ISO/DIS 18191:2025(en)

ISO/TC 147/SC 2

Secretariat: DIN

Date: 2025-04-22

Water quality — Determination of pH_T in sea water — Method using the indicator dye m-cresol purple

Qualité de l'eau — Détermination du pH_T dans l'eau de mer — Méthode utilisant l'indicateur coloré au pourpre de m-crésol

© ISO 2025

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Contents Page

Foreword iv

Introduction v

6.1 Flexible drawing tube 4

6.2 Spectrophotometric cell 4

6.3 Micropipette 4

6.4 High‑quality spectrophotometer 4

6.5 Temperature-control system for spectrophotometer cell 4

6.6 System to warm samples to measurement temperature 4

6.7 Thermostat bath 4

9.1 Correction of measured absorbances 5

9.2 Calculation of the pH_T of the sea water and indicator 6

Annex A (informative) Performance data 8

A.1 Interlaboratory comparison on TRIS buffer and natural sea water 8

A.2 Interlaboratory comparison on North Pacific sea water 10

Annex B (informative) Storage stability 12

Bibliography 13

Foreword

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Introduction

The greenhouse effect induced by anthropogenic carbon dioxide, CO₂, in the atmosphere is one of the serious global environmental issues. A key factor controlling the atmospheric CO₂ is its absorption into the ocean. As a result of the absorption, the pH in the upper layer of the ocean is observed to have fallen gradually, and its influence on the living organisms is a matter of concern all over the world.

On the other hand, carbon capture and storage (CCS) technology is considered as a useful means of reducing the CO₂ emissions from fossil fuel. When ocean environment such as sub-seabed aquifer is selected as a storage site, the monitoring of carbonate system including pH in sea water becomes very important. The analytical method for pH_T in sea water (the total hydrogen ion concentration pH scale) samples requires specific conditions and techniques essential to the precise and accurate determination. This International Standard describes a method for the determination of pH_T in sea water with the repeatability less than 0,003.

This method will provide international communities accurate data sets on pH_T in sea water being compatible with each other. This is the base of national and international operational observation or monitoring programs of the oceanic carbonate system as well as individual research works.

Water quality — Determination of pH_T in sea water — Method using the indicator dye m-cresol purple

WARNING — Persons using this document should be familiar with normal laboratory practice. This document does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user to establish appropriate safety and health practices and to ensure compliance with any national regulatory conditions.

IMPORTANT — It is absolutely essential that tests conducted in accordance with this document be carried out by suitably qualified staff.

Type text. This International Standard specifies a spectrophotometric determination of the pH_T of sea water on the total hydrogen ion concentration pH scale. The total hydrogen ion concentration, [H⁺]_t, is expressed as moles per kilogram of sea water. The method is suitable for assaying oceanic levels of pH_T from 7,4 to 8,2 for normal sea water of practical salinity ranging from 20 to 40.

The following documents are referred to in the text in such a way that some or all of their content constitutes requirements of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 5667‑1, Water quality — Sampling — Part 1: Guidance on the design of sampling programmes and sampling techniques

ISO 10523, Water quality — Determination of pH

ISO 22719, Water quality — Determination of total alkalinity in sea water using high precision potentiometric titration

For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminology databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https://www.iso.org/obp

— IEC Electropedia: available at https://www.electropedia.org/

3.1

total hydrogen ion concentration [H⁺]_t

total hydrogen ion concentration of sea water, [H⁺]_t includes the contribution of free hydrogen ions and the hydrogen ions bound to sulfate ions in the sea water and is defined as

(1)

where

[H⁺]_F is the free concentration of hydrogen ion in sea water;

S_T is the total sulfate concentration

K_S is the acid dissociation constant for

The pH_T is then defined as the negative of the base 10 logarithm of the hydrogen ion concentration as given in Equation (2):

(2)

3.2

practical salinity

S

practical salinity, symbol S, is a representative for the amount of dissolved salts in seawater and is defined as a polynomial function of the ratio R₁₅ of the electrical conductivity of the sea water sample at the temperature of 15 °C on IPTS‑68 and the pressure of one standard atmosphere, to that of a potassium chloride (KCl) solution, in which the mass fraction of KCl is 32,4356 × 10^-3, at the same temperature and pressure [xxx].

