The South China Sea (SCS) is located in southeastern Asia and has an average depth of approximately 1350 m. It is the largest marginal sea in the world, stretching 3.5×10⁶ km² from 23°57’N in the north to 3°00’N in the south and from 99°10’E in the west to 122°10’E in the east (Chen et al., 2006). The SCS also carries the most frequent human activity in the ocean. The study area, the NSCS, is affected by both the natural environment and year-round human activities. There are many estuaries (such as the Pearl River estuary) that introduce fresh water into the NSCS (Jiang and Wang, 2018).

In the NSCS, two typical continental shelf upwelling areas are located east of Hainan Island, the east of the Leizhou Peninsula and the southeast of Zhanjiang Bay (noted as Qiongdong), and the inshore areas are located from the Shantou Coast to the Nanri Islands of the Fujian Coast (noted as Yuedong) (Jing et al., 2009). Upwelling areas are characterized by low temperature, high salinity and high potential density. It has a significant effect on fisheries and coastal environmental protection in the NSCS (Jing et al., 2009). The summer coastal upwelling serves as a nutrient pump, which is most nourished to marine lives in the middle of July (Jiang and Wang, 2018). The broad continental shelf in the northwestern and southern areas of the SCS not only receives a large amount of clastic sediments from land and islands but also supports abundant marine lives, resulting in a high carbonate content in the surface sediments.

This study was carried out during the first cruise by Sun Yat-sen University in 2016 in the NSCS, from the Pearl River estuary down to the edge of the NSCS ( Figure 2 ). The 11 sampling sites in the NSCS were at different depths (13 m–76 m). In this area, two typical upwelling areas (Qiongdong and Yuedong) and shelf areas near the Pearl River Delta have abundant macrobenthos. Sediment was sampled using a stainless-steel box corer, and only the upper 5 cm of each sample was packed in polyethylene bags and frozen at -20°C until experimental analysis was performed.

Macrobenthos samples were collected using a stainless-steel box corer in 2016. The sediment samples were chosen with a 10 cm *10 cm *15 cm steel box and filtered through a 65-mesh sieve. Later, macrobenthos samples were sorted, measured, weighed and then stored frozen (-20°C) until further onboard processing. The macrobenthos samples included many kinds of macroinvertebrates, such as crabs, shrimps, echinoderms and polychaetes. The macrobenthos samples were washed and freeze-dried for later study. Part of the macrobenthos samples was ground into fine and homogeneous powder and tested to determine TN content and nitrogen isotopes. Some crabs, shrimps, echinoderms and polychaetes were acidified with 1 mol/L hydrochloric acid to remove the carbonate, and the macrobenthos samples were then rinsed with deionized water and freeze-dried. Finally, approximately 0.5 mg of powdered macrobenthos samples was tested to determine the TOC content and δ¹³C.

The sediment samples for the elemental and stable isotopic analyses were also freeze-dried, homogenized and ground into powder. The sediment samples were divided into two subsamples. One subsample for the TOC and δ¹³C analyses was treated with 4 mol/L hydrochloric acid to remove carbonate, subsequently rinsed with deionized water (6–8 times), and then freeze-dried. The other subsample was used for TN and δ¹⁵N analyses, and the bulk samples were not acidified before analysis.

The TOC and TN contents of the samples, including the macrobenthos samples and surface sediment samples, were determined by a Delta Plus Advantage isotope ratio mass spectrometer. δ¹³C and δ¹⁵N were measured by a Delta Plus Advantage isotope ratio mass spectrometer after the removal of the carbonate and drying at the laboratory in the South China Sea Institute of Oceanology, China Academy of Sciences. The results are reported in parts per mil (‰):

where δ (‰) is δ¹³C (‰) or δ¹⁵N (‰); R_sample and R_reference are the isotopic ratios of the benthos and surface sediment samples and reference. For carbon, the reference was the Pee Dee belemnite (PDB), and for nitrogen, it was atmospheric nitrogen gas (δ¹⁵N = 0‰). The analytical precision was 0.1‰ for δ¹³C and 0.15‰ for δ¹⁵N based on replicate measurements of a reference standard.

