Dynamics of phosphorus fractions in surface soils of different flooding wetlands before and after flow-sediment regulation in the Yellow River Estuary, China

Journal Pre-proofs Research papers Dynamics of phosphorus fractions in surface soils of different flooding wetlands before and after flow-sediment regulation in the Yellow River Estuary, China Junhong Bai, Lu Yu, Xiaofei Ye, Zibo Yu, Dawei Wang, Yanan Guan, Baoshan Cui, Xinhui Liu PII: DOI: Reference:

S0022-1694(19)30991-6 https://doi.org/10.1016/j.jhydrol.2019.124256 HYDROL 124256

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Journal of Hydrology

Received Date: Revised Date: Accepted Date:

5 July 2019 30 September 2019 17 October 2019

Please cite this article as: Bai, J., Yu, L., Ye, X., Yu, Z., Wang, D., Guan, Y., Cui, B., Liu, X., Dynamics of phosphorus fractions in surface soils of different flooding wetlands before and after flow-sediment regulation in the Yellow River Estuary, China, Journal of Hydrology (2019), doi: https://doi.org/10.1016/j.jhydrol.2019.124256

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Dynamics of phosphorus fractions in surface soils of different flooding wetlands before and after flow-sediment regulation in the Yellow River Estuary, China Junhong Bai, Lu Yu, Xiaofei Ye, Zibo Yu, Dawei Wang, Yanan Guan, Baoshan Cui, Xinhui Liu State Key Laboratory of Water Environment Simulation, School of Environment, Beijing Normal University, Beijing 100875, PR China Abstract: To investigate the dynamics of phosphorus fractions and their influencing factors in the surface soils of estuarine wetlands experiencing different hydrological conditions before and after flow-sediment regulation, soil samples were collected in wetlands (tidal flooding wetlands (TFW), freshwater restoration wetlands (FRW) and freshwater flooding wetlands (FFW) of the Yellow River Estuary in each month from April to October of 2012. Our results showed that the average contents of organic phosphorus (OP) occurred in the following order: FRW soils (60.05 mg/kg)>TFW soils (38.72 mg/kg)>FFW soils (27.56 mg/kg), and accounted for less than 12% of total phosphorus (TP). In contrast to the pattern for OP, FRW soils contained lower inorganic phosphorus (IP) levels than TFW and FFW soils from April to August (p<0.05). After the flow-sediment regulation, the TP, OP, moderately labile OP (ML-OP) ferrous/aluminum-bound IP (Fe/Al-P), and occluded IP (Oc-P) in the three wetlands decreased. The soluble and loosely bound IP (S/L-P) contents in TFW decreased, while the S/L-P contents in FRW increased. The levels of phosphorus fractions were affected by water and salt conditions, soil texture, exchangeable mineral element contents, and nutrient status. The Fe/Al-P and Oc-P in the three wetland soils

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Corresponding author. Prof. Junhong Bai. Email: [email protected] 1

were released, and the increase in S/L-P in FRW after the flow-sediment regulation might increase the risk of eutrophication in the coastal waters. The findings of this study could contribute to providing basic data regarding phosphorus fractions in different flooding estuarine wetlands of the Yellow River Estuary and guiding flow-sediment regulations and freshwater restoration to enhance the ecological functions of estuarine wetlands. Keywords: Phosphorus fractions; Influencing factors; Flow-sediment regulation; Freshwater input; Estuarine wetlands

1 Introduction Phosphorus levels in wetland soils are excellent indicators of phosphorus migration, physicochemical (precipitation/dissolution and adsorption/desorption) and biological/biochemical (mineralization/immobilization) processes (Bünemann, 2015; Ye et al., 2019). The spatial and temporal distribution characteristics of phosphorus can indirectly reflect the productivity, eutrophication risk, and functions of wetland ecosystems (Gao et al., 2016). Therefore, the spatial and temporal dynamics of phosphorus in wetland soils have received considerable attention over the past years. The total phosphorus (TP) in soils includes various fractions with different availabilities that can transform into each other (Gao et al., 2019). According to Ivanoff et al. (1998), organic phosphorus (OP) fractions include labile organic phosphorus (L-OP), moderately labile organic phosphorus (ML-OP), and non-labile organic phosphorus (NL-OP); this categorization has been widely applied to analyze OP fractions in many studies (Ngo et al., 2013; DeBruler et al., 2019). A modified sequential extraction method as described in Bai (2017) was used in calcareous soils, which divides soil inorganic phosphorus (IP) into four fractions, including soluble and loosely bound phosphorus (S/L-P), iron/aluminum-bound phosphorus 2

(Fe/Al-P), occluded phosphorus (Oc-P), and calcium-bound phosphorus (Ca-P). The determination of phosphorus fractions is important for investigating the availability of phosphorus and its migration and transformation. However, limited information is available on the dynamics of soil phosphorus fractions in estuarine wetlands experiencing different flooding conditions, especially with the effects of hydraulic engineering. The Yellow River Estuary of China is formed by the deposition of the Yellow River sediment as it enters the sea, which has been severely affected by sediment deposition and land-ocean interactions (Gao et al., 2016). Environmental degradation caused by natural threats and human activities (such as flow cut-off of the Yellow River and droughts of wetlands) is a major issue in the Yellow River Estuary of China. Some degradation control measures based on scientific knowledge must be used to reverse the wetland degradation in the Yellow River Estuary (Wang et al., 2012). To control the degradation in the Yellow River Estuarine wetland ecosystems and maintain the reservoir storage capacity, flow-sediment regulation of the Xiaolangdi Reservoir, which is one of the largest dams in the Yellow River Catchment, has been implemented annually since 2002, usually lasting for ~ 20 days from mid-June to early July (Gao et al., 2016). The flow-sediment regulation is an active driver of soil physicochemical conditions including soil water and salinity, soil texture, and soil mineral element and nutrient contents, which in turn regulate soil phosphorus fraction dynamics by affecting the processes of input and output, sorption and desorption, plant uptake and microbial transformation (Li et al., 2017b). The rapid discharge of water and sediment from the reservoir aggravated the imbalance of phosphorus in estuarine wetlands, which most likely exerted significant impacts on the increased risks of eutrophication in coastal ecosystems (Zhang et al., 2013; Gao et al., 2016). Therefore, it is necessary to investigate 3

