Effect of insect-based fish feed wastewater on lettuce growth and phytochemical content in decoupled aquaponic systems

1 Introduction

The cultivation of leafy vegetables using aquaculture wastewater represents a sustainable agricultural strategy that integrates nutrient recycling with food production systems (Alsanius et al., 2017; Graber and Ranka, 2009). Leafy vegetables are highly valued for their nutritional benefits and rapid growth rates; however, their production requires a continuous supply of nutrients. Traditionally, crop growth has relied heavily on synthetic fertilizers, but increasing attention has been directed toward the utilization of aquaculture effluents as an alternative nutrient source (Ahmed et al., 2021). Aquaculture wastewater, enriched with essential plant nutrients such as nitrogen, phosphorus, potassium, and micronutrients derived from fish feed and metabolic waste, offers considerable potential for hydroponic cultivation. In hydroponic systems, where plants are conventionally supplied with commercial nutrient solutions, fish wastewater can serve as a natural and sustainable substitute, supporting plant growth while reducing dependence on synthetic fertilizers (EUMOFA, 2021; FAO, 2020; Chatzigeorgiou et al., 2024). This approach promotes a circular bioeconomy by recycling nutrients that would otherwise be discharged into the environment, thereby reducing waste generation and production costs. Furthermore, the integration of aquaculture and hydroponics creates a synergistic relationship in which nutrient-rich wastewater supports plant development while contributing to environmental sustainability (HLPE, 2014). Therefore, evaluating the effects of aquaculture wastewater on lettuce growth, yield, and nutritional quality is essential for optimizing integrated production systems and advancing resource-efficient, environmentally friendly food production practices (Yang and Kim, 2020).

Aquaculture, the farming of aquatic organisms such as fish, has expanded rapidly worldwide in response to the increasing demand for high-quality protein sources. However, intensive aquaculture production generates substantial volumes of nutrient-rich wastewater, which, if discharged untreated, can contribute to eutrophication and environmental degradation (Tom et al., 2021). This wastewater contains considerable amounts of essential plant nutrients, particularly nitrogen and phosphorus, making it a valuable resource for crop production. The reutilization of aquaculture effluents for cultivating leafy vegetables offers an environmentally sustainable strategy to reduce reliance on synthetic fertilizers while simultaneously mitigating nutrient discharge into surrounding ecosystems (Campanati et al., 2022).

The recovery and reuse of nutrients from aquaculture wastewater align closely with the principles of the circular bioeconomy, which emphasizes resource efficiency, waste minimization, and the sustainable integration of biological systems (Barroso et al., 2014; Chiquito et al., 2022). Within this framework, aquaculture by-products are transformed into valuable agricultural inputs, creating a closed-loop production system in which waste streams from one sector serve as resources for another (Henry et al., 2015). The utilization of fish-culture wastewater for vegetable production exemplifies this concept by enhancing nutrient recycling, reducing environmental impacts, and improving the sustainability of both aquaculture and crop production systems (Das et al., 2023; Lakra et al., 2022). Furthermore, this approach supports the development of aquaponic systems, where fish and plant production are integrated to optimize water use efficiency, nutrient recovery, and food security.

The nutritional composition of aquaculture wastewater is influenced by fish diet formulation, particularly the source of dietary protein. Recent interest in sustainable aquafeeds has promoted the replacement of conventional fishmeal with alternative protein sources, including insect-derived ingredients such as black soldier fly larvae (BSFL, Hermetia illucens) and mealworm larvae (MW, Tenebrio molitor). These alternative feeds may alter nutrient excretion patterns and consequently affect the quality of wastewater used for plant cultivation. Therefore, understanding how different feed formulations influence plant performance is essential for optimizing integrated aquaculture-hydroponic systems.

In the present study, wastewater generated from blue tilapia cultured with diets containing fishmeal, BSFL, or MW was utilized for hydroponic lettuce production. Lettuce (Lactuca sativa L.) was selected as the model crop due to its rapid growth cycle, relatively low nutrient requirements, and widespread use in aquaponic systems. The study evaluated the effects of wastewater derived from different dietary protein sources on lettuce growth, chlorophyll accumulation, and antioxidant activity. Specifically, the objective was to determine whether partial replacement of conventional fishmeal with insect-based protein sources could enhance nutrient recycling and improve the productivity and quality of both fish and plant components within an integrated aquaponic production system.

