Rational Nitrogen Reduction Helps Mitigate the Nitrogen Pollution Risk While Ensuring Rice Growth in a Tropical Rice–Crayfish Coculture System

Abstract

The incorporation of aquaculture feed within a rice–crayfish coculture system significantly enhances nitrogen cycling, thereby diminishing the reliance on chemical fertilizers. However, this benefit is often overlooked in practice, and farmers continue to use large quantities of chemical fertilizers to maximize production, resulting in excessive soil fertility and water nitrogen pollution. Thus, avoiding nitrogen pollution in rice–crayfish coculture systems has become a pressing issue. In this study, we conducted a two-year experiment with two rice cultivars, and a 33.3% reduction in nitrogen fertilizer in a rice–crayfish coculture system (RC), to systematically analyze the overall nitrogen balance, rice nitrogen nutrition, and soil fertility, as compared with a rice monoculture system (RM). Our findings revealed the following: (1) Under the 33.3% reduction in nitrogen fertilizer, the nitrogen surplus in the rice–crayfish coculture system was comparable to that in the rice monoculture, and was controlled at an environmental safety level. (2) Nitrogen utilization efficiency and the accumulation of nitrogen in the rice–crayfish coculture were comparable to those in the rice monoculture. The nitrogen cycle in this system was able to provide the nitrogen required for rice growth after nitrogen fertilizer reduction. (3) The rice–crayfish coculture significantly improved the overall soil fertility and the effectiveness of soil nitrogen nutrition. Furthermore, cutting off the application of nitrogen fertilizer after the mid-tillering stage effectively controlled the total nitrogen content in soil after rice maturity. In conclusion, reducing nitrogen fertilizer in a rice–crayfish coculture system is feasible and beneficial. It ensures rice production, reduces the risk of excessive nitrogen surplus and surface pollution, and promotes a greener, more environmentally friendly paddy field ecosystem.

1. Introduction

In the context of coordinated development in production, environment, ecology, and biodiversity in agriculture, exploring nitrogen balance in crop systems is a necessary agricultural technology. Historically, applying large quantities of nitrogen fertilizers was common practice to ensure food production, resulting in significant nitrogen loss and environmental pollution. However, with the enhancement of social and cultural standards and continuous advancements in agricultural science and technology, alongside growing concern for agroecology, agricultural emissions, and surface pollution, more agricultural workers are acutely aware of this predicament. The urgent necessity to resolve crop production nitrogen management issues is increasingly apparent. Reducing nitrogen fertilizer usage and enhancing its efficiency in cropping systems represent the most effective methods for addressing such problems [1]. Data indicate that China’s nitrogen fertilizer consumption has increased linearly since 1961, with a notable decrease beginning in 2019 [2]. This suggests that recent efforts to reduce nitrogen fertilizer in China have become increasingly effective. However, the extensive use of nitrogen fertilizer over the past six decades has significantly compromised the health of Chinese soils. Consequently, reinforcing a rational nitrogen application system for crops, particularly for staple crops like rice and maize, is of paramount importance. Peng et al. [3] found that the implementation of site-specific nitrogen management strategies resulted in a 32% reduction in nitrogen fertilizer usage and a 5% increase in grain yield when compared to the traditional practices employed by farmers. Furthermore, Abebe et al. [4] demonstrated that maize yield with a nitrogen application of 92 kg ha⁻¹ was significantly higher than with 110 kg ha⁻¹. In conclusion, reasonable nitrogen fertilizer application is crucial for crop production and may even promote crop growth.

The integrated rice farming model represents a typical production model that can enhance nutrient efficacy and nitrogen fertilizer utilization efficiency [5]. As a flood-loving crop, rice production spaces can be developed and utilized twice. The irrigation environment in fields provides a habitat for aquatic economic animals like fish and shrimp, which thrive in shallow water [6]. Examples include rice–pink fish systems [7], rice–fish cultures [8], rice–crayfish cultures [9], rice–crab cultures [10], and rice–turtle cultures [11]. Numerous studies have shown that the moderate incorporation of aquaculture in rice fields can alleviate the land fatigue associated with rice monoculture, diversify outputs, and enhance nutrient utilization efficiency in the water–soil–crops–animals system [12,13,14,15]. Additionally, it can control weed damage, reduce rice pests and diseases, improve soil fertility and biodiversity, and increase grain yield [16]. Consequently, in the current context of scarce agricultural resources and outdated traditional agricultural technology, replacing traditional high-consumption agriculture with efficient, eco-friendly agriculture is unavoidable [17].

The rice–crayfish coculture (RC) model, combining rice production with crayfish farming, has gained significant interest in recent years due to its high output and returns [18]. The commercial value of crayfish has stimulated robust growth in China’s crayfish farming industry. Recent reports indicate that in 2022, China’s crayfish farming area was approximately 1.87 million ha, with a production of approximately 2.89 million tons. Of this, the RC model accounted for approximately 1.57 million ha, about 83.93% of the total crayfish farming area. The total output value of the Chinese crayfish industry in 2022 was approximately CNY 458 billion, with the aquaculture industry contributing around CNY 96 billion [19]. The RC model is expanding from the plains of the middle and lower Yangtze River to the surrounding provinces and cities, and even nationwide. Most provinces, except a few without suitable habitats, are actively developing this model [20]. However, the accelerated expansion of the RC model risks diverging from its original green ecological objective. In practice, farmers often increase feeding efforts to boost crayfish production, leading to the eutrophication of water bodies and increased surface source pollution [21]. Studies show that algae cultivation, residual feed and fertilizer, and fecal matter accumulation in paddy water bodies elevate nitrogen concentrations during crayfish farming [22,23]. Ling et al. [24] studied the rice–crayfish rotation model in the Jianghan Plain and found that total nitrogen had the highest isobaric loading ratio, making it a key pollutant in this model. Si et al. [25] indicated that the total nitrogen discharged from crayfish aquaculture wastewater under long-term RC reached 9.7 kg ha⁻¹. Wenyu [26] found a 157.51% average increase in nitrogen discharge in Qianjiang City’s RC model, posing a risk of agricultural surface-source pollution. Consequently, optimizing the nitrogen balance is a pressing concern for the RC model.

