Study of cork (from Quercus suber L.)-wine model interactions based on voltammetric multivariate analysis

The cork from Quercus suber L. is the premium raw material used to produce wine-bottling stoppers; however, being a natural product, cork can be contaminated and attacked in different ways that could promote differences in its proprieties. A specific contamination is the defect known in the industry as “Mancha Amarela—Yellow spot” (MA). This cork shows modifications in its mechanical, structural and optical properties and is potentially able to cause off-flavours in wine. In this study, a new analytical approach is proposed for the rapid screening of cork-wine model interactions in order to determine if the cork were able to contaminate a wine. Cork samples classified as standard (S) and cork MA were put in contact with a wine model matrix. The model matrix was analysed by cyclic voltammetry and square wave voltammetry in function of different contact times. Considering that matrices in contact with cork S and MA exhibited different pH, the influence of this parameter on the distinction power was also evaluated. The data sets obtained from the different techniques were treated by principal component analysis (PCA) and PLS_Cluster. The major difference between samples S and MA is the occurrence of an important peak at ca. 580 mV in the MA voltammograms. This more positive peak may be assigned to lignin related phenolics; therefore, it can be proposed as a possible marker to follow lignin degradation.     

A principal component analysis based method to discover chemical differences in comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (GCxGC-TOFMS) separations of metabolites in plant samples

Comprehensive two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GC × GC-TOFMS) provides high resolution separations of complex samples with a mass spectrum at every point in the separation space. The large volumes of multidimensional data obtained by GC × GC-TOFMS analysis are analyzed using a principal component analysis (PCA) method described herein to quickly and objectively discover differences between complex samples. In this work, we submitted 54 chromatograms to PCA to automatically compare the metabolite profiles of three different species of plants, namely basil (Ocimum basilicum), peppermint (Mentha piperita), and sweet herb stevia (Stevia rebaudiana), where there were 18 chromatograms for each type of plant. The 54 scores of the m/z 73 data set clustered in three groups according to the three types of plants. Principal component 1 (PC 1) separated the stevia cluster from the basil and peppermint clusters, capturing 61.84% of the total variance. Principal component 2 (PC 2) separated the basil cluster from the peppermint cluster, capturing 16.78% of the total variance. The PCA method revealed that relative abundances of amino acids, carboxylic acids, and carbohydrates were responsible for differentiating the three plants. A brief list of the 16 most significant metabolites is reported. After PCA, the 54 scores of the m/z 217 data set clustered in three groups according to the three types of plants, as well, yielding highly loaded variables corresponding with chemical differences between plants that were complementary to the m/z 73 information. The PCA data mining method is applicable to all of the monitored selective mass channels, utilizing all of the collected data, to discover unknown differences in complex sample profiles.     

Optimization of two-dimensional gas chromatography time-of-flight mass spectrometry for separation and estimation of the residues of 160 pesticides and 25 persistent organic pollutants in grape and wine

Two-dimensional gas chromatography (GC × GC) coupled with time-of-flight mass spectrometric (TOFMS) method was optimized for simultaneous analysis of 160 pesticides, 12 dioxin-like polychlorinated biphenyls (PCBs), 12 polyaromatic hydrocarbons (PAHs) and bisphenol A in grape and wine. GC × GC–TOFMS could separate all the 185 analytes within 38 min with >85% NIST library-based mass spectral confirmations. The matrix effect quantified as the ratio of the slope of matrix-matched to solvent calibrations was within 0.5–1.5 for most analytes. LOQ of most of the analytes was ≤10 μg/L with nine exceptions having LOQs of 12.5–25 μg/L. Recoveries ranged between 70 and 120% with <20% expanded uncertainties for 151 and 148 compounds in grape and wine, respectively, with intra-laboratory Horwitz ratio <0.2 for all analytes. The method was evaluated in the incurred grape samples where residues of cypermethrin, permethrin, chlorpyriphos, metalaxyl and etophenprox were detected at below MRL.     

