Underway Measurement of Dissolved Inorganic Carbon (DIC) in Estuarine Waters

Journal of Marine Science and Engineering

Article Underway Measurement of Dissolved Inorganic Carbon (DIC) in Estuarine Waters

Jinpei Yan 1,2,* , Qi Lin 1,2, Seng Chee Poh 3, Yuhong Li 1,2 and Liyang Zhan 1,2

1 Key Laboratory of Global Change and Marine Atmospheric Chemistry, MNR, Xiamen 361005, China; [email protected] (Q.L.); [email protected] (Y.L.); [email protected] (L.Z.) 2 Third Institute of Oceanography, Ministry of Natural Resources, Xiamen 361005, China 3 Faculty of Science and Marine Environment, Universiti Malaysia Terengganu, Terengganu 21030, Malaysia; [email protected] * Correspondence: [email protected]

Received: 20 August 2020; Accepted: 29 September 2020; Published: 30 September 2020

Abstract: Dissolved inorganic carbon (DIC) is an important parameter of the marine carbonate system. Underway analyses of DIC are required to describe spatial and temporal changes of DIC in marine systems. In this study, we developed a microvolume flow detection method for the underway determination of DIC in marine waters, using gas-diffusion flow analysis in conjunction with electrical 1 conductivity (EC) measurement. Only an acid carrier reagent (0.2 mol.L− ) and an ultrapure water acceptor are required for the DIC monitoring system. In this system, a sampling loop (100 µL) is used to quantify the injection sample volume, allowing micro-sample volume detection. The water sample reacts with the acid reagent to convert carbonate and bicarbonate species into CO2. The water sample is then carried into a gas-diffusion assembly, where the CO2 diffuses from the sampling stream into the acceptor stream. CO2 in the acceptor is detected subsequently by an electrical conductivity. The limit of DIC detection using ultrapure water is 0.16 mM. A good repeatability is obtained, with a relative standard deviation (RSD) of 0.56% (1 mM, n = 21). The time interval for detecting one sample is 5 min. During the observation period, measurements can be switched between standard solutions and water samples automatically. Accuracy and precision of the instrument is sufficient for the underway observation of marine DIC in estuarine waters.

Keywords: dissolved inorganic carbon (DIC); underway measurement; conductivity detector

1. Introduction

2 Dissolved inorganic carbon (DIC), which is typically comprised of CO3 −, HCO3− and CO2 (aq) [1], is a major constituent of sea water and an important indicator of ocean acidification [2–6]. DIC concentrations range from less than 20 µM in poorly acidic buffered waters to more than 5000 µM in highly alkaline hard waters. However, DIC ranges from about 100 to 10,000 µM in most freshwater, and averages about 2400 µM in the ocean. The carbonate systems can be described by four main parameters, such as DIC, pH, total alkalinity (TA), and pCO2. Measuring two of this parameters allows the calculation of the other two based on the thermodynamic equations of the carbonate system [7,8]. The determination of pH using glass electrodes is often affected by the variable ionic strength of seawater. Hence, colorimetric methods have been developed for the measurement of both pH and TA, to improve accuracy and precision [9–11]. Compared with the other three carbonate system parameters, DIC can be detected with the greatest accuracy and precision [3]. Coulometric and nondispersive infrared (NDIR)gas analysis methods are commonly used to measure DIC in seawater [12,13]. High accuracy and precision for DIC measurement with NDIR, as well

J. Mar. Sci. Eng. 2020, 8, 765; doi:10.3390/jmse8100765 www.mdpi.com/journal/jmse J. Mar. Sci. Eng. 2020, 8, 765 2 of 11 as coulometric detection, has been reported in previous studies [7,14]. Though NDIR gas analysis has been used for rapid measurements of DIC [15], neither of these approaches is practicable for the underway measurement of the large number of samples that are required to adequately describe spatial and temporal changes in dissolved carbonate species in marine systems [16]. High time-resolution and accurate observation techniques are required to understand the changes in ocean acidification and carbonate species. Rapid flow injection analysis for total DIC was first developed by Hall and Aller (1992) [17]. For the natural waters, the flow injection analysis method had a linear calibration range for DIC of 0.1–20 mM, with repeatability of 1% RSD. Recently, a gas-diffusion flow analysis system has been developed for the online determination of DIC [16,18–20]. Various techniques have been 4 used to detect carbonate species or CO2, such as photometric detection, C D detection, a bulk acoustic wave detection, and a tungsten oxide sensing etc. [16,21,22]. The gas-diffusion approach has been widely used to measure carbonate species in aqueous solutions. Though the gas-diffusion analysis in combination with different detectors can be used for the rapid and sensitive determination of DIC, large sample volume flow rates are often needed to meet the accuracy and detection limit. In this case, large amounts of indicator solution and acid solution are consumed. For example, gas-diffusion flow 1 injection with photometric detection typically consumes 60–120 mL.h− of buffered indicator solution. This level of consumption is impractical for long-term detection of DIC. However, sample volumes can be significantly reduced, by using an electrical conductivity detector, as sample volumes of only 100 µL are required. Conductivity detection is highly accurate and precise, and does not require an additional indicator solution. Hence, the measurement of electrical conductivity is considered to be a suitable method for the determination of DIC. In this study, we developed an online DIC determination method and instrument that provides fast, repeatable, and sensitive measurement of DIC in seawater. This monitoring system uses the gas-diffusion flow injection technique, in combination with an electric conductivity detector. We use a microvolume flow detection method to minimize the consumption of acceptor solution and acid reagent solution. No additional indicator solutions are needed during the detection process, reducing the reagent cost and waste disposal. The advantage of our instrument is to determine DIC in seawater rapidly and continuously. The gas-diffusion system in combination with electric conductivity detection can be used to provide continuous, underway DIC measurements with high spatial and temporal resolution along the cruise tracks and in estuarine areas within a short time. Hence, the DIC instrument is beneficial for underway and field DIC measurements.