2 Materials and methods

2.1 Fish feed waste and plant nutrient formulation

Wastewater derived from blue tilapia (Oreochromis aureus) culture was collected from the Aquaculture Laboratory of York College of Pennsylvania, York, PA, USA. Three wastewater treatments were generated based on fish diets: (1) a conventional fishmeal-based diet (FM), (2) a diet in which 50% of the fishmeal was replaced with black soldier fly larvae meal (50% BSFL), and (3) a diet in which 50% of the fishmeal was replaced with mealworm larvae meal (50% MW). A commercially available hydroponic nutrient solution (Miracle-Gro 12-4–8 Liquid All-purpose Food, Model #1001501) served as the control treatment. A total of four nutrient treatments (Control, FM, 50% BSFL, and 50% MW) were evaluated for lettuce production (Table 1).

Table 1. Ingredients and proximate composition of experimental diets: Control with 10% fishmeal (FM), 50% replacement with Black soldier fly larvae (BSFL) and 50% replacement with Mealworm larvae (MW).

Premix concentration in one kg = Vit A 926000 IU, Vit D3–100000 IU, Vit E 72000 IU, Vit K 1.8 g, vit B12–23 mg, Riboflavin 7.3 g, p-Pantothenic acid 37 g, Niacin 42 g, Thiamine 5.9 g, Pyridoxine 6.0 g, Folic acid 1.9 g, Ascorbic acid 210 g, Biotin 91 mg.

Mineral premix concentration in one kg = Manganese 24 g, Zinc 20 g, Iron 16 g, Copper 1.6 g, Cobalt 200 mg, Iodine 16g, Selenium 160 mg.

Energy was calculates as [(Crude protein x 5.65) + (lipid x 9.4) + (Digestible carbohydrate x 4.15)] x 10, where Digestible carbohydrates was calculated as (Dry matter – Crude protein – Crude fiber – Crude Fat – Ash).

Lettuce plants were cultivated using a half-strength Hoagland nutrient solution supplemented with the respective wastewater treatments. Half-strength Hoagland solution was used because the wastewater already contained a certain amount of plant nutrients. Therefore, reducing the Hoagland solution to half strength helped account for the nutrients supplied by the wastewater and prevented the total nutrient concentration from exceeding the recommended level for lettuce cultivation. The pH and electrical conductivity (EC) of the nutrient solutions were monitored weekly and adjusted to maintain a pH of 5.8 and an EC of 1.85 mS cm^-1 throughout the experimental period.

2.2 Hydroponic lettuce cultivation

Lettuce (Lactuca sativa L. cv Romaine) were grown in a growth room located in the Naylor Building at York College of Pennsylvania, York, PA, USA. Lettuce seeds (Johnny’s Seed Ltd., Winslow, Maine, USA) were sown into Rockwool growing cubes (Grodan, Hedehusene, Denmark) and germinated under white fluorescent lighting (200 µmol m⁻² s⁻¹) in a growth chamber. Once the seedlings reached a height of 4–6 cm, they were thinned to one seedling per cube. These cubes were then transferred to trays for deep-water culture hydroponics. In the growth room, the temperature was 21 °C ± 2.5 °C, the photosynthetic photon flux density (PPFD) was 200 ± 5 μmol m⁻² s⁻¹, the photoperiod was 16/8 h (day/night), and the relative humidity was 65 ± 5%.

After five weeks of transplanting (DAT), the lettuce plants were harvested for analysis.

2.3 Measurements

2.3.1 Plant growth

After 5 weeks of DAT, lettuce was harvested and plant growth characteristics were recorded. Plant height was measured from the tip of the youngest leaf to the surface level. Fresh mass (FM) was recorded immediately after harvest. Leaf area was measured by using a leaf area meter (LI-3100C, LI-COR, Lincoln, Nebraska, USA). Plants were then dried in an oven at 80 °C for at least 72 hours until a constant mass was reached. The dried plant tissues were weighed to determine dry biomass. Leaf tissues were analyzed for phytochemical content and antioxidant properties.

2.3.2 Chlorophyll and carotenoid analysis

Chlorophyll and carotenoid contents were measured using the method described by Lichtenthaler (1987). Briefly, 100 mg of fresh ground leaf tissue was incubated overnight in 10 mL of 80% acetone at room temperature. After centrifugation, the supernatant was collected, and absorbance was measured at 646.8, 663.2, and 470 nm using a spectrophotometer (PowerWave™ XS Microplate Reader, BioTek Instruments, Inc. Vermont, USA). The concentrations of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids were calculated following the equations (Equation 1):

