Impacts of large-scale aquaculture activities on the seawater carbonate system and air-sea CO2 flux in a subtropical mariculture bay, southern China

In this study, the variations of the seawater carbonate system parameters and air−sea CO2 flux (FCO2) of Shen’ao Bay, a typical subtropical aquaculture bay located in China, were investigated in spring 2016 (March to May). Parameters related to the seawater carbonate system and FCO2 were measured monthly in 3 different aquaculture areas (fish, oyster and seaweed) and in a non-culture area near the bay mouth. The results showed that the seawater carbonate system was markedly influenced by the biological processes of the culture species. Total alkalinity was significantly lower in the oyster area compared with the fish and seaweed areas, mainly because of the calcification process of oysters. Dissolved inorganic carbon (DIC) and CO2 partial pressure (pCO2) were highest in the fish area, followed by the oyster and non-culture areas, and lowest in the seaweed area. Oysters and fish can have indirect influences on DIC and pCO2by releasing nutrients, which facilitate the growth of seaweed and phytoplankton and therefore promote photosynthetic CO2 fixation. For these reasons, Shen’ao Bay acts as a potential CO2 sink in spring, with an average FCO2 ranging from −1.2 to −4.8 mmol m −2 d−1. CO2 fixation in the seaweed area was the largest contributor to CO2 flux, accounting for ca. 58% of the total CO2 sink capacity of the entire bay. These results suggest that the carbonate system and FCO2 of Shen’ao Bay were significantly affected by large-scale mariculture activities. A higher CO2 sink capacity could be acquired by extending the culture area of seaweed.


INTRODUCTION
Mariculture is developing rapidly in coastal waters and is one of the fastest-growing food-producing sectors in the world. Mariculture production comprised 28.7 million t in 2016, accounting for approximately 40% of global aquaculture production (FAO 2018). The rapid expansion in the number of aquaculture species in coastal waters has contributed to an increased release of dissolved inorganic carbon (DIC) into aquatic ecosystems. Intensive-culture animals (e.g. fish and oysters) act as carbon dioxide (CO 2 ) gen-erators through respiratory processes taking place within inner bays with slow flow velocity (Jiang et al. 2015, Morris & Humphreys 2019. This results in higher seawater bicarbonate (HCO 3 − ) and hydrogen ion (H + ) concentrations, in combination with a lower carbonate (CO 3 2− ) concentration, leading to a decreased surface ocean pH (Caldeira & Wickett 2003, Orr et al. 2005. However, whether shellfish aquaculture acts as a CO 2 source or sink is still being debated. One view is that farmed bivalves can take up larger amounts of DIC through calcification than the amount released into seawater by © The authors 2021. Open Access under Creative Commons by Attribution Licence. Use, distribution and reproduction are unrestricted. Authors and original publication must be credited. Publisher: Inter-Research · www.int-res.com *Corresponding author: qizhanhui@scsfri.ac.cn Impacts of large-scale aquaculture activities on the seawater carbonate system and air−sea CO 2 flux in a subtropical mariculture bay, southern China respiration, resulting in lower total alkalinity (TA), HCO 3 − and CO 3 2− due to calcium carbonate (CaCO 3 ) precipitation (Li et al. 2021). Another view is that TA and the buffering capacity of carbonate chemistry in aquaculture waters are reduced by the rapid calcification processes of fast-growing calcifying organisms (Mos et al. 2015); lower TA de creases the relative calcite saturation state for a given partial pressure of CO 2 (pCO 2 ) (Mos et al. 2015), causing the release of CO 2 into the atmosphere (Fine et al. 2017).
