# The fate of carbon, nitrogen and phosphorus in freshwater polyculture mesocosms using both nutrient-balance and stable-isotope assessment

S Nahon, Christophe Jaeger, Christophe Menniti, Philippe Kerhervé, Marc Roucaute, Joël Aubin

► To cite this version:

S Nahon, Christophe Jaeger, Christophe Menniti, Philippe Kerhervé, Marc Roucaute, et al. The fate of carbon, nitrogen and phosphorus in freshwater polyculture mesocosms using both nutrient-balance and stable-isotope assessment. Aquaculture Reports, 2025, 43, pp.102865. 10.1016/j.aqrep.2025.102865. hal-05078618

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# The fate of carbon, nitrogen and phosphorus in freshwater polyculture mesocosms using both nutrient-balance and stable-isotope assessment

Sarah Nahon<sup>a, *</sup>, Christophe Jaeger<sup>b</sup>, Christophe Menniti<sup>c</sup>, Philippe Kerherve<sup>c</sup>, Marc Roucaute<sup>b</sup>, Joel Aubin<sup>b</sup>

## ARTICLE INFO

### Keywords:

Common carp

Largemouth bass

Roach

Macrophyte

Phytoplankton

Multi-trophic aquaculture

## ABSTRACT

Freshwater polyculture can increase nutrient use by rearing species from different trophic levels. The aim of this study was to quantify nutrient balances and determine the food sources of fish using nitrogen (N) and phosphorus (P) nutrient balances and carbon (δ¹³C) and N (δ¹⁵N) stable isotopes. The contributions of food sources to fish diets were estimated using stable-isotope mixing models. Three treatments were compared in mesocosms: fish-only (F treatment: common carp, roach and largemouth bass), fish-macrophyte (FM treatment) and macrophyte-only (M treatment). Fish were fed with commercial pellets. The results showed that adding macrophytes to the fish did not improve water quality. Chlorophyll-α concentration in F and FM treatments was significantly higher than that in treatment M (8.1, 8.3 and 0.6 µg l⁻¹, respectively). Regardless of the treatment, sediment contained most of the total N and P at harvest (93.5 and 89.7 %, respectively). Carp, roach and bass had a similar specific growth rate in F and FM treatments (1.19, 0.36, 0.94 % day⁻¹, respectively). Carp consumed 90 % commercial feed and 8 % macrobenthic invertebrates, while roach consumed 50 % commercial feed and supplemented their diet with macrophytes (15 %), macrobenthic invertebrates (12 %) and large zooplankton (11 %). Juvenile roach and bass had higher δ¹⁵N values than carp and roach did (12.31, 14.55, 7.54, 10.44 ‰, respectively). Bass consumed mainly juvenile roach (59 %), followed by macrobenthic invertebrates (25 %). Combining the nutrient balances and stable isotopes showed that sediment was the main nutrient pool, but that organisms in the mesocosms consumed little sediment organic matter.

## 1. Introduction

Aquaculture provides ca. half of the world's aquatic food supply, and its contribution is expected to increase by 15 % over the next decade (FAO, 2024). The sector's acceptability depends on developing practices that decrease environmental impacts. In continental areas, freshwater fish are produced mainly in semi-intensive ponds (FAO, 2022), which range from non-fed to fed production systems. Excess particulate and dissolved nutrients from uneaten feed and metabolic waste from fish are released directly into the environment, which cause eutrophication crises because nitrogen (N) and phosphorus (P) limit primary production in freshwater (Abell et al., 2011; Wetzel, 2001).

Fish polyculture in ponds is an age-old practice that is considered environmentally acceptable (i.e., compatible with natural ecosystems and sustaining human quality of life) (Aubin et al., 2019; Xie et al.,

2013). It consists of rearing several fish species together that use different trophic and/or spatial niches in the same pond (Milstein, 1992; Thomas et al., 2021). Polyculture ponds mimic a simplified natural ecosystem because they contain secondary species to improve the performance of the main reared species. Fish polyculture is based on associating compatible species to avoid detrimental interactions and promote complementary use of available resources (Thomas et al., 2021). In semi-intensive polyculture ponds, fish are fed commercial pellets but also consume natural food sources available in the pond. Farmers provide additional food sources to increase fish density and the overall productivity of the system (Billard, 1999). In freshwater ponds, common carp (*Cyrpinus carpio*), which represents ca. 10 % of continental aquaculture production worldwide, can be associated in Europe with other omnivorous fish species, such as roach (*Rutilus rutilus*) or rudd (*Scardinius erythrophthalmus*), and carnivorous fish species such as perch

<footnote><sup>a</sup> MARBEC, Univ Montpellier, CNRS, Ifremer, IRD, INRAE, Sète, France</footnote>

<footnote><sup>b</sup> INRAE, Institut Agro, SAS, Rennes 35000, France</footnote>

<footnote><sup>c</sup> UMR 5110 CEFREM, CNRS, Université de Perpignan Via Domitia, Perpignan 66860, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: sarah.nahon@inrae.fr (S. Nahon).</footnote>
(Perca fluviatilis) or largemouth bass (Micropterus salmoides). In semi-intensive ponds, common carp usually consume only commercial feed, while roach and rudd consume commercial feed and supplement their diet with natural food sources (i.e., plankton and macrobenthic invertebrates) (Nahon et al., 2020). Sexually mature roach and rudd produce juveniles, which prey on plankton and invertebrates, and support the production of piscivorous fish (Nahon et al., 2024). Adding macrophytes (i.e., spermatophytes and macroalgae), which capture dissolved N and P, is another way to decrease concentrations of inorganic nutrients and improve water quality (Cunha et al., 2019; Jaeger et al., 2021). Moreover, macrophytes can serve as additional food sources for herbivorous or omnivorous fish and refuges to increase the biomass of macrobenthic invertebrates available for carnivorous fish (Chopin et al., 2001; Roucaute et al., 2024).

The sustainability of polyculture depends on appropriate combinations and proportions of reared organisms to meet chemical and biological processes (Aubin et al., 2019). Nutrient balances consider nutrient inputs to and outputs from each compartment of the entire system (Schneider et al., 2005). Nonetheless, nutrient balances only quantify nutrient pools; they do not consider nutrient fate or the relations among compartments (Azevedo et al., 2011). Carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotope analyses can trace nutrient flows in aquatic ecosystems based on the assumption that the isotopic signatures of consumers reflect those of the food sources they assimilated (Fry and Sherr, 1989). While δ¹³C values of consumer tissues are usually similar to those of their diets (DeNiro and Epstein, 1978), δ¹⁵N values are enriched from prey to consumers and are usually used to estimate a consumer's position in the trophic web (Post, 2002). Individual differences in δ¹³C and δ¹⁵N values in a species or a community assemblage are used as a proxy to study ecological niches that describe the habitat diversity and trophic diversity used by organisms (Bearhop et al., 2004; Jackson et al., 2011; Layman et al., 2007).