2. Measurement System

2.1. DIC Analysis System The online DIC observation system (Figure1a) is consisting of two injectors (Liyuan Analysis Instrument Co. Ltd., Beijing, China), a six-port valve and a triple-injection valve (Liyuan Analysis Instrument Co. Ltd., Beijing, China), a plunger pump (Liyuan Analysis Instrument Co. Ltd., Beijing, China), a gas-diffusion assembly, a CO2 removal cell (anion suppressor), and an electrical conductivity (EC) detector (Liyuan Analysis Instrument Co. Ltd., Beijing, China). The scheme of the gas-diffusion assembly (Figure1b) consists of an outer tube (1 /8 inch id, 20 mm length, Teflon) through which the acidified water sample stream flows. The inner tube is used as membrane (1/16 inch od, AF2400 membrane), through which the acceptor stream flows. In this study, two inner tubes were used in the gas-diffusion assembly to enhance the diffusion surface area and accelerate the diffusion of CO2into the acceptor stream. T-piece connectors allow the donor stream to flow across the outside of the membrane, and allow the acceptor stream to flow across the inside of the membrane, seen in Figure1b. In this study, the flow velocities of the donor and acceptor streams are 1 1 about 0.6 m.s− and 2.5 m.s− , respectively. J.J. Mar. Mar. Sci. Sci. Eng. Eng. 20202020, ,88, ,x 765 FOR PEER REVIEW 33 of of 11 11

（a）

（b） Donor

Peek Tee Outer tubing Teflon Acceptor

InnerInner tubing tubiing AF2400 AF2400 membranemembrance

Figure 1. The schematic image of the DIC instrument, (a) DIC measurement system and image of DIC Figure 1. The schematic image of the DIC instrument, (a) DIC measurement system and image of DIC instrument, (b) Scheme of gas-diffusion assembly. instrument, (b) Scheme of gas-diffusion assembly. 1 -1 For DIC measurement, the acid reagent (H3PO4, 0.2 mol.L− , flow rate 0.2–2.0 mL.min ) is first injectedFor intoDIC themeasurement, six-port valve, the usingacid reagent a syringe (H pump.3PO4, 0.2 The mol.L acid− reagent1, flow rate passes 0.2–2.0 continuously mL.min-1) through is first injectedthe sampling into the loop six-port (100 valve,µL), then using flows a syringe intothe pump. gas-di Theffusion acid reagent assembly passes during continuously the detection through and thestandby sampling periods. loop When (100 sampleµL), then injection flows into starts, the the gas-diffusion six-port valve assembly is switched during to the the sample detection injection and standbyposition. periods. At the same When time, sample thesample injection (1 starts, mL) is the pumped six-port into valve the six-port is switched valve to and the passes sample though injection the position.sampling At loop. the Whensame time, the sampling the sample injection (1 mL) ends, is pumped the six-port into the valve six-port position valve is switched and passes back though to the theoriginal sampling position. loop. Then, When the the acid sampling reagent injection passes though ends, the the six-port sampling valve loop position again and is switched is mixed withbackthe to the original position. Then, the acid reagent passes though the sampling loop again and is mixed with water sample. The water sample is acidified and the carbonate and bicarbonate are converted into CO2. the waterIn the sample. diffusion The assembly, water sample the acceptor is acidified stream and and the the carbonate sampling and stream bicarbonate flow in oppositeare converted directions into CO2. to improve the efficiency of CO2 acceptance. The acidified sample is carried by the acid reagent to the In the diffusion assembly, the acceptor stream and the sampling stream flow in opposite gas-diffusion assembly, where CO2 diffuses through the membrane and is absorbed by the acceptor. directionsFinally, the to acceptor improve solution the efficiency is detected of CO by2 electricalacceptance. conductivity. The acidified In this sample study, is ultrapurecarried by water the acid was reagent to the gas-diffusion assembly, where CO2 diffuses through1 the membrane and is absorbed by used as an acceptor stream with a flow rate of 0.5 to 2 mL.min− . Generally, the air CO2 dissolved theinto acceptor. the acceptor Finally, may the change acceptor the acceptor solution background is detected conductanceby electrical andconductivity. impact the In baseline this study, drift. ultrapure water was used as an acceptor stream with a flow rate of 0.5 to 2 mL.min−1. Generally, the To minimize the potential effect of CO2 in the atmosphere on the acceptor stream, a CO2 removal cell air(anion CO2 suppressor) dissolved into is used the inacceptor the measuring may change system. the Theacceptor carbonate background species containedconductance in the and ultrapure impact the baseline drift. To minimize the potential effect of CO2 in the atmosphere on the acceptor stream, a CO2 removal cell (anion suppressor) is used in the measuring system. The carbonate species

