe value-added products for both agriculture and aquaculture uses.

Conclusions
This study investigated the concentrations and potential risk of PAHs present in Chrysomya megacephala larvae (CML) fed with food waste and in Oujiang color common carps. The pretreatment can reduce the concentrations of ∑PAHs and 5,6-ring PAHs in dish waste. The most abundant PAH compound in the CML fed with food waste was BaP. There was no correlation between the ∑PAH concentrations in the CML and the culture substrate ratio. Appropriately improving the breeding density can effectively reduce the bioconcentration effect of the CML on the PAHs in food waste. The CML could excrete excessive 5,6-ring PAHs by its immune system and detoxification function during the bioconversion of food waste. Based on the PAH concentrations, the results indicated no potential ecological risk when the food waste residues were applied as organic fertilizers for agricultural production. Using CML protein to replace fish meal partially or wholly as a feed protein source can achieve better yield and similar product quality, and it is safe to consume Oujiang color common carps grown with the CML fish feed.