Conversely, for seaweed, photosynthetic fixation of dissolved CO 2 leads to a concurrent decrease in DIC and pCO 2 and to an increase in pH (Zou et al. 2004, Jiang et al. 2014, Han et al. 2013. Although respiration of seaweed also releases CO 2 into seawater, the photosynthesis rate is higher than the respiration rate, and the released CO 2 is balanced by that taken up through photosynthesis (Zhang et al. 2012). Seaweed species that are commercially profitable, such as Saccharina japonica and Gracilaria lemaneiformis, have been widely cultured in China for over 30 yr. The photosynthetic CO 2 fixation rates of seaweed could be depressed in seawater with elevated pH, especially in the presence of high biomass and limited seawater exchange. For example, G. lemaneiformis exhibited high affinity for DIC and carbon-saturated maximum photosynthesis in seawater with a pH of 8.0 or lower, whereas the DIC affinity and the photosynthetic ability to use HCO 3 − were dramatically re duced in seawater with a pH >8.0 (Zou et al. 2004).
Furthermore, pCO 2 is sensitive to TA, pH, salinity and temperature conditions; thus any mariculture activity that alters these parameters could affect seawater pCO 2 and air−sea CO 2 exchange. In addition to these direct influences, the release of ammonium (NH 4 + ) increases TA, while the nitrification of NH 4 + and remineralization of organic particles (e.g. animal feces and feed residue) decrease TA (Schlesinger 1997, Wolf-Gladrow et al. 2007). Overall, the uptake of nitrate (NO 3 − ) and NH 4 + by seaweed modifies TA (Brewer & Goldman 1976, Goldman & Brewer 1980. These findings suggest that the interactions between mariculture and the seawater carbonate system are very complex and that the analysis of their comprehensive effects should be based on species-and sitespecific investigations. Previous studies have investigated the effects of aquaculture fish, shellfish, seaweed and/or their combination on the seawater carbonate system, mostly based on the physiological characteristics of cultivated organisms either under controlled conditions such as in respiratory bottles or mesocosms (Han et al. 2013, Jiang et al. 2014, Fang et al. 2020, or in the open ocean (e.g. seaweed beds [Delille et al. 2000], seagrass beds [Buapet et al. 2013, Hendriks et al. 2014, Challener et al. 2016] and coral reefs [DeCarlo et al. 2017, Lønborg et al. 2019). However, to date there has been limited research on the effects of large-scale aquaculture on the seawater carbonate system, air− sea CO 2 flux (F CO 2 ) and underlying biogeochemical mechanisms in coastal aquaculture waters .
Shen'ao Bay, located in the northern South China Sea, is a typical subtropical drowned valley bay. It has been exploited for aquaculture for more than 40 yr and is one of the largest aquaculture production sites in China, with suspended longline cultures of oysters and seaweed and finfish floating cages throughout the bay. To date, the bay-scale influences produced by intensive aquaculture on the seawater carbonate system and air−sea F CO 2 in subtropical bays remain unclear. In this study, the carbonate system characteristics of surface seawater in different cultivation areas were investigated, and direct influences of physiological processes and the indirect influences of hydrographic factors were examined in order to assess the impact of large-scale aquaculture activities on the seawater carbonate system and air− sea F CO 2 , as well as to further understand the interactions between aquaculture and the marine environment at a bay scale.

Study area
The study was conducted in Shen'ao Bay, Nan'ao Island, Shantou City, Guangdong Province, China, from March to May 2016. The area is a 13.3 km 2 coastal embayment (23°27'−23°29' N, 117°04'− 117°0 7' E) with large-scale mariculture since the 1980s (Fig. 1). The bay is surrounded on 3 sides by mountains, has no surface runoff input and is affected by northeasterly winds in spring, which cause waters outside the bay to flow into the bay through a wide north-facing mouth that meets Zhelin Bay (see Section 2.2). Water depth ranges from 1 to 10 m, with an average depth of 4.3 m. The bay contains suspended longline cultures of Pacific oyster Crassostrea gigas and seaweed Gracilaria lema neiformis and floating cages of Hong Kong grouper Epinephelus akaara and yellow grouper E. awoara. Fish cultivation occurs mainly between the eastern edge of the bay and Lieyu Island, oysters are cultivated at the bottom of the bay, and seaweed cultivation is mostly located in the central area. The 3 aquaculture areas have relatively independent positions in the bay.