The values of pH_T are determined by adding an indicator to sea water. For the sulfonephthalein indicators such as m‑cresol purple, the reaction of interest at sea water pH_T is the second dissociation as given in Equation (3):

(3)

where I represents the indicator, which is present at a low level in a sea water sample. The total hydrogen ion concentration of the sample can then be determined as given in Equation (4):

(4)

The principle of this approach uses the fact that the different forms of the indicator have substantially different absorption spectra. Thus the information contained in the composite spectrum can be used to estimate [I^2‑] / [HI^-].

At an individual wavelength, λ, the measured absorbance, A_λ, in a cell with a pathlength L is given by the Beer‑Lambert law as:

(5)

where B_λ corresponds to the background absorbance of the sample and e is an error term due to instrumental noise. Provided that the values of the extinction coefficients: ε_λ (HI^-) and ε_λ (I^2-) have been measured as a function of wavelength, absorbance measurements made at two or more wavelengths can be used to estimate the ratio [I^2-] / [HI^-].

In the case that only two wavelengths are used, and provided that the background can be eliminated effectively by a subtractive procedure, Equation (5) can be rearranged to give (assuming no instrumental error)

(6)

where the numbers 1 and 2 refer to the wavelengths chosen. For the best sensitivity, the wavelengths corresponding to the absorbance maxima of the base (I^2-) and acid (HI^-) forms, respectively, are used. The various terms ε are the extinction coefficients of the specified species at wavelengths 1 and 2, respectively.

Use only reagents of recognized analytical grade.

5.1 m‑cresol purple, containing no spectrophotometric impurities.

NOTE 1 Liu, X., Patsavas, M.C. and Byrne, R.H. (2011), Reference [13], showed the indicator can be characterized and purified using the HPLC system. The wavelength of isosbestic point for HI^- / I^2- of the pure m‑cresol purple,
λ_isos (HI^- / I^2-) depends on the following equation: λ_isos( HI^- / I^2- ) = 490,6 – 0,10 t, where t is the temperature in degrees Celsius. That for H₂I / HI^- is also λ_isos( H₂I / HI^- ) = 482,6 – 0,10 t.

NOTE 2 Liu, X., Patsavas, M.C. and Byrne, R.H. (2011), Reference [13], and Patsavas, M.C., Byrne, R.H. and Liu, X. (2013), Reference [16], describe the purification method of m‑cresol purple.

NOTE 3 It is highly recommended to use purified m‑cresol purple, however in the case it isn’t available, it is required to use the correction procedure described in Douglas, N.K., and Byrne, R.H. (2017), Reference [11].

5.2 Solution of pure m‑cresol purple

A concentrated (at least 2 mmol/l) pure indicator solution of known is prepared either in ultrapure water or in a 0.7M NaCl ionic background. The pH value of the dye solution is adjusted to be in the range 7,9 ± 0,1 pH units - chosen to match pH_T measurements from an oceanic profile by adding HCl or NaOH as needed; this implies that for m‑cresol purple A₁/A₂ ≈ 1,6.

NOTE The absorbance ratio of a concentrated indicator solution can be measured using a cell with a short pathlength (0,5 mm).

5.3 Ultrapure water, of resistivity about 18 MΩ cm.

To be used in the preparation of the pure m‑cresol purple solution and in rinsing of equipment (e.g. cells).

Usual laboratory equipment and, in particular, the following:

Approximately, 40 cm long, sized to fit snugly over cell port. Silicone rubber is suitable. The drawing tube can be pre‑treated by soaking in clean sea water for at least one day. This minimizes the amount of bubble formation in the tube when drawing a sample.

