3,3′,5,5′-tetramethylbenzidine as multi-colorimetric indicator of chlorine in water in line with health guideline values

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3,3’,5,5’-tetramethylbenzidine as multi-colorimetric indicator of chlorine in water in line with health guideline

values

Journal: Analytical and Bioanalytical Chemistry Manuscript ID ABC-01360-2020

Type of Paper: Research Paper Date Submitted by the

Author: 10-Jul-2020

Complete List of Authors: Palladino, Pasquale; University of Florence, Chemistry 'Ugo Schiff' Torrini, Francesca; University of Florence, Department of Chemistry 'Ugo Schiff'

Scarano, Simona; University of Florence, Chemistry Minunni, Maria; University of Florence, Chemistry

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July 10th_{, 2020}

Dears Editor,

we are sending the original manuscript entitled: “3,3’,5,5’-tetramethylbenzidine as multi-colorimetric indicator of chlorine in water in line with health guideline values” by Pasquale Palladino, Francesca Torrini, Simona Scarano, Maria Minunni, which we would like you consider for publication in Analytical and Bioanalytical Chemistry as Research paper.

We believe that our manuscript is of interest for the ABC readership because it contributes to the field of the colorimetric determination of free chlorine in water by using 3,3’,5,5’-tetramethylbenzidine (TMB), which is the safer chromogenic substitute of benzidine in clinical chemistry assays.

Here the method has been successfully applied in tap water and appears superior to a common monochromatic commercial test kit based on DPD (N,N-diethyl-p-phenylenediamine) for visual sensing and quantification of chlorine in swimming pools where chlorine persistence is crucial for sanitizing, pointing out the significance of this work in human healthcare, especially in these times of pandemic due to Sars-Cov2.

TMB is converted by chlorine to a colored molecule though a pH-dependent multi-step oxidation process where the chromaticity of TMB is directly proportional to chlorine content and even naked-eye sensing appears possible in buffer able to mask potential redox active ions, e.g. Fe(III).

The key novelty of this work lies in the appropriate setting of reaction conditions, showing for the first time that it is possible to finely tune the TMB color changes to cover exactly the range of chlorine concentration recommended by international health agencies for treatment of drinking water and swimming pools, between a minimum level of 0.2-0.5 mg L-1 _{(expressed as Cl}

2), for an effective

disinfection, and a maximum residual concentration of 4-5 mg L-1_{, to avoid eye/nose irritation and}

stomach discomfort. Therefore, the improvement of the current detection limit of chlorine concentration in water, well below guideline concentrations, is clearly out of the scope of this paper. Furthermore, the method here described could pave the way to develop a practical tool to test home-made sanitizing solutions necessarily prepared by serial dilution of household chlorine bleach during supply shortage for public health emergency events.

Our manuscript has been written according to the Instructions for Authors, but we are ready to modify anything you will suggest. Furthermore, hereby we confirm that it has not been published previously by any of the authors and is not under consideration for publication in another journal. Thank you for your consideration.

Corresponding author: Pasquale Palladino, PhD

Department of Chemistry 'Ugo Schiff' University of Florence

50019 Sesto Fiorentino (FI), Italy

Email: pasquale.palladino@unifi.it

Phone: +39 055 457 3283

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3,3’,5,5’-tetramethylbenzidine as multi-colorimetric indicator of chlorine in water in line with health guideline values

Pasquale Palladinoa_{*, Francesca Torrini}a_{, Simona Scarano}a_{, Maria Minunni}a

a_{Department of Chemistry ‘Ugo Schiff’, University of Florence, Via della Lastruccia 3-13, 50019}

Sesto Fiorentino, Italy

Abstract

Sanitizing solutions against bacterial and viral pathogens are of utmost importance in general, and in particular in these times of pandemic due to Sars-Cov2. They frequently consist of chlorine-based solutions, or in the direct input of a certain amount of chlorine in water supply systems and swimming pools. Colorimetry is one of the techniques used to measure the crucial persistence of chlorine in water, including household chlorine test kits commonly based on colorimetric indicators. Here we show a simple and cheap colorimetric method based on 3,3’,5,5’-tetramethylbenzidine (TMB), commonly used as chromogenic reagent for enzyme-linked immunosorbent assays. TMB is converted by chlorine to a colored molecule though a pH-dependent multi-step oxidation process where the chromaticity of TMB is directly proportional to chlorine content. This molecule offers several advantages over other commonly used reagents in terms of safety, sensitivity and, peculiarly, hue modulation, giving rise to the detection of chlorine in water with a multi-color change of the indicator solution (transparent/blue/green/yellow). Moreover, through the appropriate setting of reaction conditions, such coloration is finely tunable to cover the range of chlorine concentration recommended by international health agencies for treatment of drinking water and swimming pools and to test home-made solutions prepared by dilution of household bleach during health emergency events such as during the current pandemic.

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Keywords

3,3’,5,5’-tetramethylbenzidine (TMB); Multi-color indicator; Naked-eye sensing; Chlorine; Water treatment; Sanitizing solution.

Abbreviations TMB, 3,3’,5,5’-tetramethylbenzidine; HRP, Horseradish peroxidase; DPD,

N,N-diethyl-p-phenylenediamine; OTO, o-tolidine; EPA, United States Environmental Protection Agency; WHO, World Health Organization; ELISA, Enzyme-Linked ImmunoSorbent Assay.

Introduction

Chlorine and chlorine-based compounds are widely used for oxidative inactivation of several disease-causing viruses and bacteria in water treatments, for food preservation, and during other cleaning procedures [1,2]. In all cases, the presence of chlorine residual is required to ensure the protection from recontamination. The range of chlorine concentrations recommended by international health or environmental agencies depends on the application, spanning several orders of magnitude from drinking water to sanitizing or disinfecting solutions. In detail, chlorine can be added to drinking water or swimming pools to achieve a minimum level of 0.2-0.5 mg L-1 _{(expressed as Cl}

2) for an

effective disinfection and residual concentration but should stay below 4-5 mg L-1_{to avoid eye/nose}

irritation and stomach discomfort [3,4]. Whereas, sanitizing solutions applied to reduce germs for example on food contact surfaces or mouthed toys are obtained by dilution of concentrated chlorine bleach to 50-200 mg L-1_._{Finally, disinfecting concentration for toilet or diapering areas should be}

larger than 2400 mg L-1_{. The standard analytical methods for the determination of chlorine in natural}

and treated waters are based on electrochemical techniques, iodometry, and absorption spectrophotometry [5,6]. However, several kits for household chlorine test are present on the market. Here we introduce a peculiar use of 3,3’,5,5’-tetramethylbenzidine (TMB) for the colorimetric