Previous studies have primarily focused on the impact of the RC model on soil fertility, soil microbiology, and greenhouse gases. However, there has been limited research on the effects of nitrogen fertilizer reduction on nitrogen cycling and balance in this system. Understanding the nitrogen cycle and balance in the soil–crop system can help establish criteria for nitrogen fertilizer reduction and improve nitrogen use efficiency, ensuring food security while reducing energy consumption, environmental damage, and promoting sustainable agriculture. This study addresses these issues by conducting a two-year nitrogen reduction fertilization + rice–crayfish coculture field trial. Our hypothesis is whether a reduction in nitrogen fertilizer in the RC system could be compensated by crayfish culture. The objective is to confirm the feasibility of nitrogen reduction fertilization in the RC system by analyzing differences in overall nitrogen balance, nitrogen accumulation in rice, nitrogen transfer, and soil fertility compared to the rice monoculture system.

2. Materials and Methods

2.1. Site Description

In 2021 and 2022, field trials were conducted in Lingao County, located in the Hainan Province of China (19°38′ N, 109°37′ E). Lingao County is characterized by a tropical monsoon climate, with high temperatures and abundant light throughout the year. The total precipitation during the experimental period was 1433.0 mm and 1486.1 mm in 2021 and 2022, respectively, with average daily temperatures of 28.8 °C and 26.6 °C, respectively. Prior to the application of the basal fertilizer, soil samples were collected from a depth of 20 cm in the tillage layer in order to determine the physicochemical properties. The soil properties are presented in

Table 1. Basic physicochemical properties of soil in experimental field.

Treatment	pH	Organic Matter (g kg−1)	Total Nitrogen (g kg−1)	Total Phosphorus (g kg−1)	Available Phosphorus (mg kg−1)	Available Potassium (mg kg−1)
RM	5.07	3.62	2.75	0.31	62.59	46.28
RC	5.02	3.98	3.43	0.37	60.58	50.10

Note: RM indicates rice monoculture system; RC indicates rice–crayfish coculture system.

.

2.2. Experimental Design

The experiment was designed as a randomized block design with two cultivation models in the main plots, namely the rice–crayfish coculture system (RC) and the rice monoculture system (RM), and two varieties in the sub-plots, namely YXYLS and SXHN, with each treatment replicated four times for a total of 16 plots. The area of the rice monoculture experimental field was about 500 m², and the area of the rice–crayfish experimental field was about 1560 m² (including the crayfish ditch area). Wet direct seeding was used for sowing, utilizing seed bands. The seeds were sown directly without germination, and the row spacing for rice was 25 cm. The water management maintained shallow water irrigation after rice seedling establishment and flooded water was kept after tillering. The fertilizer application for the two-year field experiment was consistent for both treatments, but the nitrogen fertilizer application was different, as detailed in

Table 2. Nitrogen fertilizer application rate by growth period.

Treatment	Total Amount Applied	Period and Proportion of Application
RM	120 kg N ha−1	Base: Tillering: Panicle Initiation: Heading = 2: 2: 1: 1
RC	80 kg N ha−1	Base: Tillering = 1: 1

Note: RM indicates rice monoculture system; RC indicates rice–crayfish coculture system.

. P and K fertilizers were the same in both treatments, with P₂O₅ applied once as the base fertilizer at 60 kg P ha⁻¹, and KCl applied once as the base fertilizer at 100 kg K ha⁻¹. Weeds, diseases, and insects were intensively controlled throughout the growing season in both years. The crayfish were released after the mid-tillering stage of rice growth. The daily management of crayfish culture was based on feeding 2–7% of the crayfish mass.

2.3. Data Collection

2.3.1. Soil Sampling

Soil samples were collected using the five-point sampling method at the mid-tillering (MT), panicle initiation (PI), heading (HD), and physiological maturity (PM) stages from a depth of 0–20 cm, following the five-point sampling method in 2022. After air-drying, the soil was sieved through 100- and 20-mesh sieves for further use. The soil’s organic matter, dissolved organic carbon, pH, electrical conductivity, total nitrogen, total phosphorus, and available potassium contents were measured. Soil pH was determined by a pH meter with a water–soil ratio of 2.5: 1. Soil electrical conductivity was measured by a portable conductivity meter with a water–soil ratio of 5: 1. Soil organic matter was determined by the potassium dichromate volumetric method with external heating. Soil available potassium was measured using nitric acid extraction followed by flame photometry. Soil calcium content was determined using ammonium acetate extraction followed by flame photometry. Soil dissolved organic carbon was extracted using 0.5 mol L⁻¹ potassium sulfate and measured using a TOC analyzer (TOC-L CPH) [27]. Soil total nitrogen was determined by H₂SO₄-H₂O₂ decoction, soil ammonium nitrogen and nitrate nitrogen were extracted by leaching with 1 mol L⁻¹ potassium chloride, and then the content of soil total nitrogen, ammonium nitrogen, and nitrate nitrogen were determined by a fully automated intermittent chemical analyzer.

2.3.2. Plant Sampling

Rice plant samples from each growth stage were dried, weighed, ground, and sieved through a 60-mesh screen. Plant total nitrogen was determined by H₂SO₄-H₂O₂ digestion, followed by measurement using an automatic intermittent chemical analyzer (DeChem-Tech, Hamburg, Germany).