As δ¹⁵N values provide an indication of the trophic position of a consumer in the food chain, the trophic level was estimated with the following formula:

where δ¹⁵N_base is the nitrogen stable isotope ratio of the primary consumers. In this study, δ¹⁵N_base was the lowest value of δ¹⁵N (6.87‰) of the primary consumers (Polychaetes). δ¹⁵N_consumer is the nitrogen stable isotope ratio of the consumers, and δ¹⁵N is the enrichment value in the process of trophic grade transfer (the average value is approximately 3.4‰) (Post, 2002).

Eco-exergy indicators include the eco-exergy (Ex) and structural eco-exergy (Exst) indicators:

where Ex (kJm^-3) is the total eco-exergy of a community; K is the total number of components selected; β_k is a conversion factor for component k (Park et al., 2001; Jørgensen, 2002; Ludovisi and Poletti, 2003); C_k is the concentration of component k, which is calculated with the biomass per unit of volume or area; Exst (kJg^-1) is the total structural eco-exergy of a community, and C_t is the total concentration or biomass of organic components in the system.

Species abundance is a widely used index in the measurement of biodiversity because it is intuitive and easy to calculate. However, the Shannon index, Simpson index and Margalef index are more commonly used in the biodiversity index system, and these indices are often smaller than the species abundance. The commonly used biodiversity indices calculated in this study are as follows ( Table 1 ):

The macrobenthos could be classified according to β-values (Jørgensen et al., 2005) and could also reflect the trophic level of the organism. The β-values of polychaetes, brittle stars, sea urchins, crabs, shrimps, molluscs and demersal fishes in this study were 133, 91, 91, 232, 232, 310 and 499, respectively. To simplify the calculation process, we hypothesize that all the macrobenthos feed on sedimentary organic matter, thus the average organic carbon content of the macrobenthos with the same β-values could be measured. The average organic carbon contents of the macrobenthos were used in formulas (4) and (5), while the required organic carbon content could be obtained by conversion. The required sediment weight in the sampling sites was calculated with the organic carbon content of the sediment:

where Fs is the required sediments to maintain ecological balance (g); Xs is the TOC content in the sediment (%); X_b is the average value of the TOC contents in the macrobenthos (%) according to the β-values; F_b is the dry weight of each macrobenthic organism (g), without bones or shells; and R is the average conversion efficiency, generally approximately 10% (Christensen and Pauly, 1992; Ren et al., 2020).

OLS was used to investigate the relationships among TOC, TN and the mean grain size in the surface sediments, as shown in Figure 3 ; the relationship between TOC and TN in the macrobenthos and TOC and TN in the surface sediments, as shown in Figure 4 ; and the relationship between Ex and the weight of required sediments using the average TOC content of macrobenthos, as shown in Figure 5 . We used Origin 9.0 software to draw Figures 3 – 5 in the OLS regression method and then used Adobe Photoshop 10.1 for postprocessing.

Five carbon/nitrogen parameters were also detected in 4 species of the 31 biological individuals: the TOC, TN, δ¹³C, δ¹⁵N and TOC/TN ratios. The contents of TOC and TN in the macrobenthic organisms ranged from 1.05% to 71.08% and from 0.29% to 11.36%, with average values of 27.01% and 5.61%, respectively. The δ¹³C values ranged from -16.68 ‰ to -21.85‰, and the δ¹⁵N values ranged from 6.97‰ to 14.53‰, with mean values of -19.37‰ and 9.59‰, respectively. The TOC/TN ratios were between 3.55 and 6.97, with a mean value of 5.10.

Five carbon/nitrogen parameters were detected in more than 6 sections of the surface sediment samples: the TOC, TN, δ¹³C, δ¹⁵N and TOC/TN ratios. The grain size parameters of the surface sediments were also detected at the corresponding stations.