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water are removed before entering the electrical conductivity detector. In fact, the CO2 trap is beneﬁcial to minimize the system drift. Furthermore, we also used a single point correction method to check the system drift. A triple valve was used to automatically switch the water samples and standard solutions injection. The standard seawater was determined automatically after water samples were measuring for a certain time. Hence, we can check the DIC instrument performance easily.

2.2. Performance of the DIC System with Standard Solutions To detect DIC in standard solutions, the triple valve was switched to the standard sample injection position, then the standard samples were pumped into the six-port valve by the sample injector. The injector and the sampling loop were flushed two to three times with the standard solutions before measurement. The standard solutions were detected from low to high DIC concentrations. Previous studies have reported that the measurement sensitivity can be improved, when NaOH was used as an acceptor stream [23]. However, the background conductivity would also increase significantly when NaOH was used as an acceptor stream. It is the case that NaOH acceptor stream may improve the CO2 gas-diffusion, as CO2 is more easily absorbed by acceptor streams with high pH, the linear calibration range was limited (0.2–2 mM) using a NaOH acceptor [24]. Typically, DIC concentrations average about 2.4 mM in the ocean [2,3], which is beyond the linear range of 0.2–2 mM. Hence, in this study, we used ultrapure water as the acceptor stream for EC detection, which has a constant background conductivity of 0.37 µs/m. Different standard solutions (0.2, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.0, 9.0, 10.0 mM) of NaHCO3 were selected for DIC calibration. Every standard solution sample was measured for five times to obtain mean values. The ultrapure water acceptor calibration data can be fitted to a quadratic function for the range from 0.1 to 10 mM (y = ((4.70 10 5 4.56 10 7)*x2 + (0.015 1.52 10 4)*x, R2 = 0.999, × − ± × − ± × − Figure2). Please note that good linear correlation was present for the DIC concentration below 5 mM using water acceptor (R2 = 0.996, Figure S1). The linear range for NaOH acceptor was ~4 mM, with R2 of 0.998 (Figure S1). For low DIC concentration determination, both water and NaOH acceptors were applicable. In this study, water acceptor was used, because water can be better used than NaOH solution in the field.The detection limit of the DIC instrument with ultrapure water was 0.16 mM, based on the regression method, seen in Figure S2, which was a substantial improvement over the photometricJ. Mar. Sci. Eng. detection2020, 8, x FOR method PEER REVIEW (limitof detection, 0.42 mM) [16]. 5 of 11

2 10 y=(4.70E-5±4.56E-7)*x +(0.015±1.52E-4)*x R2=0.999

5 Standard concentration (mM) concentration Standard

0 100 200 300 μ Signal intensity ( s)

Figure 2. Correlation between signal intensity and standard solutionsolution concentration.concentration.

2.3. Comparisons with other DIC Determination Systems Simultaneous analyses of seawater samples from Xiamen coastal regions were performed with both C4D and EC detectors. H3PO4 solution with 0.2 mol/L and ultrapure water were used as carrier and acceptor streams, respectively, for both C4D and EC detection. The DIC concentrations detected using EC ranged from 1.91 to 1.96 mM, with an average of 1.94 ± 0.013 mM (n = 52, RSD 0.60%, Figure 3). The EC detection results were well consistent those determined using C4D, with an average of 1.93 ± 0.014 mM (n = 24, RSD 0.72%, Figure 3). The accuracy of the DIC instrument was also tested using standard sea water, seen in Figure S5. The standard sea water samples were measured three times to obtain the mean values of DIC concentration. The accuracy in standard seawater with 0.5 mM of DIC is about 0.002 mM, while the accuracy in standard seawater with 5 mM of DIC is about 0.008 mM. Measured DIC concentrations correlated well with the standard seawater DIC concentration. It indicated that the DIC detection with EC was accurate and precise.