The aim of the present study was to estimate N and P balances and identify the food sources used by fish in a polyculture system with or without macrophytes. The study was performed in small closed systems to better understand relations among the trophic compartments in a simplified polyculture pond. We hypothesized that macrophytes in this kind of system (1) capture N and P nutrients from fish waste and (2) are used directly as food sources by omnivorous fish (carp and roach).

## 2. Materials and methods

### 2.1. Experimental design

The experiment was performed for seven months (February-September 2018) in the facilities of the Aquatic Ecology and Ecotoxycology unit (U3E, Rennes, France, 48°07'3"N, 1°47'33"W, https://doi.org/10.15454/1.5573930653786494E12) of the French National Research Institute for Agriculture, Food and the Environment (INRAE). Nine circular outdoor mesocosms were used to mimic a natural

freshwater pond (Fig. 1). The mesocosms, consisting of polyester glass fiber tanks each containing 9.6 m³ of water (3.5 m diameter × 1.0 m deep), were filled with tap water. Tap water was added three times during the experiment to compensate for evaporation and maintain the initial water level. In each mesocosm, a water pump (15 m³ h⁻¹) was placed to circulate and oxygenate water through two perforated baskets filled with pozzolan as a biological filter. The baskets and pump were placed on clay bricks. The bottom of each mesocosm was covered with 3 cm of sand, which was covered by 3 cm of sediment that had been extracted from a natural pond (i.e., containing associated flora and fauna and having a natural biochemical composition). A net was stretched over the top of each mesocosm to prevent bird predation.

Three treatments were compared in triplicate: fish-only (F treatment), fish-macrophyte (FM treatment) and macrophyte-only (M treatment). Treatment F contained a fish assemblage of 10 common carp (C. carpio), 4 sexual mature roach (R. rutilus) and 1 largemouth bass (M. salmoides). Common carp was chosen for its market value and was the target species of the system. Its sediment-stirring activity induces pond bioturbation that helps to cycle nutrients. Sexual mature roach was chosen for its ability to reproduce easily, producing a large number of fry that eat phytoplankton and zooplankton (Nahon et al., 2020, 2024). Fry are potential prey of carnivorous species. Carp and roach have a similar trophic level (3.1 and 3.0, respectively, Fishbase, Christensen and Pauly, 1992) and are omnivorous; they eat benthic organisms, plants and zooplankton. Bass are considered strictly carnivorous (trophic level 3.8). At stocking, the mean individual wet weight of carp, roach and bass were 18.1 ± 4.7, 68.1 ± 28.8 and 3.8 ± 0.5 g, respectively, representing a mean initial biomass of 150.8 ± 5.3, 226.9 ± 57.6 and 3.2 ± 0.5 kg ha⁻¹, respectively, per mesocosm. Fish densities were within the range of semi-intensive aquaculture systems according to previous studies (Jaeger and Aubin, 2018, Jaeger et al., 2021), and in accordance with the usual densities applied by fish farmers in the same area. Treatment FM contained the same fish assemblage as F, 2 pickerel weed (Pontederia cordata) plants (one in each basket), 1 watercress (Nasturtium officinale) plant and 2 water lily (Nuphar lutea) plants. These plant species were chosen for their ornamental or edible interest. Densities were determined according to their ability to develop in ponds (Jaeger and Aubin, 2018). Treatment M contained the same macrophytes assemblage as FM treatment. Fish in F and FM treatments were fed commercial pellets daily (32 % crude protein, 1.5 % P and 18.1 MJ of gross energy kg⁻¹) at the rate of 3.6 ± 0.8 % of live weight day⁻¹. Other macrophytes, especially filamentous algae, grew naturally in all treatments. The development of fish polyculture coupled to plant production is of interest as a form of ecological intensification (Jaeger and Aubin, 2018).

### 2.2. Physical, chemical and biological monitoring of water

Once per week, water temperature (multiparameter Templight HOBO Data Loggers, 64 K UA-002-64), pH (WTW 340i, SenTix® Plus 41 probe), conductivity (WTW 340i, TetraCon® 325 probe), dissolved

<figure><img src="image_2.png" /><figcaption>Fig. 1. Side view of a mesocosm representing the setup used for each treatment.</figcaption></figure>
oxygen (HACH HQ30, LDO probe®) and transparency (Secchi disk) were measured in each mesocosm. Water was sampled once per month, as well as each time that water was added to compensate for evaporation, and at the end of the experiment (three samples, one per each one-third draining of a mesocosm). Each time, 250 ml of water was collected from each mesocosm to analyze nutrient and chlorophyll a (chl a) concentrations. Drops of sulfuric acid (25 N) were immediately added to the sample, which was stored at 3°C until analysis. Water was analyzed by Spectro colorimetry analyses (automatic SmartChem-200®, AMS Alliance, France) to determine the concentrations of: phosphate-P (PO₄-P, detection limit (dl) = 0.013 mg l⁻¹) and total P (TP, dl = 0.013 mg l⁻¹, ISO 6878:2004); ammonia-N (NH₄-N, dl = 0.04 mg l⁻¹, ISO 11372:1997); nitrite-N (NO₂-N, dl = 0.01 mg l⁻¹) and nitrate-N (NO₃-N, dl = 0.1 mg l⁻¹, ISO 13395:1996); and total N (TN, dl = 0.1 mg l⁻¹, ISO 11905-1:1997). Concentrations below the detection limit were considered as trace. Total chl a concentration, a proxy of phytoplankton concentration, was measured once per month using a fluorometer (PhytoPAM®, Walz Company, Germany).

## 2.3. Survival rates, growth performance of fish and feed-conversion ratio

For each mesocosm and each fish species, survival rate (%) were calculated as follows:

$$ \text{Survival rate} = \frac{\text{Number of fish at stocking}}{\text{Number of fish at harvest}} \times 100 $$

For each mesocosm, each fish was individually weighed at stocking and harvest, and the specific growth rate (SGR, % day⁻¹) of each species was calculated as follows:

$$ \text{SGR} = \left( \frac{\ln(\text{Wh}) - \ln(\text{Ws})}{\text{no. of days}} \right) \times 100 $$

where Wh and Ws are the mean wet weight (g) at harvest and stocking, respectively.

