1 INTRODUCTION

The Changjiang (Yangtze) River is the largest river in China, and the large input of fresh water brings rich nutrients to the river's estuary (Guo et al., 2020). This, along with the convergence of the Taiwan warm current, the Northern Jiangsu coastal current, the Yellow Sea water mass and other current systems provides the environment and energy required to form the variety of fish spawning and breeding grounds in the Changjiang River estuary (Li et al., 2015).

In recent years, the Changjiang River estuary has faced increasing ecological pressure due to the influence of human development activities and engineering construction (Lin et al., 2018). A large number of studies have been conducted on the ecological environment of the Changjiang River estuary, including the changing of water quality (Yang and Xu, 2015; Liu et al., 2016; Lu et al., 2017; Chen and Zhu, 2018), pollution of the sediment (Bi et al., 2017; Li et al., 2019), and the relationship between biological communities and environmental factors (Jiang et al., 2006; Liao et al., 2017; Yan et al., 2019; Zhang et al., 2019). These studies have improved our understanding of the ecosystem status of the Changjiang River estuary, which will assist in the rational use of estuary resources, protection and improvement of the ecological environment and ultimately in the realization of sustainable development of the Changjiang River basin. Previous studies have shown that eutrophication of the Changjiang River estuary has resulted in deteriorating water quality (Yang and Xu, 2015; Chen and Zhu, 2018; Yan et al., 2019), increased outbreaks of red tide (Liu et al., 2013), lower species numbers, and biomass of the benthic community (Zhang et al., 2019), and a decrease in the diversity of fish community and lower numbers of medium-sized benthic fishes (Li et al., 2007). Therefore, the Changjiang River estuary ecosystem is regarded as being in a poor state and in urgent need of ecological protection and restoration.

Ecosystem health has attracted extensive attention with increasingly serious ecological and environmental problems (Costanza and Mageau, 1999; Sun et al., 2017; Li et al., 2018b). Multiple definitions of ecosystem health have emerged due to the diverse scientific disciplines showing an interest, with no consensus reached yet. However, researchers generally believe that a healthy ecosystem is a stable and sustainable ecosystem (Sun et al., 2011). The calculation of an ecosystem health metric is the most basic and fundamental part of ecosystem health research. The biotic integrity index (IBI) is generally regarded as a good index of the ecosystem health status of a river and has been widely used by scholars (Karr, 1981; Zhang et al., 2017; Cooper et al., 2018; Zhu et al., 2019). IBI considers species composition, species diversity, and functional structure characteristics of a community in a natural habitat, as well as the community's ability to maintain its ecological balance and structural integrity and its ability to adapt to environmental changes (Karr and Dudley, 1981). Karr (1981) first proposed the application of IBI in the early 1980s to evaluate the water environment quality and ecosystem status using fish as index organisms. Deegan et al. (1997) further proposed the concept of the estuarine biotic integrity index (EBI) based on changes in the estuarine fish community, and of selecting eight metrics from the total of 12 metrics in EBI, including total number of species, species dominance, fish abundance (quantity or biomass), numbers of estuarine spawning species, estuarine nursery species and estuarine sedentary species and the proportions of benthic fish and abnormal or diseased species. Hughes et al. (2002) used EBI metrics such as fish abundance, biomass, total species, species dominance, life history, and proportion of species in each living area to successfully evaluate the ecosystem status of 16 estuaries in Cape Cod and 36 sites in Buzzards Bay, Massachusetts, USA. Breine et al. (2007) presented a novel approach to identify an optimal combination of candidate metrics for the creation of a fish-based estuarine biotic index for defining the ecological status of an estuarine area. Within the development of the aforementioned metrics, the key idea was that the index development should simultaneously minimize two prediction errors, namely falsely declaring the status of a site as disturbed (Type I error) and the reverse, falsely declaring a disturbed site as undisturbed (Type II error). Liu et al. (2018) identified ten EBI metrics to evaluate the health status of the marine areas around Shanghai, namely total ichthyoplankton, benthic fish, and pelagic fish species numbers, percentages of pollution sensitive, pollution resistant, omnivorous, insectivorous, and carnivorous species, sampled number of fish individuals, and percentage of natural hybrid species. Most recent studies have focused mainly on the evaluation of the status quo of estuary ecosystem health, and there remains a lack of knowledge on the interdecadal variability in ecosystem health. Analysis of long-term changes in ecosystem health can further our understanding of the internal mechanisms driving ecosystem health and can guide efforts to restore degraded ecosystems.