2.2 C4D detection, RSD 0.72%, n=24 EC detection, RSD 0.60%,n=52 2.1

2.0

1.9

1.8 Concentration (mM) Concentration

1.7

1.6 2018/10/31 08:30 2018/10/31 10:30 2018/10/31 12:30

Date and time Figure 3.Comparison of DIC determination results with EC detection method and C4D detection method.

Four seawater samples with different DIC concentrations were analyzed using the titration detection method and the underway DIC measurement system. The measurement of DIC using

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2 10 y=(4.70E-5±4.56E-7)*x +(0.015±1.52E-4)*x R2=0.999

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The 1 mM standard solution was measured continuously to determine the repeatability (RSD%) Standard concentration (mM) concentration Standard performance of the instrument. The RSD for EC detection with an ultrapure water acceptor stream 0 was about 0.56% (1 mM, n = 21; Figure S1). The detection time for one sample was estimated to be 0 100 200 300 5 min (Figure S2), i.e., the system is capable of processing 12 samples per hour, which is suﬃcient for μ the underway observation of marine DIC, andSignal which intensity will provide ( s) DIC concentration data with high spatial and temporalFigure 2. resolution.Correlation between signal intensity and standard solution concentration.

2.3.2.3. ComparisonsComparisons withwith otherother DICDIC DeterminationDetermination SystemsSystems SimultaneousSimultaneous analysesanalyses ofof seawaterseawater samplessamples fromfrom XiamenXiamen coastalcoastal regionsregions werewere performedperformed withwith both C4D and EC detectors. H PO solution with 0.2 mol/L and ultrapure water were used as carrier both C4D and EC detectors. H33PO4 solution with 0.2 mol/L and ultrapure water were used as carrier 4 andand acceptoracceptor streams,streams, respectively,respectively, for for both both C CD4D andand EC EC detection. detection. TheThe DICDIC concentrationsconcentrations detecteddetected usingusing ECEC ranged from from 1.91 1.91 to to 1.96 1.96 mM, mM, with with an anaver averageage of 1.94 of 1.94 ± 0.0130.013 mM mM(n = 52, (n RSD= 52, 0.60%, RSD 0.60%,Figure ± 4 Figure3). The3 ).EC The detection EC detection results results were well were consistent well consistent those thosedetermined determined using usingC4D, with C D, an with average an average of 1.93 of 1.93 0.014 mM (n = 24, RSD 0.72%, Figure3). The accuracy of the DIC instrument was also tested ± 0.014± mM (n = 24, RSD 0.72%, Figure 3). The accuracy of the DIC instrument was also tested using usingstandard standard sea water, sea water, seen in seen Figure in Figure S5. The S5. standa Therd standard sea water sea samples water samples were measured were measured three times three to timesobtain to the obtain mean the values mean of values DIC concentration. of DIC concentration. The accuracy The in accuracy standard in seawater standard with seawater 0.5 mM with of DIC 0.5 mMis about of DIC 0.002 isabout mM, while 0.002 mM,the accuracy while the in accuracy standard in seawater standard with seawater 5 mM with of DIC 5 mM is about of DIC 0.008 is about mM. 0.008Measured mM. MeasuredDIC concentrations DIC concentrations correlated correlated well with well the with standard the standard seawater seawater DIC DIC concentration. concentration. It Itindicated indicated that that the the DIC DIC detection detection with with EC EC was was accurate accurate and and precise. precise.

2.2 C4D detection, RSD 0.72%, n=24 EC detection, RSD 0.60%,n=52 2.1

2.0

1.9

1.8 Concentration (mM) Concentration

1.7

1.6 2018/10/31 08:30 2018/10/31 10:30 2018/10/31 12:30

Date and time FigureFigure 3. 3.ComparisonComparison of of DIC DIC determination determination results results with with EC detection EC detection method method and C4D and detection C4D detection method. method. Four seawater samples with different DIC concentrations were analyzed using the titration detection methodFour and seawater the underway samples DIC with measurement different system.DIC concentrations The measurement were of analyzed DIC using using titration the detection titration hasdetection been described method and in detail the byunderway Johnsonn DIC (1985) measurement [12] and Ji (2002)system. [25 The]. Each measurement sample was of determined DIC using for five times by both instruments. The results obtained using the underway DIC system were well correlated with those obtained using the titration method (Figure4). The relationship between the two sets of measurements can be described using the following linear regression (R2 = 0.998; slope = 1.001).

2 DICED = (1.001 0.004)DICTR, R = 0.998 (1) ± J. Mar. Sci. Eng. 2020, 8, x FOR PEER REVIEW 6 of 11

titration detection has been described in detail by Johnsonn (1985) [12] and Ji (2002) [25]. Each sample was determined for five times by both instruments. The results obtained using the underway DIC system were well correlated with those obtained using the titration method (Figure 4). The relationship between the two sets of measurements can be described using the following linear regression (R2 = 0.998; slope = 1.001).