the spatial and seasonal distributions of soil phosphorus fractions in estuarine wetlands before and after the flow-sediment regulations to protect water quality and wetland ecosystem health. Owing to the intense changes in hydrological and salt conditions, estuarine wetlands experiencing different flooding conditions were formed in the study area, i.e., tidal flooding wetlands (TFW), freshwater restoration wetlands (FRW), and freshwater flooding wetlands (FFW) (Bai et al., 2017). Meanwhile, the implementation of the flow-sediment regulations further enhanced the complexity of soil environmental conditions in the wetlands of the Yellow River Estuary (Wang et al., 2017). Hydrological dynamics have significant effects on the environmental behaviors of phosphorus in wetland soils (Dupas et al., 2015). It is important for water quality protection and for the improvement of the productivity and function of the whole estuarine wetland ecosystem to identify the effects of the flow-sediment regulations on the phosphorus fractions in estuarine wetlands experiencing different flooding conditions. Moreover, it is necessary to identify the key environmental factors that affect the phosphorus fractions in estuarine wetland soils to provide a theoretical basis for guiding and managing the flow-sediment regulation schemes. The primary objectives of this study were (1) to investigate the spatial and temporal distributions of OP, IP and their fraction contents in the three estuarine wetland soils; (2) to identify the changes in phosphorus fractions before and after the flow-sediment regulation; and (3) to reveal the key environmental factors influencing the phosphorus fractions during the flow-sediment regulation, thus providing basic theoretical support for the design of the flow-sediment regulation scheme.

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2 Materials and methods 2.1 Site description In this study, three typical estuarine wetlands experiencing different flooding conditions (TFW, FRW, and FFW) in the Yellow River Estuary, where the reed (Phragmites australis) is the dominant species, were selected as the sampling sites (Fig. 1). TFW is located in the intertidal zone with the lowest terrain of the three sampling sites. Irregular semidiurnal tides with a reciprocating current that is almost parallel to the coast are important hydrological characteristics in TFW (Gao et al., 2016; Zhang et al., 2018). FFW is located near the south of the Yellow River, and FFW was affected by the Yellow River runoff so that the soil salinity was lower compared with the others (Bai et al., 2017). The hydrological conditions of FFW are significantly influenced by the flow-sediment regulations. FFW soils remain flooded from the beginning of the flow-sediment regulations to the end of summer (August). With the implementation of flow-sediment regulations, freshwater restoration was also implemented in the Yellow River Delta National Nature Reserve to control soil degradation and to improve productivity (Bai et al., 2017). Four reservoirs and one aqueduct were set up in the core area of the Yellow River Delta National Nature Reserve. Freshwater was delivered to the restored wetlands during the period of flow-sediment regulations. At the same time, a dike that is 9 m long, 3.4 m wide and 1.5 m high was built to avoid tidal erosion. Therefore, FRW are continuously flooded by the freshwater inputs, and they are also important wetlands to restore wetland productivity and improve ecosystem function in this region.

2.2 Sample collection and analysis The surface soil (0-10 cm) samples with three replicates were collected from each of the three wetlands in each month from April to October 2012. Because of difficulties in sampling, soil samples in 5

July and September were not taken in FRW. A total of 57 soil samples were collected in the study period. Meanwhile, a datalogger with temperature probes (CR1000, Campbell Scientific, USA) was used to record soil temperature; another three soil cores (100 cm3) in each wetland were collected in each sampling month for the determination of soil water content (WC) and bulk density (BD). All soil samples were put in polyethylene bags and brought to the laboratory immediately. One part of the fresh soil samples was air-dried, and the visible plant litter, coarse root materials, and stones were removed. Some air-dried soil samples were used for soil particle size analysis, and others were ground with a mortar and pestle until all particles passed through a 0.149 mm nylon sieve for the determination of soil chemical properties. The remaining fresh soil samples were immediately stored at 2-4 °C for the determination of soil microbial biomass carbon (MBC) and nitrogen (MBN) and phosphatase. The BD and WC contents were measured by drying at 105 °C for 24 h in an oven. Soil electrical conductivity (EC) and pH were measured in a suspension of 1:5 soil/water (5 g soil:25 ml water) using an electric conductivity meter (Mettler Toledo, USA) and pH meter (Hach Company, Loveland, CO, USA), respectively. Cl- and SO42- were determined by ion chromatography (Dionex DX600) in the supernates of the 1:5 soil/water (m/v) suspensions. Soil texture was measured on a Laser Size Analyzer (Microtrac S3500) and classified into clay (0.01 μm-0.002 mm), silt (0.002–0.02 mm), and sand (0.02–2 mm) (Bai et al. 2017). Soil organic matter (SOM) was determined using dichromate oxidation (Nelson and Sommers, 1982). Total nitrogen (TN) and total carbon (TC) were determined in a CHNOS Elemental Analyzer (Vario EL, German). Soil samples were digested in a HClO4-HNO3-HF mixture. The digested solution was analyzed for TP, aluminum (Al), calcium (Ca), and iron (Fe) by inductively coupled plasma 6