In this study, we selected suitable metrics for the evaluation of EBI in the Changjiang River estuary based on the methods of Karr (1981), Deegan et al. (1997), Hughes et al. (2002), and Liu et al. (2018), calculating the EBI for the Changjiang River estuary for the spring periods of 1986, 1999, 2007, and 2016 based on ichthyoplankton data to evaluate changes in ecosystem health for the past 30 years. The results can provide a scientific basis for the protection and restoration of the Changjiang River estuary ecosystem, and furthermore, can offer a reference for the assessment of the ecosystem health of estuaries in China with an index of biological integrity.

2 MATERIAL AND METHOD 2.1 Data source

A sampling survey to determine ichthyoplankton diversity was carried out in the Changjiang River estuary (30°30'N-32°00'N, 121°20'E-123°00'E) in May 2016. According to the Specifications for Oceanographic Survey (China National Standard GB/T12763.6-2007), a large plankton net (280-cm net length, 80-cm net mouth inner diameter, 0.5-m2 net mouth area, and 0.505-mm net mesh) was used in horizontal trawl, whereas a shallow water type I plankton net (50-cm mouth diameter, CQ14 bolting-silk and 0.505-mm hole diameter) was used for vertical trawl from the bottom to the surface. The samples were fixed and stored in 99.7% anhydrous ethanol. After returning to the laboratory, samples were initially identified under stereomicroscope, after which the species of larvae and juveniles were identified and counted by DNA barcode technology based on mitochondrial COI gene sequence (Ko et al., 2013; Zhou et al., 2015; Liu et al., 2017). Survey data collected by the Institute of Oceanology of the Chinese Academy of Sciences (Yang et al., 1990; Zhu et al., 2002; Liu and Xian, 2009) over the same month (May) over different years (1986, 1999, and 2007) were collected for comparative analysis (Fig. 1).

By referencing the Latin-Chinese Dictionary of Fish Names by Classification System (Wu et al., 2012) and the Fishes of Jiangsu Province (Ni and Wu, 2006), uniform naming was conducted for ichthyoplankton with the same Latin scientific names and different Chinese scientific names in different years or for ichthyoplankton of the same species but with both different Latin and Chinese scientific names.

2.2 Setting and calculation of evaluation metrics

The current study selected suitable metrics for the evaluation of EBI in the Changjiang River estuary based on the methods of Karr (1981), Deegan et al. (1997), Hughes et al. (2002), and Liu et al. (2018), and considering the properties of the estuarine habitat in the study area. The total of 12 evaluation metrics were divided into three categories, namely species composition, trophic structure and fish population status (Table 1). The species composition category contained eight metrics, namely numbers of total species, estuarine nursery species, estuarine spawning species, estuarine resident species, benthic species, pelagic species and intolerant species, proportion of tolerant species. The trophic structure considered included the proportion of omnivorous species, insectivorous species and carnivorous species. The fish population status was represented by the proportion of natural hybrids. Among the evaluation metrics, the Fishes of the Changjiang River estuary (Zhuang et al., 2006) and the Fishes of Jiangsu Province (Ni and Wu, 2006) were referred to for further details on the determination of estuarine spawning species (Ⅰa), estuarine nursery species (Ⅰb), and estuarine sedentary species (Ⅰc). The FishBase website and relevant literature (Zhang and Wu, 2005; Zhuang et al., 2006; Wu and Zhong, 2008) were referred to for further details on the determination of trophic types [omnivorous (IIa), insectivorous (IIb) and carnivorous (IIc)], pollution tolerance types and whether the sampled species were natural hybrids.

Studies in recent years, fishery resources determined in years with less human interference were selected as a reference. The status of the species and population structures of ichthyoplankton are influenced by adult fish (Xiao et al., 2017; Tucker et al., 2018). Before the 1980s, the development and utilization of fishery resources in the Changjiang River estuary were within a sustainable range, and the fish community remained largely intact (Cheng et al., 2006; Li et al., 2007). Therefore, the current studies had set the fish community reference data as that collected in 1986. By referring to the reference dataset, the datasets collected for subsequent years were divided into three categories of "Good", "Fair" or "Poor", and were assigned 5 points, 3 points or 1 point, respectively. The sum of the assigned scores of each attribute for each year was the calculated EBI for that year. According to the points assignment method, the assignment criteria for each index of the EBI of the Changjiang River estuary was obtained (Table 1).