J. Mar. Sci. Eng. 2020, 8, 765 6 of 11 = 1.001 ± 0.004, = 0.998 (1)

Measurement 2.0 Average

1.5

1.0

EC detection (mM) EC detection Slope=1.001, R =0.998 0.5

0.0 0.0 0.5 1.0 1.5 2.0 Titration detection (mM)

FigureFigure 4.4. The relationship of DIC determinationdetermination betweenbetween titrationtitration detectiondetection andand thisthis study.study.

2.4.2.4. ShipboardShipboard MeasurmentsMeasurments OnboardOnboard DICDIC measurementsmeasurements werewere carriedcarried out by RR/V/V “Discovery” in Terengganu river estuary, MalaysiaMalaysia (Figure(Figure5 5).). The The TerengganuTerengganu River River basin basin is is situated situated on on the the east east coast coast ofof PeninsularPeninsular Malaysia.Malaysia. TheThe TerengganuTerengganu RiverRiver isis approximatelyapproximately 6565 kmkm inin lengthlength andand flowsflows fromfrom HuluHulu TerengganuTerengganu toto thethe mouthmouth ofof thethe river river at at Kuala Kuala Terengganu Terengganu and and discharges discharges into into the the southern southern part part of the of Souththe South China China Sea. TheSea. Terengganu The Terengganu Estuary Estuary (5.34◦ N, (5.3 103.134° N,◦ E) 103.13° is a sheltered E) is a estuary sheltere whered estuary the constructed where the breakwater constructed at thebreakwater river mouth at the shelters river mouth the estuary shelters from the direct estuary influence from direct of the influence open sea. of Additionally, the open sea. a Additionally, hydroelectric dama hydroelectric located at the dam upper located stretches at the of upper catchment stretches area of is catchment regulated byarea the is freshwater regulated runoby theff capacityfreshwater in therunoff estuary. capacity The tidalin the regime estuary. in thisThe area tidal is regime dominated in this by diurnalarea is dominated tide; with the by mean diurnal tidal tide; range with being the approximatelymean tidal range 1.60 being m, while approximately the tidal range 1.60 m, during while the the spring tidal range and neapduring tide the are spring 2.40 mand and neap 0.80 tide m, respectivelyare 2.40 m and [26 0.80]. The m, residence respectively time [26]. of TerengganuThe residence estuary time of is Teren relativelygganu short estuary (5–10 is h),relatively due to short high volume(5–10J. Mar. h),Sci. of dueEng. freshwater 2020to high, 8, x FORvolume discharged PEER of REVIEW freshwater upstream. discharged upstream. 7 of 11 In this study, DIC analyses were performed over 24 h while the ship was anchored in the Terengganu Estuary (at 5.34° N, 103.13° E). Temporal variability in carbon and nitrogen, as well as the magnitudes of the carbon and nitrogen biogeochemical fluxes, were measured during the diurnal tidal cycle. Water samples were pumped from the sea using a peristaltic pump deployed on the R/V “Discovery”. In addition, the seawater samples were collected using Niskin bottles. Asonde was used to measure temperature, salinity and pH, as well as chlorophyll-a.

FigureFigure 5.5. MapMap showingshowing thethe observationobservation regionregion onon thethe TerengganuTerengganu river river estuary, estuary, Malaysia. Malaysia.

3. ResultsIn this and study, Discussions DIC analyses were performed over 24 h while the ship was anchored in the Terengganu Estuary (at 5.34◦ N, 103.13◦ E). Temporal variability in carbon and nitrogen, as well as theApplication magnitudes of On of Board the carbon Sea Water and DIC nitrogen Observation biogeochemical ﬂuxes, were measured during the diurnal The DIC instrument was installed on R/V “Discovery” (University Malaysia Terengganu, Figure S6). DIC, pH and salinity were measured simultaneously during a diurnal tidal cycle from 4th to 5th August 2019. Surface sea water DIC and salinity, as well as the tidal levels, are illustrated in Figure 6. The large data gap in the field test was caused by the peristaltic pump deployed on the R/V “Discovery”, but was not caused by the DIC monitoring instrument. In this study, sampleswere pumped from the seawater using a peristaltic pump. However, the peristaltic pump broke down during the low tide. Hence we could not get the water samples during that period. Variations in DIC concentration were consistent with the variations in salinity and tidal levels in the estuary. DIC concentration profile well reflected the whole tidal cycle, indicating that the temporal resolution of the underway DIC observation instrument was sufficient for estuarine seawater analysis. High DIC levels, as well as salinity value were present in high tide. DIC concentrations and salinity values decreased from 1.98 mM to 0.51 mM and from 30.2 to 4.6, respectively, as the tidal level decreased from 2.1 m to 0.8 m. A similar relationship between DIC and salinity was also observed during a cruise on the Yarra River estuary, SE Australia, on 19 May 2011 [16]. They observed variations in salinity indicated that the Terengganu Estuary is dominated by seawater at high tide and by freshwater at low tide.

