Field investigation

This study was performed between from May 2017 and October 2019 at Xinsheng Aquaculture Professional Cooperative (121° 0′ 56″ N, 30° 58′ 17″ E) in Qingpu District, Shanghai, Eastern China. This region has a subtropical monsoon climate with a mean monthly air temperature of 17.6 ± 2.3 °C and mean monthly precipitation of 126.9 ± 24.6 mm.

Each RC paddy (667 m2) had a rice-growing area (80% of the total area), aquaculture area (10%) and ridge area (10%; Fig. 4A). In the aquaculture area, a 1.2 m deep ditch was dug to provide a more comfortable habitat for the crayfish and eels. The ridge had a height of 40 cm, and it was covered with a high-density polyethylene film to prevent the aquatic animals from escaping. Every May, rice (Oryza sativa L., Qing-Xiang-Ruan-Geng) seedlings were transplanted from a nursery into the paddies at a planting density of 20 × 20 cm (one seedling on each hill). Moreover, the juvenile crayfish weighing 1.5 ± 0.3 g were released into the paddies according to the standard of 45,000 juveniles per hectare, and the crayfish were allowed to self-propagate inside the rice paddies. A total of nine RC paddies were divided into three groups according to the rearing density of the eels: control group (C), low-density group (LD) and high-density group (HD) with rearing densities of 0, 6000 and 12,000 ind. ha−1, respectively. The LD and HD groups were supplemented with juvenile eels at a density of 2000 and 4000 ind. ha−1 in June 2018 and 2019. The average weights of juvenile eels in 2017, 2018 and 2019 were 21.4 ± 1.8, 24.1 ± 0.9 and 26.8 ± 1.1 g, respectively. All juvenile crayfish and eels were purchased from Shanghai Xiangsheng Aquaculture Cooperative. In the aquaculture area, floating plants, such as duckweed (Lemna minor L.) and foxtail (Myriophyllum spicatum L.), covered one-third of the water surface. The soil contained 20.6–23.7 g kg−1 of organic matter, 0.7–1.2 g kg−1 of total N and 0.31–0.37 g kg−1 of total P.

Figure 4

Photographs of the paddies in the field investigation (A) and plots in the mesocosm experiment (B).

Only basal fertilizer was used for rice cultivation, and it contained 587 kg ha−1 of urea (46.4% N), 625 kg ha−1 of superphosphate and 150 kg ha−1 of potassium chloride. Every day, 500 g of commercial fish diet (5.83% N) was applied, and no pesticides or herbicides were used in the paddies.

In late August, the mature crayfish and eels were collected using ground cages to measure the aquatic product yields. The immature crayfish and eels were returned to the paddy fields during the collection. After the rice was harvested, the rice grains were air-dried and weighed to estimate the rice yield. The N content of the rice grains and aquatic animals was determined using the semi-micro Kjeldahl method33. Before testing, rice grains, crayfish and eels were weighted, dried at 65 °C and ground. Then, all the samples were digested with concentrated sulphuric acid (H2SO4) and hydrogen peroxide.

Water samples were collected every month during the co-culture period. Three duplicate 500 mL water samples were collected from 0 to 10 cm below the surface in the aquaculture area; the three subsamples were combined to obtain one sample per paddy. In the laboratory, the total N content of the water was analysed using UV spectrophotometry after digestion by alkaline potassium persulfate oxidation.

Soil samples were collected after the rice-planting period. In each paddy, three samples were collected from a rice-planting area of 0.25 m × 0.25 m × 0.10 m. All the soil samples were air-dried, ground, passed through a 0.15 mm sieve and digested with K2SO4–CuSO4–Se solution. Then, the semi-micro Kjeldahl method was used to test the total N content of the soil.