atomic absorption spectrometry (ICP-AAS). The exchangeable iron (Fe0) and exchangeable aluminum (Al0) contents were determined after extraction with acid ammonium oxalate (0.175 mol/L) at pH=3, and the exchangeable calcium (Ca0) content was determined after extraction with ammonium acetate (1.0 mol/L) at pH=8.5 (Makoto et al., 2012). Microbial biomass carbon and nitrogen (MBC and MBN) were measured by the chloroform-fumigation extraction method (Vance et al., 1987). The soil phosphatase activity (including acid and alkaline phosphatase (ACP and AKP, respectively)) was determined using the P-nitrobenzene colorimetric method (Dick et al., 2000). The soil properties of these soil samples are listed in Table 1. Phosphorus extracted from nonignited soils with 0.5 mol/L H2SO4 was considered to be IP, whereas the increment in H2SO4-extractable phosphorus after ignition (500 °C, 1 h) was assumed to be OP (Saunders and Williams, 1955). The OP fractions (i.e., L-OP, ML-OP, and NL-OP) were measured using the OP grading system proposed by Ivanoff et al. (1998) (Ivanoff et al., 1998). The IP fractions (i.e., S/L-P, Fe/Al-P, Oc-P, and Ca-P) were determined using modified sequential extractions as described in Bai (2017).

2.3 Statistical analysis One-way analysis of variance (ANOVA) followed by Duncan’s test were used to compare the differences in soil properties among the three wetlands and between different sampling months. Differences were considered to be significant when p<0.05. Pearson correlation analysis and redundancy analysis (RDA) were used to identify the relationships among various fractions of phosphorus and environmental variables in the three wetland soils. ANOVA and Pearson correlation analysis were conducted using the SPSS 20.0 software package, and the Origin 8.6 software package was used to create the figures. RDA was performed using the Canoco 4.5 7

software package. Structural equation modeling (SEM) was used to further identify key environmental drivers of the changes in soil phosphorus fraction levels during the flow-sediment regulations. It was conducted using the SPSS Amos 21.0 software package. The best-fit model was derived using the maximum likelihood based on model fit. Nonsignificant paths have been removed from the models to save degrees of freedom for model testing.

3 Results 3.1 Dynamics of OP and OP fractions in surface soils of three wetlands 3.1.1 OP levels The spatial and temporal changes in the OP contents in surface soils of the three wetlands experiencing different flooding conditions are shown in Fig. 2. The average contents of OP decreased in the order from FRW soils (60.05 mg/kg) > TFW soils (38.72 mg/kg) > FFW soils (27.56 mg/kg), and in all cases the OP accounted for less than 12% of TP (Fig. S1). The OP content in TFW was significantly higher than that in FFW in July (p<0.05); there were no significant differences in OP content among TFW and FFW wetland soils in other months (p>0.05). The OP levels in FRW soils were higher than those in TFW and FFW soils in June and August (p<0.05). In TFW and FFW soils, the OP contents decreased from April to August and then increased in September and October. However, in FFW soils, OP levels exhibited a slight fluctuation from April to June (p>0.05) and then decreased and reached the lowest level in October (p<0.05). Higher levels of OP in FRW and FFW were observed before the flow-sediment regulation (from April to June) than after the regulation (from July to October) (p<0.05). 3.1.2 OP fractions Fig. 3 shows the dynamics of the OP fractions in the surface soils of the three wetlands 8

experiencing different flooding conditions. L-OP contents exhibited a decline in FFW soils in the whole growing season, and L-OP contents were higher before the flow-sediment regulation (from April to June) than after the regulation (from July to October) (p<0.05). The L-OP contents in TFW soils showed a decreasing tendency was similar to that of FFW before the regulation and then increased after August, with higher L-OP levels than FFW soils in September (p<0.05). Comparatively, L-OP contents in FRW soils presented S-shape changes with time, with the highest levels in May and October and the lowest levels in August. Generally, L-OP levels were below 5 mg/kg in the three wetland soils, accounting for less than 9% of the OP (Fig. S2). They reached the lowest levels in summer (June to August). ML-OP contents varied from 0.56 to 30.13 mg/kg in the three wetlands from April to October, accounting for 2.58% to 60.06% of OP (Fig. S2) and significantly positively correlated with OP (p<0.01; Fig. S4). The ML-OP contents generally decreased in the three wetland soils in the studied period. Higher ML-OP levels were observed before regulation (April to June) than after regulation (July to October) in the three wetlands (p<0.05). The contents of ML-OP also decreased significantly from June to July (p<0.05) in TFW and FRW. Compared with FFW soils, TFW soils contained higher ML-OP contents in May to September. FRW soils showed higher ML-OP levels in May and June and lower ML-OP levels in August and October (p<0.05). The NL-OP contents ranged from 3.38 to 63.55 mg/kg in the three wetlands during the growing season, accounting for 38.78 to 97.42% of OP (Fig. S2). Significant positive correlations between NL-OP and OP were also observed in three wetland soils (p<0.01; Fig. S4). NL-OP contents showed a fluctuation in TFW and FFW soils in the growing season, with lower levels in August (p>0.05). Compared with TFW and FFW soils, FRW soils generally contained higher 9

NL-OP contents during the growing season, except in October. The NL-OP contents in FRW soils increased significantly in August (after the flow-sediment regulation) (p<0.05).

3.2 Dynamics of IP and IP fractions in surface soils of three wetlands 3.2.1 IP levels IP was the dominant phosphorous fraction in the three wetlands, with IP/TP ratios ranging from 91.80 to 96.18% in TFW soils, 93.09 to 98.95% in FFW soils, and 88.19 to 97.39% in FRW soils (Fig. S1). The dynamics of IP contents in surface soils of the three wetlands are shown in Fig. 4. IP contents in TFW and FFW soils generally showed a slight decreasing tendency during the whole growing season, reaching the lowest level in September and then slightly increasing in October. However, IP levels in FRW soils generally showed an “S” tendency and reached the lowest level in August and the highest level in October. FRW soils contained lower IP levels than TFW and FFW soils from April to August but exhibited higher levels in October compared with TFW and FFW soils (p>0.05). No significant differences in IP levels were observed between FFW and TFW soils (p>0.05), except for higher levels in FFW soils in April and May (p<0.05). 3.2.2 IP fractions The dynamics of IP fractions in surface soils from the three wetlands are illustrated in Fig. 5. Generally, S/L-P levels were low in wetland soils, accounting for less than 0.3% of total OP (Fig. S3). The S/L-P contents in TFW soils (0.89 to 1.68 mg/kg) showed an “increasing before decreasing” tendency from April to October, whereas they exhibited a “decreasing before increasing” tendency in FRW (0.86 to 1.53 mg/kg) and FFW (0.88 to 1.67 mg/kg) soils during the growing season. Therefore, before the regulation (from April to June), higher levels of S/L-P in TFW soils were observed compared with after the regulation (from July to October) (p<0.05). The S/L-P contents in FRW 10