2.0 DIC 2 Tide 30 Salinity 1.5

20 1.0 1 Salinity Tidal levels (m)

DIC concentration (mM) DIC concentration 10 0.5

0.0 0 0 2019/8/4 09:00 2019/8/4 21:00 2019/8/5 09:00 Date and time Figure 6. Time series of DIC, salinity and tidal height determined during the observation in Kuala Terengganu on 4–5 August 2019.

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tidal cycle. Water samples were pumped from the sea using a peristaltic pump deployed on the R/V “Discovery”.Figure In 5. addition, Map showing the seawater the observation samples region were on collected the Terengganu using Niskin river estuary, bottles. Malaysia. Asonde was used to measure temperature, salinity and pH, as well as chlorophyll-a. 3. Results and Discussions 3. Results and Discussion Application of On Board Sea Water DIC Observation Application of On Board Sea Water DIC Observation The DIC instrument was installed on R/V “Discovery” (University Malaysia Terengganu, Figure S6). DIC,The DICpH and instrument salinity were was installed measured on simultaneously R/V “Discovery” during (University a diurnal Malaysia tidal cycle Terengganu, from 4th Figureto 5th AugustS6). DIC, 2019. pH Surface and salinity sea water were DIC measured and salinity, simultaneously as well as duringthe tidal a levels, diurnal are tidal illustrated cycle from in Figure 4th to 6.5th The August large 2019.data gap Surface in the sea field water test DIC was and caus salinity,ed by asthe well peristaltic as the tidalpump levels, deployed are illustrated on the R/V in “Discovery”,Figure6. The but large was data not gap caused in the by field the testDIC was monitoring caused by instrument. the peristaltic In this pump study, deployed sampleswere on the pumpedR/V “Discovery”, from the butseawater was not using caused a peristaltic by the DIC pu monitoringmp. However, instrument. the peristaltic In this study,pump sampleswerebroke down duringpumped the from low thetide. seawater Hence we using could a not peristaltic get the pump.water samples However, during the peristalticthat period. pump Variations broke in down DIC concentrationduring the low were tide. consistent Hence we with could the not variations get the waterin salinity samples and during tidal levels that period. in the estuary. Variations DIC in concentrationDIC concentration profile were well consistent reflected withthe whole the variations tidal cycle, in salinityindicating and that tidal the levels temporal in the resolution estuary. DIC of theconcentration underway profileDIC observation well reflected instrument the whole was tidal sufficient cycle, indicating for estuarine that theseawater temporal analysis. resolution High of DIC the levels,underway as well DIC as observation salinity value instrument were present was suffi incient high for tide. estuarine DIC co seawaterncentrations analysis. and Highsalinity DIC values levels, decreasedas well as salinityfrom 1.98 value mM were to 0.51 present mM and in high from tide. 30.2 DIC to 4.6, concentrations respectively, and as salinitythe tidal values level decreased decreased fromfrom 1.982.1 m mM to to0.8 0.51 m. mMA similar and from relationship 30.2 to 4.6, between respectively, DIC and as the salinity tidal levelwas also decreased observed from during 2.1 m toa cruise0.8 m. on A similarthe Yarra relationship River estuary, between SE DICAustralia, and salinity on 19 was May also 2011 observed [16]. They during observed a cruise variations on the Yarra in salinityRiver estuary, indicated SE Australia,that the onTerengganu 19 May 2011 Estuary [16]. Theyis dominated observed variationsby seawater in salinityat high indicatedtide and thatby freshwaterthe Terengganu at low Estuary tide. is dominated by seawater at high tide and by freshwater at low tide.

2.0 DIC 2 Tide 30 Salinity 1.5

20 1.0 1 Salinity Tidal levels (m)

DIC concentration (mM) DIC concentration 10 0.5

0.0 0 0 2019/8/4 09:00 2019/8/4 21:00 2019/8/5 09:00 Date and time FigureFigure 6. TimeTime series series of of DIC, salinity and tidal height determined during the observation in Kuala Terengganu on 4–5 August 2019.2019.