Experimental design and sampling procedure
The selected station grid included 9 stations placed in the fish cage area (S1−S3, referred to as fish area), oyster raft areas (S4−S6, oyster area) and seaweed raft area (S7−S9, seaweed area). Three additional stations (S10−S12), where no culture activities were practiced and which were located near the bay mouth, were chosen as reference sites (non-culture area). The distance between the non-culture area and the closest seaweed area was ca. 1.5 km, and that between the non-culture area and Zhelin Bay mouth ca. 6 km. Zhenlin Bay is a large fish cage culture location in China. However, southward currents are a recurring event in spring and, as Shen'ao Bay is characterized by a 'V' shape, with its mouth opening northwards toward Zhelin Bay, Zhelin Bay waters are forced southwards and enter Shen'ao Bay. Thus, nonculture areas in Shen'ao Bay during spring may also be impacted by water coming from Zhelin Bay. However, it was difficult to find non-culture stations to be used as re ference areas for Shen'ao Bay be cause of the high intensity of aquaculture activities af fecting the entire bay. The stations in each area were laid out in a grid, and the distance between adjacent stations in the non-culture, fish, oyster and seaweed areas was ca. 0.6, 0.2, 0.5 and 0.8 km, re spectively. The size of each culture area was calculated based on the GPS positioning data recorded at peripheral stations, and the non-culture, fish, oyster and seaweed areas measured ca. 2, 1, 2 and 5 km 2 , respectively.
Three monthly survey cruises were conducted in spring 2016, from March to May. The culture period of G. lemaneiformis is from late November to May, harvest is no later than the end of May; after that the water temperature is too high for this species. During this period, the water temperature is also suitable for the cultured fish and oysters in the bay. Their growth and metabolism rates are the highest in these months of the year, so their physiological and ecological influences on the seawater carbonate system and air−sea F CO 2 are also most evident at this time.
The irregular semidiurnal tidal current in this bay can cause temporal variations in the seawater chemistry of the same station. To minimize the effects of tidal currents on sampling, the collection of samples took place each month between 10:00 and 14:00 h on the same day of the lunar tidal cycle. Each month, one surface water sample at each station was collected at a depth of 0.5 m with a 5 l acidcleaned erect plexiglass sampler. For each sample, a 500 ml subsample for TA analysis was immediately filtered using a Whatman GF/F filter and filled with a 0.02% volume-saturated HgCl 2 solution; it was then sealed and stored in a cooler. Two 1 l subsamples were filtered through 2 separate Whatman GF/F filters; the filters were used for the determination of chlorophyll a (chl a) and for particulate organic matter (POM) analysis, and the filtrate was used for dissolved inorganic nutrient concentration analysis, including NO 3 − , NH 4 + , nitrite (NO 2 − ) and phosphate (PO 4 3− ). All Whatman GF/F filters were pre-combusted at 450°C for 4 h. Before filtering, the sampled water was pre-filtered through a 100 µm sieve to remove zooplankton.