soils increased significantly after the flow-sediment regulation (p<0.05). However, no significant differences in S/L-P contents were observed among FFW soils before and after regulation (p>0.05). Fe/Al-P contents accounted for 0.9 to 2.1% of IP in the three wetlands in the growing season, with an average value of 1.3% (Fig. S3). Generally, FFW soils contained higher Fe/Al-P contents (8.81 to 11.91 mg/kg) than TFW soils (6.15 to 8.68 mg/kg) and FRW soils (5.73~9.14 mg/kg), and no significant differences in Fe/Al-P contents were observed between FRW soils and TFW soils. The Fe/Al-P contents in FFW soils showed a small change in the growing season, with higher contents in April and lower contents in August and September. However, an increasing tendency was observed in TFW and FRW soils before the flow-sediment regulation (p>0.05), and then Fe/Al-P contents decreased significantly after the flow-sediment regulation (p<0.05). Oc-P contents accounted for 7.1 to 22.5% of the IP in the three wetlands in the growing season, with an average value of 14.2% (Fig. S3). Generally, Oc-P levels were higher in TFW soils (90.02 to 125.10 mg/kg) than in FFW (61.41 to 124.96 mg/kg) and FRW soils (46.57 to 93.59 mg/kg), and no significant differences were observed between FFW and FRW soils. The Oc-P contents in the TFW soils exhibited little change during the growing season, whereas the Oc-P contents in the FRW and FFW soils exhibited a slight decrease from April to August and then a slight increase until October. A significant decrease in Oc-P was observed in FRW soils from June to August (p<0.05). In addition, Oc-P in FFW decreased significantly after the flow-sediment regulation (from July to October; p<0.05). The Ca-P contents accounted for 47.8 to 81.9% of the IP in the three wetlands in the growing season, with an average value of 55.45% (Fig. S3), so IP was dominant in the Ca-P fraction in the three wetland soils. Generally, FRW soils contained higher Ca-P levels (355.29~453.03 mg/kg) than 11

TFW (326.05 to 363.99 mg/kg) and FFW (339.93~382.05 mg/kg) soils. The Ca-P contents showed a small fluctuation and reached their lowest levels in the TFW and FFW soils in the growing season in September, whereas in the FRW soils, Ca-P levels exhibited an increasing tendency, with higher Ca-P levels after the flow-sediment regulation than before the regulation (p<0.05).

3.3 Relationships between phosphorus fractions and soil properties in three wetlands RDA (Fig. 6) and correlation analysis (Table S1) identified the relationships between different phosphorus fractions and soil environmental factors. In TFW, the eigenvalues of the four ordination axes were 0.587, 0.197, 0.118, and 0.066, and the first two axes could explain 78.5% of the effects of soil environmental factors on phosphorus fractions (Fig. 6a). The first RDA axis was mainly positively correlated with soil salinity (EC and Cl-) and silt percentage and was mainly negatively correlated with soil nutrients (SOM and TN), clay percentage, MBN and Ca0. The second RDA axis was mainly positively correlated with soil TC and Fe and was mainly negatively correlated with soil BD, Cl-, SOM and Al. The soil TP and IP fractions existed in quadrants one and four, whereas the OP fractions presented in quadrants three and four, as shown in Fig. 6a. Among them, TP showed significantly positive correlations with EC and Cl- and negative correlations with TN and Ca0 (p<0.05; Table S1). OP was significantly positively correlated with BD and SOM (p<0.01; Table S1). There was a significant positive correlation between IP and silt, and IP showed significant negative correlations with SOM and Ca0 (p<0.05; Table S1). After the flow-sediment regulation, the sampling sites moved from quadrant four to quadrant two. Higher levels of TP, ML-OP, and S/L-P were observed before the flow-sediment regulation (from April to June) compared to after the regulation (from July to October) (p<0.05). The contents of ML-OP and Fe/Al-P also decreased significantly from June to July (p<0.05), accompanied by a decrease in 12

Cl- and sand percentage and an increase in clay percentage in environmental factors from June to July (p<0.05). Moreover, there was an increasing trend of soil WC, Ca, and Ca0 after the regulation. In FRW, the eigenvalues of the four ordination axes were 0.456, 0.397, 0.127, and 0.015, and the first two axes could explain 85.3% of the effects of soil environmental factors on phosphorus fractions (Fig. 6b). The first RDA axis was mainly positively correlated with soil pH, sand percentage, and temperature and was mainly negatively correlated with soil SOM, TC, silt percentage, and Al. The second RDA axis was mainly negatively correlated with soil salinity (EC, Cl- and SO42-), nutrients (SOM and TN), TC, and mineral elements (Ca0, Al0, Fe0). Soil TP and IP fractions existed in quadrants two and three, and Ca-P and S/L-P existed in quadrant one, whereas OP fractions presented in quadrant four (OP and NL-OP), quadrant two (L-OP) and quadrant three (ML-OP), as shown in Fig. 6b. Among them, TP showed significantly positive correlations with EC and Al and negative correlations with pH and temperature (p<0.05; Table S1). OP was significantly positively correlated with soil salinity (Cl- and SO42-), TN, and mineral elements (Ca0, Al0, and Fe0) and negatively correlated with soil WC and clay percentage (p<0.05; Table S1). IP showed significant positive correlations with soil WC and Al and negative correlations with soil pH and temperature (p<0.05; Table S1). All sampling sites in FRW could be clearly discriminated as before the regulation (from April to June) and after the regulation (August and October). After the regulation, the sampling sites moved from quadrants three and four to quadrants one and two. Higher levels of OP, ML-OP and Fe/Al-P and lower levels of S/L-P were observed before the regulation compared with after the regulation (from July to October) (p<0.05). The contents of ML-OP, Fe/Al-P and Oc-P also decreased significantly, and NL-OP, S/L-P and Ca-P contents 13