Surface sea water samples were also collected and analyzed at two hours intervals, simultaneously. These measurements were compared with the underway DIC analyses. During the measurement period, standard seawater samples (2.02 mM) were also measured automatically every two hours by the DIC instrument (Figure7), for the purposes of quality checking and data calibration.The analyses of the seawater standards during the observation period indicated that the instrument was performing well. Our results showed that the surface water DIC concentrations were consistent with those made underway (Figure7). Thus, the tidal cycle had a signiﬁcant e ﬀect on seawater DIC concentrations in the Terengganu Estuary. J. Mar. Sci. Eng. 2020, 8, x FOR PEER REVIEW 8 of 11 J. Mar. Sci. Eng. 2020, 8, x FOR PEER REVIEW 8 of 11 Surface sea water samples were also collected and analyzed at two hours intervals, simultaneously.Surface sea These water measurements samples were were also compar collecteded with and the underwayanalyzed DICat two analyses. hours During intervals, the measurementsimultaneously. period, These standard measurements seawater were samples compar (2.02ed mM) with werethe underway also measured DIC analyses.automatically During every the twomeasurement hours by period,the DIC standard instrument seawater (Figure samples 7), fo(2.02r the mM) purposes were also of measured quality checking automatically and everydata calibration.Thetwo hours by analysesthe DIC of instrument the seawater (Figure standards 7), foduringr the thepurposes observation of quality period checking indicated and that datathe instrumentcalibration.The was performinganalyses of well.the seawater Our results standards showed during that the the surface observation water DICperiod concentrations indicated that were the consistentinstrument with was thoseperforming made well.underway Our results (Figure showed 7). Thus, that the the tidalsurface cycle water had DIC a significant concentrations effect were on seawaterJ.consistent Mar. Sci. Eng.DIC with2020 concentrations ,those8, 765 made inunderway the Terengganu (Figure Estuary.7). Thus, the tidal cycle had a significant effect8 of on 11 seawater DIC concentrations in the Terengganu Estuary.

2.0 2.0 Underway determination Standard Underway sea determination water Surface Standard sea sea water water samples 1.5 Surface sea water samples 1.5

1.0 1.0 DIC concentration (mM) DIC concentration (mM) 0.5 0.5

2019/8/4 09:00 2019/8/4 21:00 2019/8/5 09:00 2019/8/4 09:00 2019/8/4Date 21:00 and time 2019/8/5 09:00 Date and time Figure 7. Relationship between surface sea water samples DIC detection and underway DIC observationFigureFigure 7. 7.Relationship Relationship results. between between surface surface sea watersamples sea water DIC samples detection DIC and underwaydetection DICand observation underway results. DIC observation results. 2 Strong positive linear correlation between DICDIC concentration andand salinitysalinity waswas presentpresent ((RR2 = 0.95,0.95, FigureStrong 88),), indicatingindicating positive thatlinearthat DICDIC correlation concentrationsconcentrations between inin DI thethCe concentration estuarineestuarine areaarea and werewere salinity determineddetermined was present byby thethe (R raterate2 = 0.95, ofof mixingFigure between 8), indicating seawater that andDIC freshwater. concentrations in the estuarine area were determined by the rate of mixing between seawater and freshwater.

2.0 2.0

1.5 1.5

1.0 1.0 Slope=0.04, R2=0.95 Slope=0.04, R2=0.95 DIC concentration (mM) concentration DIC

DIC concentration (mM) concentration DIC 0.5 0.5

0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 Salinity Salinity FigureFigure 8. CorrelationCorrelation between between DIC DIC and and salinity. salinity. Figure 8. Correlation between DIC and salinity. To further clarify the impact of tidal level on seawater DIC, water samples were collected at variousTo depths further (0.5, clarify 3.0, and the impact5.0 m) over of tidal two-hour level onintervals. seawater Each Each DIC, water water water sample sample samples was was determined determinedwere collected three three at times,various and depths the mean (0.5, 3.0,of the and three 5.0 m) measurements over two-hour was intervals. retained. Each The water variations sample in wasDIC determinedconcentration three at differentditimes,ﬀerent and depthsdepths the mean werewere of associatedassociated the three with withmeasurements tidal tidal level. level. DICwas DIC concentrationsretained. concentrations The variations were were greatest greatest in DIC at at aconcentration deptha depth of 5.0of 5.0 m, at followeddifferent bydepths those were at a depth associated of 3.0 mwith and tidal those level. at a depthDIC concentrations of 0.5 m (Figure were9). This greatest indicated at a depth the relative of 5.0 impact of seawater increased with water depth. Please note that the discrepancy in DIC concentrations between the surface water and bottom water was small at high tide, but DIC concentration in the bottom water was much greater than that in the surface water at low tide. This indicated that in the estuary, seawater stratiﬁcation was obvious at low tide, but freshwater and seawater were well mixed at high tide. J. Mar. Sci. Eng. 2020, 8, x FOR PEER REVIEW 9 of 11

m, followed by those at a depth of 3.0 m and those at a depth of 0.5 m (Figure 9). This indicated the relative impact of seawater increased with water depth. Please note that the discrepancy in DIC concentrations between the surface water and bottom water was small at high tide, but DIC concentration in the bottom water was much greater than that in the surface water at low tide. This indicated that in the estuary, seawater stratification was obvious at low tide, but freshwater and J.seawater Mar. Sci. Eng.were2020 well, 8, 765 mixed at high tide. 9 of 11

0.5m 2.0 3.0m 2 5.0m Tide

1.5

1.0 1 Tidal levels (m)