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determination of chlorine in aqueous solution. TMB is still one of the most commonly used chromogenic reagent for enzyme-linked immunosorbent assays (ELISAs) 46 years after its first synthesis as safer substitute for carcinogenic benzidine in the detection of occult blood [7-10]. This molecule gives rise to non-colored solution in reduced state that becomes blue upon oxidation (diamine). Further oxidation leads to a green stage, which is a mixture of the blue stage and the yellow endpoint (diimine). Notably, the blue colored product has been characterized as the diamine/diimine charge-transfer complex in equilibrium with the cation-radical and diamine/diamine mixture, as reported in Fig. 1 [11,12]. This reaction sequence is sensitive to several factors, like pH [12], solvent, temperature [13,14], and catalyst [10,15,16]. The application of TMB as an analytical reagent outside the field of clinical chemistry, together with ion-masking salts conferring high tolerance limits for potential interfering ions, started long time ago thanks to good stability of the reaction product and high sensitivity of such analytical methods [17-20]. In particular, the reactions reported in Fig. 1 have been used for quantitative measurements of chlorine in water [17-21], under organic matter-rich conditions [22], or generated by myeloperoxidase of neutrophils [23-25], reaching an enhanced degree of automation, reduced reagent consumption and waste production [19] . Although a rigorous control of pH is not necessary, the pH range determines the color development. In detail, pH 1-2 allows to follow the yellow TMB-diimine formation only (Fig. 1C) [17-19,21], whereas pH 4-6 has been used to track the blue TMB charge-transfer complex formation (Fig. 1B) [20]. In both conditions, the color intensity is directly proportional to the total amount of the chlorine present in the solution, reporting a limit of detection as low as 2 µg L-1_{at pH 1.5 and 4 µg L}-1_{at pH 4.5 [18].}

TMB presents several advantages in terms of sensitivity and safety over the other reagents used for colorimetric analysis of chlorine in water like o-tolidine (OTO), syringaldazine, N,N-diethyl-p-phenylenediamine (DPD), and dopamine [17,18,26].Here we report the experimental tuning of TMB concentration, according to one-to-one stoichiometry for oxidant/substrate system [11], to achieve the visual and spectroscopic detection of chlorine by means of the color change of solution from blue to yellow (Fig. 1) like a universal indicator that covers the entire range of chlorine concentrations

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recommended for drinking water and swimming pools treatment [3,4]. This method appears superior to common household chlorine test kits for drinking water and swimming pools, which are based on monochromatic indicators, and could offer a useful tool to check home-made chlorine-based sanitizing solutions, which become rare during public health emergency events due to large demand.

Fig. 1 Here Materials and method

Reagents and materials

Disposable low-binding 96-microplates were purchased from Sarstedt (Germany). Absorption suprasil® quartz cuvettes of 10 mm pathlength and 1400 µL chamber volume were provided by Hellma GmbH & Co. KG (Germany). Sodium hypochlorite solution was purchased from Merck KGaA (Germany) (Lot number: K50436114, originally containing 120 g L-1_{of active chlorine as}

calculated from iodometric titration) and used to prepare stock solutions based on the intrinsic UV-absorbance of ClO-_{[27]. DPD1 free chlorine test kit was purchased from Powehaus24}® _{GmbH & Co.}

KG (Germany). TMB and other chemicals were purchased from Sigma Aldrich (Milan, Italy). A stock solution of about 1 mM TMB has been prepared in ethanol/H2O 1:1 v/v and left overnight at

Room Temperature (RT) until completely dissolved before storing it at 4 °C in a dark bottle. All reagents were used as received without any further purification. MilliQ water with a resistivity of 18.2 M cm at 25.0 °C was degassed and used in preparation of all the solutions (Merck Millipore, Italy).

UV-Vis Spectroscopy

Optical measurements have been performed on a UV-visible spectrophotometer Evolution 201 from Thermo Scientific at 25.0 °C, by using quartz cells also for determination of hypochlorite concentration of stock solution, and on a microplate absorbance visible reader iMark™ from Bio-Rad

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at room temperature, by using 96-well polystyrene microplates. Acetic acid/sodium acetate 200.0 mM in presence of tetrasodium diphosphate 40.0 mM (Na4P2O7), referred to hereinafter as buffer 1,

or disodium dihydrogen diphosphate 20.0 mM (Na2H2P2O7), referred to hereinafter as buffer 2, have

been chosen to stabilize TMB solution around pH 4.5. Spectrophotometric measurements have been carried out by filling the quartz cuvette with 700 µL of TMB 42.3 10-6_{M plus 700 µL of freshly}

prepared H2O or tap-water samples containing chlorine in the range 0.25 – 5.00 mg L-1 with three

replicas for each chlorine concentration. After the analysis of each chlorine concentration, the cuvette has been emptied and thoroughly washed. Analogously, microplates experiments have been performed adding 150 µL of freshly prepared H2O or tap-water samples to each well containing

chlorine in the range 0.25 – 5.00 mg L-1_{with 4 replicas for each chlorine concentration. All the wells}

have been preloaded with 150 µL of TMB 42.3 10-6_{M, which is equimolar to 3.00 mg L}-1_{of active}

chlorine for drinking water or swimming pool simulation. Finally, DPD1 chlorine test kit has been used following the manufacturer instructions. Data were analyzed with OriginLab Corporation software.

Results and discussion

Chlorine titration experiments

Titration curve of TMB with H2O2 (50 10-6 M at pH 5.0) in presence of horseradish peroxidase

catalyst (HRP) has been elsewhere reported [11], showing that the blue charge-transfer complex formation and subsequent destruction is nearly symmetrical about the midpoint. The endpoint has been found to correspond to about equimolar peroxide and TMB, obtaining the full conversion to diimine and thus confirming a one-to-one stoichiometry for this catalyzed TMB/oxidant reaction. Analogously, here we follow spectrophotometrically all the stages of reaction in Fig. 1 exploiting the

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full potential of the catalyst-free TMB reaction with active chlorine by titration, in polystyrene plates or quartz cuvettes, in mild acidic buffer that controls chlorine speciation and TMB reactivity.