The TOC and TN contents in the sediments were between 0.11% and 0.84% and between 0.011% and 0.088%, with average values of 0.30% and 0.033%, respectively. Similarly, the contents of δ¹³C and δ¹⁵N ranged from -25.6‰ to -21.4‰ and from 4.58‰ to 7.48‰, with average values of -22.61‰ and 6.07‰, respectively. TOC/TN ranged from 7.46 to 11.98, with an average value of 9.31.

The results of grain size analysis showed that the surface sediment grains in the NSCS could be divided into those mainly consisting of sand, silt, and clay, with relatively low gravel content. The mean grain size (Mz) in the sediments of the NSCS was between 1.03Φ and 7.33Φ, with an average value of 4.53Φ.

The trophic levels of most macrobenthos on the northern shelf of the SCS ranged from 2.0 to 4.4, and the trophic levels of most macrobenthos were between 2.0 and 3.4. The average trophic levels in sampling sites B1, E3, F2, G4, H2, H4, I2 and I2-A were 2.81, 2.57, 2.75, 2.75, 2.87, 4.22, 2.74 and 2.55, respectively. The trophic levels of fish (2.8), crabs (2.7), shrimp (2.4–3.0), molluscs (2.4–3.0), most polychaetes (2.4–2.9) and some brittle stars (2.8) were similar. Different macrobenthos at different sites had similar trophic levels as a result of food sources similar to those found by other researchers (Loc'H et al., 2008; Kiyashko et al., 2014). The trophic levels of most of the brittle stars (2.0–2.1) and one polychaete (2.0) were low. However, the trophic levels of a few polychaetes had high values of 4.4 and 4.2, which might be related to the consumption of bacteria with relatively high δ¹⁵N values. The trophic level of sea urchins was high at 3.9 as a result of foods including insoluble sedimentary organic matter with relatively high δ¹⁵N values.

As shown in Figure 6 , the eco-exergy indicators and diversity indices of all the stations were estimated and reflected the structure and function of benthos from the perspective of thermodynamics and community structure, respectively (Tang et al., 2015). The results showed that the regional variations in Ex and Exst had different trends from site B1 to site I2-A and from north to south. Figure 6A shows that the Ex values of the macrobenthic communities in H4 and I2 were higher than those in the other sites, which means that Ex is higher in the southern part of the study area (in the area northeast of Hainan Island) than in the northern part (the area near the Pearl River Estuary, Guangdong Province). However, the spatial distribution of Exst ( Figure 6A ) is different from that of Ex, and the Exst values of sites B1 and F2 are higher than the average value, i.e., the values of Ex and Exst are generally inconsistent in the study area. In addition, the values of the biodiversity indicators basically showed an upward trend from sections B to I, which is the same as the trend of Ex ( Figure 6B ).

The average TOC contents were assumed to be 29.8%, 4.47%, 1.05%, 29.53%, 36.9%, and 27.23% in polychaetes (133), brittle stars (91), sea urchins (91), crabs (232) and shrimps (232), molluscs (310), and demersal fishes (499), respectively. The required sediment weights of the eleven sites calculated with TOC contents of the macrobenthos ranged widely from 2.2 g to 968.2 g used in formulas (4) and (5) ( Figure 7 ). The required sediment weight of the Site H4 was the highest (968.2g), while the average value of all sites was only 138.2g ( Figure 7 ).

The organic carbon in the sediments is from dead organisms (Meyers, 1997). The TOC and TN contents in the sediments are positively correlated with the mean grain size ( Figure 3 ), and the correlation coefficients R of the TOC and TN contents with the mean grain size in the surface sediments are 0.65 and 0.70, respectively, indicating that sediment particles can affect the preservation of TOC and TN. However, there are other factors affecting TOC and TN, such as the upper water column environment and biological perturbation. Macrobenthos play crucial roles in the functioning of marine ecosystems, such as the decomposition of organic matter and the transfer and recycling of nutrients within food webs (Xu et al., 2019).