Sea surface salinity (SSS) and sea surface temperature (SST) were measured at a depth of 0.5 m using a YSI meter (YSI Professional Plus 6600, Yellow Springs Instrument Company). The accuracy of SSS and SST measurements was ± 0.1 and ± 0.15°C, respectively; pH was measured at a depth of 0.5 m using a pH meter (Thermo Scientific Orion 320P-01, Thermo Electron) calibrated on the US National Bureau of Standards scale. The precision of pH measurements was ± 0.01 pH units. SSS, SST and pH were measured twice at each station, and the average values were used in the analysis. In each in the fish culture area, S4−S6 in the oyster culture area, S7−S9 in the seaweed culture area, and S10−S12 in the non-culture area. The red arrows represent the direction of the current subsample, TA was measured in triplicate by Gran titration with 0.1 M HCl, using an alkalinity titrator (AS-AlK2, Apollo SciTech). The accuracy of the TA measurements was determined using the Certified Reference Material (Batch 158, A. G. Dickson, Scripps Institution of Oceanography, California, USA). The reproducibility of TA values was better than ± 5 µmol kg −1 . DIC, HCO 3 − , CO 3 2− , CO 2 and pCO 2 were obtained from SSS, SST, pH and TA using the CO2_ SYS_XLS calculation program (Pierrot et al. 2006). Uncertainties in the measurements of pH and TA may result in probable errors of ± 4 µmol kg −1 and ± 2 µatm in the computation of DIC and pCO 2 , respectively (Millero 2007 were determined using standard spectrophotometric methods. The filters used for the determination of POM were dried at 60°C until constant weight was reached, and then the filter weight losses were measured after an ignition phase of 4 h at 450°C. Chl a was extracted from a 10 ml solution of 90% acetone following 24 h in darkness at 4°C (Parsons et al. 1984), and its concentration was determined with a Turner Design 10 fluorometer. Air−sea F CO 2 was calculated using the following equation: is the gas transfer velocity of CO 2 . We computed k using the parameterization given by Wanninkhof (2014), which uses short-term winds k = 0.251 × u 10 2 / (Sc/660) 1/2 , where u 10 is the wind speed at a height of 10 m from the water surface level (m s −1 ), Sc is the Schmidt number of CO 2 at in situ temperature and salinity, which has a 20% uncertainty. α (mol kg −1 atm −1 ) is the solubility coefficient of CO 2 calculated after Weiss (1974). ΔpCO 2 is the pCO 2 difference between surface seawater and the atmosphere. In this study, the values of atmospheric pCO 2 were downloaded from www.cmdl.noaa.gov (Climate and Meteorological Diagnostics Laboratory, NOAA) and corrected for water vapor pressure (Takahashi et al. 2002). Positive magnitudes of F CO 2 indicate a flux from water to air and vice versa.

Statistical analysis
Since the samples were collected at the same station each month, we adopted a sampling design with dependent repeated measures. In this context, a 2way repeated-measures ANOVA (rmANOVA) with 4 sampling areas and 3 sampling months was performed to test the separated effects of areas, months and their interaction on the seawater carbonate sys-tem parameters and hydrographic factors. The rm -ANOVA assumption of sphericity was evaluated using Mauchly's criterion. The probabilities of the interactive effect of area and month on the seawater carbonate system parameters and some hydrographic parameters (pH, NO 2 − , PO 4 3− and POM) were higher than the Bonferroni-corrected level of p, suggesting that the interactions were not significant. Subsequently, the data from the above-mentioned parameters were analyzed by two 1-way ANOVAs, one examining the effect of area combined across all months and the other examining the effect of month across all areas. The residual parameters with the interactive effect of area and month factors were further analyzed with a 1-way ANOVA to test all 12 treatments (4 areas × 3 months). The LSD multiple comparisons test was used to determine the difference among areas and months.
Tests for homogeneity and normality were run using Levene's test and the Kolmogorov-Smirnov test prior to the ANOVA. All parameters in each area and each month were normally distributed and had equal variances. Correlation analysis (Pearson's correlation coefficients) was used to test the relationships between carbonate system parameters and hydrographic factors. Given the high number of tests conducted within 2-way rmANOVA and correlation analysis, p-values were adjusted by applying a Bonferroni correction to the number of analyzed parameters to reduce the risk of a Type I error. Sixteen parameters affected by areas and months were adjusted at 0.003125 (0.05/16; number of 2-way rmANOVAs performed), and the carbonate system correlations with environment were adjusted at 0.001111 (0.05/[5 carbonate system parameters × 9 hydrographic factors]). The above calculations and statistical analyses were performed in SPSS 19.0 for Windows.