The N2O flux rate was measured using the static chamber method34. The size of the chamber was 1.0 m × 1.0 m × 1.0 m. The N2O samples were collected every half month between 8:30 and 10:30 AM from June to October. In each paddy, four gas samples were collected using 40 mL vacuum tubes at 10 min intervals (0, 10, 20 and 30 min after chamber closure). All samples were analysed with gas chromatography (GC 2010; Shimadzu, Kyoto, Japan). The N2O flux rate was calculated using the following equation:

$$F = rho times h times left[ {{273}/left( {{273} + T} right)} right] times {text{d}}C{text{/d}}t$$


where F is the N2O flux rate (μg N m−2 h−1); ρ, density of N2O at the standard state (μg m−3); h, height of the chamber (m); T, average temperature in the chamber during gas collection and dC/dt, concentration variation rate of N2O.

The ammonia volatilization flux was measured with a continuous airflow enclosure method35. The NH3 flux was measured every half month from 09:00 to 11:00 AM during the rice-planting period. NH3 was absorbed using boric acid, and 0.01 M H2SO4 was used to titrate the solution to determine the rate of NH3 volatilization. The ammonia volatilization flux was calculated using the following equation:

$$F = 14 times V times C times A^{ – 1} times t^{ – 1}$$


where F denotes the ammonia volatilization flux (mg N m−2 h−1); V, volume of H2SO4 titrated (L); C, concentration of H2SO4 (mol L−1); A, area of the chamber base (m2) and t, continuous measurement time.

In this study, all the data were shown as mean ± standard error of the mean (SEM) values. One-way ANOVA and Tukey’s test (SPSS V.16.0) were used to compare the differences of the yields and total N content among the three groups and three investigated years.

Mesocosm experiment

Between May and October 2019, the mesocosm experiment was conducted at Shanghai Academy of Agricultural Sciences. Each mesocosm consisted of an experimental plot (1.2 m × 1.2 m × 0.6 m) covered with a high-density polyethylene film (Fig. 4B). In each experiment plot, 30 kg of soil from Xinsheng Aquaculture Professional Cooperative was used to construct a rice-planting platform and an aquaculture ditch (40 cm in depth). The platform area was about three-fourth of the cross-sectional area of the plot.

A total of six mesocosms were constructed: three experimental plots (RCE) and three control plots (RC). In each plot, the rice seedlings were planted in hills (one seedling per hill) within rows in May, with 20 cm between rows and 20 cm between hills in the same row for the experimental and control plots. The fertilizers used in each plot contained 84.5 g of urea (N content, 46.8%; 15N abundance, 10.15%), 90 g of superphosphate and 15 g of potassium chloride. The duckweed was planted in the aquaculture area, and it covered 30% of the aquaculture zone. Mudsnails (Cipangopaludina cathayensis, 500 g) were added to each plot. After a month, 12 crayfish were cultured in each simulated paddy, and two eels were reared in each experiment plot. The proportion of crayfish and eels was set according to that in the LD group of field investigation. The crayfish feed was supplied once every day, and the daily allowance was about 3% of the estimated crayfish weight in each mesocosm. The rice and aquatic products were harvested in October.

Rice, crayfish and eel samples were collected to measure the total N content and 15N abundance. The total N content of the soil and organism samples were measured using the semi-micro Kjeldahl method after digestion with concentrated H2SO4 and hydrogen peroxide. The 15N abundance was measured in all samples by using the MAT-271 isotope mass spectrometer (Finnigan MAT, California). The accumulation of N in rice, crayfish and eels from N fertilizer was calculated using the following equations:

$${text{Percentage of accumulated N from fertilizer NDFF}});( % ) = {text{A}}% ;{text{E of the organism sample/A}}% ;{text{E of the fertilizer sample}} times 100$$


$${text{Amount of accumulated N from fertilizer}} = {text{organism N accumulation amount}} times {text{NDFF}}$$


$${text{N use efficiency NUE}});(% ) = {text{amount of N accumulated by the organism accumulated from N fertilizer/total N content of the fertilizer}} times 100$$


where A% E is the difference between the 15N abundance of the samples or 15N-labelled fertilizers and natural abundance of 15N.

The independent-samples t-test was used to determine the differences in total N, N use efficiency and percentage of N derived from fertilizer between RCE and RC at 95% confidence level by using SPSS 16.0 (P value < 0.05 was considered statistically significant).

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