Trilinearity deviation ratio: A new metric for chemometric analysis of comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry data

Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC × GC–TOFMS) is a well-established instrumental platform for complex samples. However, chemometric data analysis is often required to fully extract useful information from the data. We demonstrate that retention time shifting from one modulation to the next, Δ²t_R, is not sufficient alone to quantitatively describe the trilinearity of a single GC × GC–TOFMS run for the purpose of predicting the performance of the chemometric method parallel factor analysis (PARAFAC). We hypothesize that analyte peak width on second dimension separations, ²W_b, also impacts trilinearity, along with Δ²t_R. The term trilinearity deviation ratio, TDR, which is Δ²t_R normalized by ²W_b, is introduced as a quantitative metric to assess accuracy for PARAFAC of a GC × GC–TOFMS data cube. We explore how modulation ratio, M_R, modulation period, P_M, temperature programming rate, T_ramp, sampling phase (in-phase and out-of-phase), and signal-to-noise ratio, S/N, all play a role in PARAFAC performance in the context of TDR. Use of a P_M in the 1–2 s range provides an optimized peak capacity for the first dimension separation (500–600) for a 30 min run, with an adequate peak capacity for the second dimension separation (12–15), concurrent with an optimized two-dimensional peak capacity (6000–7500), combined with sufficiently low TDR values (0–0.05) to facilitate low quantitative errors with PARAFAC (0–0.5%). In contrast, use of a P_M in the 5 s or greater range provides a higher peak capacity on the second dimension (30–35), concurrent with a lower peak capacity on the first dimension (100–150) for a 30 min run, and a slightly reduced two-dimensional peak capacity (3000–4500), and furthermore, the data are not sufficiently trilinear for the more retained second dimension peaks in order to directly use PARAFAC with confidence.     

Comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry combined with solid phase microextraction as a powerful tool for quantification of ethyl carbamate in fortified wines. The case study of Madeira wine

An analytical methodology based on headspace solid phase microextraction (HS-SPME) combined with comprehensive two-dimensional gas chromatography—time-of-flight mass spectrometry (GC × GC–ToFMS) was developed for the identification and quantification of the toxic contaminant ethyl carbamate (EC) directly in fortified wines. The method performance was assessed for dry/medium dry and sweet/medium sweet model wines, and for quantification purposes, calibration plots were performed for both matrices using the ion extraction chromatography (IEC) mode (m/z 62). Good linearity was obtained with a regression coefficient (r²) higher than 0.981. A good precision was attained (R.S.D. <20%) and low detection limits (LOD) were achieved for dry (4.31 μg/L) and sweet (2.75 μg/L) model wines. The quantification limits (LOQ) and recovery for dry wines were 14.38 μg/L and 88.6%, whereas for sweet wines were 9.16 μg/L and 99.4%, respectively. The higher performance was attainted with sweet model wine, as increasing of glucose content improves the volatile compound in headspace, and a better linearity, recovery and precision were achieved. The analytical methodology was applied to analyse 20 fortified Madeira wines including different types of wine (dry, medium dry, sweet, and medium sweet) obtained from several harvests in Madeira Island (Portugal). The EC levels ranged from 54.1 μg/L (medium dry) to 162.5 μg/L (medium sweet).     

Analysis of bacterial fatty acids by flow modulated comprehensive two-dimensional gas chromatography with parallel flame ionization detector/mass spectrometry

Comprehensive two-dimensional gas chromatography (GC × GC) offers an interesting tool for profiling bacterial fatty acids. Flow modulated GC × GC using a commercially available system was evaluated, different parameters such as column flows and modulation time were optimized. The method was tested on bacterial fatty acid methyl esters (BAMEs) from Stenotrophomonas maltophilia LMG 958^T by using parallel flame ionization detector (FID)/mass spectrometry (MS). The results are compared to data obtained using a thermal modulated GC × GC system. The data show that flow modulated GC × GC-FID/MS method can be applied in a routine environment and offers interesting perspectives for chemotaxonomy of bacteria.     