2.3.3. Rainwater Sampling

Rainwater samples were collected several times by using a 50 mL disposable plastic centrifuge tube (Thermo Scientific, Waltham, MA, USA), and after the samples were collected, they were placed in a refrigerator at less than 4 °C for cold storage (storage time not exceeding 24 h), or subjected to concentrated sulfuric acid acidification to pH ≤ 2 to be stored for a longer period of time. The total nitrogen of rainwater was determined by the alkaline potassium persulphate–copper–zinc–sulfate hydrazine reduction method using an automatic intermittent chemical analyzer. Rainfall data were obtained from a nearby national meteorological station (Danzhou 59845, China).

2.3.4. Plant Nitrogen Accumulation

For each period of rice, 1 m of direct seeded rice was taken from each replicated plot, with 4 replicates of each variety, washed and put into an oven at 105 °C for 1 h to kill the greening, then adjusted to 75 °C for drying, and dried to a constant weight, and then the samples were processed and determined with reference to the method outlined in Section 2.3.2, and the nitrogen concentration was determined in units of g kg⁻¹. The total amount of plant nitrogen accumulation is the sum of the multiplication of the biomass and nitrogen concentration of each part of the stem, leaf, and panicle. The equations for calculating the contributions of the stem and leaf to grain nitrogen accumulation are as follows:

$$\begin{gathered} \mathrm{T}\mathrm{h}\mathrm{e} \mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{n}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n} \mathrm{a}\mathrm{c}\mathrm{c}\mathrm{u}\mathrm{m}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=\mathrm{A}\mathrm{b}\mathrm{o}\mathrm{v}\mathrm{e}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d} \mathrm{b}\mathrm{i}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s} \mathrm{o}\mathrm{f} \mathrm{e}\mathrm{a}\mathrm{c}\mathrm{h} \mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t} \mathrm{p}\mathrm{a}\mathrm{r}\mathrm{t}\times \\ \mathrm{n}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{o}\mathrm{f} \mathrm{e}\mathrm{a}\mathrm{c}\mathrm{h} \mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t} \mathrm{p}\mathrm{a}\mathrm{r}\mathrm{t} \end{gathered}$$

(1)

$$\begin{gathered} \mathrm{N}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{b}\mathrm{u}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{s}\mathrm{t}\mathrm{e}\mathrm{m} \mathrm{a}\mathrm{n}\mathrm{d} \mathrm{l}\mathrm{e}\mathrm{a}\mathrm{f} \mathrm{t}\mathrm{o} \mathrm{g}\mathrm{r}\mathrm{a}\mathrm{i}\mathrm{n}\left(\mathrm{N}\mathrm{C}\mathrm{A}\right)=\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n} \mathrm{a}\mathrm{c}\mathrm{c}\mathrm{u}\mathrm{m}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t} \mathrm{a}\mathrm{t} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{P}\mathrm{M} \mathrm{s}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e}- \\ \mathrm{n}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n} \mathrm{a}\mathrm{c}\mathrm{c}\mathrm{u}\mathrm{m}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t} \mathrm{a}\mathrm{t} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{H}\mathrm{D} \mathrm{s}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e} \end{gathered}$$

(2)

2.3.5. Nitrogen Balance

The nitrogen input sources included chemical nitrogen fertilizer, irrigation water nitrogen, wet deposition nitrogen, crayfish feed nitrogen, seed nitrogen, juvenile crayfish nitrogen, and adult crayfish nitrogen. Specific measurements and reference methods are shown in Table 3.

Nitrogen surplus and nitrogen use efficiency were calculated based on the following formulas [28,29]:

$$N_{inp}=N_{fer}+N_{fee}+N_{irr}+N_{wet}+N_{see}+N_{juv}$$

(3)

$$\begin{gathered} N_{out}=(\mathrm{A}\mathrm{b}\mathrm{o}\mathrm{v}\mathrm{e}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d} \mathrm{b}\mathrm{i}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s}\times \mathrm{n}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{o}\mathrm{f} \mathrm{a}\mathrm{b}\mathrm{o}\mathrm{v}\mathrm{e}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d} \mathrm{b}\mathrm{i}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s})+ \\ (\mathrm{C}\mathrm{r}\mathrm{a}\mathrm{y}\mathrm{f}\mathrm{i}\mathrm{s}\mathrm{h} \mathrm{p}\mathrm{r}\mathrm{o}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\times \mathrm{n}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{o}\mathrm{n} \mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{o}\mathrm{f} \mathrm{c}\mathrm{r}\mathrm{a}\mathrm{y}\mathrm{f}\mathrm{i}\mathrm{s}\mathrm{h}) \end{gathered}$$

(4)

$$N_{sur}=N_{inp}-N_{out}$$

(5)

$$NUE={\displaystyle \frac{N_{out}}{N_{inp}}}\times 100 \%$$

(6)

In these formulas, $N_{sur}$ and $NUE$ refer to nitrogen surplus and nitrogen utilization efficiency, respectively. $N_{fer}$, $N_{fee}$, $N_{irr}$, $N_{wet}$, $N_{see}$, and $N_{juv}$ refer to nitrogen inputs from chemical fertilizers, crayfish feed, irrigation water, wet deposition, rice seed, and juvenile crayfish, respectively. $N_{inp}$ and${ N}_{out}$ refer to the total amount of nitrogen that is inputted and outputted through different nitrogen pathways.

Table 3. Determination of nitrogen input sources and reference methods.