Principal component analysis (PCA), performed using the package 'vegan' in R (R Core Team 2016), was used to identify the key variables with the highest influence on seawater environmental characteristics. Before conducting the PCA, all parameters were scale standardized with zero mean and unit variance, so that they all had equal weight in the analysis.

SSS and SST
SSS in Shen'ao Bay during spring ranged from 25.7 to 31.7. There were significant differences in SSS among different areas and months (2-way rmANOVA, p < 0.003125). SSS in April and May was significantly lower than that in March (1-way ANOVA, p < 0.05). This was mainly due to the heavy rains that occurred in April and May. The overall variation trend of SSS increased moving from the outside to the inside of the bay, and the SSS in the fish area was significantly higher than that in the other areas (1-way ANOVA, p < 0.05) ( Fig. 2A). Significant (2-way rmANOVA, p < 0.003125) differences in SST were found between months rather than between areas. SST in Shen'ao Bay gradually increased from 16.4°C in March to 25.9°C in May (Fig. 2B).

pH, nutrients, POM and chl a
pH values were significantly different among areas and months (2-way rmANOVA, p < 0.003125). Over the 3-mo period (March to May), seawater pH values were significantly higher in April than in the other 2 months (1-way ANOVA, p < 0.05), and they were significantly higher in the seaweed area than in the other 3 areas for the entire period (March to May) (1-way ANOVA, p < 0.05) (Fig. 3A).
Dissolved inorganic nitrogen (DIN) in Shen'ao Bay was mainly composed of NO 3 − (ca. 70%) and NH 4 + (ca. 20%). There were significant differences in NO 3 − and NH 4 + concentrations among areas, months and their interactions; the PO 4 3− concentration was significantly different only between months (2-way rm -ANOVA, p < 0.003125). The average concentrations of NO 3 − , NH 4 + and PO 4 3− were lowest in April and highest in March. In March and May, they were significantly higher in the fish, oyster and non-culture areas than in the seaweed area (1-way ANOVA, p < 0.05) (Fig. 3B,C,E).
POM concentrations were significantly different among areas and months (2-way rmANOVA, p < 0.003125). The lowest and highest POM concentrations were recorded in March and April, respectively. POM concentration in the seaweed area was the lowest, and it was significantly lower than in the other 3 areas (1-way ANOVA, p < 0.05), which did not show any significant differences between themselves (oneway ANOVA, p > 0.05) (Fig. 3F).
Chl a concentration presented significant differences among areas, months and their interaction (2-way rmANOVA, p < 0.003125). In April and May, chl a concentration in the non-culture and seaweed areas showed the highest and lowest values, respectively, and was significantly higher and lower than in the other areas, respectively (1-way ANOVA, p < 0.05). Chl a concentrations were significantly different between all months in the non-culture, fish and oyster areas; whereas in the seaweed area, the difference was significant only between March and May (1-way ANOVA, p < 0.05) (Fig. 3G).

Carbonate system parameters
Seawater carbonate system parameters presented significant differences among areas and months (2-way rmANOVA, p < 0.003125). TA in the oyster area was significantly lower than that in the fish and seaweed areas (1-way ANOVA, p < 0.05) (Fig. 4A). The highest and lowest DIC concentrations were ob served in March and April, respectively. DIC concentration in the fish area was significantly higher than that in the other areas throughout the study period, while DIC concentration in the seaweed area in March and April was significantly lower than that in the other areas (1-way ANOVA, p < 0.05) (Fig. 4B). The calculated HCO 3 − and CO 2 concentrations showed a variation pattern similar to that of DIC, while CO 3 2− showed an opposite pattern ( Fig. 4C−E).