increased significantly from June to August (p<0.05). Meanwhile, there was an increasing trend of soil WC and a decreasing trend of soil SOM after regulation. This trend was accompanied by a decrease in soil salinity (Cl- and SO42-), Ca0 contents, and AKP and an increase in soil pH and sand percentage in environmental factors from June to August (p<0.05). In FFW, approximately 84.8% of the variance could be explained by the first two axes, and their eigenvalues were 0.719 and 0.129, respectively (Fig. 6c). The first RDA axis was mainly positively correlated with soil TN and mineral elements and was mainly negatively correlated with soil WC. The second RDA axis was mainly positively correlated with soil silt percentage and negatively correlated with soil TN, microbial biomass (MBC and MBN), and mineral elements. Soil phosphorus fractions were mainly present in quadrant four, as shown in Fig. 6c. Among them, TP showed significantly positive correlations with soil TN and mineral elements (Al and Ca0) and negative correlations with soil WC (p<0.01; Table S1). OP was significantly positively correlated with soil TC, TN, MBN, and mineral elements (Ca, Al, Fe, and Ca0) (p<0.05) and negatively correlated with soil WC (p<0.01; Table S1). For IP, there was a significant positive correlation with soil Al and a significant negative correlation with soil ACP (p<0.05; Table S1). After the regulation, the sampling sites moved from quadrants one and four to quadrants two and three. Before the regulation (from April to June), higher levels of TP, Oc-OP, OP, L-OP and ML-OP were observed compared with after the regulation (from July to October) (p<0.05). This shift was accompanied by an increase in soil WC, clay percentage and AKP in environmental factors from June to August (p<0.05). Moreover, there was a decreasing trend of soil TC and TN after the regulation. SEM was used to further identify key environmental drivers of soil phosphorus fraction 14

levels in the three wetlands (Fig. 7). The model was significant (chi-square p-value>0.05) and had an acceptable goodness-of-fit index (GFI=0.807) and root mean square error of approximation (RMSEA=0.032) after the nonsignificant paths were removed. The SEM revealed that soil salinity, nutrient status, and soil texture had direct effects on soil OP contents, while soil exchangeable mineral element contents and nutrient status had direct effects on soil IP contents. Among these factors, nutrient status had the greatest influence on OP and IP, with standardized coefficients of 1 and 0.9, respectively, indicating that OP and IP increased with increasing soil nutrients. Soil WC could indirectly affect soil phosphorus fraction contents by affecting soil salinity and exchangeable mineral elements. The change in WC was accompanied by a change in soil texture because of the influence of flow-sediment regulation (p<0.05). Moreover, there were significant correlations between soil salinity, nutrient condition, and texture (p<0.05). Soil exchangeable mineral element contents were significantly correlated with soil nutrients (p<0.05). OP and IP fractions can transform into each other. This model could explain 61% and 46% of the variance in OP and IP, respectively.

4 Discussion 4.1 Changes in phosphorus levels in three wetlands For the three wetlands, TP existed predominantly in the IP fraction (especially Ca-P) in three wetlands, and IP accounted for 88.19 to 98.95% of TP, consistent with the results of Qu et al. (2018). Those results because the Yellow River Estuary is dominated by calcareous soils (Ye et al., 2014). OP accounted for less than 12% of TP. Lu et al. (2013) also observed comparable phosphorus levels in sediments in the northern part of China. L-OP levels accounted for the least proportion of OP and ML-OP contents, ranging from 0.56 to 30.13 mg/kg, which was consistent with the results of 15

Zhu et al. (2017) and Lu et al. (2013). NL-OP accounted for 38.78 to 97.42% of OP. This is consistent with the results of Zhu (2017), who reported that soil OP was predominantly in the NL-OP fraction in the Yeyahu wetland of China. Therefore, OP levels showed the same dynamics as NL-OP. Moreover, the contents of OP and IP showed opposite dynamics because OP and IP could transform into each other through mineralization and immobilization (Bünemann, 2015). FRW soils contained higher OP levels and lower IP levels than TFW and FFW soils from April to August. Meanwhile, FRW soils contained higher SOM levels than TFW and FFW soils. The plants grew best in FRW, leading to the most absorption of IP by plants and causing phosphorus to exist in the form of NL-OP in SOM (Schachtman et al., 1998).

4.2 Effects of environmental factors on phosphorus fractions before and after regulation The phosphorus fractions in the different flooding wetlands showed inconsistent dynamics before and after flow-sediment regulation because there were differences in environmental factors among the three wetlands. Salt stress strongly limits the productivity of the coastal soils in the Yellow River Estuary (Luo et al., 2017). Soil salinity decreased significantly due to freshwater input after the flow-sediment regulation, leading to an increase in reed biomass and an increase in the amount of assimilated nutrients. Accordingly, the phosphorus absorbed by plants could not be returned to the soil quickly (Luo et al., 2017). Therefore, the large decrease in many phosphorus fractions in the three wetlands after the regulation was largely caused by plant growth and absorption. The TP contents in TFW and FFW soils decreased significantly after the regulation, and the TN contents also showed a decreasing trend because both nitrogen and phosphorus are essential nutrients for plant growth (Verhoeven et al., 1996). 16