Phase equilibria of liquid (water + butyric acid + oleyl alcohol) ternary system

(Liquid + liquid) equilibrium (LLE) data for the ternary system of (water + butyric acid + oleyl alcohol) at T = (298.15, 308.15, and 318.15) K are reported. Complete phase diagrams were obtained by determining solubility and the tie-line data. The reliability of the experimental tie lines was confirmed by using Othmer-Tobias correlation. The UNIFAC method was used to predict the phase equilibrium data. The phase diagrams for the ternary mixtures including both the experimental and correlated tie lines are presented. Distribution coefficients and separation factors were evaluated for the immiscibility region. A comparison of the solvent extracting capability was made with respect to distribution coefficients, separation factors, and solvent-free selectivity bases for T = (298.15, 308.15, and 318.15) K. It is concluded that oleyl alcohol may serve as an adequate solvent to extract butyric acid from its dilute aqueous solutions.     

Characterization of new types of stationary phases for fast and ultra-fast liquid chromatography by signal processing based on AutoCovariance Function: A case study of application to Passiflora incarnata L. extract separations

In this study, a comparative investigation was performed of HPLC Ascentis^® (2.7 μm particles) columns based on fused-core particle technology and Acquity^® (1.7 μm particles) columns requiring UPLC instruments, in comparison with Chromolith™ RP-18e columns. The study was carried out on mother and vegetal tinctures of Passiflora incarnata L. on one single or two coupled columns. The fundamental attributions of the chromatographic profiles are evaluated using a chemometric procedure, based on the AutoCovariance Function (ACVF). Different chromatographic systems are compared in terms of their separation parameters, i.e., number of total chemical components (m_tot), separation efficiency (σ), peak capacity (n_c), overlap degree of peaks and peak purity. The obtained results show the improvements achieved by HPLC columns with narrow size particles in terms of total analysis time and chromatographic efficiency: comparable performance are achieved by Ascentis^® (2.7 μm particle) column and Acquity^® (1.7 μm particle) column requiring UPLC instruments. The ACVF plot is proposed as a simplified tool describing the chromatographic fingerprint to be used for evaluating and comparing chemical composition of plant extracts by using the parameters D% – relative abundance of the deterministic component – and c_EACF – similarity index computed on ACVF.     

Profiling food volatiles by comprehensive two-dimensional ga schromatography coupled with mass spectrometry: Advanced fingerprinting approaches for comparative analysis of the volatile fraction of roasted hazelnuts (Corylus avellana L.) from different origins

This study examined how advanced fingerprinting methods (i.e., non-targeted methods) provide reliable and specific information about groups of samples based on their component distribution on the GC × GC chromatographic plane. The volatile fractions of roasted hazelnuts (Corylus avellana L.) from nine different geographical origins, comparably roasted for desirable flavor and texture, were sampled by headspace-solid phase micro extraction (HS-SPME) and then analyzed by GC × GC-qMS. The resulting patterns were processed by: (a) “chromatographic fingerprinting”, i.e., a pattern recognition procedure based on retention-time criteria, where peaks correspondences were established through a comprehensive peak pattern covering the chromatographic plane; and (b) “comprehensive template matching” with reliable peak matching, where peak correspondences were constrained by retention time and MS fragmentation pattern similarity criteria. Fingerprinting results showed how the discrimination potential of GC × GC can be increased by including in sample comparisons and correlations all the detected components and, in addition, provide reliable results in a comparative analysis by locating compounds with a significant role. Results were completed by a chemical speciation of volatiles and sample profiling was extended to known markers whose distribution can be correlated to sensory properties, geographical origin, or the effect of thermal treatment on different classes of compounds. The comprehensive approach for data interpretation here proposed may be useful to assess product specificity and quality, through measurable parameters strictly and consistently correlated to sensory properties and origin.     

Comprehensive two-dimensional gas chromatography improves separation and identification of anabolic agents in doping control