The TOC and TN contents of macrobenthos (such as polychaetes, crabs and shrimps) and their corresponding sediments are shown in Figure 4A . The TOC and TN contents of the macrobenthos are widely different. The TOC contents of the macrobenthos ranged from 1.05% to 50.1%, and the highest TOC content was 71.08%. The TN contents of the macrobenthos ranged from 0.29% to 11.36% ( Figure 4A ). The TOC and TN contents in the macrobenthos in study area are similar to the TOC (4.1%–51.6%) and TN (0.7%–14.7%) contents in benthic invertebrates on a continental margin in the NW Atlantic (Parzanini et al., 2018). The TOC and TN contents in the macrobenthos are much higher than those in the sediments. The TOC and TN contents of the surface sediments are concentrated similarly in a small range (red square) ( Figure 4 ). The TOC (0.11%–0.84%) and TN (0.01%–0.088%) contents in the surface sediments are close to the TOC (0.06%–1.02%) and TN (0.03%–0.19%) contents in the Pearl River Estuary and on the SCS shelf (Hu et al., 2006), and TOC (0.1%–1.0%) and TN (0.02%–0.15%) contents on the South Yellow Sea shelf (Liu et al., 2020). These values are lower than TOC (0.5%–1.9%) and TN (0.03%–0.2%) in the Mississippi Estuary and on the adjacent shelf (Bianchi et al., 2002), and TOC (1.13–1.97%) and TN (0.14%–0.2%) in the Mackenzie Estuary and on the adjacent shelf (Goni et al., 2000).

The correlation analysis shows that the correlation coefficient R of the TOC and TN contents in the macrobenthos is 0.95 as the confidence level is 0.01 (2-tailed) ( Figure 4A ). The correlation coefficient R of the TOC and TN contents in the surface sediments is 0.98 with the confidence level of 0.01 (2-tailed) ( Figure 4B ). There is a positive correlation between the TOC and TN contents in the sediments. This is similar to the results of other studies (Meyers, 1994; Hu et al., 2006; Jia et al., 2013; Liu et al., 2020).

The material and energy flow of the food web in macrobenthic ecosystems is key to revealing the structure and function of benthic ecosystems. In the process of macrobenthic metabolism, organisms also tend to utilize light isotopes (¹²C and ¹⁴N) to participate in metabolism, such as respiration and excretion, resulting in the bioaccumulation of heavy isotopes (¹³C and ¹⁵N) (Deniro and Epstein, 1978). Therefore, trophic pathways (food sources and trophic levels) are established by ¹³C and δ¹⁵N of the main macrobenthic species in the aquatic ecosystem, which have been widely used in previous studies (Cai et al., 2002; Carabel et al., 2006; Sokolowski et al., 2012).

The δ¹³C values (-21‰ ~ -18‰) and δ¹⁵N values (7‰ ~ 11‰) of most polychaetes, crabs, shrimps, fishes, brittle stars and molluscs in the study area are shown in Figure 8 , and species with similar stable isotope ratios may have similar food compositions and living habits. The δ¹³C values of macrobenthic organisms in the NSCS show marked similarities with those found in the continental shelf of the Bay of Biscay and in the Yap Trench in the Pacific Ocean. There are also points of similarities between the δ¹³C values of macrobenthic organisms in the NSCS and those in the Chongming tidal flat, China, and in the Sea of Japan. A significant discrepancy in the δ¹³C values of macrobenthic organisms indicates that the food composition and carbon flow of the NSCS and those in a continental margin in the Northwest Atlantic may be different ( Table 2 ).

The minimum δ¹³C values of the macrobenthos in the Chongming tidal flat (-23.6‰) and in the Sea of Japan (-23.9‰) might be related to the input of terrigenous organic matter. The δ¹³C values of terrigenous organic matter are usually low, ranging from -26‰ to -28‰ (Meyers, 1997). The δ¹³C of consumers can reflect the δ¹³C value of food, and the enrichment degree of δ¹³C is 0–1‰ moving up the food chain (Fanelli et al., 2011). The δ¹³C value of the macrobenthos is higher than the δ¹³C value of food, as it accumulates along the food chain. On the other hand, the minimum δ¹³C value (-20.1‰) of the benthos in the continental margin and marine trench is less affected by terrigenous materials (Guo et al., 2018; Parzanini et al., 2018). The lowest value of the benthos (-21.85‰) in the SCS may be related to the δ¹³C value (-22.35‰) in the sediments. A similar relationship is also found for the lowest δ¹³C value in the benthos (-23.9‰) and that (-22.9‰) in sediments and particulate organic matter (POM) (-24.3‰) in the Sea of Japan (Kiyashko et al., 2014).