S/L-P is the smallest IP pool but has the highest activity and is easily absorbed by plants (Darilek, 2010). The contents of S/L-P in TFW soil decreased significantly to meet the needs of fast-growing plants after the regulation. However, there was a significant increase in the S/L-P contents and a significant decrease in the AKP activities in FRW soils after the regulation. This could be explained by the abundance of bioavailable IP (such as S/L-P) in FRW soils that could be absorbed and utilized by plants and microorganisms such that the microorganisms tend to produce less AKP (Richardson and Simpson, 2011). Phosphorus in FRW was easily released to the overlying water after the flow-sediment regulation because of the increase in S/L-P (Bai et al., 2017). The increased fresh water input effectively increased soil WC after the flow-sediment regulation in the three wetlands. The redox condition became reductive in the soil after the regulation, since the oxygen supply diminishes with the amount of water (Mitsunobu et al., 2006). Fe/Al-P includes phosphorus associated with oxides and hydroxides of Fe and Al (Wang et al., 2013). The Oc-P are those that are bound in iron plaques of calcium phosphate, iron phosphate, aluminum phosphate, and other phosphate forms (Yu et al., 2015). Phosphorus can be released from these phosphate forms when anoxic conditions prevail, since phosphorus is liberated from the surface of reduced Fe and Al oxides and hydroxides or released from the iron plaque because of the reduction of Fe2O3 (Wu et al., 2015; Li et al., 2017a). Therefore, the contents of Fe/Al-P in TFW and FRW soils and the contents of Oc-P in FRW and FFW soils decreased significantly after the regulation. The contents of Ca and Ca0 in TFW soils increased after the regulation due to the input of Ca from the fresh water and sediment inputs, while an increase in Ca-P contents was not observed in TFW. Meanwhile, the contents of Ca-P in FRW soils were significantly increased after the regulation, while a decrease in Ca0 contents and an increase in pH were observed. 17

Previous studies have shown that fresh water input caused a decrease in the salinity and an increase in the alkalinity of the soil (Bai et al., 2015; Wang et al., 2016). This was caused by the release of Ca+, Mg+, and other metal cations after fresh water input. Cationic adsorption sites previously occupied by metal cations in the soil were replaced by hydrolyzed hydrogen ions so that the soil pH increased (Chen et al., 2015). The increased pH value is conducive to the combination of Ca0 and IP in soil to form Ca-P. Therefore, the contents of Ca-P in estuarine wetland soils could be controlled by soil alkalinity. OP can be mineralized into IP under microbial oxidation and extracellular enzymatic processes to satisfy the needs of plant and microbial growth (Bünemann, 2015). ML-OP in the three wetlands decreased after the regulation because ML-OP is the main source of OP mineralization (Zhu et al., 2017). Additionally, a decreasing trend of SOM was observed in FRW after the regulation, since SOM can be decomposed by microorganisms driven by the need for energy and can be mineralized by extracellular enzymes secreted by plants and microorganisms, which may be associated with the mineralization of carbon, nitrogen and phosphorus (Weintraub and Schimel, 2003). Therefore, SOM and OP showed the same trend of variability. Moreover, AKP activity increased significantly in FFW after the regulation, which is consistent with the significant decrease in OP, L-OP and ML-OP in FFW, because AKP is characterized as a typically inducible enzyme that hydrolyzes phosphorus-containing anhydrides (Zheng et al., 2019). The flow-sediment regulation had different effects on the soil texture of estuarine wetlands experiencing different flooding conditions. The higher flow velocity of the fresh water input in FRW resulted in a significant increase in the percentage of soil sand (Ahfir et al., 2017). A large amount of sand was trapped in the FRW and more clay particles were deposited in FFW and TFW 18

due to lower fresh water flow velocity, resulting in a significant increase in the percentage of clay in these two wetlands. Soil phosphorus dynamics could be greatly influenced by soil particle size distribution through its effects on phosphorus sorption and desorption capacities (Olsen and Watanabe, 1957). Gérard’s study showed that in most soils, clay minerals should be considered as important phosphate-binding constituents due to their high specific surface area (Gérard, 2016). Meanwhile, phosphorus could be carried with clay sediments upstream (Sonzogni et al., 1982). Bai et al. (2019) also showed that the water rich in IP from the upstream areas of the Yellow River Estuary introduced by flow-sediment regulation significantly replenished the sediment IP pool of TFW and FFW. After flow-sediment regulation, the NL-OP in FRW soils increased significantly, which may have been caused by the upstream input of organic residue during regulation (Zhao et al., 2018). However, in this study, increases in phosphate contents in FFW and TFW were not observed. This was associated with the fact that the flow-sediment regulation could affect the amount of phosphorus assimilation of plants by affecting their growth (Schachtman et al., 1998). Moreover, phosphorus levels in FFW and TFW soils were higher than those in water so that it promoted the release of soil phosphorus into water, even though the flow-sediment regulation induced an impulse delivery of phosphorus (Wang et al., 2017). Additionally, the increase in water quantity with the changes in soil physicochemical properties (e.g., soil pH and EC) after the input of fresh water and sediments could influence the adsorption and desorption of phosphorus at the interface between soil and water, the mineralization and immobilization of phosphorus, and phosphorus leaching (Gaind and Nain, 2015; Mullane et al., 2015; Bai et al., 2017). Phosphorus could also be transported between wetlands and outside areas with the input and output of fresh water and sediments (Johannesson et al., 2015). 19