N Input	Calculation Method	References
Chemical nitrogen fertilizer	120 N kg ha−1 (RM), 80 N kg ha−1 (RC)
Irrigation water nitrogen	The determination method was consistent with the method for determining total nitrogen in rainwater samples
Wet deposition nitrogen	Section 2.3.3
Crayfish feed	Feed total nitrogen was determined by the same method as for the analysis of rice plant samples, with a feed nitrogen content of 46.6 g kg−1, calculated at 5.8% moisture content
Seed nitrogen	Seed nitrogen carryover was calculated as the multiplication of seed dosage and seed nitrogen content, and the nitrogen content of rice seeds was estimated at 1.3% of seed mass	[30]
Juvenile crayfish	Juvenile crayfish were estimated at 6.0 N kg ha−1	[31]
Adult crayfish	Adult crayfish were calculated at 56.2 g kg−1 nitrogen content and 77.9% moisture content	[31]

Note: RM indicates rice monoculture system; RC indicates rice–crayfish coculture system.

Table 3. Determination of nitrogen input sources and reference methods.

N Input	Calculation Method	References
Chemical nitrogen fertilizer	120 N kg ha−1 (RM), 80 N kg ha−1 (RC)
Irrigation water nitrogen	The determination method was consistent with the method for determining total nitrogen in rainwater samples
Wet deposition nitrogen	Section 2.3.3
Crayfish feed	Feed total nitrogen was determined by the same method as for the analysis of rice plant samples, with a feed nitrogen content of 46.6 g kg−1, calculated at 5.8% moisture content
Seed nitrogen	Seed nitrogen carryover was calculated as the multiplication of seed dosage and seed nitrogen content, and the nitrogen content of rice seeds was estimated at 1.3% of seed mass	[30]
Juvenile crayfish	Juvenile crayfish were estimated at 6.0 N kg ha−1	[31]
Adult crayfish	Adult crayfish were calculated at 56.2 g kg−1 nitrogen content and 77.9% moisture content	[31]

Note: RM indicates rice monoculture system; RC indicates rice–crayfish coculture system.

2.3.6. Statistical Analysis

The data were subjected to statistical processing using Excel (Microsoft Excel 2019). The final graphical representation of the data was produced using Origin 2022. To determine whether the variances between treatment groups were homogeneous, we performed a homogeneity of variance test prior to conducting the Analysis of Variance (ANOVA). Specifically, Levene’s test was used to assess variance homogeneity across groups. If the homogeneity of variance assumption was violated (p < 0.05), a Welch ANOVA was applied instead to account for unequal variances. Post hoc comparisons were performed using the LSD test for equal variances, or the Games–Howell test when variances were unequal.

3. Results and Discussion

3.1. Systemic Nitrogen Balance

The nitrogen balance of the two systems in 2021 and 2022 is shown in

Table 4. Nitrogen balance analysis of the rice–crayfish coculture system and rice monoculture system in 2021.

Cropping System	Rice Monoculture		Rice–Crayfish
	YXYLS	SXHN	YXYLS	SXHN
Inputs (kg ha−1)
Chemical nitrogen fertilizer	120.00	120.00	80.00	80.00
Irrigation water nitrogen	31.31	31.31	34.72	34.72
Wet deposition nitrogen	16.95	16.95	16.95	16.95
Crayfish feed	-	-	69.22	69.22
Seed nitrogen	0.47	0.62	0.47	0.62
Juvenile crayfish	-	-	6.00	6.00
Total input	168.73	168.88	207.36	207.51
Mean total input	168.81 ± 0.11 B		207.44 ± 0.11 A
Outputs (kg ha−1)
Plant removal	148.50 ± 15.86 a	132.79 ± 4.68 a	160.98 ± 23.97 a	144.08 ± 22.17 a
Crayfish removal			18.63	18.63
Total output	148.50 ± 15.86 bc	132.79 ± 4.68 c	179.61 ± 23.97 a	162.71 ± 22.17 ab
Mean total output	140.65 ± 11.11 B		171.16 ± 11.95 A
Nitrogen balance
Nitrogen surplus (kg ha−1)	20.23 ± 15.86 a	36.09 ± 4.68 a	27.75 ± 23.97 a	44.80 ± 22.17 a
Mean nitrogen surplus (kg ha−1)	28.16 ± 11.22 A		36.28 ± 12.06 A
NUE (%)	88.01 ± 9.40 a	78.63 ± 2.77 a	86.62 ± 11.56 a	78.41 ± 10.68 a
Mean NUE (%)	83.32 ± 6.64 A		82.52 ± 5.81 A

Note: The presence of different letters in the same row indicates that the difference between the two groups reaches a statistically significant level at the 0.05 level of the LSD test. “±” indicates standard deviation.

,

Table 5. Nitrogen balance analysis of the rice–crayfish coculture system and rice monoculture system in 2022.

Cropping System	Rice Monoculture		Rice–Crayfish
	YXYLS	SXHN	YXYLS	SXHN
Inputs (kg ha−1)
Chemical nitrogen fertilizer	120.00	120.00	80.00	80.00
Irrigation water nitrogen	24.45	24.45	21.50	21.50
Wet deposition nitrogen	17.54	17.54	17.54	17.54
Crayfish feed	-	-	69.22	69.22
Seed nitrogen	0.47	0.62	0.47	0.62
Juvenile crayfish	-	-	6.00	6.00
Total input	162.46	162.61	194.73	194.88
Mean total input	162.54 ± 0.11 B		194.81 ± 0.11 A
Outputs (kg ha−1)
Plant removal	133.59 ± 7.18 a	114.10 ± 26.79 a	133.49 ± 16.10 a	108.87 ± 18.04 a
Crayfish removal			21.69	21.69
Total output	133.59 ± 7.18 ab	114.10 ± 26.79 b	155.18 ± 16.10 a	130.56 ± 18.04 ab
Mean total output	123.85 ± 13.78 A		142.87 ± 17.41 A
Nitrogen balance
Nitrogen surplus (kg ha−1)	28.87 ± 7.18 b	48.51 ± 26.79 ab	39.55 ± 16.10 ab	64.32 ± 18.04 a
Mean nitrogen surplus (kg ha−1)	38.69 ± 13.89 A		51.94 ± 17.52 A
NUE (%)	82.23 ± 4.42 a	70.17 ± 16.47 a	79.69 ± 8.27 a	67.00 ± 9.26 a
Mean NUE (%)	76.20 ± 8.53 A		73.35 ± 8.98 A

Note: The presence of different letters in the same row indicates that the difference between the two groups reaches a statistically significant level at the 0.05 level of the LSD test. “±” indicates standard deviation.

and

Table 6. Interannual comparisons and analysis of variance (ANOVA) of nitrogen balance analyses in two systems.