For each mesocosm, the mean feed-conversion ratio (FCR) of the fish assemblage was calculated as follows:

$$ \text{FCR} = \frac{\text{Feed distributed (kg)}}{\text{Total net weight gain for all fish during the experiment (kg)}} $$

For each mesocosm, the mean nutrient feed-conversion ratio (XFCR) of the fish assemblage was calculated for N and P as follows:

$$ \text{XFCR} = \frac{\text{X in feed distributed (g)}}{\text{Total net X gain for all fish during the experiment (g)}} $$

Where X is N or P (g)

## 2.4. Nitrogen and phosphorus nutrient balances

For each mesocosm, nutrient balances for TN and TP were calculated as follows:

$$ \sum X_{\text{stocking}} = \sum X_{\text{harvest}} + X_{\text{unaccounted}} $$

where X is TN or TP (g)

The nutrients input included those in (1) commercial feed, (2) fish, (3) macrophytes, (4) sediment and sand and (5) water inputs. The nutrients at harvest included those in (1) fish, (2) macrophytes, (3) accumulated sediments (i.e., mixed sediment and sand, deposits on pozzolan and clay bricks) and (4) water outflow (i.e., drained water). The quantities of N and P were calculated by multiplying the total weight or volume of each input or output by its N or P concentration, respectively.

TN and TP concentrations of commercial feed were 5.12 and 1.12 g kg⁻¹, respectively, according to the feed ingredients described by the manufacturer. During the 7-month experiment, 6501.6 g of feed were distributed to the fish per treatment with fish (i.e., 2167.2 g per mesocosm with fish). According to the literature, common carp, roach and largemouth bass contained 0.083, 0.105 and 0.096 g g⁻¹ of N and 0.018, 0.027 and 0.023 g g⁻¹ of P, respectively (Ren et al., 2018; Schreckenbach et al., 2008). TN and TP concentrations of macrophytes were measured according to Dumas method (ISO 16634-1:2008) and by spectrometry (ICP-AES radial, ISO 6878:2004), respectively. The dry weight of macrophytes was measured at stocking and harvest after being dried at 50°C. TN and TP concentrations of all sediment compartments were measured according to Dumas method (Thermofisher EA flash 1112, dl = 0.005 g 100 g⁻¹, ISO 6878:2004) and Spectro colorimetry analyses (automatic SMARTCHEM-200, AMS Alliance, dl = 0.013 mg 100 g⁻¹, ISO 10694:1995), respectively. At stocking, quantities of sediment and sand were estimated based on the density and volume of each. At harvest, quantities of mixed sediment and sand, as well as sediment deposits on pozzolan and clay bricks, were weighed after being dried at 50°C. The nutrient balances did not include the dynamics of macrobenthic invertebrates, whose biomass was assumed to be negligible in the mesocosms, nor nutrient inputs from rainwater or nutrient losses through evaporation (Nhan et al., 2008).

## 2.5. Carbon and nitrogen stable-isotope analysis

At harvest, for F and FM treatments, fish, commercial feed and food sources (i.e., macrophytes, suspended particulate organic matter (SPOM), sediment organic matter (SOM), large zooplankton, macrobenthic invertebrates and juvenile roach) in the mesocosms were sampled to determine the food preferences of the three fish species. Juvenile roach that had resulted from natural reproduction in the mesocosm were only found in FM treatment. All fish were individually euthanized by immersing them in a water bath using a lethal concentration of benzocaine (Nahon et al., 2017), according to European Union regulations for the protection of animals used for scientific purposes (i. e., Directive 2010/63/EU). After death, white dorsal muscle tissue was sampled from above the lateral line of each fish. All macrophytes in FM and M treatments were manually collected. SOM was collected at two locations in each mesocosm using sediment corers. Water samples containing SPOM were collected from each mesocosm, pre-filtered through a 200 µm mesh to remove large particles and then filtered through pre-combusted (5 h at 450°C) Whatman GF/F filters. Large zooplankton were sampled in each mesocosm using a 200 µm mesh net pulled horizontally around each mesocosm, removed from the cod-end and then concentrated into containers. Macrobenthic invertebrates were collected using a 500 µm mesh net, separated and then identified before being placed into tubs. See Table S1 for the number of replicates analyzed for each compartment. All samples were carefully rinsed with ultrapure water (milliQ®; Merck Millipore, Molsheim, France), frozen and then freeze-dried. Before stable-isotope analysis, samples were ground to a fine homogeneous powder using a grinding mill (Precellys®, Bertin Technologies, Montigny-le-Bretonneux, France).

Analyzing δ¹³C values of animal tissues may require lipid extraction when C:N ratios exceed 3.5, since lipids are naturally δ¹³C-depleted (DeNiro and Epstein, 1977; Post et al., 2007). However, since none of the C:N ratios of fish exceeded 3.5, ca. 1 mg of each fish sample was weighed and packed untreated into a tin capsule to analyze C and N stable isotopes simultaneously. To remove carbonate, SOM, SPOM and large zooplankton were acidified with 1 N HCl and rinsed with ultrapure water. The acidified samples were then analyzed to determine δ¹³C values, while untreated samples were analyzed to determine δ¹⁵N values. Macrobenthic invertebrates and macrophytes were packed untreated into tin capsules.

The δ¹³C and δ¹⁵N values of samples were determined via elemental analyzer/isotope ratio mass spectrometry in continuous-flow mode using an isotope ratio mass spectrometer (Isoprime GVI, Elementar, Langenselbold, Germany) interfaced with an elemental analyzer (EuroEA 3000, Eurovector, Pavia, Italy). The δ¹³C/¹²C or δ¹⁵N/¹⁴N ratios are expressed in conventional delta notation in per mil (‰) relative to the levels of δ¹³C in Vienna Pee Dee Belemnite and δ¹⁵N in the atmosphere, respectively. Repeated measurements of alanine yielded a precision of ± 0.11 ‰ and ± 0.12 ‰ for δ¹³C and δ¹⁵N values, respectively. Working standards (alanine, wheat flour and corn flour) as well as reference standards (IAEA-N-1, IAEA-N-2, IAEA-CH₃ and USGS24 from the
National Institute of Standard and Technology, Gaithersburg, Maryland, USA) were used for multipoint calibration.

## 2.6. Statistical analysis, stable-isotope mixing models and standard ellipse areas

Mean water parameters were compared among F, FM and M treatments using a non-parametric Kruskal-Wallis test (KW) followed by post-hoc Dunn's multiple-comparison test with Bonferroni's adjustment method, since the data were not normal or homoscedastic even after log-transformation (tested using the Shapiro-Wilk and Bartlett's test, respectively).

The mean individual weight of carp, roach and bass were compared between F and FM treatments using nested analysis of variance (ANOVA) with the factors “treatment” and “mesos cosm nested in treatment”, the latter of which was removed when it had no significant effect (i.e., *p* ≈ 1). If necessary, the mean individual weight was log-transformed before analysis to obtain normal and homoscedastic residuals (tested using the Shapiro-Wilk and Bartlett's test, respectively).

The survival rate of the three fish species and the mean SGR of carp and roach, FCR, NFCR and PFCR of the fish assemblage were compared between F and FM treatments using a non-parametric Mann-Whitney test (MW), but the SGR and weight of bass could not be analyzed statistically because the experiment contained only two individuals. For the nutrient balance, the mean TN or TP of each compartment was compared among F, FM and M treatments using a non-parametric Kruskal-Wallis test followed by Dunn's multiple-comparison test with Bonferroni's adjustment method.