The SEM showed that soil WC, salinity, texture, exchangeable mineral element contents, and nutrient status were the key factors influencing soil phosphorus dynamics (Fig. 7). Generally, soil WC increased and soil salinity decreased in the three wetlands, so that the flow-sediment regulation improved the growth of plants in the Yellow River Estuary (Wang et al., 2016). However, nutrient status (e.g., SOM and TN) had the greatest effects on OP and IP fractions in the different flooding wetlands (LeBauer and Treseder, 2008). The primary productivity of the three wetlands was nearly limited by nitrogen (the molar ratio of N:P is 2.20±1.09), especially in TFW and FFW (the molar ratio of N:P<2.29). The nitrogen contents were not increased after the flow-sediment regulation, and thus the primary productivity in the three wetlands was still under nitrogen-limited status. Additionally, the soils in the three wetlands were under anaerobic reduction conditions caused by the excessive soil WC after the regulation, which likely caused the release of some IP fractions (Fe/Al-P and Oc-P) from the wetland soils to the water, thus increasing the migration of wetland phosphorus and possibly causing eutrophication in the coastal waters (Smith, 2003). Moreover, Bai et al. (2019) showed that during the plant growing period (from August to September), phosphorus uptake by plants peaked in TFW and FFW. Therefore, more research on the effects of the timing, flow rate and duration of flow-sediment regulation on soil phosphorus fraction dynamics, environmental factors, microorganisms and functional genes is still needed to determine the mechanisms influencing soil phosphorus transformation in estuarine wetlands. A flow-sediment regulation scheme to meet the needs of plant growth rhythms should be proposed, which would be beneficial for improving phosphorus retention and the ecological restoration of estuarine wetlands. Other ecological restoration measures, such as the addition of nitrogen fertilizer, can be combined with flow-sediment regulations and freshwater restoration to 20

further restore vegetation and productivity in estuarine wetlands.

5 Conclusions We investigated the dynamics of soil phosphorus fractions in estuarine wetland soils experiencing different flooding conditions before and after flow-sediment regulation. The results showed that TP dominantly existed in the IP fraction (especially Ca-P) in three wetlands, and OP accounted for less than 12% of TP. FRW soils contained higher OP levels and lower IP levels than TFW and FFW soils from April to August. After the flow-sediment regulation, S/L-P in TFW, TP in TFW and FFW, and ML-OP in all three wetlands decreased, mainly due to the freshwater input effectively reducing soil salinity and thus promoting plant growth and the absorption of nutrients. Fe/Al-P and Oc-P decreased in the three wetland soils because continuous anaerobic conditions were formed after regulation and promoted their release. Generally, the levels of phosphorus fractions were affected by water and salt conditions, soil texture, exchangeable mineral element contents, and nutrient status. The findings of this study can contribute to providing basic data regarding phosphorus fractions in estuarine wetland soils and guiding freshwater restoration and flow-sediment regulation to enhance the ecological functions of estuarine wetlands. However, the amount of phosphorus migration, including the phosphorus adsorbed by plants and inputted by litter, the phosphorus adsorbed and desorbed at the interface between soil and water, the phosphorus leached into the subsoil, and the phosphorus input and output by flow and sediment, should be measured quantitatively so that the effects of the flow-sediment regulation on the phosphorus dynamics in estuarine wetlands can be further analyzed. Fe/Al-P and Oc-P in the three wetlands was released and S/L-P in FRW was increased after the regulation, which might increase the risk of eutrophication in the coastal waters. Therefore, a flow-sediment regulation scheme to 21

meet the needs of plant growth should be taken into consideration; such a scheme would be beneficial to control phosphorus fate and the water quality of estuarine wetlands.

Acknowledgements This study was financially supported by the National Basic Research Program of China (no. 2017YFC0505906) and the National Natural Science Foundation of China (no. 51639001), Project supported by the Fund for Innovative Research Group of the National Natural Science Foundation of China (no. 51721093) and the Fundamental Research Funds for the Central Universities.

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Table 1 Physical and chemical properties of the three wetland soils. Site

WC (%)

BD (g/cm3)

EC (ms/cm)

Cl(g/kg)

SO42(g/kg)

pH

Clay (%)

Silt (%)

TFW FRW FFW

45.5±6.4a 42.3±7.8a 34.2±5.4b

1.2±0.1b 1.1±0.2c 1.3±0.1a

2.1±0.7a 1.5±0.8b 0.6±0.2c

2.1±0.8a 1.6±0.9b 0.4±0.2c

0.7±0.2b 1.6±1.1a 0.3±0.1c

8.8±0.2a 8.8±0.7a 9.0±0.2a

7.1±2.0a 3.5±1.7b 1.4±1.0c

49.7±2.7a 51.0±14.4a 36.9±7.0b

Site

Sand (%)

SOM (g/kg)

TC (g/kg)

TN (mg/kg)

TP (g/kg)

Al (g/kg)

Ca (g/kg)

Fe (g/kg)

TFW FRW FFW

43.3±3.0b 45.5±14.4b 61.7±7.4a

10.1±1.4b 16.4±8.4a 6.6±2.0c

24.0±0.8a 23.1±6.4a 19.3±2.7b

590.0±32.4b 886.2±575.3a 534.6±120.7b

667.2±24.5a 664.3±29.0a 680.4±39.0a

67.4±4.6a 64.5±5.6a 64.4±6.0a

62.8±2.2a 52.2±4.1b 55.0±6.0b

36.4±1.4a 34.2±2.6b 32.3±3.6c

Site

Al0 (g/kg)

Ca0 (g/kg)

Fe0 (g/kg)

MBC (mg/kg)

MBN (mg/kg)

AKP

ACP

(mg/(gh))

(mg/(gh))

Temp (℃)

TFW FRW FFW

0.4±0.0b 0.5±0.1a 0.4±0.1c

27.7±3.6a 18.9±3.7b 20.1±5.5b

1.1±0.1b 2.1±0.5a 1.1±0.2b

263.6±201.5b 543.7±416.4a 236.0±256.8b

38.5±44.9b 79.9±35.3a 41.5±29.2b

0.7±0.3a 0.9±0.6a 0.7±0.3a

0.4±0.1a 6.0±21.5a 0.5±0.3a

24.3±3.0a 23.6±2.9a 24.6±7.3a

Note: TFW-Tidal flooding wetlands; FRW-Freshwater restoration wetlands; FFW-Freshwater flooding wetlands. ab Different letters present significant differences among different wetland soils (p < 0.05). One-way analysis of variance (ANOVA) followed by a Duncan test were used to compare the differences in soil properties.