Item	Rice Monoculture			Rice–Crayfish
	2021	2022	ANOVA	2021	2022	ANOVA
Inputs (kg ha−1)
Mean total input	168.81 ± 0.11	162.54 ± 0.11	***	207.44 ± 0.11	194.81 ± 0.11	***
Outputs (kg ha−1)
Mean total output	140.65 ± 11.11	123.85 ±13.78	Ns	171.16 ± 11.95	142.87 ± 17.41	ns
Nitrogen balance
Mean nitrogen surplus	28.16 ± 11.22	38.69 ± 13.89	Ns	36.28 ± 12.06	51.94 ± 17.52	ns
Mean NUE (%)	83.32 ± 6.64	76.20 ± 8.53	Ns	82.52 ± 5.81	73.35 ± 8.98	ns

Note: According to the LSD test, “***” indicates a significant difference at the 0.001 level, and “ns” indicates no significant difference. “±” indicates standard deviation.

. This study systematically compared the nitrogen balance between rice–crayfish coculture (RC) and rice monoculture (RM) systems. The nitrogen inputs included chemical fertilizer nitrogen, irrigation water nitrogen, wet deposition nitrogen, crayfish feed, seed nitrogen, and juvenile crayfish. The mean total nitrogen input of the RC system was significantly higher than that of the RM system in both years, specifically 22.89% (2021) and 19.85% (2022) higher. Fertilizer reduction techniques reduced the proportion of chemical fertilizer nitrogen in the total nitrogen input to the RC system. However, this reduction was not as significant as the nitrogen input from feeding during crayfish farming, leading to a higher mean total nitrogen input in the RC system. The crayfish aquaculture aspect contributed significantly to the overall nitrogen balance in the RC system due to the nitrogen content from crayfish feed and juvenile crayfish. The introduction of crayfish farming presents a unique nitrogen cycling challenge since uneaten feed and crayfish excreta add to the total nitrogen load in the system, potentially increasing nitrogen surplus and the risk of environmental pollution [32]. The higher nitrogen removal through crayfish harvesting only partially offsets the additional nitrogen input, creating a complex nitrogen cycling dynamic between the rice and crayfish components.

The mean total nitrogen output, including plant removal and crayfish removal, was also higher in the RC system than in the RM system, with the difference being significant in 2021 (18.85% higher) but not in 2022. Despite these differences, the nitrogen surplus and nitrogen utilization efficiency (NUE) exhibited no significant difference between the two systems in both years, suggesting that the system can sustain nitrogen input while maintaining productivity. However, the additional nitrogen input through crayfish feed may present challenges if not managed properly, as nitrogen losses through volatilization or leaching could occur [33]. The careful management of feed input and integration of nutrient cycling in crayfish farming is crucial for optimizing nitrogen efficiency in the RC system.

Combining data from 2021 and 2022, the reduction in irrigation water nitrogen led to a lower total nitrogen input by 6.09% in 2022 compared to 2021 for the RC system. The total nitrogen output for both years was influenced by the total nitrogen uptake of the crop, resulting in lower total nitrogen outputs for both the RC and RM systems in 2022 than in 2021, with average reductions of 15.50% and 14.00%, respectively. The elevated total nitrogen output of RC in 2021 resulted in a diminished systematic nitrogen surplus and elevated nitrogen use efficiency (NUE), leading to a 30.14% lower average nitrogen surplus and a 12.50% higher average NUE in 2021 compared to 2022. Additionally, there were significant differences in the nitrogen requirements of distinct rice varieties [34]. Consequently, the differential nitrogen uptake of rice varieties influenced the total nitrogen output, with YXYLS exhibiting significantly higher total nitrogen output than SXHN. This resulted in a significantly higher nitrogen surplus in SXHN compared to YXYLS, while the NUE of YXYLS was significantly higher than that of SXHN.

The calculation of the nitrogen balance represents a potentially useful method for the prediction of environmental risk [35,36]. The estimation of the systemic nitrogen balance is facilitated by the use of nitrogen surplus. The reduction in systemic nitrogen surplus is beneficial for minimizing environmental pollution whilst maintaining soil fertility [37]. In this study, the introduction of crayfish aquaculture increased the total nitrogen input to the RC system, while the application of nitrogen fertilizer reduction techniques reduced the total nitrogen input. Despite this, the nitrogen surplus of the RC system was slightly higher than that of the RM system. The nitrogen surplus for the RC system ranged from 28.16 kg ha⁻¹ to 51.93 kg ha⁻¹, comparable to the rice season in a rice–vegetable rotation in Hainan Province (43 kg ha⁻¹) as reported by Zhao et al. [38]. This value meets the international threshold of 80 kg ha⁻¹ recommended by the European Nitrogen Panel for each crop season [39]. Furthermore, in both the RC and RM systems, microbial communities play a critical role in mediating nitrogen cycling. In particular, microbial stimulation within the RC system may enhance organic matter decomposition and nutrient mineralization, which could explain the relatively higher nitrogen inputs and outputs observed in this system. The presence of organic inputs from crayfish feed and farming residues likely promotes microbial activity, particularly nitrogen-fixing bacteria and denitrifiers, which contribute to improved nitrogen cycling and soil fertility. Additionally, soil microorganisms facilitate the conversion of organic nitrogen into plant-available forms, which supports nitrogen uptake by rice and crayfish alike. However, in the RM system, lower organic matter input may suppress microbial activity, leading to slower nutrient cycling and less efficient nitrogen utilization [40,41].