The δ¹³C and δ¹⁵N values of fish and their potential food sources were compared between F and FM treatments using nested ANOVA with the factors “treatment” and “mesos cosm nested in treatment”, the latter of which was removed when it had no significant effect (i.e., *p* ≈ 1). The mean δ¹³C and δ¹⁵N values of carp and roach were compared between F and FM using ANOVA with the factors “species”, “mesos cosm” and their interaction. If necessary, data were log-transformed before analysis to obtain normal and homoscedastic residuals (tested using the Shapiro-Wilk and Bartlett's test, respectively).

The contributions of food sources to fish diets were estimated using Bayesian stable-isotope mixing models (simmr package; Parnell et al., 2013) of R software v. 4.3.1 (R Core Team, 2023). The C and N trophic-discrimination factors were 1 ‰ and 4.8 ‰, respectively, for both carp and roach (Mill et al., 2007) and 1 ‰ and 3.5 ‰, respectively, for bass (Suhareva et al., 2021). The Bayesian standard ellipse area (SEAB) occupied by a species in δ¹³C and δ¹⁵N spaces was calculated for carp and roach reared in F or FM treatments using the SIBER package (Jackson et al., 2011) of R. The SEAB metric is a bivariate equivalent of the standard deviation that contains the mean isotopic niche of the core population. SEAB metrics were chosen instead of convex hull metrics (Layman et al., 2007), which include outlier individuals, due to their robustness to differences in sample size and to circumvent the bias caused by small sample sizes. SEAB sizes and overlap between species and treatments were then compared using a Bayesian approach based on Markov-chain Monte Carlo (with 100 000 draws) (Jackson et al., 2011). We calculated the proportion, and thus probability, that the SEAB for one group was smaller than that for another group. All statistical analyses were performed and graphs were generated using R. Regardless of the test, all differences were considered significant at *p* < 0.05.

# 3. Results

## 3.1. Physical, chemical and biological parameters of water

The mean temperature in F, FM and M treatments did not differ significantly (KW, *p* ≥ 0.05, Table 1). The mean pH, dissolved oxygen and transparency in F and M treatments were significantly lower than that in M treatment (KW, *p* < 0.05). The mean conductivity in F and FM

<caption>**Table 1**</caption>

Mean (± SD) physical, chemical and biological parameters of water during the seven months of the experiment in the fish-only (F), fish-macrophyte (FM) and macrophyte-only (M) treatments. *n* indicates the number of replicates.

<table><thead><tr><th rowspan="2">Parameter</th><th rowspan="2">n</th><th colspan="3">Treatments</th></tr><tr><th>F</th><th>FM</th><th>M</th></tr></thead><tbody><tr><td>Temperature (°C)</td><td>96</td><td>18.6 ± 5.9</td><td>18.5 ± 6.0</td><td>18.6 ± 6.1</td></tr><tr><td>pH</td><td>96</td><td>7.9 ± 0.3<sup>a</sup></td><td>7.9 ± 0.3<sup>a</sup></td><td>8.7 ± 0.7<sup>b</sup></td></tr><tr><td>Conductivity (mS cm<sup>-1</sup>)</td><td>96</td><td>517 ± 61<sup>a</sup></td><td>511 ± 61<sup>a</sup></td><td>444 ± 42<sup>b</sup></td></tr><tr><td>Dissolved oxygen (mg l<sup>-1</sup>)</td><td>96</td><td>8.44 ± 2.75<sup>a</sup></td><td>8.61 ± 2.70<sup>a</sup></td><td>9.48 ± 1.99<sup>b</sup></td></tr><tr><td>Transparency (cm)</td><td>96</td><td>60.3 ± 32.7<sup>a</sup></td><td>61.3 ± 33.0<sup>a</sup></td><td>98.7 ± 3.1<sup>b</sup></td></tr><tr><td>PO<sub>4</sub>-P (mg l<sup>-1</sup>)</td><td>27</td><td>0.03 ± 0.02<sup>a</sup></td><td>0.03 ± 0.03<sup>a</sup></td><td>0.01 ± 0.01<sup>b</sup></td></tr><tr><td>TP (mg l<sup>-1</sup>)</td><td>27</td><td>0.04 ± 0.03<sup>a</sup></td><td>0.04 ± 0.03<sup>a</sup></td><td>0.01 ± 0.01<sup>b</sup></td></tr><tr><td>NH<sub>4</sub>-N (mg l<sup>-1</sup>)</td><td>27</td><td>0.09 ± 0.05</td><td>0.09 ± 0.05</td><td>0.07 ± 0.04</td></tr><tr><td>NO<sub>2</sub>-N (mg l<sup>-1</sup>)</td><td>27</td><td>tr</td><td>tr</td><td>tr</td></tr><tr><td>NO<sub>3</sub>-N (mg l<sup>-1</sup>)</td><td>27</td><td>0.35 ± 0.35</td><td>0.40 ± 0.38</td><td>0.23 ± 0.27</td></tr><tr><td>TN (mg l<sup>-1</sup>)</td><td>27</td><td>1.22 ± 0.48<sup>a</sup></td><td>1.24 ± 0.64<sup>a</sup></td><td>0.69 ± 0.21<sup>b</sup></td></tr><tr><td>chl a (µg l<sup>-1</sup>)</td><td>27</td><td>8.1 ± 10.1<sup>a</sup></td><td>8.3 ± 12.8<sup>a</sup></td><td>0.6 ± 0.6<sup>b</sup></td></tr></tbody></table>

PO₄-P: phosphate-phosphorus; TP: total phosphorus; NH₄-N: ammonia-nitrogen; NO₂-N: nitrite-nitrogen; NO₃-N: nitrate-nitrogen; TN: total nitrogen; chl a: total chlorophyll a. Lowercase letters indicate significant differences among treatments F, FM and M (Kruskal-Wallis test followed by post hoc Dunn's multiple-comparison test, *p* < 0.05). tr: concentration lay below the detection limit (0.001 mg l⁻¹).

treatments was significantly higher than that in M treatment (KW, *p* < 0.05).

The mean PO₄-P and TP concentrations in F and FM treatments were significantly higher than those in M treatment (KW, *p* < 0.05). The mean concentration of each of the three forms of dissolved N did not differ significantly (KW, *p* ≥ 0.05) among the treatments, but the mean TN concentration in F and FM treatments was significantly higher than that in M treatment (KW, *p* < 0.05), as was the mean chl a concentration (13 times higher, KW, *p* < 0.05).