In detail, molecular chlorine (Cl2) in mostly present at pH < 2, hypochlorous acid (HOCl) dominates

the equilibrium at 4 < pH < 6, and hypochlorite (ClO-_{) is predominant at pH > 8 [25,27]. Buffering}

the sample solutions in one of these pH ranges can limit the variability of results due to different reactivity of chlorine species. However, mild acidic buffer guarantees the best conditions to measure the chlorine content in real samples by tracking all the non-catalytic oxidation stages of TMB (Fig. 1). In fact, it has been demonstrated that a pH below 2 prevents the formation of the blue TMB charge-transfer complex formation (Fig. 1B) in favor of yellow TMB-diimine formation only (Fig. 1C). Analogously, it has been reported that the charge-transfer complex formation is significantly reduced at pH above 6. Therefore, we have performed experiments stabilizing TMB solution at pH 4.5 with diphosphate salts (see Materials and method), thus preventing interference from redox active ions like Fe(III) potentially present in water supply systems [17-20]. Differently from previously reported single-color intensity estimation with TMB (either yellow or blue) or DPD (magenta) [17-22,25], this method allows the naked-eye quantification of chlorine by multi-color changes of TMB solution. This approach for the first time proposes TMB as a universal indicator for the selected range of chlorine concentrations, from a minimum blue level for an effective disinfection, through an optimal green level, up to the safe yellow guideline level corresponding to equimolar chlorine and TMB (see below).

TMB reaction with active chlorine in H2O and tap water

Fig. 2A shows preliminary experiments of color development for TMB test solution in buffer 1 (pH 4.5, see Materials and method) with chlorine samples in H2O in quartz cuvettes. The main

representative absorbances selected from UV-visible spectra at 25.0 °C have been acquired in a kinetic rate mode and reported in Fig. 2B. In detail, after 30 seconds of incubation necessary for

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sample preparation, three fixed wavelengths (280 nm, 450 nm and 655 nm) have been recorded every 10 s for a total accumulation time of 300 s. The solution of a fixed amount of TMB 42.3 10-6_M_is

initially transparent due to UV absorbance only of diamine ( max 280 nm, Fig. 1A) that decreases

along with chlorine accumulation (Fig. 2C). Simultaneously, the solution develops a blue color of increasing intensity in the presence of chlorine from 0.25 mg L-1_{to 1.00 mg L}-1_{. This visible band}

corresponds to the blue diamine/diimine charge-transfer complex ( max 655 nm, Fig. 1B). The titration

curve at 655 nm shows the formation and subsequent disappearance of the blue product (Fig. 2C). Higher concentrations of chlorine lead to the appearance of green color, due to blue/yellow absorbance mixing. A yellow solution due to yellow diimine form of TMB ( max 450 nm, Fig. 1C) is

obtained at higher chlorine concentration up to 3.00 mg L-1_{of Cl}

2. Finally, the solution becomes

orange when chlorine reaches the 5.00 mg L-1_{(70.5 10}-6_{M), which is the guideline value that could}

lead to potential health effects from long-term exposure [3,4]. Data show a good reproducibility, obtaining a mean coefficient of variation of 4.72 % for absorbance values at 280 nm, 3.91 % at 450 nm, and 3.52 % at 655 nm over the entire range of chlorine concentrations after 0 seconds, for example (Table 1). All the reactions appear complete within 1 minute in agreement with previous observations of blue color development obtained in similar conditions but with a large excess of TMB [20]. The time evolution plots of main absorbance bands are reported in Fig. 2D showing quite stable colors at 25.0 °C. Similar results were obtained in buffer 2, however absorbance values appear less stable after 15 minutes (data not shown). The same experiments performed by using tap water for chlorine dilutions instead of H2O have been reported in Fig. 3, showing a limited matrix effect (likely

due to foreign ions) on colors appearance and intensity along chlorine titration with respect to H2O

alone, confirming the good reproducibility, obtaining, for example, a mean coefficient of variation of 3.18 % for absorbance values at 280 nm, 3.23 % at 450 nm, and 2.66 % at 655 nm after 0 seconds (Table 1). The complete dataset, for H2O or tap water, in dependence of time, chlorine concentration,

and max is reported in the Supplementary material (SI).

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Table 1 Here Fig. 2 Here Fig. 3 Here

Comparison of TMB method with a DPD1 chlorine test kit

96-well polystyrene microplates have been used for the optimization of the method, enabling the experimental procedure described for quartz cuvettes to be carried out on 96 samples simultaneously, with minor volume consumption and thus more replicates for each chlorine concentration. Furthermore, taking the advantage of distinct visible max absorbances for TMB oxidation products

by reaction with chlorine, we have used a filter-based plate reader instead of a spectrophotometer preliminarily used for greater flexibility due to wavelength scanning capability from UV to visible. In detail, DPD-containing tablets from a commercial test kit have been compared to TMB solution in buffer 1 here formulated for the naked-eye sensing of chlorine concentration in water samples. Fig. 4A shows a polystyrene microplate containing Cl2 in H2O, including the range of concentrations

recommended for drinking water treatment, about 0.25-3.00 mg L-1_{[3,4], in presence of a fixed}

amount of DPD or TMB. DPD solutions have been prepared mimicking a swimming pool test; i.e. using one DPD tablet for 13 mL of water, which is the volume of the commercial test tube. Such chlorine solutions present a magenta color with intensity depending on chlorine content and here ideally representing the color reference scale for a chlorine sample test with DPD. However, it seems difficult to distinguish the analyte concentration solely by naked-eye comparison of color intensities. On the contrary, the same chlorine solutions give different colors in presence of TMB, from light blue to bright yellow through green hues, depicting the color reference scale for a chlorine test with this TMB formulation, which allows an easier visual sensing and estimation of chlorine content, behaving

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like a universal indicator within the chosen range of chlorine concentration. The same results have been obtained by using tap water for chlorine dilutions (Fig. 4B). The representative absorbances of the main molecular species and colors are reported in Figs. 4C and 4D for H2O and tap water

solutions, respectively. As expected, the absorbances of TMB solution recorded at 450 nm (yellow) and 655 nm (blue) follow the trend observed in quartz cuvettes with optimal results in terms of reproducibility expressed as percent coefficient of variation (%CV in Table 2). In particular, the titration curve at 655 nm shows the formation and subsequent disappearance of the blue product with a bell-shaped trend symmetrical about the midpoint corresponding to 0.50 mol chlorine/mol of TMB; i.e. 1.50 mg L-1_{(Fig. 4C), as theoretically expected and previously shown for another oxidant system}

[11]. The yellow diimine absorbance ( max 450 nm) reaches the maximum when the oxidant

concentration is equimolar with TMB, i.e. 3.00 mg L-1_{of Cl}

2 chosen as maximum safe concentration

for active chlorine in water, whereas at same chlorine concentration the blue color contribution from charge-transfer complex returns to the minimum level and the green color disappears from the plate with it (Figs. 4A and 4B). Conversely, the absorbance of DPD solutions recorded at 555 nm (magenta) shows a monotone increase with chlorine concentration, as expected (Figs. 4C and 4D), presenting also a reproducibility worse than TMB (Table 2). Finally, in agreement with previous studies describing the better precision, sensitivity and safety of TMB over the other reagents used for colorimetric analysis of chlorine [17,18], here we report a comparison at t 0 s of the proposed method based on TMB, and commercial method based on DPD. The analytical parameters have been obtained on three chlorine concentration ranges by using the piecewise linear fitting implemented in the Originlab software (see SI Fig. S1). In detail, Table 3 shows the best detection limit for the linear fitting of TMB blue color development ( max655 nm) for lower chlorine concentrations (0.00-1.50 mg