The calculation of the EBI can be defined as:

The highest possible EBI is 60 points, and the higher the score gets, the higher the biological integrity of ichthyoplankton in the study area and the more stable ecosystem is. Microsoft Office Excel was used for EBI data processing, regression equation was obtained, and reliability test was carried out.

2.3 EBI evaluation criteria and grading

By referring to Karr (1981), the current studies set nine score ranges for EBI relating to nine classifications, namely Excellent (57-60), Excellent- Good (53-56), Good (48-52), Good-Fair (45-47), Fair (39-44), Fair-Poor (36-38), Poor (28-35), Poor- Very Poor (23-27) and Very Poor (≤22). Table 2 lists the EBI values, the corresponding EBI levels and the feature descriptions of the main EBI levels. The manifestation of the Excellent (57-60) level indicated an ichthyoplankton community undisturbed by humans, in which all ichthyoplankton categories appeared, including the presence of the most pollution sensitive fish, a balanced trophic structure, an absence of natural hybrids and few infected individuals. The manifestation of the Good (48-52) level indicated an ichthyoplankton community with abundance somewhat below expectation, particularly for pollution sensitive species, a trophic structure indicating stress in the environment and the presence of a few hybrid individuals. The manifestation of the Fair (39-44) level indicated signs of additional deterioration, including lower ichthyoplankton abundance and fewer pollution sensitive forms. The manifestation of the Poor (28-35) level indicated fewer species, the absence of sensitive ichthyoplankton and a higher proportion of hybrids. The manifestation of the Very Poor (≤22) level indicated by a low abundance and species number present, and the presence of mostly very tolerant forms, with a high proportion of hybrids.

3 RESULT AND DISCUSSION 3.1 Ecological habits of the ichthyoplankton

During spring, 60 species of ichthyoplankton from 27 families were collected in the Changjiang River estuary. Among them, 50 species, 4 genera, and 6 families were identified (Table 3). The 60 species collected were divided into the four ecological categories according to their ecological habits and distribution, namely, freshwater species, coastal species, brackish water species, and offshore species (Table 3). The majority were coastal and brackish water species, collectively constituting 55% of the total.

A total of 29 species of ichthyoplankton from 16 families, 20 species of 15 families, 17 species of 13 families, and 19 species of 10 families were identified in 1986, 1999, 2007, and 2016, respectively. The diversity of ichthyoplankton showed a slow decreasing trend at the level of family over the different years. Species diversity at 29 species was highest in 1986, following which it dropped sharply to 20 species in 1999, and then stabilized to 17 in 2007 and 19 in 2016 (Fig. 2).

The total number of species in the ichthyoplankton community dropped from 29 in 1986 to 20 in 1999, thereafter remaining stable at around 20 in 2007 and 2016 (Fig. 2). Over the four periods, rare species, such as Protosalanx chinensis, those that occur very infrequently in the community (Shen and Shi, 2002), were identified to have had a great influence on the ichthyoplankton community structure in the Changjiang River estuary. Compared with more common species, these rare species have weak adaptabilities to the environment. Under more favorable environmental conditions (Shen and Shi, 2002), the abundance of diversity of the rare species increased. Under a deteriorating environment (Shen and Shi, 2002), these rare species struggle to adapt and will escape or die, thereby resulting in a rapid decline in the diversity of ichthyoplankton community (Shan et al., 2005). The changes in the structure of the ichthyoplankton community are closely related to those of the adult fish community (Xiao et al., 2017). In recent years, there has been a decline in the diversity of the fish community in the nearshore waters of the Changjiang River estuary. The species composition changed from a dominance of medium-sized benthic fish species (Trichiurus japonicus and Larimichthys polyactis) in the 1960s to a relatively simple community structure dominated by small-sized pelagic fish species (Psenopsis anomala) in the 21^st century (Cheng et al., 2006). The fish community structure has shown an increasing simplification over time (Shen et al., 2013; Wu et al., 2019), with large individuals decreasing (Cheng et al., 2006; Zhang et al., 2019), whereas small fish species with short life cycles, such as Chaeturichthys stigmatias, dominated (Shi et al., 2011; Shen et al., 2013). According to statistics, the dominant ichthyoplankton species in the 21^st century are mostly small and medium-sized fishes such as Coilia mystus (Liu and Xian, 2010).