## 3.2. Survival rates, growth performance of fish and feed-conversion ratio

Survival rates of the three fish species did not differ significantly among F and FM treatments (MW test, *p* > 0.05, Table 2). At harvest, the mean weight of carp and roach in F and FM treatments did not differ significantly (ANOVA, *p* ≥ 0.05, Table 2). The mean weight of bass in F and FM treatments was 32.3 and 25.5 g, respectively. The mean SGR of carp in F and FM treatments did not differ significantly (MW test, *p* ≥ 0.05) but that of roach was significantly lower in F treatment than that in FM treatment (Table 2, MW test, *p* < 0.05). The mean SGR of bass was 0.93 ± 0.29 and 0.95 ± 0.10 % day⁻¹ in F and FM treatments, respectively. The mean FCR in F and FM treatments did not differ significantly (MW test, *p* ≥ 0.05).

## 3.3. Nitrogen and phosphorus nutrient balances

At stocking, no significant difference was observed among F, FM and M treatments in mean TN (1588 ± 400, 1454 ± 242 and 1460 ± 760 g, respectively, KW test, *p* ≥ 0.05) or mean TP (261.6 ± 23.5, 261.8 ± 16.8 and 242.5 ± 21.9 g, respectively, KW test, *p* ≥ 0.05, Table S2). The sediment compartment (i.e., sediment and sand) in F, FM and M treatments contained most of the TN (91.6 ± 0.1, 90.6 ± 1.7 and 98.2 ± 0.2 %, respectively) and TP (90.5 ± 0.9, 79.2 ± 10.3 and 99.3 ± 5.1 %, respectively) (Fig. 2). The second-largest compartment for mean TN and TP was feed in F treatment (6.2 ± 0.1 and 8.3 ± 0.8 %, respectively) and FM treatment (6.8 ± 1.3 and 7.3 ± 1.3 %, respectively) and water inputs in M treatment (1.5 ± 0.2 and 6.2 ± 0.1 %, respectively). The mean TN in fish did not differ significantly between F and FM treatments (13 ± 1 and 10 ± 1 g, respectively), while the mean TP in fish was significantly higher in F treatment than that in FM treatment (3.1 ± 0.2 and 2.4 ± 0.3 g, respectively, KW test, *p* < 0.05, Table S2). Mean TN and TP in fish and macrophytes at stocking
<table><caption>**Table 2**
Growth performances (mean ± SD) of fish reared in the fish-only (F) and fish-macrophyte (FM) treatments. n indicates the number of replicates.</caption><thead><tr><th rowspan="2">Species</th><th colspan="3">Survival rate (%)</th><th colspan="3">Individual wet weight at harvest (g)</th><th colspan="3">Specific growth rate (% day<sup>-1</sup>)</th><th colspan="3">Feed-conversion ratio</th></tr><tr><th>n</th><th>F</th><th>FM</th><th>n</th><th>F</th><th>FM</th><th>n</th><th>F</th><th>FM</th><th>n</th><th>F</th><th>FM</th></tr></thead><tbody><tr><td>Cyprinus carpio</td><td>3</td><td>70 ± 10</td><td>70 ± 26</td><td>21</td><td>230.1 ± 72.2</td><td>250.6 ± 110.0</td><td>3</td><td>1.17 ± 0.06</td><td>1.22 ± 0.15</td><td>3</td><td>1.30 ± 0.07</td><td>1.20 ± 0.07</td></tr><tr><td>Rutilus rutilus</td><td>3</td><td>83 ± 29</td><td>83 ± 29</td><td>10</td><td>164.5 ± 44.7</td><td>132.4 ± 22.3</td><td>3</td><td>0.32 ± 0.03<sup>a</sup></td><td>0.40 ± 0.04<sup>b</sup></td><td></td><td></td><td></td></tr><tr><td>Micropterus salmoides</td><td>3</td><td>67 ± 58</td><td>67 ± 58</td><td>2</td><td>32.3 ± 15.6</td><td>25.5 ± 4.9</td><td>2</td><td>0.93 ± 0.29</td><td>0.95 ± 0.10</td><td></td><td></td><td></td></tr></tbody></table>

Letters indicate significant differences between the two treatments (Mann-Whitney test, *p* < 0.05).

<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Representation of the mass balance of total nitrogen (TN) and total phosphorus (TP) in the fish-only (F), fish-macrophyte (FM) and macrophyte-only (M) treatments, expressed as a percentage of TN or TP (mean ± SD).</figcaption></figure>

represented less than 1 % of TN or TP in the entire system, regardless of the treatment.

At harvest, the mean TN in fish did not differ significantly between F and FM treatments (51 ± 2 and 51 ± 4 g, respectively, KW test, *p* ≥ 0.05), nor mean TP did in fish (11.6 ± 0.6 and 11.6 ± 1.1 g, respectively, post-hoc test, *p* ≥ 0.05, Table S2). The mean increase in fish biomass in F and FM treatments represented 38 ± 2 and 41 ± 3 g of TN, respectively, and 8.5 ± 0.6 and 9.2 ± 0.8 g of TP, respectively. The means NFCR were 0.39 ± 0.02 and 0.39 ± 0.03 and the mean PFCR were 0.42 ± 0.03 and 0.42 ± 0.04 in F and FM treatments, respectively. Values did not differ significantly between F and FM treatments (MW test, *p* ≥ 0.05). The mean TN in macrophytes in F, FM and M treatments was 6 ± 11, 4 ± 1 and 41 ± 11 g, respectively, while the mean TP in macrophytes was 2.1 ± 3.6, 1.6 ± 0.5 and 14.1 ± 5.1 g, respectively. The mean change in macrophyte biomass in F, FM and M treatments represented a change of 6 ± 1, -1 ± 3 and 37 ± 11 g of TN, respectively, and 2.1 ± 3.6, -0.3 ± 1.2 and 12.6 ± 5.1 g of TP, respectively. The mean TN in water did not differ significantly between F and FM treatments (14 ± 2 and 17 ± 1 g, respectively, post-hoc test, *p* ≥ 0.05), but it did differ significantly between FM and M treatments (5 ± 1 g, post-hoc test, *p* < 0.05). Likewise, the mean TP in water did not differ significantly between F and FM treatments (0.5 ± 0.3 and 0.8 ± 0.5 g, respectively, post-hoc test, *p* ≥ 0.05), but it did differ significantly between FM and M treatments (0.1 ± 0.1 g, post-hoc test, *p* < 0.05). At harvest, the sediment in F, FM and M treatments still contained most of

the mean TN (52.3 ± 12.7, 73.0 ± 25.7 and 49.3 ± 30.2 %, respectively) and mean TP (90.7 ± 4.8, 95.3 ± 5.2 and 88.4 ± 12.0 %, respectively). The sediment lost a mean of 624 ± 203, 256 ± 284 and 714 ± 452 g of TN in F, FM and M treatments respectively, but gained 0.6 ± 15.5 and 47.9 ± 49.9 g of TP in F and FM treatments, respectively, and lost 26.6 g of TP in M treatment. A similar pattern was observed for the water, which lost a mean of 7 ± 3, 5 ± 1 and 17 ± 2 g of TN in F, FM and M treatments, respectively, but gained 0.39 ± 0.31 and 0.65 ± 0.45 g of TP in F and FM treatments, respectively, and lost 0.04 ± 0.03 g of TP in M treatment. No significant difference was observed at harvest, among F, FM and M treatments in mean TN (902 ± 209, 1134 ± 494 and 766 ± 430 g, respectively) or mean TP, (251.5 ± 17.9, 297.7 ± 61.0 and 228.5 ± 31.6 g, respectively).