L-1_{) in H}

2O and tap water. Moreover, the sensitivity represented by the slope (m) of the calibration

curve, and the reproducibility reported as %CVmean appear both superior for TMB (both at max655

nm and 450 nm) over DPD method, in H2O and tap water, within the working range for chlorine

determination (0.00-3.00 mg L-1_). 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Fig. 4 Here Table 2 Here Table 3 Here

Conclusion

This work contributes to the field of the colorimetric determination of free chlorine in water by using TMB, which is the safer chromogenic substitute of benzidine in clinical chemistry assays, already shown to have a precision and sensitivity higher than other common colorimetric methods and offering the further advantage of hue modulation upon oxidation. The latter feature of TMB solutions have been exploited here for spectroscopic detection of chlorine in water, and even naked-eye sensing appears possible through the recognition of typical color of each oxidation stage of TMB in diphosphate-based buffer at pH 4.5, which is also able to mask potential redox active ions, e.g. Fe(III), commonly present in drinking water and swimming pools. The key novelty of this work lies in the appropriate setting of reaction conditions, showing for the first time that it is possible to finely tune the TMB colors change to cover exactly the range of chlorine concentration recommended by international health agencies for treatment of drinking water and swimming pools. The assay relies on the simple tuning of TMB final concentration to be equimolar to chlorine concentration value with limited potential health effects from long-term exposure. Therefore, obtaining a multi-colorimetric indicator of chlorine in water working between minimum value, to achieve an effective disinfection, and maximum health guideline value, to avoid irritations or discomfort. Consequently, the improvement of the current detection limit of chlorine concentration in water, well below guideline concentrations, is clearly out of the scope of this paper. In detail, TMB solutions produce a yellow color at the maximum safe concentration of chlorine, a green color at intermediate and recommended optimal chlorine values, and a blue color at the prescribed lower end concentration of chlorine in

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water. The method has been also successfully applied in tap water and appears superior to a commercial test kit for visual sensing and quantification of chlorine in swimming pools that are instead based on monochromatic chromophores like DPD. Finally, this analytical method could offer a practical tool to test home-made sanitizing solutions, and their “shelf life”, necessarily prepared by serial dilution of household chlorine bleach during supply shortage for public health emergency events such as pandemic influenza, as we did here to prepare a stock solution from concentrated chlorine bleach bottle, pointing out the significance of this work in human healthcare. A different modulation of TMB assay for a direct measure of higher chlorine concentration will be the subject of future work.

Funding information This study was supported by a grant for the scientific program PLABAN (grant

D53D16002290009) from the Regione Toscana.

Ethics declarations

Conflict of interest The authors declare that they have no conflict of interest.

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[21] Zhang J, Yang X. A simple yet effective chromogenic reagent for the rapid estimation of bromate and hypochlorite in drinking water. Analyst. 2013; https://doi.org/10.1039/c2an36287b [22] Yamaoka H, Nakayama‐Imaohji H., Horiuchi I, Yamasaki H, Nagao T, Fujita Y, Maeda H,

Goda H,. Kuwahara T. Tetramethylbenzidine method for monitoring the free available chlorine and microbicidal activity of chlorite‐based sanitizers under organic‐matter‐rich environments. Lett Appl Microbiol. 2016; https://doi.org/10.1111/lam.12506

[23] Andrews PC, Krinsky NI. Quantitative determination of myeloperoxidase using tetramethylbenzidine as substrate. Anal Biochem. 1982; https://doi.org/10.1016/0003-2697(82)90185-3

[24] Freitas M, Lima JL, Fernandes E. Optical probes for detection and quantification of neutrophils’ oxidative burst. A review. Anal Chim Acta. 2009; https://doi.org/10.1016/j.aca.2009.06.063

[25] Kettle AJ, Albrett AM, Chapman AL, Dickerhof N, Forbes LV, Khalilova I, Turner R. Measuring chlorine bleach in biology and medicine. Biochim Biophys Acta. 2014; https://doi.org/10.1016/j.bbagen.2013.07.004

[26] Palladino P, Torrini F, Scarano S, Minunni M. Colorimetric analysis of the early oxidation of dopamine by hypochlorous acid as preliminary screening tool for chemical determinants of neuronal oxidative stress. J Pharmaceut Biomed Anal. 2020; https://doi.org/10.1016/j.jpba.2019.113016

[27] Feng Y, Smith DW, Bolton JR. Photolysis of aqueous free chlorine species (HOCl and OCl) with 254 nm ultraviolet light. J Environ Eng Sci. 2007; https://doi.org/10.1139/s06-052

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Figure captions

Fig. 1. The reaction sequence of TMB oxidation by chlorine (this work), or by H2O2 in presence of

peroxidase catalyst (HRP) [11]. (A) Non-colored reduced state (diamine); (B) Blue charge-transfer complex (diamine/diimine); (C) Yellow two-electron oxidation product (diimine); (D) One-electron oxidation product (cation-radical). The most representative UV-Vis spectra for the TMB species upon oxidation by chlorine (this work) are reported on the right.

Fig. 2. Reaction of TMB solution in buffer 1 (pH 4.5) with active chlorine in H2O at 25.0 °C. (A)

Color development for TMB oxidation by chlorine in quartz cuvettes. (B) Absorbance at 280 nm, 450 nm and 655 nm. (C) Time evolution plot for the TMB absorbance at different wavelengths after reaction with chlorine. The absorbances are reported as mean value of three replicates for each chlorine concentration.

Fig. 3. Reaction of TMB solution in buffer 1 (pH 4.5) with active chlorine in tap water at 25.0 °C.

(A) Color development for TMB oxidation by chlorine in quartz cuvettes. (B) Absorbance at 280 nm, 450 nm and 655 nm. (C) Time evolution plot for the TMB absorbance at different wavelengths after reaction with chlorine. The absorbances are reported as mean value of three replicates for each chlorine concentration.

