Assessment of benthic ecological quality status of small estuaries using multiple biotic indices in East China Sea

1、Introduction：

Estuaries are transitional environments between rivers and oceans, characterized by openness, ecological vulnerability, and high sensitivity to disturbances (Huang et al., 2024). With the progress of industriali- zation and urbanization, human activities are exerting increasing pres- sure on estuarine ecosystems. In the highly urbanized Port River Estuary in south Australia, the core activity areas of dolphins are increasingly constrained due to estuary pollution and human activities (Newman et al., 2025). In the Guadalquivir Estuary in southwestern Spain, heavy metals undergo secondary enrichment as a result of sediment resus- pension and tidal dynamics (Dona,zar-Aramendía et al., 2025). In the estuarine areas of New Zealand, researchers assessed pollution status by

measuring trace element accumulation and related stress indicators in the mollusks, which highlighted the potential value of mollusks in assessing the ecological quality status (EcoQs) in coastal settings worldwide (De Silva et al., 2024). Damage to the ecological health of estuaries decreases their ecosystem service capacity. In recent years, assessments of estuarine EcoQs have received increasing attention from the scientific community, stakeholders and policy-makers (Dong et al., 2023; Mulik et al., 2017; Ouyang et al., 2022; Puente et al., 2022; Yan et al., 2020).
Among the various biological components, macrobenthos are regarded as a preferred and effective indicator for assessing the EcoQs of estuaries. They inhabit the interface between the water column and sediment, thereby integrating environmental changes occurring in both

subsystems over time. Moreover, macrobenthic communities consist of diverse taxonomic groups with varying mobility and life history strate- gies, including slow-moving and long-lived species that exhibit differ- ential sensitivities to stressors. These characteristics are potential indicators of temporary fluctuations and chronic disturbances (Blanchet et al.,2008; Zhang, 2017; Jin et al.,2024; Sun et al.,2023). The dynamic patterns of macrobenthic communities, which respond rapidly to human activities and environmental stressors (Pinto et al., 2008), are widely used to evaluate the status of coastal and estuarine ecosystems (Dong et al., 2023; Villna… s and Norkko,2011; Xu et al., 2020; Yan et al., 2017). Many macrobenthos-based biotic indices have been proposed and developed to assess the EcoQs of bays, estuaries and coastal areas, such as AZTI's Marine Biotic Index (AMBI), multivariate-AMBI (M-AMBI), benthic index (BENTIX), Benthic Opportunistic Polychaete Amphipoda index (BOPA), Shannon - Wiener diversity index (H,), Feeding evenness index (jFD), Benthic Quality index (BQI), etc. Based on the Shannon - Wiener diversity index and Pielou's evenness index, Gamito and Furtado (2009) have proposed an index called jFD, which focus on feeding groups of macrobenthos, to assess the benthic EcoQs. Studies in Europe have demonstrated that the AMBI and M-AMBI can effectively detect habitat degradation in different estuaries (Borja et al., 2000; Borja et al., 2007; Khedhri et al.,2017). Taking the Seine Estuary in France as a case study, Dauvin (2007) discusses the application of multiple indices such as AMBI, BENTIX, and BQI in a comprehensive assessment of EcoQS in transitional waters, while also highlighting the limitations of relying on a single index. Thus, in this study, assessment of the estuarine ecosystem was based on five benthic indices (H,, AMBI, M-AMBI, BENTIX, BOPA) that are widely used in ecological quality status. These indices are developed based on different profiles and perspectives of macrobenthic communities, thereby enabling a more comprehensive and integrated assessment of ecological quality at the same location when using them synthetically. Similar studies have been conducted in globally recog- nized research hotspots such as the Seine Estuary in France (Dauvin, 2007), the Yangtze River Estuary (Yan et al., 2020), and Bohai Bay near the Yellow River Estuary (Dong et al., 2023). However, few studies have focused on small estuaries undergoing rapid urbanization and industrialization.
The coastal area of the western Pacific hosts numerous small macro- tidal estuaries, which serve not only as crucial socio-economic founda- tions for the region but also as representative examples of analogous ecosystems globally (Liu et al., 2022). Oujiang River and Aojiang River systems drain into the East China Sea, representing exemplars of this category of estuarine ecosystems. The estuaries of the Oujiang and Aojiang rivers and their surrounding waters are both significant for community livelihoods and marine biodiversity (Li et al., 2023). These nutrient-rich areas serve as essential habitats for macrobenthos, plankton, and fish by providing favorable conditions for feeding, spawning, and sheltering (Song et al., 2023; Xu, 2008), thereby deliv- ering a variety of ecosystem services. Furthermore, they contribute significantly to economic production. However, in recent years, the Oujiang and Aojiang estuaries have experienced significant ecological fluctuations and degradation due to industrial, aquacultural, and infrastructure development, especially reclamation projects and port construction (Chen et al., 2016; Qiu et al., 2012; Wang et al., 2014; Wang et al., 2016; Yu et al., 2023; Zhao et al., 2015). These anthropo- genic stressors have resulted in a decline in biodiversity and the overall health of the coastal ecosystems (He and Silliman, 2019; Yadav and Gjerde, 2020). Although preliminary studies on macrobenthic commu- nities have been conducted in the Oujiang and Aojiang estuaries (Hu et al., 2016; Wang et al., 2016), comprehensive assessments of benthic ecological quality in these regions remain scarce.
Therefore, the study aimed to use data from July 2023 to January 2024 to assess the recent benthic ecological status in adjacent areas of the Oujiang and Aojiang estuaries using different benthic indices (1) to evaluate the overall benthic EcoQs in the study area, (2) to investigate spatial and seasonal patterns in benthic EcoQs, and (3) to examine the