Factors influencing the seawater carbonate system
Pearson's correlation analysis was conducted to determine the correlations existing between seawater carbonate system parameters and hydrographic factors. For example, seawater carbonate system parameters were significantly affected by SSS (p < 0.001111; Table 1). In order to examine the contribution of hydrographic factors to the variations of the seawater carbonate system and remove the contribution derived from SSS changes, the seawater carbonate system parameters were normalized to a constant salinity of 35 as nTA, nDIC, nHCO 3 − , nCO 3 2− and nCO 2 (e.g. nTA = TA × 35/SSS [salinity in situ]) (Millero et al. 1998). Subsequently, the correlations between the normalized seawater carbonate system param eters and hydrographic factors were estimated by a Pearson's correlation analysis. After salinity normalization, nTA, nCO 3 2− and nCO 2 were significantly correlated with salinity; there was no significant correlation between salinity and nDIC and nHCO 3 − ( Table 2). Area had a significant effect on the distribution of SSS values ( Fig. 2A), mainly because of river inputs flowing into Zhelin Bay and domestic sewage. Thus, salinity was not a relevant factor to be used to make comparisons in this study. The key factors were re lated to physiological activities such as photosynthesis in seaweed, respiration and excretion in fish and oysters and calcification in oysters. Pearson's correlation analysis showed that the distribution profiles of nTA were significantly affected by pH, nutrients (NO 3 − , NH 4 + and PO 4 3− ), POM and chl a; nDIC had a strong positive correlation with  Table 2). Seawater spatial characteristics were further clarified by PCA, in which the first 2 principal components (PCs) accounted for 84.8% of the total variability. Variability along the first axis was mainly explained by a decrease in nCO 2 and nHCO 3 − in the order of seaweed area, oyster area and fish area, and a concomitant in crease in pH and nCO 3 2− (Fig. 5). PC1 was as sociated with variables such as nutrients, nCO 2 , pH, nCO 3 2− and nTA, showing the direct relationship of C, N and P with the physiological metabolic activities of the different culture species. PC2 mainly represents the de pendence on nDIC, nHCO 3 − , POM and chl a, showing the indirect influence of culture species on the seawater carbonate system.

pCO 2 and air−sea F CO 2
pCO 2 and air−sea F CO 2 were affected by both area and month (2-way rmANOVA, p < 0.003125). From March to May, atmospheric pCO 2 values ranged from 407.7 to 409.6 µatm, and the pCO 2 values in the surface water of Shen'ao Bay ranged from 56.2 to 309.2 µatm, which was lower than the atmospheric pCO 2 , indicating that the bay was acting as a sink for atmospheric CO 2 . The calculated air−sea F CO 2 ranged from −1.2 to −4.8 mmol m −2 d −1 , with the lowest and highest CO 2 sink capacities observed in the fish and seaweed areas, respectively. The CO 2 sink capacities of the 4 areas were highest in April and lowest in March, with the seaweed area value being significantly higher than that of the other areas (1-way ANOVA, p < 0.05) (Fig. 6).

DISCUSSION
There were significant differences in the seawater carbonate system parameters and air−sea F CO 2 between areas and over months in Shen'ao Bay, suggesting that mariculture activities modify the seawater carbonate system at the bay scale. The presence of culture species can affect the seawater carbonate system directly and indirectly. The photosynthesis of Gra cilaria lemaneiformis absorbed CO 2 from the seawater, leading to decreased DIC, HCO 3 − and pCO 2 , and increased pH and CO 3 2− in the seaweed area. Conversely, the respiration of a large number of fish and oysters released CO 2 into seawater, following the reaction CH 2 O + O 2 → CO 2 + H 2 O → HCO 3 − + H + → CO 3 2− + 2H + , causing an increase in seawater DIC, HCO 3 − and pCO 2 , and a decrease in pH, which did not occur in the non-culture and seaweed areas, particularly in April.