These should be made of optical glass with a 10 cm pathlength, two ports and stoppers. A sufficient number of cells are needed to collect all the samples that will be analysed from a particular cast.

NOTE A flow through cuvette with a 10 cm pathlength is also available. Sample bottles of at least 200 ml with air tight caps are needed to use the cuvette.

A micropipette is used to add the indicator to the cell. It should be of ~0,1 cm³ capacity with a narrow tube attached to act as a nozzle.

For work of the highest sensitivity and precision, a double‑beam spectrophotometer is desirable. However, good results can be obtained with a high‑quality single‑beam instrument.

Commercially manufactured, thermostated spectrophotometer compartments that can accommodate 10 cm cells are rarely available and one will probably have to be custom‑made. Equipment need to be maintained in a close room with controlled temperature, and all the materials, solutions and samples need to be maintained in the same room for at least 2-h prior to start the test. The temperature should be regulated to within 0,1 °C.

Although it is possible to warm up the cells containing samples in the sealed bags in a thermostat bath, this is inconvenient. It is much better to build a custom‑made thermostated compartment that can hold approximately 12 cells at once without getting them wet.

A thermostat bath is used to control the temperature of both the cell compartment and the system to ±0,05 °C

Sea water should be collected from the sampler bottle immediately after opening, and before a significant amount of water is removed, to ensure representative sampling. This is necessary to minimize exchange of CO₂ with the air space in the sampler which affects all carbon parameters except total alkalinity. It is desirable that the carbon samples be collected before the sampler bottle is half empty and within 10 min of it being first opened.

Rinse the sample bottle — If the bottle is not already clean, rinse it twice with 30 cm³ to 50 cm³ of sample to remove any traces of a previous sample.

Fill the sample bottle — Fill the bottle smoothly from the bottom using a drawing tube which extends from the sampler bottle drain to the bottom of the sample bottle. It is critical to remove any bubbles from the draw tube before filling. Overflow the water by at least a full bottle volume. The air space within the sample bottle is kept to a minimum, References [9] and [10]. It is allowed to draw the sample directly from sampler bottle into the optical cell with two ports.

It is recommended that the pH analysis must be performed immediately after sampling, although storage experiments showed that the pH of sea water is stable up to 24 h even in the case of coastal water (see Annex B). However, while awaiting analysis, store the samples in the refrigerator or icebox (not frozen). The stability has not been assessed in case of important primary productivity (e.g. during a plankton bloom): in this case, it could be recommended to poison the seawater.

In the case of optical cell with two ports, warm sample cell to 25,0 ± 0,1 °C by placing a number of cells in a thermostated compartment (see 6.6) for at least 1h. Clean and dry the exterior of the cell before placing it in the thermostated sample compartment of the spectrophotometer.

Measure and record the absorbances at three wavelengths: at the wavelengths corresponding to the absorption maxima of the base (I^2-) and acid (HI^-) forms of the m‑cresol purple, 578 nm and 434 nm, respectively, and a non‑absorbing wavelength (750 nm).

NOTE 1 750 nm is chosen as a non-absorbing wavelength because it lies in a flat absorbance region, making it relatively insensitive to wavelength accuracy. This allows for the correction of any baseline shifts that may occur during the measurement process.

Remove one of the cell caps to inject m‑cresol purple: add approximately 0,05 cm³ to 0,1 cm³ of m‑cresol purple to the sample. Replace the cap and shake the cell to mix the sea water and m‑cresol purple. Absorbance values between 0,4 and 1,0 at each of the two absorbance peaks should be obtained by adding the appropriate amount of m-cresol purple.

Return the cell to the spectrophotometer, again measure and record the absorbances at three wavelengths of sea water after adding m‑cresol purple.

Cells should be positioned to maintain consistent alignment(s) between two absorbance measurements.

NOTE 2 In the case of a flow-through cuvette, warm the pH sample bottles to 25,0 ± 0,1 °C. Measure and record the reference absorbances at three wavelengths as described above. Then, add m-cresol purple to the sea water, gently mix the sample, and immediately measure and record the absorbances at three wavelengths, ensuring that the absorbance values fall between 0,4 and 1,0 at both absorbance peaks.