3.2 Attribute types of ichthyoplankton in the estuary over different periods

Three categories of ichthyoplankton were identified in the Changjiang River estuary in spring, namely estuarine spawning species, estuarine nursery species, and estuarine sedentary species. The year 1986 and 2007 were found to have the most and least nursery species at 11 and 4 species, respectively. The year 1986 showed the most spawning species at 4 species, whereas the remaining years all showed 2 species. The highest and lowest numbers of estuarine resident species were found in 1986 and 1999 at 14 and 8 species, respectively. The most and least benthic species were identified in 1986 and 2007 at 11 and 4 species, respectively. The most and least pelagic species were recognized in 1986 and 2016 at 18 and 11 species, respectively. The most and least pollution sensitive species were classified in 1986 and 2016 with 27 and 15 species, respectively. The most and least pollution tolerant species were ascertained in 2016 and 1999 at 21.1% and 5%, respectively. Ichthyoplankton groups that are categorized by feeding habits include omnivorous species, carnivorous species, and insectivorous species. The highest and lowest proportions of omnivorous species were in 2016 and 2007 at 31.6% and 17.6%, respectively. The highest and lowest proportions of carnivorous species were in 1999 and 2007 at 75% and 58.8%, respectively. The proportion of insectivorous species was highest in 2007 at 23.5%, whereas no insectivorous species was found in 1999. No natural hybrids were found (Table 3, Figs. 3-5).

The changes in ichthyoplankton species composition are driven by changes of pollution resistant species. There was a decline in pollution sensitive species. In the past 30 years from 1986 to 2016, pollution sensitive ichthyoplankton species gradually decreased in the Changjiang River estuary. In 1986, 1999, 2007, and 2016, 27, 19, 16, and 15 pollution sensitive species were identified, respectively (Fig. 4). The most obvious declines in pollution sensitive species occurred between 1986 and 1999, with a decrease of approximately 30%, mainly reflected in decreases in species of families such as Clupeidae, Salangidae, and Pleuronectiformes. The reduced number of species found in Clupeidae included Sardinops melanostictus, Sardinella zunasi, and Konosirus punctatus. The reduced number of species in Salangidae included Protosalanx chinensis and Neosalanx tangkahkeii. The reduced number of species in Pleuronectiformes included Bothidae and Cynoglossidae, such as Pseudorhombus arsius, Paralichthys olivaceus, Cynoglossus joyneri, Cynoglossus abbreviatus, and Cynoglossus gracilis (Table 3). With the increasing number of pollution tolerant ichthyoplankton species, Chelon haematocheilus in Mugilidae gradually became the dominant species in the Changjiang River estuary area. From 1986 to 2016, ten dominant species were identified, the vast majority of which were pollution tolerant species, concentrated in families Gobiidae, Clupeidae, and Engraulidae (Strydom et al., 2003; Song et al., 2018). Larimichthys polyactis in family Sciaenidae was the dominant species in the Changjiang River estuary in 1986, 1999, and 2001 (Liu and Xian, 2010), but gradually became a common species after 2001. Only Coilia mystus and Engraulis japonius in the family Engraulidae persisted as dominant species during the nearly 30 years from 1986 to 2016, but their relative importance in the index decreased to some extent over time, mainly due to the decline in their numbers (Liu and Xian, 2010). Nibea albiflora, Scomber japonicus, Salanx prognathous, and Allanetta bleekeri were the dominant species in 1986, 1986, 1999, and 2007, respectively (Table 3). The dominance of the pollution sensitive species decreased with a concurrent appearance of pollution resistant species among the dominant species. Variation in the dominant species was a significant signal of the structural changes in the estuarine ecosystem of Changjiang River estuary.