3.2. Nitrogen Concentration and Its Accumulation in Rice

The transfer of photosynthetic assimilates is of great significance for crop yield formation [42]. During the rice growing season, nitrogen is initially stored in the stem and leaves and then transferred to the grain during the grain filling stage. The nitrogen accumulation of stem and leaf reached a maximum at the HD stage, with a subsequent reduction of 13.65–36.35% at the PM stage (Figure 1). The nitrogen contribution amount of stem and leaf to grain (NCA) in RM was 15.18 kg ha⁻¹ to 44.04 kg ha⁻¹, while in RC it was 18.61 kg ha⁻¹ to 47.75 kg ha⁻¹. There was no significant difference in the average NCA between RC and RM in both years (

Table 7. Comparison of nitrogen contribution amounts of stem and leaf to grain (NCA) between rice–crayfish coculture and rice monoculture systems in 2021 and 2022.

Year	Group	Rice Varieties	Nitrogen Accumulation Amount of Stem and Leaf at the HD Stage (kg ha−1)	Nitrogen Accumulation Amount of Stem and Leaf at the PM Stage (kg ha−1)	NCA (kg ha−1)
2021	RM	YXYLS	121.17 ± 20.76 a	77.13 ± 9.40 a	44.04 ± 18.00 ab
	RM	SXHN	116.25 ± 9.43 a	77.34 ± 5.79 a	38.91 ± 9.65 ab
	Mean		118.71 ± 15.16 A	77.23 ± 7.23 B	41.48 ± 13.65 A
	RC	YXYLS	135.99 ± 22.20 a	88.23 ± 10.73 a	47.76 ± 16.42 a
	RC	SXHN	111.33 ± 14.20 a	86.03 ± 12.10 a	25.30 ± 7.63 b
	Mean		123.66 ± 21.71 A	87.13 ± 10.65 A	36.53 ±16.87 A
2022	RM	YXYLS	81.24 ± 8.38 a	64.10 ± 3.30 a	17.14 ± 8.59 a
	RM	SXHN	74.48 ± 6.91 a	59.30 ± 13.14 a	15.18 ± 11.04 a
	Mean		77.86 ± 8.00 B	61.70 ± 8.34 C	16.16 ± 8.86 B
	RC	YXYLS	81.47 ± 5.64 a	61.75 ± 1.80 a	19.72 ± 7.31 a
	RC	SXHN	82.59 ± 3.54 a	63.98 ± 12.05 a	18.61 ± 15.41 a
	Mean		82.03 ± 4.25 B	62.86 ± 7.80 C	19.17 ± 10.80 B

Note: The presence of different letters in the same column for the same variety indicates that the difference between the two groups reaches a statistically significant level at the 0.05 level of the LSD test. “±” indicates standard deviation.

).

Although nitrogen accumulation remained stable in RC under nitrogen reduction conditions, the co-existence of crayfish in the system adds a layer of complexity. Crayfish feed and wastes act as an organic nitrogen source that may be mineralized over time, leading to slow-release nitrogen for plant uptake during critical growth stages [43]. This explains the non-significant differences in nitrogen accumulation between the two systems, as the RC system’s organic inputs from crayfish farming may supplement nitrogen demands later in the rice growing season. The crayfish system thus plays a dual role by not only providing protein through crayfish harvest but also potentially contributing to soil nitrogen through organic matter decomposition, which can buffer the nitrogen deficiencies caused by fertilizer reduction. Moreover, this slow nitrogen release could prevent nitrogen peaks that might otherwise lead to nitrogen losses through volatilization or runoff [44].

There was no significant difference in nitrogen accumulation in different rice plant organs in two rice cultivars between RC and RM, except for the leaf nitrogen accumulation in 2022 (Figure 1). However, the nitrogen concentration in different rice plant organs varied between RC and RM in the early rice growth stages in both years. The leaf nitrogen concentration in RC was significantly higher than that in RM, while the stem nitrogen concentration in RC was significantly lower than that in RM, which were consistent at MT and PI, in both cultivars and both years (Figure 2). No consistent differences in nitrogen concentration in different rice plant organs between RC and RM were observed.

In comparison to RM, the nitrogen accumulation in RC remained unchanged under the nitrogen reduction condition. However, the distribution of nitrogen concentration in each organ of the plant differed in individual periods, indicating that the RC system was sufficiently nitrogenous for the normal growth of rice. The reason for the non-deficiency of nitrogen in the RC system was primarily due to the supply of organic fertilizer (crayfish farming wastes) supplementing the deficiency of chemical fertilizer. Hou et al. [45] demonstrated that the replacement of 50% of chemical fertilizer by organic fertilizer resulted in stable yields of rice, as well as an increase in soil nitrogen retention and a reduction in potential nitrogen losses. Moreno-García et al. [46] demonstrated that 170 kg NH₄-N ha⁻¹ of pig slurry can be employed as a complete replacement of chemical fertilizer to ensure the maximum rice yield is achieved. Conversely, the application of 120 kg NH₄-N ha⁻¹ of pig slurry necessitates the addition of nitrogen fertilizer to achieve the maximum yield. In this study, crayfish feed and juvenile crayfish brought 75.22 kg N ha⁻¹ into the RC system, and only 20.16 kg N ha⁻¹ was taken away by crayfish harvest across two years; the remaining nitrogen was used as organic fertilizer for rice plants. Therefore, the reduction in chemical fertilizer in the RC system could be compensated by the extra 53.06 kg N ha⁻¹ from crayfish feeding (Table 4 and Table 5).