For routine samples, a single measurement is sufficient. However, multiple measurements of a known reference material are recommended to verify both the precision and accuracy of the method.

At each of the three wavelengths, subtract the absorbances measured for the background measurement (without indicator) from the corresponding absorbances measured for the system containing indicator.

In addition, the absorbance measured at a non‑absorbing wavelength is used to monitor and correct for any baseline shift due to error in repositioning the cell, instrumental shifts, etc. This assumes that the magnitude of any observed baseline shift is identical across the visible spectrum. Subtract the measured shift from the background-corrected absorbances at wavelengths 1 and 2 to obtain the final corrected absorbance value at each wavelength. These final absorbance values, corrected for background absorbances and any observed baseline shifts, are used to calculate A₁/A₂, the absorbance ratio which describes the extent of protonation of the indicator.

The difference between the baseline absorbance (sea water only) and the absorbance of the sample and indicator at 750 nm should be no greater than ±0,001. If this value is exceeded, the cell should be removed and the optical windows cleaned before the absorbances are measured again.

The pH_T of the sea water and indicator in the cell is computed from

(7)

Equation (7) can be rewritten as:

(8)

where K₂ is the equilibrium constant between the species HI^- and I^2- (expressed on the total hydrogen ion concentration scale in mol/kg), and A₁ and A₂ are the corrected absorbances measured at the wavelengths corresponding to the absorbance of 578 nm and 434 nm, respectively.

The first term of Equation (8), -log₁₀ { K₂ε₁ (I^2-) /ε₂(HI^-) }, is a function of salinity and temperature and has been determined by careful laboratory measurements.

-log₁₀ { K₂ε₁ (I^2-) / ε₂(HI^-) } = a + b / T + c lnT - dT

where parameters of a, b, c and d are in the range of 278,15 ≤ T ≤ 308,15 K and 20 ≤ S ≤ 40.

a = -246,642 09 + 0,315 971 S + 2,885 5 × 10^-4 S²

b = 722 9,238 64 – 7,098 137 S – 0,057 034 S²

c = 44,493 382 – 0,052 711 S

d = 0,0781 344

The various extinction coefficient terms ε correspond to values measured for the specified species at wavelengths 578 nm and 434 nm and are defined as follows:

ε₁(HI^-) / ε₂(HI^-) = -0,007 762 + 4,517 4 × 10^-5T

ε₂(I^2-) / ε₁(I^2-) = -0,020 813 + 2,602 62 × 10^-4T + 1,043 6 × 10^-4 ( S -35 )

NOTE The -log₁₀ { K₂ε₁(I^2-) / ε₂(HI^-) } given here and the various extinction coefficient terms ε are those from Liu, X., Patsavas, M.C. and Byrne, R.H. (2011), Reference [13].

The addition of indicator to the sea water sample will perturb the pH_T (another acid–base system has been added). Although care is taken to minimize this (by adjusting the indicator solution pH), it is desirable to correct for the addition of indicator to obtain the best pH_T measurements.

Although, in principle, the pH_T perturbation could be calculated from knowledge of the equilibrium chemistry of the sample and the indicator, it is simpler to evaluate the magnitude of the correction empirically. A pair of additions of indicator is made to each of a series of sea water samples with different pH_T values, and the change in the measured ratio (A₁/A₂) with the second addition of indicator solution is determined as a function of the measured value (A₁/A₂) determined after the first addition of indicator using a least squares procedure.

(9)

where V is the volume of indicator added at each addition. The final, corrected, absorbance ratio is given in Equation (10):

(A₁/A₂)_corr = (A₁/A₂) – V [a + b (A₁/A₂)] (10)

NOTE Absorbance at the isosbestic point wavelength for HI^-/I^2- depends on the concentration of indicator. Then, (A₁/A₂)_corr can be determined by the linear regression of (A₁/A₂) and the absorbance at the isosbestic point wavelength.