Significant similarities found in the values of δ¹⁵N in the macrobenthos from the NSCS, Chongming tidal flat, continental shelf of the Bay of Biscay, and continental margin in the Northwest Atlantic ( Table 2 ) indicate analogous trophic pathways in these locations. In contrast, the δ¹⁵N values (11.9–17.9‰) in the Yap Trench are significantly higher than those in the other locations.

The highest δ¹⁵N value (14.53‰) of polychaetes in the NSCS ( Figure 8 ) might be related to the bacteria and other microorganisms and insoluble sedimentary organic matter with relatively high δ¹⁵N values absorbed by the polychaetes (Hall and Meyer, 1998; Li et al., 2010). This occurred on the continental margin, similar to the high δ¹⁵N values of Harmothoe impar and Harmothoe derjugini (-13.7‰) in the Sea of Japan (Kiyashko et al., 2014) and the high δ¹⁵N values of Glycera rouxii (-14.26‰) on the continental shelf of the Bay of Biscay (Loc'H et al., 2008). The δ¹⁵N values of the benthos in the Yap Trench are also higher than those in this study as a result of more bacteria and other microorganisms and insoluble sedimentary organic matter with high δ¹⁵N existing in the Yap Trench (Guo et al., 2018).

Ex and Exst, as integrated ecosystem health indicators, stand out as useful biomonitoring tools, potentially helpful as indicators to support environmental decisions with regard to protection and restoration measures (Linares et al., 2018). Ex value of macrobenthos community was affected by biodiversity in the northern continental shelf of the South China Sea. Tang et al. (2018) proposed a 4-division classification method (classified as I_E, II_E, III_E, IV_E), where high/low values were divided by the average value (Ex or Exst) for all benthic communities within the defined area. In our study, the average value of Ex was 7345 KJ/m² and the average value of Exst was 263 KJ/g. Using this method, the thermodynamic structures of the benthic communities in the study area were graded and analyzed. The grading results showed that H4 in the northern continental shelf of the South China Sea was graded as I_E, I2 was II_E, five sampling sites (B1, E1, F2, G4 and H1) were III_E, and four sites (E3, F1, H2 and I2-A) were IV_E. Compared with the relatively healthy macrobenthic community in H4, the other sampling sites were in a subhealthly state, especially group IV_E, which were relatively unstable and had low energy efficiency.

By quantifying the eco-exergy grades of each site, we performed a correlation analysis between the eco-exergy grades and the average trophic level of macrobenthos. The results showed that there were positively correlations (correlation coefficient R=0.79) by using the OLS regression method.

Investigating the relationships of macrobenthic invertebrates and sedimentation in aquatic environments is challenging because fine sediments act as both natural habitats and potential pollutants at excessive levels (Longing et al., 2010; Zhao et al., 2011). Preservation of organic matter in coastal marine sediments is an important process in the global cycle of carbon, as more than 90% of the carbon buried in the oceans occurrs in continental margin sediments (Ramaswamy et al., 2008). Marine macrobenthos are also an important part of marine carbon storage. Two thermodynamic indicators, Ex and Exst, were used in this integrated assessment. The results showed that there was a significant positive correlation (correlation coefficient R=0.98) between Ex value of macrobenthic ecosystem and the weight of required sediments to support the energy maintenance ( Figure 5 ). Therefore, the average organic carbon content of macrobenthos could be calculated based on the same β value. In summary, the results would provide new ideas and useful supplements for further research on the carbon energy flow and balance of coastal ecosystems.