Microbial communities also influence nitrogen accumulation in rice by affecting nitrogen availability throughout the plant growth stages. The organic inputs from crayfish farming in the RC system stimulate microbial processes that help in breaking down complex organic compounds into ammonium and nitrate forms, which are readily taken up by the plants. This dynamic interaction between microbial activity and nitrogen availability may explain the stable nitrogen accumulation observed in the RC system despite reductions in chemical fertilizer use. In contrast, the RM system, with fewer organic inputs, may experience reduced microbial stimulation, limiting the efficient breakdown of nitrogenous compounds and consequently affecting nitrogen uptake and accumulation in rice [47].

In comparison to RM, the nitrogen accumulation in RC remained unchanged under the nitrogen reduction condition. However, the distribution of nitrogen concentration in each organ of the plant differed in individual periods, indicating that RC system was sufficiently nitrogenous for the normal growth of rice. The reason for the non-deficiency of nitrogen in RC system was primarily due to the supply of organic fertilizer (crayfish farming wastes) supplementing the deficiency of chemical fertilizer. Hou et al. [45] demonstrated that the replacement of 50% of the chemical fertilizer by organic fertilizer resulted in stable yields of rice, as well as an increase in soil nitrogen retention and a reduction in potential nitrogen losses. Moreno-García et al. [46] demonstrated that 170 kg NH₄-N ha⁻¹ of pig slurry can be employed as a complete replacement of chemical fertilizer to ensure the maximum rice yield is achieved. Conversely, the application of 120 kg NH₄-N ha⁻¹ of pig slurry necessitates the addition of nitrogen fertilizer to achieve the maximum yield. In this study, crayfish feed and juvenile crayfish brought 75.22 kg N ha⁻¹ into the RC system, and only 20.16 kg N ha⁻¹ was taken away by the crayfish harvest across two years; the remaining nitrogen was used as organic fertilizer for rice plants. Therefore, the reduction in chemical fertilizer in the RC system could be compensated by the extra 53.06 kg N ha⁻¹ from crayfish feeding (Table 4 and Table 5).

3.3. Dynamics of Soil Nutrition

To gain a deeper understanding of the dynamic changes in soil fertility in RC system, the changes in soil pH, soil organic matter, dissolved organic carbon, total nitrogen, ammonium nitrogen, and nitrate nitrogen at all fertility stages under the two systems were analyzed in 2022 (Figure 3).

The results showed that the soil pH was significantly higher in RC than that in RM at the mid-tillering (MT) and panicle initiation (PI) stages. There was no significant difference in soil organic matter between RC and RM, except for that at the heading stage (HD). Dissolved organic carbon in RC was significantly higher at the PI, HD, and PM stages than that in RM. Total nitrogen in RC was significantly higher at the PI and HD stages, and ammonium nitrogen and nitrate nitrogen in RC were significantly higher than that in RM at all the stages. Specifically, the total nitrogen of RC exhibited a significant increase of 35.14% to 62.44% at the PI stage and 23.22% to 28.39% at the HD stage. The ammonium nitrogen in RC was significantly higher, increasing from 11.81% to 20.04% at the MT stage, 28.49% to 40.55% at the PI stage, 37.06% to 57.42% at the HD stage, and 19.01% to 21.17% at the PM stage. The nitrate nitrogen of RC exhibited a significant increase of 11.00 to 15.47% at the PI stage, 12.55 to 18.42% at the HD stage, and 41.63 to 43.77% at the PM stage.

The co-existence of crayfish in the rice system directly impacts soil nutrient dynamics. The presence of organic matter from uneaten crayfish feed and metabolic waste contributes to the increase in soil organic matter and dissolved organic carbon. As crayfish excreta and feed residues break down, they release organic nitrogen into the soil, gradually increasing soil nitrogen levels, particularly in the form of ammonium and nitrate nitrogen. This contributes to the overall fertility and sustainability of the RC system [48]. The higher levels of ammonium and nitrate nitrogen in RC throughout the growth stages indicate that crayfish farming not only supports rice production but also enhances soil nitrogen availability, potentially reducing the need for synthetic fertilizers [49]. However, the long-term accumulation of organic nitrogen in the soil could increase the risk of nitrogen losses if not carefully managed, emphasizing the need for balanced nutrient cycling and feed management in RC systems [50].

After applying fertilizers, there is typically a temporary rise in soil pH, followed by a rapid increase in soil H⁺ concentration as crop inter-root ions are transported and used [51]. This contributes to a significant decrease in soil pH. As a result, long-term fertilizer inputs can cause severe soil acidification [52]. However, in the RC system, soil pH was significantly higher at the MT and PI stages compared to RM. This increase was largely attributed to lime application for pond clearing before the introduction of crayfish, which displaced H⁺, Fe²⁺, Al³⁺, Mn⁴⁺, and Cu²⁺ ions from the soil adsorption sites, thus raising soil pH [53,54]. Crayfish feed is organic matter that contains a significant quantity of carbon and nitrogen sources [55]. The daily feeding of crayfish resulted in feed residues or metabolic products becoming primary sources of increased soil organic matter. Consequently, crayfish feeding is closely associated with alterations in soil organic matter content. It was found that soil organic matter in the RC system exhibited a more pronounced increase at the PI stage, reaching significant levels at the HD stage, coinciding with the feeding period (Figure 3). Dissolved organic carbon represents the fraction of soil carbon within the soil organic carbon pool that is soluble and more readily mineralized and decomposed, making it a crucial component of soil organic carbon [56]. The total soil organic carbon content determines dissolved organic carbon quantity [57], which is influenced by both organic matter input and mineralization processes [58]. Thus, the elevation of dissolved organic carbon is relevant to crayfish farming. It was demonstrated that the observed increase in soil dissolved organic carbon resulted from the cumulative effect of crayfish feed, as evidenced by the continuous dissolved organic carbon increase throughout the growth period (Figure 3). Furthermore, the flooded paddy field environment promotes organic carbon sequestration, leading to an increase in dissolved organic carbon content over time in both the RM and RC systems [59].