Another method is to plot the ratio (A₁/A₂) as a function of the volume of dye added. This allows, by a linear extrapolation to a volume equal to zero, to compute the corrected (A₁/A₂) ratio to be used for the calculation of the pH_T.

If exactly the same volume of indicator was added in the first and second addition, the corrected (A₁/A₂) ratio can be calculated with the following equation:

(A₁/A₂) _corrected = 2 (A₁/A₂) _{first_addition} - (A₁/A₂) _{second_addition} (11)

For sea water samples with similar salinity and pH, the correction can be calculated for one sample using the double or triple dye addition method described above. This same correction can then be applied to the other measurements

1. 
   (informative)
   
   Performance data
   1. Interlaboratory comparison on TRIS buffer and natural sea water

This interlaboratory comparison (ILC) exercise has been proposed to partners and stakeholders of the marine community, in the framework of the EURAMET SapHTies project, in autumn 2023.

The objectives pursued by the ILC were the following:

— validating the capacity of the improved spectrophotometric pH_T method to meet the needs necessary for the quantification of ocean acidification and the capacity of laboratories to apply these new recommendations. The targeted pH_T expanded uncertainty is 0,006.

— proposing improvements of the current QA/QC practices implemented in the different oceanographic observation networks and target a possible completion of the ISO18191:2015 “Water quality — Determination of pH_T in sea water — Method using the indicator dye m‑cresol purple” standard.

• • 1. ILC samples

Two samples were selected for the realisation of this ILC:

1. A TRIS buffer made of a salted matrix was proposed as the best standardised current reference material to represent a sea water matrix. This commonly accepted reference material for sea water pH_T is an equimolal buffer of 2-Amino-2-(hydroxymethyl)-1,3-propanediol (TRIS) and TRIS hydrochloride (TRIS.HCl) in artificial sea water of salinity 35 (Wolfe et al., 2021, reference [21]). This buffer can be certified in terms of pH_T by potentiometric measurement made in an electrochemical cell without junction called Harned cell. It is provided in 160 ml bottles.

2. A natural sea water was also included in the ILC to collect measurement similar to the one produced in routine applications. This sample from the OSIL Company has been selected for its specific production features. The sample is free from any biological content that can disturb its stability in pH_T. It is not added with any chemical materials that might interact with the chemicals used for spectrophotometric pH_T measurements. The sample is supplied in 250 ml borosilicate bottles with limited air headspace, reducing possible pH_T evolution coming from equilibrium with air CO₂ content. Moreover, this sample has been surveyed in several carbonates studies and a preliminary test performed for the objectives of this ILC indicated promising batch homogeneity.

• • 1. Dye

A purified meta-cresol purple (m-CP) solution of 2 mM, dissolved in a basic solution (NaOH in 0,7 mol/kg NaCl) and adjusted to pH~8 was provided to the participants in the framework of the ILC. Indeed, the dye used when performing pH_T measurement is entirely correlated to the measurement obtained. It was thus crucial to provide to all, exactly the same product. This dye was specifically made by GEOMAR for the purposes of the SapHTies project and fully characterized during the lifetime of the project; its extinction coefficients were determined as a function of temperature and salinity. This dye has been produced in one batch, then aliquoted in 12 ml amber vials.

• • 1. Measurement protocol

The measurement protocol has been thoroughly described in a document distributed to the participants. Special attention was drawn on instruments and sample conditioning, sample sampling, temperature conditioning and avoiding sample pollution. The measurement itself was detailed in terms of measurement wavelengths, blanks and corrections to take care of, and a specific protocol for correcting for the dye addition in the sample has been imposed. This document was accompanied by a video to ease the realisation of the measurements: https://embed.ifremer.fr/videos/b3596a386e6a49429a4e5dd7fa33bd1b

A reporting table has been sent to each participant, requesting two types of measurements:

— Raw absorbance and temperature measurements, to assess the performances of the spectrophotometric method

— pH_T measurements at the measurement temperature and pH_T measurements reported to 25 °C, to evaluate the ability of each laboratory to estimate the pH_T.