applicability of five benthic indices (BIs) in the region. This study pro- vides new insights and suggestions for assessing the ecological status of small estuary and similar marine ecosystems, and the findings offer scientific evidence to inform management recommendations.

2、Materials and methods：

2.1. Study area
The main channel of the Oujiang River stretches 384 km and has an average annual flow of 20.27 billion m3, making it the second-largest river in Zhejiang Province. Under the combined influence of river flow and tidal currents, several shoals and islands, such as the Qidu Shoal and Lingkun Island, have formed in the estuary. Lingkun Island divides the estuary into southern and northern channels. After exiting the northern channel, the river's surface area expands rapidly as the water flows into Wenzhou Bay (Zheng et al., 2008; Liu et al., 2022). The main channel of the Aojiang River is 81 km and has an average annual flow of 2 billion m3. It is one of the seven major river systems in Zhejiang Province that flow independently into the sea. The north and south shores of the Aojiang estuary are characterized by broad, low-lying muddy tidal flats, and the estuary itself has an asymmetrical trumpet shape (Fan et al., 2017). Both estuaries are mountain rivers with regular semidiurnal tides and average tidal ranges over 4 m that are classified as strong tide estuaries.
2.2. Sampling design
Macrobenthos sampling in the Oujiang and Aojiang estuaries was carried out in July 2023 (summer) and January 2024 (winter). The study area in the Aojiang estuary is located within the latitudes 27.5o - 28oN and the longitudes 120.4o - 120.72oE, with 6 intertidal stations (AT1 - AT6) and 8 subtidal stations (AS1 - AS8). The study area in the Oujiang estuary is located within the latitudes 27.92o - 28oN and the longitudes 120.81o - 121.1oE, with 8 intertidal stations (UT1 - UT8) and 10 subtidal stations (US1 - US10, Fig. 1).
Intertidal macrobenthic surveys were conducted during the low tide of spring tides. In the intertidal zone, samples were collected from the high tide zone (1 site), the middle tide zone (3 sites), and the low tide zone (1 site). A quantitative sampling frame of 0.25 m × 0.25 m × 0.3 m was used to sample the tidal flat section. Two parallel samples and extensive qualitative samples were widely collected at each station. At each subtidal station, two macrobenthos samples were collected with the Van Veen grab sampler, which has a sampling area of 0.1 m2. The biological samples were separated from the sediment using a 500 μm mesh sieve and subsequently stored in a 5 % formaldehyde solution (Liu et al., 2024). In the laboratory, the biological samples were identified to the finest taxonomic level possible, grouped by classification, and counted under a binocular stereoscopic microscope. The samples were subsequently weighed using an electronic analytical balance, and results were recorded to three decimal places.
Subtidal surface sediment samples (0–5 cm) were collected with the Van Veen grab sampler to measure sediment environmental variables. However, sediment samples were only collected in the summer. Sedi- ment samples brought back to the laboratory were freeze-dried in a freeze dryer. A portion of the freeze-dried sample was used for particle size analysis; the volume mean diameter (Md) was analyzed with a laser diffraction particle size analyzer (Jin et al., 2024). Another portion of the sample was ground using a ball mill and subsequently sieved through a 100-mesh sieve. Mercury (Hg) concentrations were deter- mined through atomic fluorescence spectroscopy. Copper and chro- mium concentrations were quantified using graphite furnace atomic absorption spectrophotometry, while zinc concentration was quantified using flame atomic absorption spectrophotometry (Athira et al., 2024).
Seawater transparency was determined using the visual transparency disc method. The pH was determined with a pH meter, while chemical oxygen demand (COD) was measured through the acidified potassium permanganate method (Goh and Lim, 2017). Dissolved oxygen (DO) was determined using iodometric titration. Active phosphate (SRP), nitri- te–nitrogen, nitrate–nitrogen, and ammonium–nitrogen concentrations were determined using the ascorbic acid reduction phosphomolybde- num blue method for PO4-P (Lin et al., 2024), the diazo azo method for NO2-N, the zinc_cadmium reduction method for NO3-N, and the sodium hypobromite oxidation method for NH4-N (Xu et al., 2022). Suspended particulate matter (SPM) was quantified using the gravimetric method (Jin et al., 2024).