It has been demonstrated that 80% to 97% of the nitrogen present in the surface layer of soil is found in organic matter [60]. Consequently, the dynamic trend of soil total nitrogen is more similar to that of soil organic matter (Figure 3). The majority of soil nitrogen is inert and unavailable for plant uptake. The biologically significant nitrogen is known as effective nitrogen [61]. The majority of soil effective nitrogen is present in the forms of NH₄⁺-N and NO₃⁻-N. Therefore, the quantity of soil ammonium nitrogen and nitrate nitrogen has a significant impact on the normal growth of crops. The performance of soil ammonium nitrogen in different fertility periods was comparable to that of nitrate nitrogen. Both soil ammonium nitrogen and nitrate nitrogen in RC were significantly higher than that in RM from the PI stage (Figure 3). This indicates that the soil effective nitrogen content of RC is higher than that of RC, and that RC is conducive to improving the effectiveness of soil nitrogen nutrients. This finding is consistent with the study of Si [59]. Moreover, Microbial communities are key to understanding the differences in soil nutrient dynamics between the RC and RM systems. The increased levels of total nitrogen, ammonium nitrogen, and nitrate nitrogen observed in the RC system may be due to the stimulation of microbial activity by the organic matter introduced through crayfish feed. Soil microorganisms, particularly nitrifying and denitrifying bacteria, play essential roles in converting organic nitrogen to inorganic forms that are available for plant uptake. The enhanced microbial activity in the RC system likely contributes to higher soil nutrient availability, particularly at the panicle initiation and heading stages. This is consistent with previous studies showing that organic matter inputs promote microbial diversity and activity, leading to improved nutrient cycling and availability in integrated farming systems [62]. Conversely, the RM system, with fewer organic inputs, likely experiences reduced microbial stimulation, which may limit soil nutrient turnover and availability, leading to lower nitrogen utilization efficiency [63].

The implementation of RC led to a notable enhancement in soil pH, soil organic matter, dissolved organic carbon, total nitrogen, ammonium nitrogen, and nitrate nitrogen at the specific stages of growth, particularly at the PI and HD stages, as compared to RM. This resulted in a notable enhancement of the overall fertility of the soil and the effectiveness of soil nitrogen nutrients. Furthermore, the cessation of nitrogen fertilizer application at the MT stage of rice led to the more efficient utilization of soil nitrogen nutrients at the PI and HD stages, thereby enabling more effective control of the soil total nitrogen content after rice maturity.

4. Limitations

Despite providing valuable insights into nitrogen dynamics within rice–crayfish coculture (RC) and rice monoculture (RM) systems, this study has several limitations that warrant consideration.

First, this study only spanned two years (2021 and 2022), which may not capture the full variability of environmental conditions such as temperature, rainfall, and soil properties over a longer period. These factors can significantly influence nitrogen cycling, crop yield, and soil fertility, limiting the generalizability of the findings to different climatic conditions or geographic locations. Second, this study focused on key nitrogen inputs and outputs; other potentially influential factors, such as soil microbial activity, water management practices, and nutrient leaching, were not explicitly measured. These could provide a more comprehensive understanding of nutrient dynamics, particularly regarding nitrogen losses through denitrification or leaching, which could impact environmental assessments. Third, this study relied on the assumption that the nitrogen contributions from crayfish farming waste remain consistent across different growth stages. However, nutrient release from organic waste is a dynamic process that may vary based on feeding practices, crayfish health, and environmental factors, introducing potential variability into the results. Additionally, the comparison between the RC and RM systems was based on specific rice varieties (YXYLS and SXHN), which may not represent the performance of other rice varieties commonly grown in different regions. Therefore, the extrapolation of these findings to other rice varieties or farming systems should be performed with caution. Finally, the experimental setup did not fully address the long-term sustainability of nitrogen reduction techniques, especially regarding potential cumulative effects on soil health and crop yield over multiple cropping seasons. These limitations highlight the need for further research that incorporates long-term monitoring, broader environmental assessments, and tests with diverse rice varieties under different agroecological conditions.

5. Conclusions

Excessive nitrogen inputs have become an urgent problem for the development of rice–crayfish coculture (RC) systems in pursuit of improved production. However, this study found that compared with the conventional rice monoculture (RM) system, the soil fertility of the RC system as a whole could still be significantly increased under a 33.3% reduction in nitrogen fertilizer, which ensured the accumulation of nitrogen and the production of rice grains. The measure of nitrogen fertilizer reduction also effectively controlled the soil total nitrogen content at the PM stage. In addition, the measure of nitrogen fertilizer reduction effectively reduced the nitrogen input of the RC system, resulting in the nitrogen surplus and NUE of the RC system being comparable to that of RM, which fell within the environmental safety range.

In conclusion, the application of nitrogen reduction fertilization technology, which is guaranteed to provide adequate rice nitrogen nutrition, effectively stimulates systematic nitrogen cycling, reduces the amount of soil nitrogen residue, reduces the risk of excessive systematic nitrogen surplus and surface source pollution, and further protects the ecological environment. Consequently, this study concluded that nitrogen reduction fertilization technology is essential in an RC system, and a 33.3% reduction in nitrogen fertilizer in the tropics is the optimal choice of nitrogen reduction fertilization amount in an RC system. Nevertheless, the standard of nitrogen reduction fertilization may vary depending on the amount of fertilizer and feed nitrogen inputs, as well as other factors. Therefore, the actual standard of nitrogen reduction fertilization should be improved in light of practice.