To ensure reliable statistical analysis, participants were asked to return at least 5 individual measurements each.

• • 1. Assessment of the reference value of TRIS buffer

The material has been characterised with Harned cells with a reference value of 8,096 0 ± 0,005 4 (k = 2; 25 °C) for a shell life of 2 months. Homogeneity tests, stability tests as well as the evaluation of the total uncertainty budget were performed according to (Capitaine et al. 2023, reference [6]).

• • 1. Homogeneity, stability and metrological traceability of natural sea water

Several bottles from the production batch of natural sea water included in the ILC, have been tested for homogeneity and stability. Concerning homogeneity, it should be noted that spectrophotometric measurements, due to their own repeatability, are limited to demonstrate the homogeneity of the batch. Taking in account the measurement variations that can be observed within one bottle, the material was considered homogeneous for the purpose of the ILC.

The stability of the batch has been surveyed for several months, before, over and after the course of the ILC. Despite the variability of measurements, a global trend of increasing pH_T was observed. This increase was less than 0,001 pH_T unit over the 2 weeks of the ILC.

Natural sea water pH_T measurement cannot currently qualify for any recognised way of traceability to the International Standard of Unit (SI). Several limitations are part of this lack of traceability, one of them being the technological inapplicability of the recognized Harned cell primary method on sea water matrix.

• • 1. Assessment of the pH_T spectrophotometric method performance according to NF ISO 5725-2

In order to assess the performances of the practical realisation of the pH_T spectrophotometric method itself, a unique post-processing was applied on raw data reported by laboratories (raw absorbances together with temperature measurements). In this way, variations due to modelling were reduced to a minimum.

• • • 1. TRIS buffer

The Cochran test indicated no isolated laboratory. The Grubbs test performed on laboratories averages indicated no isolated laboratory. The statistical analysis of all results collected for the TRIS buffer is presented in Table A.1.

• • • 1. Natural sea water

The Cochran test indicated no isolated laboratory. The Grubbs test performed on laboratories averages indicated no isolated laboratory. The statistical analysis of all results collected for the natural sea water sample is presented in Table A.1.

Table A.1 — Performance data for TRIS buffer and natural sea water

• 1. Interlaboratory comparison on North Pacific sea water

An interlaboratory trial was organised by Dr. Shuichi Watanabe and supervised by Dr. Koh Harada, Convenor of ISO/TC 147/SC 2/WG 67 with the assistance of Dr. Yoshiyuki Nakano and Mr. Koichi Goto and was performed in spring 2014. A total of 11 laboratories from 8 countries participated (Japan: 3, France: 2, Canada: 1, Korea: 1, Norway: 1, Portugal: 1, Sweden: 1, UK: 1). Two participants did not adhere to the method description. Their results were excluded from the statistical evaluation of the interlaboratory trial.

Five bottles of 500 ml sea water sample which were subsampled from one large container were sent to each laboratory. The sea water was collected in the western North Pacific and its salinity was 34,399.

The results are given in Table A.2.

Table A.2 — Performance data for sea water pH

1. 
   (informative)
   
   Storage stability

The storage stability of sea water sample for pH_T was confirmed by sequential experimentation over 24 h. Sea water samples were collected at the coastal sea of Aomori Prefecture, Japan by a plastic bucket. Salinity of the sea water was 33,532. Sea water samples 1 were sampled in 250 ml glass bottles from a bucket. Samples 2 were filtered through 25 mm-diameter Whatman GF/F filter in laboratory and sampled in 250 ml glass bottles. Samples 3 were sampled in 250 ml glass bottles from a bucket and poisoned with 100 μl of over saturated solution of mercury chloride. These samples were stored in a refrigerator and got out an hour and a half before analysis.

The results are given in Table B.1.

Table B.1 — Storage stability of sea water pH_T

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