Decrease in the numbers of estuarine sedentary species and estuarine nursery species also affected the changes in species composition (Fig. 3). Change in estuarine sedentary species was obvious from the reduction in the species of the genus Cynoglossidae in 1999, such as Cynoglossus joyneri, Cynoglossus abbreviates, and Cynoglossus gracilis among the estuarine benthic species (Table 3). The drop in these species indicates changes to the benthic habitat over the last 30 years. Liu et al. (2012) similarly identified alterations in the benthic habitat showing that macrobenthos of the Changjiang River estuary underwent three stages in the past 30 years, which were a stable stage (before the 1990s), a disturbed stage (1990-2005) and a slow recovery stage (2005- 2010). In the past 30 years, the group breeding strategy of macrobenthos in the Changjiang River estuary has changed. More specifically, K-type species has been replaced by r-type species. K-type, which is the dominant breeding strategy of low birth rate, long life span, large individual, adapting to stable habitat environment, has a perfect protection mechanism for offspring. While r-type, which is with high birth rate, short life, small individual, wide adaptability, high reproductive capacity, and often lack of the mechanism of offspring protection, must make full use of resources to increase reproduction and give full play to their intrinsic growth rate (Shen and Shi, 2002). Therefore, macrobenthos have to adapt to the increasingly unstable natural environment in the Changjiang River estuary in recent years (Ye et al., 2004). The reversion of observation results would be difficult to happen in a short period of time. During the period of 1999-2007, the number of estuarine nursery species decreased by approximately 50%. The changes to the estuarine nursery species can be grouped into two periods, 1986-1999 and 2007-2016. The species that disappeared after 1999 were mainly in the family Clupeidae, such as Sardinops melanostictus and Sardinella zunasi, in the family Bothidae such as Pseudorhomus arsius and Paralichthys olivaceus, and in Scombridae such as Scomberomorus niphonius. Only Larimichthys polyactis of the family Sciaenidae and Pampus argnteus of the family Stromateidae appeared during both periods of 1986-1999 and 2007-2016 (Table 3). The prey items of larval and juvenile fish were dominated by zooplankton such as copepods (Wang et al., 2018). Jiang et al. (2006) showed a negative correlation between larva number and planktonic copepods. Since 1990s natural factors (runoff etc.) enhanced by anthropogenic activities (pollution discharge, eutrophication, large-scale water conservancy projects, beach reclamation, etc.) have led to dramatic plankton loss in Changjiang River estuary (Wang et al., 2004). In the late 1990s, plankton community in the Changjiang River estuary changed, with the number of phytoplankton species in the late 1990s 35% lower than that in the early 1990s, and the number of zooplankton species in the late 1990s 67% lower than that in the early 1990s. The number of dominant species of phytoplankton and zooplankton reduced, with the most dominant species among both the phytoplankton and zooplankton accounting for > 50% of the total (Wang et al., 2004).

3.3 Temporal changes in biological integrity in the Changjiang River estuary

The EBI values of the Changjiang River estuary in 1986, 1999, 2007, and 2016 were calculated to be 50, 38, 36, and 32, respectively (Fig. 6), corresponding to EBI levels of "Good", "Fair-Poor", "Fair-Poor" and "Poor", respectively, indicating that biological integrity initially declined and then stabilized. The temporal changes in the metrics of species composition (M1-M8) were similar to that of the total EBI, i.e. a downward trend from 1986 to 1999 followed by stabilization. In contrast, trophic structure and fish health status remained stable.