No significant difference was observed among F, FM and M treatments in mean unaccounted TN (686 ± 213, 319 ± 281 and 694 ± 445 g, respectively, KW test, *p* ≥ 0.05) or mean unaccounted TP (10.1 ± 17.9, -35.9 ± 49.5 and 14.0 ± 38.3 g, respectively, KW test, *p* ≥ 0.05). Mean unaccounted TN was the second-largest compartment in F, FM and M treatments (43.2 ± 13.3, 22.0 ± 24.8 and 47.5 ± 29.7 %, respectively). In F treatment, the mean percentage of unaccounted TP (3.9 ± 6.3 %) was similar to that in harvested fish (4.4 ± 0.3 %). In M treatment, the mean percentage of unaccounted TP (5.8 ± 14.4 %) was similar to that in macrophytes (5.8 ± 1.9 %). In FM treatment, the mean percentage of unaccounted TP was negative (-12.1 ± 15.8 %).
### 3.4. δ¹³C and δ¹⁵N values of fish and their potential food sources

In F and FM treatments, δ¹³C and δ¹⁵N values varied among the potential food sources for fish (Fig. 3). In F treatment, δ¹³C values of food sources ranged from -29.02 ± 0.16 ‰ (SOM) to -25.35 ± 0.13 ‰ (commercial feed), while δ¹⁵N values ranged from 3.69 ± 0.45 ‰ (commercial feed) to 8.76 ± 0.53 ‰ (large zooplankton). In FM treatment, δ¹³C values of food sources ranged from -29.22 ± 1.09 ‰ (SPOM) to -24.75 ± 3.02 ‰ (macrophytes), while δ¹⁵N values ranged from 3.69 ± 0.45 ‰ (commercial feed) to 9.24 ± 1.60 ‰ (macrophytes). The δ¹³C and δ¹⁵N values of each potential food source did not differ significantly between F and FM treatments (ANOVA, *p* > 0.05).

For reach and carp, δ¹³C and δ¹⁵N values did not differ significantly between F and FM treatments (ANOVA, *p* > 0.05). In both treatments, carp had significantly lower δ¹³C and δ¹⁵N values than roach (Fig. 3, Table 3). Compared to commercial feed, carp were enriched by 1.26 ± 0.22 ‰ and 3.57 ± 0.22 ‰ in δ¹³C and δ¹⁵N, respectively, while roach were enriched by 1.55 ± 0.51 ‰ and 6.75 ± 0.76 ‰ in δ¹³C and δ¹⁵N, respectively. The SEA~B~ of carp and roach did not overlap in F or FM treatments (Fig. 4). Roach in FM treatment had larger ellipses than carp

in F and FM treatments and than roach in F treatment (Table 3, Fig. 4). In both treatments, mixing models indicated that the common carp diet was 90 % commercial feed. The roach diet was ca. 50 % commercial feed; 40 % of a mixture of macrophytes, macrobenthic invertebrates and large zooplankton; and 5 % each of SPOM and SOM (Fig. 5).

Juvenile roach and bass had lower δ¹³C and higher δ¹⁵N values than carp and roach did. Bass were depleted by 1.13 ± 0.00 ‰ in δ¹³C and enriched by 2.03 ± 0.26 ‰ in δ¹⁵N compared to juvenile roach. In both treatments, mixing models indicated that the bass diet was ca. 60 % juvenile roach and 22 % macrobenthic invertebrates (Fig. 5).

## 4. Discussion

### 4.1. Water quality, macrophytes and phytoplankton biomass in the mesocosms

The presence of fish and the distribution of commercial feed influenced water quality in the mesocosms. According to the water parameters measured, F and FM treatments had similar mean water quality during the experiment, even though FM treatment included

<figure><img src="image_4.png" /><figcaption>**Fig. 3.** δ¹³C and δ¹⁵N values (mean ± SD) of fish and their potential food sources in the fish-only (F) and fish-macrophyte (FM) treatments.</figcaption></figure>
<caption>**Table 3**</caption>

$\delta^{13}\text{C}$ and $\delta^{15}\text{N}$ values (mean ± SD) and Bayesian standard ellipse area ($\text{SEA}_B$) of fish reared in the fish-only (F) and fish-macrophyte (FM) treatments.

<table><thead><tr><th rowspan="3">Species</th><th colspan="6">Treatments</th></tr><tr><th colspan="3">F</th><th colspan="3">FM</th></tr><tr><th>δ<sup>13</sup>C (‰)</th><th>δ<sup>15</sup>N (‰)</th><th>SEA<sub>B</sub> (‰<sup>2</sup>)</th><th>δ<sup>13</sup>C (‰)</th><th>δ<sup>15</sup>N (‰)</th><th>SEA<sub>B</sub> (‰<sup>2</sup>)</th></tr></thead><tbody><tr><td>Cyprinus carpio</td><td>-24.40<br>± 0.39<sup>a</sup></td><td>7.82<br>± 0.54<sup>a</sup></td><td>0.62<sup>A</sup></td><td>-24.09<br>± 0.22<sup>a</sup></td><td>7.26<br>± 0.58<sup>a</sup></td><td>0.38<sup>A</sup></td></tr><tr><td>Rutilus rutilus</td><td>-23.86<br>± 0.19<sup>b</sup></td><td>10.13<br>± 0.50<sup>b</sup></td><td>0.27<sup>A</sup></td><td>-23.74<br>± 0.71<sup>b</sup></td><td>10.58<br>± 0.96<sup>b</sup></td><td>1.94<sup>B</sup></td></tr><tr><td>Micropterus salmoides</td><td>-25.09<br>± 1.21</td><td>14.95<br>± 0.93</td><td>n.c.</td><td>-25.39<br>± 0.01</td><td>14.34<br>± 0.26</td><td>n.c.</td></tr><tr><td>Juvenile R. rutilus</td><td>n.d.</td><td>n.d.</td><td>n.c.</td><td>-24.26<br>± 0.04</td><td>12.31<br>± 0.40</td><td>n.c.</td></tr></tbody></table>

Lowercase letters indicate significant differences (Mann-Whitney test, *p* < 0.05) in $\delta^{13}\text{C}$ or $\delta^{15}\text{N}$ values between *C. carpio* (n = 15) and *R. rutilus* (n = 10). *M. salmoides* and juvenile *R. rutilus* were excluded from the analysis since n = 2. Uppercase letters indicate the probability that $\text{SEA}_B$ were larger based on 100 000 Monte Carlo draws. n.d.: not detected (fish absent). n.c.: not calculated due to too few individuals.