DICconcentration (mM) 0.5

0.0 0 2019/8/4 09:00 2019/8/4 19:00 2019/8/5 05:00 Date and time

Figure 9. Observations of DIC samples at different water depth. Figure 9. Observations of DIC samples at different water depth. 4. Conclusions 4.Conclusions An underway DIC measurement instrument was developed using gas-diffusion flow in combination withAn electrical underway conductivity DIC measurement (EC) detector, whichinstrument was suitable was developed for the measurement using gas-diffusion of DIC in estuarineflow in waterscombination with highwith spatial electrical and conductivity temporal resolution. (EC) detector, A microvolume which was flowsuitable technique for the withmeasurement 100 µL was of achievedDIC in estuarine for DIC determination. waters with high A high spatial efficiency and AF2400temporal membrane resolution. gas-diffusion A microvolume device wasflow constructed. technique Onlywith 100 an acid µL was carrier achieved stream for and DIC an ultrapuredetermination. water acceptorA high efficiency stream were AF2400 required membrane for the DICgas-diffusion detection. Thedevice limits was of constructed. detection for theOnly DIC an instrumentacid carrier with stream ultrapure and an water ultrapure was 0.16 water mM. acceptor Repeatability stream was were also high,required with for an the RSD DIC of 0.56% detection. (1 mM, The n =limits21). Theof detection detection for time the for DIC one instrument sample was with estimated ultrapure to be 5water min, equivalentwas 0.16 mM. to approximately Repeatability 12was samples also high, per with hour. an The RSD instrument of 0.56% switches(1 mM, n automatically = 21). The detection between time the standardfor one sample solution was and estimated seawater samplesto be 5 min, during equi underwayvalent to use. approximately High accuracy 12 andsamples precision per ofhour. the DICThe observationinstrument instrumentswitches automatically provides high between spatial and the temporal standard resolution solution DIC and measurements. seawater samples during underwayThe underway use. High DIC accuracy instrument and precision was used of to the measure DIC observation seawater DIC instrument in the Terengganu provides river high estuary, spatial Malaysia.and temporal This resolution method was DIC used measurements. to obtain DIC data with high temporal resolution, and to clarify the impactThe of underway the diurnal DIC tidal instrument cycle on seawater was used DIC. to Changes measure in seawater DIC concentrations DIC in the were Terengganu well correlated river withestuary, variations Malaysia. in salinity This method and tidal wa levels used in to the obtain estuary. DIC The data DIC with concentration high temporal profile resolution, obtained and in this to studyclarify accurately the impact reflected of the diurnal the entire tidal tidal cycle cycle, on indicating seawater that DIC. the Changes temporal in resolution DIC concentrations of the underway were DICwell analysiscorrelated instrument with variations was su inffi salinitycient for and DIC tidal detection level in in the estuarine estuary. waters. The DIC concentration profile obtained in this study accurately reflected the entire tidal cycle, indicating that the temporal Supplementaryresolution of the Materials: underwayThe DIC following analysis are availableinstrument online was at http:sufficient//www.mdpi.com for DIC detection/2077-1312 in/8 /estuarine10/765/s1, Figure S1: Correlation between DIC concentration and Signal intensity, Figure S2: The signal spectrum of the DIC instrumentwaters. of different DIC concentration, Figure S3: Time series of repeatability detection with standard samples, Figure S4: Spectrograms of the repeatability detection with 1mM standard sample, Figure S5: Measurement resultsSupplementary of standard Materials: seawaterThe using following the DIC are instrument, available Figureonline S6:at www.mdpi.com/xxx/s1, Application of the underway Figure DIC S1: detectionCorrelation in estuarinebetween DIC waters. concentration and Signal intensity, Figure S2: The signal spectrum of the DIC instrument of Authordifferent Contributions: DIC concentration,Conceptualization, Figure S3: Time J.Y. series and of Q.L.; repeatability methodology, detection J.Y.; validation, with standard Q.L., samples, and S.C.P.; Figure formal S4: analysis,Spectrograms J.Y.; investigation,of the repeatability J.Y.; resources, detection S.C.P. with and 1mM L.Z.; standard data curation, sample, Y.L. Figure and S.P.;S5: Measurement writing—original results draft of preparation, J.Y.; writing—review and editing, J.Y. and S.C.P.; project administration, J.Y.; funding acquisition, J.Y. and L.Z. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by “Qingdao National Laboratory for marine science and technology, grant number QNLM2016ORP0109” and “Project of Xiamen Development Institute, grant number K200602”, and China-ASEAN Maritime Cooperation Fund Project “Monitoring and conservation of the coastal ecosystem in the South China Sea”. J. Mar. Sci. Eng. 2020, 8, 765 10 of 11

Acknowledgments: The authors gratefully acknowledge R/V “Discovery” for the assistance of onboard DIC monitoring. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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