Fig. 4. TMB prepared in buffer 1 versus DPD test kit for chlorine detection by microplate visible

reader. Chlorine concentration ranges from 0.25 to 5.00 mg L-1_{with four replicas for each chlorine}

solution in H2O (A) or tap water (B). Each well of a 96-well polystyrene microplate contains 300 µL

of sample solution with a final concentration of 1 tablet of DPD for 13 mL of water, as required by original commercial test, or TMB equimolar to 3.00 mg L-1_{of Cl}

2 in buffer. The absorbances at 450

nm and 655 nm (TMB), and 555 nm (DPD) for chlorine in H2O (C) or tap water (D) are reported as

mean value of four replicates for each chlorine concentration.

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Fig. 1. The reaction sequence of TMB oxidation by chlorine (this work), or by H2O2 in presence of peroxidase catalyst (HRP) [11]. (A) Non-colored reduced state (diamine); (B) Blue charge-transfer complex

(diamine/diimine); (C) Yellow two-electron oxidation product (diimine); (D) One-electron oxidation product (cation-radical). The most representative UV-Vis spectra for the TMB species upon oxidation by chlorine (this

work) are reported on the right. 180x119mm (300 x 300 DPI) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Fig. 2. Reaction of TMB solution in buffer 1 (pH 4.5) with active chlorine in H2O at 25.0 °C. (A) Color development for TMB oxidation by chlorine in quartz cuvettes. (B) Absorbance at 280 nm, 450 nm and 655

nm. (C) Time evolution plot for the TMB absorbance at different wavelengths after reaction with chlorine. The absorbances are reported as mean value of three replicates for each chlorine concentration.

180x119mm (300 x 300 DPI) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Fig. 3. Reaction of TMB solution in buffer 1 (pH 4.5) with active chlorine in tap water at 25.0 °C. (A) Color development for TMB oxidation by chlorine in quartz cuvettes. (B) Absorbance at 280 nm, 450 nm and 655 nm. (C) Time evolution plot for the TMB absorbance at different wavelengths after reaction with chlorine.

The absorbances are reported as mean value of three replicates for each chlorine concentration. 180x119mm (300 x 300 DPI) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Fig. 4. TMB prepared in buffer 1 versus DPD test kit for chlorine detection by microplate visible reader. Chlorine concentration ranges from 0.25 to 5.00 mg L-1 with four replicas for each chlorine solution in H2O (A) or tap water (B). Each well of a 96-well polystyrene microplate contains 300 µL of sample solution with a

final concentration of 1 tablet of DPD for 13 mL of water, as required by original commercial test, or TMB equimolar to 3.00 mg L-1 of Cl2 in buffer. The absorbances at 450 nm and 655 nm (TMB), and 555 nm

(DPD) for chlorine in H2O (C) or tap water (D) are reported as mean value of four replicates for each chlorine concentration. 180x119mm (300 x 300 DPI) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Table 1. Coefficient of variation (%CV) for Absorbance values vs [Cl2] from Fig. 2A (H2O) and 3A (Tap water)

Time 0 (s) H2O Tap water

[Cl2] mg L1 %CV280nm %CV450nm %CV655nm %CV280nm %CV450nm %CV655nm 0.00 0.92 4.89 1.55 0.29 8.25 1.06 0.25 1.00 3.73 2.34 3.51 0.50 7.58 0.50 0.71 4.80 2.50 0.88 3.11 4.23 1.00 1.00 9.58 8.08 2.02 4.16 4.72 1.50 4.86 5.77 1.69 1.11 9.99 3.93 2.00 6.02 2.86 1.06 5.03 0.69 2.69 2.50 1.72 9.18 1.07 0.47 0.77 0.62 3.00 14.5 3.35 1.91 1.27 2.51 0.05 3.50 2.37 0.49 2.70 1.07 2.58 1.98 4.00 1.44 1.00 3.39 5.85 3.15 1.39 4.50 15.9 0.77 5.60 13.8 2.42 3.31 5.00 6.17 0.54 10.3 2.87 0.60 0.30 %CVmean 4.72 3.91 3.52 3.18 3.23 2.66 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Table 2. Coefficient of variation (%CV) for Absorbance values vs [Cl2] from Fig. 4C (H2O) and 4D (Tap water)

Time 0 (s) H2O Tap water

DPD TMB DPD TMB [Cl2] mg L-1 %CV555nm %CV450nm %CV655nm %CV555nm %CV450nm %CV655nm 0.00 5.89 5.29 5.41 14.7 7.64 2.56 0.25 11.0 17.9 1.11 16.9 17.5 6.54 0.50 18.4 6.05 4.07 16.2 9.79 1.78 1.00 11.1 10.7 2.66 15.3 9.43 1.57 1.50 10.9 0.48 0.71 10.2 3.77 2.53 2.00 8.86 0.81 1.83 7.94 2.21 1.45 2.50 8.19 1.28 4.58 7.83 7.62 12.0 3.00 2.57 1.25 13.1 8.46 0.75 8.25 3.50 6.20 0.93 16.6 2.84 1.38 9.59 4.00 3.92 0.97 18.0 1.32 2.44 29.2 4.50 5.68 1.81 4.88 4.84 0.64 2.92 5.00 3.60 0.66 10.3 3.44 1.38 17.9 %CVmean 8.03 4.01 6.94 9.16 5.38 8.02 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Table 3. Comparison of the proposed (TMB) and commercial (DPD) methods at t 0 s. The analytical parameters have been obtained

on three chlorine concentration ranges by using the piecewise linear fitting implemented in the Originlab software.

Calibration curve: 𝐴𝑏𝑠𝜆𝑚𝑎𝑥= 𝐴𝑏𝑠0𝜆𝑚𝑎𝑥+ 𝑚 ×[𝐶𝑙2]; 𝐴𝑏𝑠0𝜆𝑚𝑎𝑥𝑖𝑠 𝑡ℎ𝑒 𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡 𝑎𝑡 [𝐶𝑙2]= 0, 𝑎𝑛𝑑 𝑚 𝑖𝑠 𝑡ℎ𝑒 𝑠𝑙𝑜𝑝𝑒

TMB DPD

H2O Tap water H2O Tap water H2O Tap water

[Cl2]

(mg L-1₎

Analytical

parameters max 450 nm (yellow) max 655 nm (blue) max 555 nm (magenta)