macrophytes. However, few of the macrophytes in FM treatment survived, likely due to fish behavior, since common carp and roach are known to damage macrophyte communities (Körner and Dugdale, 2003; Miller and Crowl, 2006). Nonetheless, in all treatments, we observed spontaneous development of a few macrophytes and filamentous macroalgae that grew from seeds in the sediment. A previous study exhibited that planted lagoon, including similar plant species (i.e., watercress, water lily, manna grass and pickerel weed), provides ecosystem services on nutrients cycling but plants were protected from fish degradation (Jaeger et Aubin, 2018). In F and FM treatments, N and P concentrations

in the water were 2 and 4 times as high, respectively, as those in M treatment. Bioturbation of sediment due to fish feeding activity increased nutrient concentrations in the water, along with turbidity, as observed by Rahman et al. (2008). Thus, common carp influence water quality greatly, which influences water transparency and nutrient concentrations (Bajer and Sorensen, 2015; Chumchal et al., 2005; Fischer et al., 2013). Feeding commercial feed (i.e., fish waste and unconsumed feed) likely also increased N and P concentrations in the water, but to a lesser extent than fish bioturbation (Jaeger and Aubin, 2018).

The greater bioavailability of N and P in F and FM treatments than that in M treatment increased the development of phytoplankton greatly. Previous studies have observed that the presence of fish increases phytoplankton growth due to the additional nutrients excreted by fish and fish bioturbation activity, which releases nutrients from the sediment into the water column (Adánek and Marsálek, 2013; Fischer et al., 2013). In M treatment, the mean phytoplankton concentration was extremely low (92 % lower than that in F and FM treatments), likely due to the relative lack of nutrients and greater transparency of the water (40 % greater than that in F and FM treatments), which increased macrophyte development. Moreover, macrophytes are known to decrease phytoplankton biomass through competition for nutrients and allelopathic inhibition (Amorim and Moura, 2020; Jeppesen et al., 2000).

The higher chl *a* concentration, a proxy of phytoplankton concentration, observed in F and FM treatments than that in M treatment was not associated with an increase in the mean dissolved oxygen concentration (11 % lower in F and FM treatments than that in M treatment), as previously observed in a freshwater environment (Seki et al., 1980). The lower dissolved oxygen concentration was likely due to the high activity of heterotrophic organisms that degraded organic matter from fish waste, or perhaps the presence of more submerged macrophytes (which

<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Individual $\delta^{13}\text{C}$ and $\delta^{15}\text{N}$ values of *Cyprinus carpio* and *Rutilus rutilus* in the fish-only (F) and the fish-macrophyte (FM) treatment enclosed by the standard ellipse area ($\text{SEA}_B$).</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Estimated contributions of food sources to the diets of fish (*Cyprinus carpio*, *Rutilus rutilus* and *Micropterus salmoides*) using Bayesian stable-isotope mixing models in the fish-only (F) and fish-macrophyte (FM) treatments.</figcaption></figure>

have more efficient photosynthesis than phytoplankton, Sand-Jensen, 1997) in M treatment than in F or FM treatments.

## 4.2. The fate of nitrogen and phosphorus in polyculture mesocosms

In order to investigate the fate of nutrients and fish nutrition in freshwater polyculture ponds, mesocosms are valuable tools that mimics natural environments (Macaulay et al., 2025). In this study, mesocosms were used rather than natural ponds especially for a better estimation of N and P in sediment. Quantification of N and P in nutrient balances requires to estimate an accurate volume of sediment that is not possible in natural ponds. Mesocosm also allow to have robust replicate with exactly the same size and the same initial environmental conditions. Experimental natural ponds often have different initial environmental parameters that result from their use in previous experiments. Thus, thanks to the experimental conditions in mesocosms, since P is a conservative element, the estimated phosphorus balances obtained, as well as nitrogen, were confidently considered as well balanced. Indeed, the mean percentage of unaccounted TP was near zero and varied from -12.1-5.8 % of mean TP at harvest. The inaccuracy was due mainly to analytical error. In comparison, Flickinger et al. (2020) observed mean percentages of -1.7-50.2 % of mean TP at harvest. The estimated N balances were less well balanced. The percentage of unaccounted N, being 22-47 % of mean TN at harvest, was mainly explained by the effectiveness of the pozzolan biofiltration, in which bacteria mineralized the N, emitting nitrogenous gases into the atmosphere (Hu et al., 2012, Liu et al., 2023).

In F and FM treatments, the percentage of N in the sediment decreased from stocking to harvest, which means that fish waste did not accumulate in the sediment. That was explained in part by the fact that the reared organisms assimilated feed efficiently. The N in the sediment likely decreased due to fish bioturbation too. The N released into the water column was assimilated by phytoplankton and macrophytes to produce biomass. Potentially, it is also due to the N from sediment used by bacteria in denitrification, emitting gases into the atmosphere (Boyd and McNevin, 2024). In contrast, the percentage of P in the sediment changed little from stocking to harvest, and P is highly adsorbed by sediment (Boyd, 1985; Shrestha and Lin, 1996). In M treatment, macrophytes represented 2.8 % and 5.8 % of TN and TP, respectively. In both treatments at harvest, fish represented ca. 3 % and 4 % of TN and TP, respectively, which was similar to percentages observed for fish in

semi-intensive freshwater ponds (3-25 % of TN and 5-18 % of TP; Nhan et al., 2008; Pouil et al., 2019). The nutrient balances at stocking and harvest indicated that sediment in the mesocosms was the main pool of N and P, containing more than 90 % of TN and TP. These results differed from those observed in semi-intensive pond aquaculture of fish or shrimp (50-76 % and 53-83 % of TN and TP at stocking, respectively; Casillas-Hernández et al., 2006; Pouil et al., 2019; Barbosa et al., 2024). Previous studies of nutrient balances have shown that 20-50 % of N and P added to ponds in feed and fertilizers is recovered in reared organisms, while the rest accumulates in the water or sediment (Hu et al., 2021; Pouil et al., 2019). This is in accordance with the results observed in the present work, with 39-42 % of N and P added to mesocosms in feed is recovered in fish and 2-4 % of N and P added to mesocosms in feed is recovered in macrophytes. It means that macrophytes production should have increased 23 and 13 times more to recover the remaining N and P from feed, respectively. The nutrient balances indicated that N and P recovered by macrophytes represented less than 0.5 % of TN and 1.0 % of TP in both F and FM treatments. In this study, macrophytes stored 8 times as much N and P in M treatment as in F and FM treatments. So, assuming that planted macrophytes would have recovered as much nutrients as macrophytes which grew up in M treatment, it would not be enough to recover the remaining N and P from feed. Initially, the aim was to cultivate edible and ornamental macrophytes that would be harvested progressively. In this way, the production of macrophytes would have been improved in stimulating plant growth as well as the nutrient recovery. Unfortunately, only unmarketable macrophytes grew up, which should have been harvested.