TA is a measure of the acid neutralization capacity of seawater. This parameter is particularly useful because it can be used to calculate the concentrations of various chemical species characterizing the carbonate system (e.g. CO 2 , HCO 3 − and CO 3 2− ). Animal respiration and seaweed photosynthesis can influence DIC concentration by either producing or  absorbing CO 2 , but have almost no effect on TA (Redfield et al. 1963). Oyster calcification processes, via the reaction known as Ca 2+ + 2HCO 3 − → CaCO 3 + CO 2 + H 2 O, lead to higher CO 2 concentrations and lower TA. However, DIC concentration in the oyster area was significantly lower than that in the fish area. A decreased DIC has also been reported in bivalve farms area in Sanggou Bay (Li et al. 2021). The ratio of released CO 2 /precipitated CaCO 3 during calcification was largely dependent on the buffering capacity of the surrounding seawater (Frankignoulle et al. 1995), and was estimated by calculating the temperature as 0.8 − 8.3/10 3 × SST (Frankignoulle et al. 1994); in the present study, it ranged from 0.58 to 0.67. Previous studies have shown that oysters can fix larger amounts of DIC by calcification than the amount released into seawater through respiration (Li et al. 2021). The reduced DIC concentration in the oyster area should result in a relatively low pCO 2 level. However, the oyster area did not contribute to a significant reduction in pCO 2 and F CO 2 when compared with the non-culture area. Thus, whether oyster calcification favors the fixation of CO 2 from the atmosphere needs to be further investigated.
In addition to these direct influences, culture species can also exert indirect influences, through excretion or absorption of nutrients, and thus further influence phytoplankton production, which has a similar function to that of seaweed. Pearson correlation analysis results (Table 2) showed a significant relationship between the carbonate system and nutrients. In particular, it showed that the close relationship between C, N and P was regulated by different biological processes.
Because seaweed and phytoplankton compete for nutrients and carbon sources, the large biomass of G. lemaneiformis depresses phytoplankton production in the seaweed area. For this reason, the lowest chl a concentration was recorded in this area in April and May. This was probably also due to the water temperature during this period (20−26°C) being suitable for the growth of G. lemaneiformis (Yang et al. 2006, Zou & Gao 2013. The competition for nutrients from the seaweed decreased the biomass of phytoplankton; whereas in the colder March conditions, the growth of G. lemaneiformis was lower than that in April and May. This could be a possible reason for the higher chl a found in the seaweed area in March. Furthermore, the exhaustion of gaseous CO 2 in seawater limited the growth of both seaweed and phytoplankton. In such conditions, seaweed and phytoplankton absorbed HCO 3 − using the proton pump mechanism and carbonic anhydrase (Tortell et al. 1997), causing a significantly lower HCO 3 − concentration in the seaweed area. For fish and oysters, the excretion of dissolved nutrients leads to high chl a and intense primary productivity (Ho et al. 2010, Gong et al. 2011. Higher phytoplankton consumes more CO 2 , and this could be a possible explanation for the lower pCO 2 observed in these areas. However, this was inconsistent with what was observed in the bivalve culture area of Sanggou Bay -another intensive aquaculture bay specializing in scallops and oysters in northern China -where pCO 2 ranged from 427 to 862 µatm (Li et al. 2021) and the lower average annual chl a concentration ranged from 0.44 to 6.89 µg l −1 (Jiang et al. 2017).