29

Figure captions Fig. 1. Location map of sampling sites in the Yellow River Estuary, China. Note: TFW-Tidal flooding wetlands; FRW-Freshwater restoration wetlands; FFW- Freshwater flooding wetlands. Fig. 2. Dynamics of organic phosphorus (OP) in the surface soils of three estuarine wetlands. Note: The dashed bar represents the period for the flow-sediment regulation.

ab

Different

lowercase letters indicate significant differences in OP levels among different months in the same wetland (p < 0.05).

AB

Different uppercase letters indicate significant differences among different

wetland soils in the same sampling month (p < 0.05). Fig. 3. Dynamics of organic phosphorus (OP) fractions in the surface soils of three estuarine wetlands. Note: (a) Labile organic phosphorus (L-OP); (b) Moderately labile organic phosphorus (ML-OP); (c) Non-labile organic phosphorus (NL-OP). The dashed bar represents the period for the flow-sediment regulation. ab Different lowercase letters indicate significant differences in each OP fraction among different months in the same wetland (p < 0.05).

AB

Different uppercase letters

indicate significant differences among different wetland soils in the same sampling month (p < 0.05). Fig. 4. Dynamics of inorganic phosphorus (IP) in surface soils of three estuarine wetlands. Note: The dashed bar represents the period for the flow-sediment regulation.

ab

Different

lowercase letters indicate significant differences in IP levels among different months in the same wetland (p < 0.05).

AB

Different uppercase letters indicate significant differences in IP levels

among different wetland soils in the same sampling month (p < 0.05). 30

Fig. 5. Dynamics of inorganic phosphorus (IP) fractions in surface soils of three estuarine wetlands. Note: (a) Soluble and loosely bound P (S/L-P); (b) Iron/aluminum-bound P (Fe/Al-P); (c) Occluded P (Oc-P); (d) Calcium-bound P (Ca-P). The dashed bar represents the period for the flow-sediment regulation.

ab

Different lowercase letters indicate significant differences in each IP

fraction among different sampling months in the same wetland (p < 0.05).

AB

Different uppercase

letters indicate significant differences in each IP fraction among different wetland soils in the same sampling month (p < 0.05). Fig. 6. Redundancy analysis (RDA) results of soil phosphorus fractions and environmental variables in the wetlands of the Yellow River Estuary experiencing different flooding conditions. Note: (a) Tidal flooding wetland (TFW); (b) Freshwater restoration wetland (FRW); (c) Freshwater flooding wetland (FFW). The number next to the sample point indicates the sampling time. Fig. 7. The structural equation model (SEM) shows the key environmental drivers of soil phosphorus fractions in the three wetlands. Note: The width of the arrows and the numbers along the arrows indicate the strength of the standardized coefficients. The solid lines indicate positive coefficients, and the dashed lines indicate negative coefficients. The numbers in the OP and IP boxes are their R2 values, which indicate the proportion of variance explained.

31

Fig. 1

32

100

OP (mg/kg)

80

60

40

aA

aA aA

aA

aA abA

aA

abB

abA

abA

abA

abcA abcB

20

bcA bcB

TFW

0 Apr

FRW

May

abA

bB

cB

FFW Jun

bA

Jul

month

Fig. 2

33

Aug

Sep

Oct

ML-OP (mg/kg)

L -OP (mg/kg)

70

(a)

FFW

60 50 aA aA 4 3 abA 2 1 0 70 60

aA

aA

bB

bcA bB

bA

bA bA

bA

aA abAB

bA

bB

bAB

bB

cB

(b)

50 aA

40 30 20

aA aA abA

10 0 70 NL-OP (mg/kg)

FRW

TFW

60 50 40 30 20

aA aB bC

cB

bcA bA bB

aA

bA bA

aAB aB

aA aA

10 0

dA cB

bcC

cdA cB

bB

(c) bA

dA

aB

Apr

May

aA

aB aB

Jun

aA

Jul Month

Fig. 3

34

aB

aA aA

aB

Aug

Sep

aA aA cA

Oct

720 680

aA

aA

aB

aB

IP (mg/kg)

abA

640

abA aA

aA abA

600 cC

abA

aA bA

aA

abA aA

aA

bcA cB

560 TFW

FRW

FFW

520

Apr

May

Jul

Jun

Month

Fig. 4

35

Aug

Sep

Oct

Fe/Al-P (mg/kg)

S/L -P (mg/kg)

480 400 2.5 2.0 1.5 1.0 0.5 0.0 480

FRW

FFW

aA aA

aA

abB bB

abA

abA

abA

bA bA

bA

abA

bA bA

abA

bA

abA

bA

bA

abA bA bA

(b) 400 15 10 5 0 480

Oc-P (mg/kg)

TFW

(a)

aA abB abB

Ca-P (mg/kg)

400 320

abA aA aA

bB

bB bB

abA

bA

abA

abA

abA

abA abB bB

(c)

aA abAB aB

aA bB bB

50 0 480

abB abC

400 100

abA

bcB

bA

cB

bcB

abA abB bcC

abA cB

(d) abA aA cA

aA cA aA bcB

cA

aA

bA

aB bA

abA abA

abA

abC

Jun

Jul

Aug

abB bcB

cA

5 0

Apr

May

Month

Fig. 5

36

Sep

Oct

Fig. 6

37

Fig. 7

38

Highlights 

FRW contained higher OP levels and lower IP levels than TFW and FFW.



Some P fractions decreased after the regulation, mainly due to plant absorption.



Key environmental factors influencing P fractions during the regulation were revealed.



Eutrophication risk in estuarine wetlands might increase after the regulation.

39