(1) Chl a = 12.25 × A663− 2.79 × A647

Chl b = 21.50 × A647− 5.10 × A663

Car = (1000 × A470− 1.82 × Chl a− 85.02 × Chl b)/198

2.3.3 Antioxidant capacity measurement

The antioxidant capacity was determined using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay, as described by Naznin et al. (2015). Briefly, 1 mL of the sample extract was mixed with 3 mL of a freshly prepared DPPH solution in 100% methanol and incubated in the dark at room temperature (22–25 °C) for 4 h to allow the reaction to reach completion. Following incubation, the absorbance of the reaction mixture was measured at 517 nm using a UV–Vis spectrophotometer (PowerWave™ XS Microplate Reader, BioTek Instruments, Inc. Vermont, USA), with methanol serving as the blank. The DPPH radical scavenging activity was calculated based on the reduction in absorbance relative to the control according to the following equation (Equation 2):

(2) RSA (%) = [ (A0 − Ax)/A0 ] × 100

2.3.4 Statistical analysis

Statistical analyses were performed using Minitab statistical software (version 17; Minitab Inc., State College, PA, USA). Data were subjected to one-way analysis of variance (ANOVA) to determine whether significant differences existed among the experimental treatments. When the ANOVA indicated significant treatment effects, mean comparisons were performed using Tukey’s multiple-comparison test with simultaneous 95% confidence intervals. Differences among treatment means were considered statistically significant at p < 0.05. The results are presented as mean ± standard error (SE). Microsoft Excel was used for data organization, calculation of descriptive statistics, and preparation of tables and figures.

3 Results

3.1 Plant growth characteristics

The insect-based fish wastewater treatments significantly influenced lettuce plant growth characteristics. Morphologically, all plants exhibited a compact leaf arrangement and a rich dark green color when cultivated in the 50% MW and 50% BSFL treatments. In contrast, plants grown in the FM treatment displayed a looser leaf arrangement, while those in the control group were smaller in size (Figure 1).

In Table 2, it is observed that 50% MW treatment led to an increase in plant height, leaf area, leaf number, fresh weight, and dry weight. The height of lettuce is significantly greater in 50% MW treatment than that of the control group (p< 0.05), although it did not differ significantly from the heights observed in the FM and 50% BSFL treatments. In addition, no significant differences were observed between FM and BSFL treatments.

Table 2. Growth characteristics of the lettuce grown in an aquaponic system using different wastage fish feed.

The standard deviation is represented by bars (n = 8). Values with different letters differ significantly (p < 0.05).

3.2 Chlorophyll fluorescence

The chlorophyll fluorescnce (Fv/Fm) values are consistent across all treatments, indicating that the various treatments did not significantly affect the maximum quantum efficiency of photosystem II (Figure 2).

3.3 Pigment analysis

Table 3 shows that the 50% MW treatment was the most effective at enhancing pigment levels, including chlorophyll a, chlorophyll b, and total chlorophyll content, indicating improved photosynthetic potential in plants under this treatment. However, the 50% BSFL treatment resulted in the highest total carotenoid content, suggesting potential antioxidant benefits.

Table 3. Pigment content of the lettuce grown using different wastage fish feed.

The standard deviation is represented by bars (n = 8). Values with different letters differ significantly (p < 0.05).

3.4 Antioxidant capacity

When lettuce was cultured in different fish waste water, the scavenging capacity of the lettuce was influenced (Figure 3). The antioxidant capacity in lettuce was significantly higher when plants were cultured in 50% BSFL waste water compared to control. The FM and 50% MW showed lower antioxidant capacity compared to 50% BSFL but there is no significant difference.

4 Discussion

The enhanced growth observed in lettuce supplied with MW wastewater is consistent with previous reports demonstrating positive effects of insect-based aquafeeds on plant production in aquaponic systems. Ogbaudu (2023) reported that replacing fishmeal with mealworm meal significantly improved lettuce growth, yield, and antioxidant properties. Similarly, Romano et al. (2023) found that incorporating black soldier fly larvae (BSFL) meal into tilapia diets enhanced nutrient recycling efficiency and improved the growth of both fish and associated crops such as stevia and lavender. Jones et al. (2024) also observed greater biomass production of lettuce, and basil when plants were cultivated using wastewater generated from fish fed BSFL-based diets compared with conventional fishmeal diets. Collectively, these studies support the hypothesis that insect-based fish feeds can improve nutrient cycling and plant productivity in integrated aquaculture systems.