## 4.3. Fish growth and food sources used in polyculture mesocosms

Despite mesocosms are still simplified systems, and biodiversity of potential food sources of fish is probably restricted compared to natural ponds, the SGR and FCR of fish in the mesocosms lay in the same order of magnitude as those observed previously in a semi-intensive polyculture pond (Jaeger and Aubin, 2018). During the 7-month experiment, the mean weight of carp, roach and bass increased by ca. 13, 2 and 7.5 times, respectively. Fish growth performances did not differ significantly between F and FM treatments. The δ¹³C and δ¹⁵N values of carp and roach clearly showed that commercial pellets were the main food source for both species. Carp had isotopic values more similar to those of commercial pellets than roach did. In both treatments, roach occupied a
higher trophic level than carp, as observed in previous studies (Nahon et al., 2020; 2024). The mixing models confirmed that the carp diet was 90 % commercial pellets, regardless of the treatment. Schultz et al. (2012) observed that carp reared in ponds preferentially consumed high-quality food sources, such as commercial pellets, over natural food sources. In both treatments, the roach diet was ca. 50 % commercial pellets and 50 % natural food sources from the mesocosm (i.e., macrobenthic invertebrates, macrophytes, SPOM and large zooplankton). Both fish species are able to shift their diet depending on which prey are available in their environment (Jones and Waldron, 2003; Schiemer and Wieser, 1992). Roach can consume a large percentage of macrophytes and adapt its trophic behavior to the food sources available (Brabrand, 1985). The SEA~B~ of carp and roach did not overlap, which confirmed that carp and roach can be reared in the same feeding environment, with little trophic competition (Nahon et al., 2020; Saowakoon et al., 2021). The larger SEA~B~ of roach in FM treatment than that in F treatment was likely due to certain individuals specializing on food sources such as macrophytes or macrobenthic invertebrates.

Regardless of the treatment, carp and roach consumed little SOM (< 5 % of the diet), even though the sediment was the main pool of TN and TP. In contrast, commercial pellets, the main food source for carp and roach, represented only ca. 6 % of TN and 8 % of TP.

Juvenile roach were enriched by 1.79 ‰ in ¹³C and 3.96 ‰ in ¹⁵N compared to large zooplankton, which suggests that large zooplankton contributed greatly to their diet. However, only two juvenile roach were found in the three FM mesocosms at harvest, which means that its estimated diet, and that of bass, must be interpreted with caution. Because largemouth bass feed on juvenile fish and macrobenthic invertebrates (Costantini et al., 2018). We hypothesized that (1) adult roach spawned in each mesocosm, (2) bass consumed most or all of the juvenile roach, and (3) δ¹³C and δ¹⁵N values of juvenile roach were similar among treatments, like those of all compartments analyzed in this study. The mixing models indicated that bass consumed mainly juvenile roach, followed by macrobenthic invertebrates, regardless of the treatment. However, these results should be confirmed with more than two juvenile individuals. Furthermore, although stable isotopes are relevant biochemical markers for estimating fish diets in freshwater environments, interpreting their results requires caution because many factors (e.g., metabolic processes, ontogeny, environmental parameters) influence isotope transfer from food sources to consumers. For instance, a fish's chemical composition reflects the composition of its diet during the few weeks before sampling, and the food sources available during sampling may differ from those throughout a given period or the entire growing period. Thus, several food sources may not have been sampled because they had been completely consumed (e.g., certain macroalgae) or were absent at the time of sampling (e.g., larvae of flying invertebrates).

## 5. Conclusion and perspectives

With the aim to improve nutrient use efficiency toward fish production while avoiding wastes into the surrounding environment, it is important to represent how the nutrients circulate through the system. Analyzing methods, like nutrient budget and stable isotopes analyze, are accurate method to understand more the use of the nutrients introduced in a complex system like fishpond polyculture. Selecting complementary fish species is crucial to ensure viable polyculture that mitigate the excess nutrients/organic matter generated by intensive monospecific. This study estimated the diets of three fish species in a fed polyculture. Carp consumed mainly commercial feed, roach showed high trophic plasticity, and largemouth bass behaved as a carnivore. Our study indicated that at fawning scale, carp, roach and bass are good candidates to be rear together in the same polyculture. Carp and roach, two omnivorous species, adapt their trophic behavior and exhibited low trophic competition. Moreover, adding mature sexual roach in polyculture is an advantage to provide juveniles that regulate plankton

biomass and are the principal food source of carnivorous species. Macrophytes were only used by roach and contributed less than 15 % to their diet. Fish bioturbation promoted the growth of phytoplankton. This study improves understanding of nutrient cycling and trophic webs in a fishpond system. In order to explore eco-friendly aquaculture systems, it may help to design and test nutrient-efficient freshwater polyculture ponds by balancing the trophic compartments and using ecosystem functions. Future studies should investigate the possibility to avoid high N loss by gas emissions in order to recover that into biomass. Mesocosm is a tool mimicking larger fishponds even if its entire complexity is not present. Nevertheless, it can be used to evaluate the relevance of some organisms. Sediment was the main nutrient pool, but organisms in the mesocosms consumed little SOM. The diversity of macrobenthic invertebrates in the sediment was not measured at stocking, even though they are a major food source for fish and consume large quantities of SOM. Adding a detritivore as another intermediate trophic level could increase the system's nutrient-use efficiency. For instance, Chironomidae larvae consume SOM and could serve as another food source for fish. We also found that the presence of fish prevented the development of macrophytes, which did not help to decrease nutrient concentrations in the water. To protect macrophytes from fish, improve water quality and support the development of invertebrates, a new experimental design should be tested in which macrophytes can grow separately from fish polyculture.

## CRediT authorship contribution statement

**Joel Aubin:** Writing – original draft, Methodology, Conceptualization. **Christophe Menniti:** Methodology. **Christophe Jaeger:** Writing – original draft, Methodology, Conceptualization. **Marc Roucaute:** Writing – original draft, Methodology, Conceptualization. **Philippe Kerherve:** Writing – original draft, Methodology, Conceptualization. **nahon sarah:** Writing – original draft, Methodology, Conceptualization.

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

We thank Antoine Gallard, Bernard Joseph, Yoann Bennevault, Caroline Gorzerino, Martine Ollitrault, Maïra Coke, Didier Azam, the entire team of the INRA-U3E experimental site and all the kind volunteers who helped to weigh and measure the fish. We also thank Laurence Carteaux, Béatrice Trinkler and Yannick Fauvel for the water and sediment analyses. We thank both reviewers for their helpful comments. These results were obtained in the IMTA-Effect project (ERA-NET COFASP), managed in France by the French National Research Agency (grant no. ANR-15-COFA-0001-01). We thank the 3 reviewers for their helpful comments.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.aqrep.2025.102865.

## Data Availability

data are available on a repository
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