Ichthyoplankton are an important part of the estuarine ecosystem (Korsman et al., 2017), as not only are they a major prey item, but they are also important consumers of secondary productivity, acting as a biomass consumer and bioenergy converter (Wan and Jiang, 2000). Ichthyoplankton therefore constitute an important link in the food chain of estuarine ecosystems between primary producers and secondary consumers. Since ichthyoplankton have weak motor capacity, they respond quickly to environmental changes (Marshall et al., 2019). This sensitivity to environmental changes is the reason why ichthyoplankton can be used as metrics of estuarine ecosystem health. The present study focused on three broad categories of the ichthyoplankton community, namely species composition, trophic structure, and fish population status as metrics for the construction of the EBI. The purpose of the present study was to determine the temporal changes in the ecosystem health of the Changjiang River estuary by studying temporal changes in various ichthyoplankton community characteristics such as species composition and ichthyoplankton population status and their responses to environmental change (Karr et al., 1986). The results of the current study show that over the past 30 years, the ecosystem health of the Changjiang River estuary has transferred from "Good" in the mid-1980s to "Poor" since the beginning of the 21 st century. The most obvious manifestation of the declining health of an ecosystem is the destruction of biological integrity. Many past studies which focused on the ecosystem health of the Changjiang River estuary from other perspectives, came to a similar conclusion of a decline in the ecosystem health of the Changjiang River estuary in recent years. Zhou et al. (2011) using the Pressure-State-Response Index structure model, concluded that the marine area was in a compromised state of health from 1996 to 2005. Ye et al. (2007) evaluated ecosystem health from 2002 to 2004 using 30 indices from three categories (physical and chemical indices, ecological indices, and socio-economic indices), and found that the marine area was generally in a compromised state of health from 2002 to 2004. Many researchers use the Ecopath model to explore changes in the internal trophic structure and energy flow of an ecosystem, and found that the ecosystem of the Changjiang River estuary remained in an immature development stage from 2000 to 2016, with low stability, weak ability to counter interference and poor ecosystem health (Lin et al., 2009; Han et al., 2016; Xu et al., 2018).

The ecosystem status of the estuarine area is concurrently affected by human activities and natural changes. Changes in ecosystem health of the Changjiang River estuary is affected by many factors, including fishing (Li et al., 2007; Ruzicka et al., 2019), rainfall-runoff (Liu et al., 2012), environmental pollution (Wang et al., 2004) and beach reclamation (Li et al., 2018a). According to Crosby et al. (2018), climate change presents an increasing threat to estuary ecosystem services they provide, especially when coupled with other anthropogenic stressors. Fish abundance declined overall from 1987 to 2016, with simultaneous changes in catch per unit effort (CPUE) observed across multiple species including the commercially important winter flounder (Pseudopleuronectes americanus). As fish can serve as effective indicator of estuarine health, these changes suggest a negative shift in the health of this Long Island Sound embayment (Norwalk Harbor, Connecticut). The present study similarly found that changes to the ecological health of the Changjiang River estuary over the past 30 years were mainly related to overfishing, environmental pollution, and changes in the rainfall-runoff of the Changjiang River, which reduced the stability and the sustainability of the ecosystem. A trade-off between fishing intensity and the diversity of fishes exists (Washington, 1984). Fishing capacity statistics shown in the fishery statistical yearbook of the East China Sea indicate that there has been a progressive increase in fishing capacity in the East China Sea from 1951 to 2002, particularly from the early 1980s (Li et al., 2007). In addition, fishing nets were changed to be less selective of fishes, resulting in a sharp increase in the numbers of juvenile fish caught (Han et al., 2016). Environmental pollution directly resulted in the degradation of high quality habitat, which posed a serious threat to the survival of many species (Waltham et al., 2020). In the past 30 years, there has been an increase in eutrophication of the Changjiang River estuary (Yan et al., 2019), with obvious increases in contents of active phosphate and inorganic nitrogen from 1987 to 2005, and stabilization thereafter. The variety of organic pollutants increased from 115 in 1986 to 308 in the early 2000s (Kong et al., 2007). Sediment runoff data for the Datong station collected over many years has shown that sediment transport to the Changjiang River has dropped sharply from the 1950s to 2004 (Yang and Xu, 2015), following which inputs stabilized at a low input. There was a decrease in the self-cleaning capacity of the seawater in the Changjiang River estuary with increasing sediment runoff (Shan et al., 2004), leading to a weakening of the self-regulation capacity of the ecosystem.

4 CONCLUSION

Monitoring ichthyoplankton community is a viable alternative to physiochemical monitoring programs and traditional biological monitoring programs (phytoplankton, benthos, even nekton) for assessment of estuary biotic integrity. The results showed that temporal changes in EBI in the Changjiang River estuary from "Good" to "Fair-Poor" to "Poor", which indicates a declining biological integrity of ichthyoplankton community over the past 30 years. This result further reinforces the argument for urgent protection and restoration of the Changjiang River estuary. The EBI effectively indicated that changes in species composition were the primary reason for the significant decreases in EBI between 1986 and 1999 and for the large differences between 1986 and 2007 and between 2007 and 2016.

5 DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.