The application of comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC × GC-TOFMS) for the analysis of six anabolic agents (AAs) in doping control is investigated in this work. A non-polar–polar column configuration with 0.2 μm film thickness (d_f) second dimension (²D) column was employed, offering much better spread of the components on ²D when compared to the alternative 0.1 μm d_f²D column. The proposed method was tested on the “key” AA that the World Anti-Doping Agency (WADA) had listed at the low ng mL⁻¹ levels (clenbuterol, 19-norandrosterone, epimethendiol, 17α-methyl-5α-androstane-3α,17β-diol, 17α-methyl-5β-androstane-3α,17β-diol and 3′-OH-stanozolol). The compounds were spiked in a blank urine extract obtained by solid-phase extraction, hydrolysis and liquid–liquid extraction; prior to analysis they were converted to the corresponding trimethylsilyl (TMS) derivatives. The limit of detection (LOD) was below or equal to the minimum required performance limit (MRPL) of 2 ng mL⁻¹ defined by WADA, and the correlation coefficient was in the range from 0.995 to 0.999. The method allows choosing an ion from the full mass spectra which shows the least interference from the matrix and/or the best sensitivity; this can only be done if full scan mass spectral data are available. The advantage of GC × GC over classical one-dimensional GC (1D GC), in terms of separation efficiency and sensitivity, is demonstrated on a positive urine control sample at a concentration of 5 ng mL⁻¹. The obtained similarity to the in-house created TOFMS spectra library at this level of concentration was in the range from 822 to 932 (on the scale from 0 to 999). Since full mass spectral information are recorded, the method allows the retro-search of non-target compounds or new “designer steroids”, which cannot be detected with established GC–MS methods that use selected ion monitoring (SIM) mode.     

Study of azeotropic mixtures with the advanced distillation curve approach

Classical methods for the study of complex fluid phase behavior include static and dynamic equilibrium cells that usually require vapor and liquid recirculation. These are sophisticated, costly apparatus that require highly trained operators, usually months of labor-intensive work per mixture, and the data analysis is also rather complex. Simpler approaches to the fundamental study of azeotropes are highly desirable, even if they provide only selected cuts through the phase diagram. Recently, we introduced an advanced distillation curve measurement method featuring: (1) a composition explicit data channel for each distillate fraction (for both qualitative and quantitative analysis), (2) temperature measurements that are true thermodynamic state points that can be modeled with an equation of state, (3) temperature, volume and pressure measurements of low uncertainty suitable for equation of state development, (4) consistency with a century of historical data, (5) an assessment of the energy content of each distillate fraction, (6) trace chemical analysis of each distillate fraction, and (7) corrosivity assessment of each distillate fraction. We have applied this technique to the study of azeotropic mixtures, for which this method provides the bubble point temperature and dew point composition, completely defining the thermodynamic state from the Gibbs phase rule perspective. In this paper, we present the application of the approach to several simple binary azeotropic mixtures: ethanol + benzene, 2-propanol + benzene, and acetone + chloroform.     

Vibrational spectra and structural parameters of some XNCO and XOCN (X = H, F, Cl, Br) molecules

Ab initio calculations with full electron correlation by the perturbation method to second order and hybrid density functional theory calculations by the B3LYP method utilizing the 6-31G(d), 6-311+G(d, p), and 6-311+G(2d, 2p) basis sets have been carried out for the XNCO and XOCN (X = H, F, Cl, Br) molecules. From these calculations, force constants, vibrational frequencies, infrared intensities, Raman activities, depolarization ratios, and structural parameters have been determined and compared to the experimental quantities when available. By combining previously reported rotational constants for HNCO, ClNCO and BrNCO with the ab initio MP2/6-311+G(d, p) predicted structural values, adjusted r₀ parameters have been obtained. The r₀ values for BrNCO are: r(BrN) = 1.857(5); r(NC) = 1.228(5); r(CO) = 1.161(5) Å; BrNC = 117.5(5) and NCO = 172.3(5)°. For ClNCO the determined r₀ parameters are in excellent agreement with the previously determine r_s values, whereas those for HNCO the HNC angle is larger with a value of 126.3(5)° compared to the previous reported value of 123.9(17)°. However, considering the relatively large uncertainty in the value given initially the two results are in near agreement. Structural parameters are also estimated for FNCO and XOCN (X = H, F, Cl, Br). The centrifugal distortion constants have been calculated and are compared to the experimentally (XNCO: X = H, Cl, Br) determined values. Predicted values for the barriers of linearity are given for both the XNCO (X = H, F, Cl, Br) molecules and the results were compared to the corresponding isothiocyanate molecules. The predicted frequencies for the fundamentals of the XNCO molecules compare favorably to the experimental values but some of the predicted intensities differ significantly from those in the observed spectra. The two OCN bends for HOCN have been assigned and the frequencies for the two corresponding fundamentals of DOCN are predicted.     