In addition to growth enhancement, lettuce cultivated with MW wastewater exhibited higher chlorophyll and carotenoid contents. Chlorophyll concentration is widely recognized as an indicator of plant nutritional status and photosynthetic capacity, whereas carotenoids contribute to light harvesting and protection against photooxidative stress (Lichtenthaler, 1987). The increased accumulation of these pigments may indicate improved nutrient availability, particularly nitrogen, magnesium, and other micronutrients essential for pigment synthesis. Previous studies have reported that nutrient-rich aquaculture effluents can increase chlorophyll concentrations in leafy vegetables by providing a continuous supply of mineral nutrients in readily available forms (Goddek et al., 2019; Yang and Kim, 2020).

The higher antioxidant activity observed in lettuce grown with MW wastewater may be attributed to enhanced biosynthesis of secondary metabolites, including phenolic compounds, flavonoids, and carotenoids. Antioxidants play important roles in protecting plants from oxidative stress and in enhancing the nutritional quality of vegetables for human consumption. In aquaponic systems, nutrient availability and root-zone microbial interactions can influence the production of bioactive compounds (Saha et al., 2016). Insect-derived feed ingredients contain various bioactive compounds, including chitin and chitosan derivatives, which may indirectly stimulate microbial activity and nutrient transformation processes within aquaculture systems, thereby promoting the accumulation of antioxidant compounds in plants (Gasco et al., 2018). The elevated antioxidant capacity observed in the present study suggests that MW-based fish diets may enhance not only crop yield but also the nutritional quality of lettuce.

The PCA analysis indicated that wastewater derived from fish fed a 50% MW diet was strongly associated with plant height, leaf area, leaf number, fresh weight, dry weight, and chlorophyll-related traits, suggesting enhanced nutrient availability and photosynthetic capacity. Improved chlorophyll accumulation is often linked to greater biomass production and plant growth due to increased photosynthetic efficiency (Taiz et al., 2018; Kozai et al., 2016). In contrast, the 50% BSFL treatment was more closely associated with carotenoid accumulation, indicating a greater influence on secondary pigment metabolism and antioxidant-related responses (Hashimoto et al., 2016). The intermediate position of the fishmeal treatment reflects moderate effects on plant performance. Overall, the PCA supports previous findings that insect-based fish feeds can improve nutrient recycling efficiency and enhance the growth and quality of leafy vegetables in aquaponic systems (Goddek et al., 2019; Chen et al., 2025).

The results further highlight the importance of feed formulation as a management strategy for optimizing aquaponic production. Traditionally, research has focused on improving fish performance through feed modification; however, the present findings indicate that dietary interventions can also influence downstream plant productivity. This supports the concept of aquaponics as a circular bioeconomy system, where feed inputs affect fish growth, wastewater quality, nutrient recycling efficiency, and ultimately crop performance. By replacing a portion of fishmeal with sustainable insect protein sources, producers may improve resource-use efficiency while reducing the environmental footprint associated with fishmeal production (Makkar et al., 2014; Gasco et al., 2020).

Although the present study demonstrates promising effects of MW-derived wastewater on lettuce growth and quality, the underlying mechanisms remain insufficiently understood. Future research should characterize the nutrient composition of wastewater, including nitrate, ammonium, phosphorus, potassium, and micronutrient concentrations, as well as microbial community dynamics associated with different fish diets. Additional plant growth parameters such as biomass accumulation, leaf area, root morphology, nutrient uptake efficiency, and photosynthetic performance should also be evaluated. Furthermore, studies examining varying substitution levels of mealworm and BSFL meal are needed to identify optimal inclusion rates that maximize both fish performance and plant productivity. Such investigations will contribute to the development of more sustainable and efficient aquaponic systems that integrate insect-based aquafeeds and hydroponic crop production.

5 Conclusions

This study demonstrated that aquaculture wastewater derived from blue tilapia fed insect-based diets can serve as an effective nutrient source for hydroponic lettuce production. Among the treatments evaluated, wastewater from fish fed a diet of MW produced the greatest improvements in plant growth, significantly enhancing plant height, leaf number, chlorophyll and carotenoid concentrations, and antioxidant capacity. These findings indicate that the composition of fish feed influences wastewater quality and subsequent plant performance in integrated aquaponic systems. The incorporation of insect-derived proteins, particularly mealworm meal, into fish diets offers a sustainable strategy to improve nutrient recycling, reduce reliance on conventional fishmeal, and enhance the productivity and nutritional quality of leafy vegetables. Overall, integrating insect-based aquafeeds with hydroponic crop production represents a promising circular bioeconomy approach for improving resource-use efficiency and sustainability in both aquaculture and horticultural systems.

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Footnotes

Edited by: Xiao Yang, Chinese Academy of Agricultural Sciences, China

Reviewed by: Ashwini A. Waoo, AKS University, India

Julia Hwei Zhong Moh, University of Malaysia Terengganu, Malaysia