The possible influence of culturing G. lema nei formis on the seawater carbonate system is not negligible, since this species reaches a large biomass volume in Shen'ao Bay during spring. Based on the nutrient-H + -compensation principle and TA expression, the assimilation of 1 mol of NO 3 − during the photosynthetic process of G. lemaneiformis results in an increase of alkalinity by 1 mol (Wolf-Gladrow et al. 2007). Thus, the massive NO 3 − uptake by cultivated G. lemaneiformis could increase TA, as observed in the seaweed area. [c] [bc] [a]  (Schlesinger 1997). Therefore, nitrification of NH 4 + produces an increase in NO 3 − concentration, coupled with a decrease in TA in areas subjected to animal aquaculture (Schlesinger 1997, Wolf-Gladrow et al. 2007). At the same time, uneaten feed, feces from fish, and feces and pseudofeces from oysters are released into the water column (Giles et al. 2006), resulting in higher amounts of POM compared with seaweed areas. Remineralization of POM can introduce a considerable amount of nitrogen and phosphate into the marine environment around aquaculture farms (Kim et al. 2019, Qi et al. 2019. As a con sequence, this process may modify seawater TA and its distribution depending on the form of reactive nitrogen produced. In Shen'ao Bay, NO 3 − accounts for 70% of total DIN. It mainly derives from NH 4 + nitrification and remineralization of POM. A release of 1 mol of NO 3 − results in a 1 mol decrease in TA (Wolf-Gladrow et al. 2007). Therefore, this could be an indirect influence of fish and oysters on the de creased TA observed in their respective areas.
It is noteworthy that NO 3 − concentration in the non-culture area was also very high. A possible reason for it is that this area is located near the mouth of Shen'ao Bay, which is about 6 km away from Zhelin Bay (Fig. 1). The level of NO 3 − was high in Zhelin Bay due to the intensive fish cage culture present in that bay. A portion of the NO 3 − could have diffused from there to the non-culture area in Shen'ao Bay through seawater exchange driven by tides and northeasterly monsoon winds in early March (Du et al. 2010).
Both fish and oysters release CO 2 into seawater, but they also excrete dissolved inorganic nutrients (e.g. NH 4 + ), which stimulate intense phytoplankton productivity. Phytoplankton, in turn, consumes CO 2 and HCO 3 − , thus counterbalancing the CO 2 released by fish and oysters. Consequently, the negative values of F CO 2 observed in the 4 areas indicate that the entire Shen'ao Bay acted as a net importer of CO 2 in spring, as the CO 2 absorbed was greater than that released. Conversely, in the bivalve farming area of Sanggou Bay, F CO 2 in May was 8.90 ± 3.47 mmol m −2 d −1 , indicating that the area acted as a net source of CO 2 for the atmosphere (Li et al. 2021). This is probably correlated with primary production, due to the lower chl a concentration of that bay in spring (0.83 ± 0.45 µg l −1 ) (Jiang et al. 2017) compared with what was observed in Shen'ao Bay (5.65 ± 3.85 µg l −1 ). This hypothesis was, to some extent, confirmed by observations in Jiaozhou Bay, another mariculture bay in northern China, located at a similar latitude as Sanggou Bay. In Jiaozhou Bay, it was found that approximately half of the bay area shifted from being a CO 2 source to being a sink following a considerable increase in primary production . Although seaweeds release both particulate and dissolved organic carbon, comprising about 43.5% of seaweed production, they can be buried into sediments or exported to the deep sea, thus acting also as CO 2 sinks (Duarte et al. 2017). Therefore, it is inferred that in Shen'ao Bay, the seaweed aquaculture area represents the strongest CO 2 sink, with an approximately 25−60% greater capacity than the other areas. These results clearly demonstrated that seaweed cultivation could be an effective method to promote CO 2 sequestration from the atmosphere. Based on the extent of the 4 areas studied and the CO 2 flux at the air-sea interface, the total CO 2 absorption of Shen'ao Bay was estimated at ca. 12 Mt C per month from March to May. CO 2 absorption values observed separately in non-culture, fish, oyster and seaweed areas were ca. 6.3, 2.5, 5.6 and 19.8 Mt C, respectively. CO 2 fixation in the seaweed area was the largest contributor to the CO 2 flux, accounting for ca. 58% of the total CO 2 sink capacity of the entire bay.

CONCLUSIONS
The results of the present study suggest that the sea water carbonate system and air−sea CO 2 flux of Shen'ao Bay are strongly influenced by bay-scale mariculture activities. A higher CO 2 sink capacity could be acquired by extending the culture of seaweed such as G. lemaneiformis.