Optimization of ultrasound assisted-emulsification-dispersive liquid-liquid microextraction by experimental design methodologies for the determination of sulfur compounds in wines by gas chromatography-mass spectrometry

A new method was developed for analyzing sulfur compounds in the aroma of white wines using ultrasound assisted-emulsification-dispersive liquid-liquid microextraction coupled with gas chromatography-mass spectrometry detection. In the present work, the analytical method for simultaneous determination of seven sulfur compounds (methylmercaptoacetate, methyl(methylthio)acetate, 2-methylthioethanol, 3-methylthiopropanol, 3-methylthiohexanol, 4-methylthio-4-methyl-2-pentanone and hexanethiol) is reported. Parameters that affect the efficiency of the methodology such as extracting and dispersing solvents, sample volume, ion strength, cavitation time and centrifugation time were investigated using a fractionated factorial 2^6-1 (R = V) screening design. Then, the factors presenting significant positive effects on the analytical response (extracting volume, ion strength, cavitation time and centrifugation time) were considered in a further central composite design to optimize the operational conditions for the ultrasound assisted-emulsification-dispersive liquid-liquid microextraction procedure. Additionally, multiple response simultaneous optimization by using the desirability function was used to find the optimum experimental conditions. The best results were obtained using pH sample 4.25, extractant volume 150 μL, ionic strength 8.75% NaCl, cavitation time 20 s and centrifugation time 50 s. The use of the optimized ultrasound assisted-emulsification-dispersive liquid-liquid microextraction technique allowed to obtain the best extraction results with the minimum interference from other substances from the matrix, and it allowed to quantify the analytes in white wine samples by calibration graphs. Recoveries ranging from 91.99% to 125.87% for all sulfur compounds proved the accuracy of the proposed method in white wine samples. Method detection limits were in the range of 0.36-1.67 ng mL⁻¹ and limits of quantitation were between 0.63 and 3.02 ng mL⁻¹ for sulfur compounds in white wine samples. The proposed methodology was successfully applied for the determination concentrations of sulfur compounds in different commercial Chardonnay wine samples from Mendoza, Argentine.     

Supercritical fluid chromatography hyphenated with twin comprehensive two-dimensional gas chromatography for ultimate analysis of middle distillates

This paper reports the conditions of online hyphenation of supercritical fluid chromatography (SFC) with twin comprehensive two-dimensional gas chromatography (twin-GC × GC) for detailed characterization of middle distillates; this is essential for a better understanding of reactions involved in refining processes. In this configuration, saturated and unsaturated compounds that have been fractionated by SFC are transferred on two different GC × GC columns sets (twin-GC × GC) placed in the same GC oven. Cryogenic focusing is used for transfer of fractions into the first dimension columns before simultaneous GC × GC analysis of both saturated and unsaturated fractions. The benefits of SFC–twin-GC × GC are demonstrated for the extended alkane, iso-alkane, alkene, naphthenes and aromatics analysis (so-called PIONA analysis) of diesel samples which can be achieved in one single injection. For that purpose, saturated and unsaturated compounds have been separated by SFC using a silver loaded silica column prior to GC × GC analysis. Alkenes and naphthenes are quantitatively recovered in the unsaturated and saturated fractions, respectively, allowing their identification in various diesel samples. Thus, resolution between each class of compounds is significantly improved compared to a single GC × GC run, and for the first time, an extended PIONA analysis of diesel samples is presented.     

Qualitative drug analysis of hair extracts by comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry

A technique using comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC × GC/TOFMS) is applied to a qualitative analysis of three sample extracts from hair suspected of containing various drug compounds. The samples were also subjected to a quantitative target analysis for codeine, morphine, 6-monoacetylmorphine (6-MAM), amphetamine, methamphetamine, methylenedioxyamphetamine (MDA), methylenedioxymethylamphetamine (MDMA), methadone, and benzylpiperazine (BZP) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). GC × GC/TOFMS provided a non-specific procedure that identified various drugs, metabolites, and impurities not included in the target analysis. They included cocaine, diazepam, and methaqualone (quaalude). Comprehensive GC × GC separation was achieved using twin-stage cryo-modulation to focus eluant from a DB-5ms (5% phenyl) to a BPX50 (50% phenyl) GC column. The TOF mass spectrometer provided unit mass resolution in the mass range m/z 5–1000 and rapid spectral acquisition (≤500 spectra/s). Clean mass spectra of the individual components were obtained using mass spectral deconvolution software. The ‘unknown’ components were identified by comparison with mass spectra stored in a library database.     