𝐴𝑏𝑠0 𝜆𝑚𝑎𝑥 0.03 ± 0.02 0.03 ± 0.01 0.05 ± 0.01 0.04 ± 0.01 0.08 ± 0.01 0.10± 0.02 𝑚 (𝑚 1 _0.12_±_0.01 _0.08_±_0.02 _0.15_±_0.01 _0.17_±_0.01 _0.11_±_0.01 _0.11_±_0.03 %CVmean (n =4) 8.08 9.62 2.79 3.00 11.47 14.64 0.00-1.50 LOD (mg L-1₎ _0.05 _0.11 _0.04 _0.02 _0.13 _0.42 𝐴𝑏𝑠0 𝜆𝑚𝑎𝑥 -0.87 ± 0.07 -1.01 ± 0.06 0.48 ± 0.02 0.53 ± 0.02 0.05 ± 0.03 0.07 ± 0.07 𝑚 (𝑚 1 _0.71_±_0.04 _0.77_±_0.03 _-0.14_±_0.01 _-0.16_±_0.01 _0.13_±_0.01 _0.13_±_0.03 1.50-3.00 %CVmean (n =4) 0.95 3.59 5.06 6.05 7.63 8.60 𝐴𝑏𝑠0 𝜆𝑚𝑎𝑥 1.46 ± 0.19 1.48 ± 0.12 0.11 ± 0.05 0.10 ± 0.04 0.17 ± 0.03 0.16 ± 0.09 𝑚 (𝑚 1 -0.06 ± 0.05 -0.05 ± 0.03 -0.01 ± 0.01 -0.01 ± 0.01 0.09 ± 0.01 0.10 ± 0.22 3.00-5.00 %CVmean (n =4) 1.12 1.32 12.57 9.48 4.40 4.18 R2 _0.99 _0.99 _0.98 _0.98 _1.00 _0.98 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1

Supplementary material

Fig. S1. Piecewise linear fitting for TMB and DPD titration experiments with chlorine in H2O (A)

and tap water (B) at t 0 s. Data and fitting parameters are reported in the main text in Figure 4 and Table 3, respectively. The chlorine concentration ranges are 0.00-1.50 mg L-1_{, 1.50-3.00 mg L}-1_,

and 3.00-5.00 mg L-1_. 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Table S1.1 Coefficient of variation (%CV) for Absorbance values at 280 nm vs [Cl2] and

time from Figure 2B

[Cl2] mg L1 in H2O 0.00 1.00 2.00 3.00 4.00 5.00 Time (s) %CV280nm %CV280nm %CV280nm %CV280nm %CV280nm %CV280nm 0 0.92 1.00 6.02 14.53 1.44 6.17 12 1.00 1.04 5.90 15.54 3.42 7.01 22 0.97 1.05 5.73 15.06 5.16 5.23 32 1.00 1.02 6.01 14.96 7.94 4.89 42 1.03 1.06 6.06 14.87 10.00 1.70 52 1.06 1.05 5.77 14.68 9.85 4.10 62 1.05 1.09 5.89 11.29 10.04 4.44 72 1.03 1.36 5.96 14.70 9.22 3.64 82 1.05 1.06 6.21 14.63 9.17 7.47 92 1.09 0.78 5.79 14.84 10.60 3.84 102 1.11 1.08 5.73 14.81 10.27 3.05 112 1.06 1.10 5.24 16.72 9.43 3.46 122 1.13 1.04 5.41 14.26 0.79 2.56 132 1.13 1.09 5.56 14.20 11.19 3.24 142 1.15 0.96 6.32 13.85 11.06 3.48 152 1.16 1.62 8.65 14.26 11.54 2.02 162 1.19 1.15 6.67 14.11 9.28 3.19 172 1.18 1.22 5.31 13.97 9.18 3.22 182 1.15 1.21 5.38 14.20 9.74 3.01 192 1.17 1.14 5.47 11.55 9.63 2.62 202 1.18 1.22 5.47 13.89 10.01 2.90 212 1.19 1.16 5.40 13.55 9.50 3.34 222 1.15 1.16 5.24 13.52 10.62 3.25 232 1.20 1.09 5.29 13.85 9.68 3.16 242 1.22 1.20 6.11 13.47 9.79 1.99 252 1.22 1.21 5.06 13.58 9.02 3.84 262 1.23 1.11 5.05 13.78 9.08 3.66 272 1.30 1.01 5.04 13.62 8.55 3.95 282 1.27 1.06 5.01 13.69 8.87 3.31 292 1.25 1.06 5.02 13.94 9.12 3.10 302 1.24 0.97 5.06 14.25 8.94 2.66 %CVmean (0-300 s) 1.13 1.11 5.70 14.13 8.78 3.66 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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3

time from Figure 2B

[Cl2] mg L1 in H2O 0.00 1.00 2.00 3.00 4.00 5.00 Time (s) %CV450nm %CV450nm %CV450nm %CV450nm %CV450nm %CV450nm 0 4.89 9.58 2.86 3.35 1.00 0.54 12 3.07 7.66 2.86 5.16 0.99 0.34 22 4.78 15.69 2.24 2.92 0.59 0.28 32 5.44 13.35 2.02 2.92 0.16 0.26 42 5.63 14.97 1.53 2.79 0.65 0.20 52 13.07 14.92 1.42 2.79 0.90 0.19 62 10.51 14.06 1.31 2.83 1.33 0.18 72 6.58 13.61 1.31 2.75 1.27 0.18 82 2.25 8.36 1.20 2.77 1.43 0.17 92 9.88 13.77 0.70 2.77 1.41 0.10 102 5.27 15.56 1.25 2.79 1.21 0.18 112 2.86 13.05 1.26 2.77 1.28 0.18 122 9.81 12.95 1.64 2.72 1.37 0.24 132 1.63 13.16 1.26 2.80 1.36 0.18 142 7.58 13.31 1.10 2.79 1.18 0.16 152 14.42 13.99 0.73 2.75 1.32 0.11 162 10.35 9.24 0.33 2.75 1.32 0.05 172 0.27 12.19 0.61 2.71 1.34 0.09 182 8.97 12.06 1.20 2.72 1.35 0.18 192 1.56 11.62 1.18 2.78 1.40 0.18 202 2.59 12.09 1.11 2.77 1.16 0.26 212 2.71 12.23 1.11 2.73 1.30 0.39 222 3.75 12.09 1.14 2.75 1.27 0.17 232 0.55 11.76 1.18 2.67 1.24 0.18 242 12.90 12.37 1.04 2.66 1.32 0.16 252 3.72 11.54 0.66 2.72 1.35 0.10 262 4.36 11.86 1.13 2.69 1.35 0.17 272 9.40 12.66 1.18 2.67 1.29 0.18 282 9.13 13.32 1.18 2.69 1.35 0.18 292 1.56 12.93 1.16 2.67 1.27 0.33 302 1.35 13.48 1.23 2.71 1.35 0.19 %CVmean (0-300 s) 5.83 12.56 1.29 2.85 1.20 0.20 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Table S1.3 Coefficient of variation (%CV) for Absorbance values at 655 nm vs [Cl2] and time

from Figure 2B [Cl2] mg L1 in H2O 0.00 1.00 2.00 3.00 4.00 5.00 Time (s) %CV655nm %CV655nm %CV655nm %CV655nm %CV655nm %CV655nm 0 1.55 8.08 1.06 1.91 3.39 10.32 12 8.72 8.23 0.99 1.79 6.24 5.58 22 1.66 8.28 0.86 1.55 6.38 4.78 32 1.77 8.12 0.78 1.40 6.06 2.69 42 2.20 7.98 0.63 1.12 6.04 2.81 52 6.13 8.24 0.67 1.21 5.88 5.88 62 3.91 8.58 0.54 0.97 7.09 5.09 72 3.21 6.83 0.50 0.89 6.94 3.02 82 9.05 8.07 0.52 0.92 7.60 1.89 92 3.88 8.70 0.69 1.23 7.77 2.49 102 2.26 8.33 0.66 1.18 8.13 7.49 112 1.96 8.08 0.53 0.94 8.98 12.79 122 4.55 8.47 0.56 0.99 8.53 6.47 132 7.39 8.26 0.53 0.94 8.58 5.38 142 3.34 8.56 0.54 0.96 8.08 12.38 152 6.49 7.31 0.42 0.75 8.55 12.34 162 4.88 7.72 0.41 0.73 9.20 7.09 172 0.13 8.28 0.56 0.99 8.57 5.89 182 4.46 8.31 0.52 0.92 9.53 5.62 192 0.76 8.01 0.43 0.75 9.40 14.25 202 1.25 8.21 0.51 0.89 10.33 5.46 212 1.31 8.56 0.55 0.96 9.78 22.33 222 1.86 7.91 0.45 0.79 9.91 9.28 232 0.27 8.34 0.35 0.62 9.88 4.24 242 9.54 8.07 0.34 0.59 10.65 3.73 252 1.83 8.74 0.45 0.79 10.09 13.20 262 2.16 8.37 0.37 0.65 10.19 4.37 272 4.61 8.67 0.63 1.11 11.70 5.12 282 9.35 8.65 0.39 0.68 11.86 12.94 292 7.62 8.85 0.43 0.76 9.84 13.81 302 7.07 8.81 0.50 0.87 10.13 9.14 %CVmean (0-300 s) 4.04 8.25 0.56 1.00 8.56 7.67 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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time from Figure 3B

[Cl2] mg L1 in tap water 0.00 1.00 2.00 3.00 4.00 5.00 Time (s) %CV280nm %CV280nm %CV280nm %CV280nm %CV280nm %CV280nm 0 0.29 2.02 5.03 1.27 5.85 2.87 12 0.47 1.95 4.98 1.02 6.48 3.29 22 0.25 2.00 5.06 0.70 6.14 2.46 32 0.21 1.98 5.06 1.29 5.47 2.30 42 0.19 1.93 5.11 1.55 6.38 0.82 52 0.21 1.98 5.15 1.56 6.13 1.97 62 0.21 1.96 5.35 1.62 5.88 2.14 72 0.18 1.92 5.50 1.52 5.86 1.77 82 0.15 1.92 5.44 2.01 6.00 3.76 92 0.15 1.91 5.19 1.71 5.82 1.90 102 0.17 1.88 5.14 1.72 5.82 1.50 112 0.15 1.85 5.43 1.87 6.12 1.70 122 0.16 1.86 5.39 1.89 5.45 1.28 132 0.16 1.86 5.46 1.71 5.78 1.63 142 0.15 1.82 5.44 1.67 6.00 1.75 152 0.14 1.85 5.50 1.69 5.65 1.02 162 0.14 1.89 5.57 1.89 5.97 1.60 172 0.16 1.88 5.52 1.60 5.85 1.62 182 0.18 1.92 5.56 1.78 5.72 1.51 192 0.20 1.94 5.58 0.62 6.10 1.33 202 0.20 1.93 5.62 1.62 6.45 1.47 212 0.19 1.95 5.62 1.40 6.10 1.69 222 0.19 1.94 5.67 1.60 5.99 1.66 232 0.15 1.95 5.90 1.55 5.83 1.62 242 0.17 1.94 6.23 1.44 5.91 1.03 252 0.17 1.94 6.29 1.60 6.15 1.99 262 0.18 1.97 5.84 1.65 6.06 1.90 272 0.17 1.97 5.80 1.67 6.06 2.05 282 0.12 1.97 5.80 1.61 5.82 1.70 292 0.10 1.96 5.82 1.51 5.76 1.59 302 0.07 2.30 5.85 1.46 5.88 1.37 %CVmean (0-300 s) 0.18 1.94 5.51 1.54 5.95 1.82 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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time from Figure 3B

[Cl2] mg L1 in tap water 0.00 1.00 2.00 3.00 4.00 5.00 Time (s) %CV450nm %CV450nm %CV450nm %CV450nm %CV450nm %CV450nm 0 8.25 4.16 0.69 2.51 3.15 0.60 12 3.21 3.79 0.81 2.32 3.45 2.70 22 6.49 3.99 1.17 2.29 3.55 2.82 32 1.91 4.07 1.22 2.16 3.61 2.91 42 3.82 4.46 1.24 2.10 3.71 2.99 52 5.89 3.79 1.40 2.06 3.71 3.03 62 5.10 4.27 1.25 2.02 3.76 3.07 72 4.52 4.32 0.92 2.00 3.76 3.06 82 3.86 4.29 1.31 1.94 3.76 3.09 92 3.90 4.63 1.70 1.98 3.81 3.11 102 6.51 4.58 1.84 1.96 3.80 3.11 112 6.10 4.44 1.62 1.92 3.81 3.13 122 4.02 4.31 1.67 1.86 3.75 3.09 132 4.68 4.71 1.64 1.86 3.80 3.14 142 1.94 4.74 1.64 1.84 3.81 3.16 152 6.13 4.22 1.62 1.83 3.79 3.15 162 3.78 4.62 1.59 1.80 3.79 3.15 172 3.39 4.29 1.66 1.79 3.81 3.18 182 7.29 4.17 1.58 1.80 3.76 3.15 192 9.22 4.27 1.60 1.88 3.77 3.16 202 11.42 4.10 1.60 1.83 3.80 3.18 212 10.72 4.02 1.58 1.78 3.80 3.18 222 11.02 4.07 1.54 1.78 3.76 3.16 232 8.50 3.96 1.31 1.79 3.79 3.19 242 10.41 4.22 0.85 1.78 3.77 3.18 252 7.94 3.79 0.85 1.77 3.80 3.20 262 10.16 3.80 1.33 1.77 3.79 3.21 272 10.55 3.68 1.37 1.75 3.76 3.18 282 6.57 3.62 1.35 1.73 3.75 3.17 292 1.13 3.85 1.33 1.74 3.76 3.19 302 5.13 1.98 1.25 1.74 3.75 3.19 %CVmean (0-300 s) 6.24 4.10 1.37 1.92 3.73 3.03 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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7