Solubilities of betulin in chloroform + methanol mixed solvents at T = (278.2, 288.2, 293.2, 298.2, 308.2 and 313.2) K

Experimental solubilities of betulin in mixed solvents of chloroform (1) + methanol (2) were determined at T = (278.2, 288.2, 293.2, 298.2, 308.2 and 313.2) K. The solubilities of betulin in mixed solvents of chloroform (1) + methanol (2) increase with increasing of temperature. The curves of solubility versus solvent composition on solute-free basis went through a maximum. Experimental data of solubilities were correlated with a three-parameter equation. In addition, three crystals of betulin obtained in different compositions of chloroform (1) + methanol (2) mixtures were characterized by scanning electron microscope (SEM) and differential scanning calorimetry (DSC).     

Synthesis and reactions of silicon containing cyclic α-amino acid derivatives

A simple synthesis of a new amino acid derivative 5,6-bis(trimethylsilyl)indanylglycine via cobalt mediated [2 + 2 + 2] cycloaddition strategy is described. Co-trimerization of diyne building block containing amino acid moiety with bis(trimethylsilyl)acetylene in presence of CpCo(CO)₂ catalyst afforded the silylated indane-based α-amino acid (AAA) derivative. Electrophilic aromatic substitution reaction, ipso to the trimethylsilyl group gave highly functionalised indane-based AAA derivatives.     

Excess enthalpies and (vapour + liquid) equilibrium data for the binary mixtures of dimethylsulphoxide with ketones

Excess enthalpies (H^E), at ambient pressure and T = 298.15 K, have been measured by using a solution calorimeter for the binary liquid mixtures of dimethyl sulphoxide (DMSO) with ketones, as a function of composition. The ketones chosen in the present investigation were methyl ethyl ketone (MEK), methyl isobutyl ketone (MIBK), and cyclohexanone (CH). The H^E values are positive over the entire composition range for the three binary mixtures. Furthermore, the (vapour + liquid) equilibrium (VLE) was measured at 715 Torr for these mixtures, of different compositions, with the help of Swietoslawski-ebulliometer. The experimental temperature-mole fraction (t-x) data were used to compute Wilson parameters and then used to calculate the equilibrium vapour-phase compositions as well as the theoretical points for these binary mixtures. These Wilson parameters are used to calculate activity coefficients (γ) and these in turn to calculate excess Gibbs free energy (G^E). The intermolecular interactions and structural effects were analyzed on the basis of the measured and derived properties.     

Improvement of the analysis of the biochemical oxygen demand (BOD) of Mediterranean seawater by seeding control

Biochemical oxygen demand (BOD) is a useful parameter for assessing the biodegradability of dissolved organic matter in water. At the same time, this parameter is used to evaluate the efficiency with which certain processes remove biodegradable natural organic matter (NOM). However, the values of BOD in seawater are very low (around 2 mg O₂ L⁻¹) and the methods used for its analysis are poorly developed. The increasing attention given to seawater desalination in the Mediterranean environment, and related phenomena such as reverse osmosis membrane biofouling, have stimulated interest in seawater BOD close to the Spanish coast. In this study the BOD analysis protocol was refined by introduction of a new step in which a critical quantity of autochthonous microorganisms, measured as adenosine triphosphate, is added. For the samples analyzed, this improvement allowed us to obtain reliable and replicable BOD measurements, standardized with solutions of glucose-glutamic acid and acetate. After 7 days of analysis duration, more than 80% of ultimate BOD is achieved, which in the case of easily biodegradable compounds represents nearly a 60% of the theoretical oxygen demand. BOD₇ obtained from the Mediterranean Sea found to be 2.0 ± 0.3 mg O₂ L⁻¹ but this value decreased with seawater storage time due to the rapid consumption of labile compounds. No significant differences were found between two samples points located on the Spanish coast, since their organic matter content was similar. Finally, the determination of seawater BOD without the use of inoculum may lead to an underestimation of BOD.