time from Figure 3B

[Cl2] mg L1 in tap water 0.00 1.00 2.00 3.00 4.00 5.00 Time (s) %CV655nm %CV655nm %CV655nm %CV655nm %CV655nm %CV655nm 0 1.06 4.72 2.69 0.05 1.39 0.3 12 14.64 4.7 2.9 0.46 2.12 1.84 22 2.25 4.83 2.97 0.46 2.46 0.34 32 2.52 4.4 3.19 0.5 2.66 0.35 42 3.67 4.64 3.22 0.39 2.51 0.48 52 9.04 4.64 3.23 0.48 2.67 1.32 62 6.84 4.7 3.37 0.56 2.66 0.97 72 5.11 4.8 3.71 0.57 2.69 0.7 82 15.28 4.7 3.47 0.72 3.16 2.03 92 6.23 4.73 3.32 0.71 2.8 0.95 102 4.37 4.8 3.5 0.71 3.21 0.61 112 3.24 4.4 3.35 0.81 3.01 0.43 122 6.75 4.8 3.59 0.68 3.02 1.03 132 11.36 4.73 3.58 0.8 2.99 1.6 142 5.36 4.89 3.79 0.89 2.76 0.81 152 10.8 4.58 3.7 0.68 2.91 1.55 162 8.42 4.79 3.73 0.73 2.96 1.15 172 0.23 4.78 3.61 1.01 2.71 0.03 182 6.97 4.4 3.7 0.89 2.72 1.01 192 1.31 4.32 3.7 1.1 2.88 0.17 202 2.31 4.71 3.64 1.35 2.63 0.3 212 2.23 4.61 3.73 1.09 2.56 0.31 222 3.1 4.66 3.86 1.35 2.73 0.44 232 0.49 4.52 3.92 1.11 2.85 0.06 242 1.75 4.73 4.38 1.24 2.36 2.26 252 3.57 4.69 4.27 1.39 2.83 0.44 262 4.13 4.69 4 1.25 2.8 0.51 272 8.32 4.6 3.92 1.26 2.85 1.11 282 8.87 4.68 4.03 1.34 2.7 2.19 292 14.26 4.57 4.01 1.32 2.8 1.89 302 11.03 4.28 2.69 1.31 2.86 1.58 %CVmean (0-300 s) 5.98 4.65 3.57 0.88 2.72 0.93 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Table S3.1 Coefficient of variation (%CV) for DPD Absorbance values at

555 nm vs [Cl2] and time from Figure 4C (H2O)

0 s 30 s 60 s 120 s 240 s [Cl2] mg L1 %CV555nm %CV555nm %CV555nm %CV555nm %CV555nm 0.00 5.89 5.76 4.91 4.56 4.61 0.25 11.04 14.74 15.40 15.85 13.59 0.50 18.40 17.26 17.23 16.20 15.86 1.00 11.11 8.09 6.75 4.73 3.55 1.50 10.90 10.45 8.99 7.21 6.43 2.00 8.86 7.18 4.57 2.68 3.27 2.50 8.19 6.43 5.03 4.54 4.86 3.00 2.57 2.63 2.67 2.70 3.05 3.50 6.20 6.03 5.81 5.43 4.65 4.00 3.92 2.41 1.64 1.07 1.46 4.50 5.68 4.38 4.21 3.44 4.44 5.00 3.60 2.83 1.57 2.11 2.77 %CVmean 8.03 7.35 6.57 5.88 5.71

Table S3.2 Coefficient of variation (%CV) for DPD Absorbance values at

555 nm vs [Cl2] and time from Figure 4D (Tap water)

0 s 30 s 60 s 120 s 240 s [Cl2] mg L1 %CV555nm %CV555nm %CV555nm %CV555nm %CV555nm 0.00 11.82 10.26 10.21 9.68 8.48 0.25 9.16 8.96 8.20 7.80 6.94 0.50 11.82 10.26 10.21 9.68 8.48 1.00 9.16 8.96 8.20 7.80 6.94 1.50 11.82 10.26 10.21 9.68 8.48 2.00 9.16 8.96 8.20 7.80 6.94 2.50 11.82 10.26 10.21 9.68 8.48 3.00 9.16 8.96 8.20 7.80 6.94 3.50 11.82 10.26 10.21 9.68 8.48 4.00 9.16 8.96 8.20 7.80 6.94 4.50 11.82 10.26 10.21 9.68 8.48 5.00 9.16 8.96 8.20 7.80 6.94 %CVmean 9.16 8.96 8.20 7.80 6.94 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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9

Table S3.3 Coefficient of variation (%CV) for TMB Absorbance values at

0 s 30 s 60 s 120 s 240 s [Cl2] mg L1 %CV450nm %CV450nm %CV450nm %CV450nm %CV450nm 0.00 1.38 1.15 0.39 0.53 0.56 0.25 0.64 0.33 0.90 1.13 0.94 0.50 2.44 2.16 1.60 1.49 2.40 1.00 1.38 0.84 1.11 1.24 1.35 1.50 0.75 0.85 0.75 0.78 0.83 2.00 7.62 1.11 8.14 8.09 8.20 2.50 2.21 0.12 1.56 1.59 1.82 3.00 3.77 2.74 3.56 4.11 4.78 3.50 9.43 3.80 6.20 6.02 7.04 4.00 9.79 3.45 10.71 12.32 15.20 4.50 17.48 12.41 17.16 17.82 3.64 5.00 7.64 3.06 3.02 3.79 3.85 %CVmean 5.38 2.67 4.59 4.91 4.22 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0 s 30 s 60 s 120 s 240 s [Cl2] mg L1 %CV655nm %CV655nm %CV655nm %CV655nm %CV655nm 0.00 2.56 2.64 2.83 4.14 3.56 0.25 6.54 7.7 8.87 8.45 10.79 0.50 1.78 2.73 4.63 5.15 6.84 1.00 1.57 2.16 2.16 2.41 2.96 1.50 2.53 2.28 3.38 3.75 4.35 2.00 1.45 1.29 2.18 2.6 2.54 2.50 11.96 6.26 11.13 10.03 9.09 3.00 8.25 3.92 2.83 0.73 2.91 3.50 9.59 9.11 16.33 13.03 10.94 4.00 29.21 3.2 2.34 2.14 2.63 4.50 2.92 13 12.7 10.2 7.62 5.00 17.93 16.88 15.95 17.11 16.11 %CVmean 8.02 5.93 7.11 6.65 6.70 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Graphycal Abstract 173x73mm (300 x 300 DPI) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60