Microfluidic Chemiluminescence System with Yeast - ACS Publications

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Microfluidic chemiluminescence system with yeast Saccharomyces cerevisiae for rapid biochemical oxygen demand measurement Susana P. F. Costa, Edite Cunha, Ana M. O. Azevedo, Sarah A. P. Pereira, Ana F. D. C. Neves, Andre G. Vilaranda, André RTS Araujo, Marieta L. C. Passos, Paula C. A. G. Pinto, and Maria Lucia Marques Ferreira De Sousa Saraiva ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b04736 • Publication Date (Web): 20 Mar 2018 Downloaded from http://pubs.acs.org on March 22, 2018

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ACS Sustainable Chemistry & Engineering

Microfluidic chemiluminescence system with yeast Saccharomyces cerevisiae for rapid biochemical oxygen demand measurement Susana P. F. Costaa, Edite Cunhaa, Ana M. O. Azevedoa, Sarah A. P. Pereiraa, Ana F. D. C. Nevesa, André G. Vilarandaa, André R. T. S. Araujoa,b, Marieta L. C. Passosa, Paula C. A. G. Pintoa,c, M. Lúcia M. F. S. Saraivaa* a

LAQV, Requimte, Departamento de Ciências Químicas, Laboratório de Química Aplicada,

Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, nº 228, 4050-313 Porto, Portugal b

Unidade de Investigação para o Desenvolvimento do Interior, Instituto Politécnico da Guarda,

Av. Dr. Francisco de Sá Carneiro, nº 50, 6300-559 Guarda, Portugal c

A3D - Association for Drug Discovery and Development, Rua do Baixeiro, nº 38 Aradas, 3810-

399 Aveiro, Portugal

Authors e-mails: [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]

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ABSTRACT

A new automated chemiluminescence method resorting to sequential injection analysis (SIA) was developed to rapidly determine biochemical oxygen demand (BOD). The assay is based on the redox reaction between quinone and Baker’s yeast in the presence of organic substances. The formed active oxygen species reacted with luminol, under the catalytic action of ferricyanide, and increased chemiluminescence signal. The automation of the assay ensured a precise control of the reaction conditions and enabled a reduction of more than 75-times in the reagents consumption and effluents production comparatively to BOD5. The sampling rate was widely improved with a flow rate of 8 cycles per hour. The method was applied to determine the BOD of ionic liquids (ILs) incorporating different chemical elements and deep eutectic solvents (DESs) combining choline chloride with varying hydrogen-bond donors. Differences in BOD and biodegradability were observed between tested compounds, with DESs showing, in general, higher BOD values and greater biodegradation than ILs. The results obtained in the developed bioassay demonstrated statistical correlation with the BOD5 method. Therefore, the developed methodology is a simple, economic and high-throughput alternative screening bioassay to the conventional method, with the potential to preliminarily assess the potential biodegradability of chemicals in the environment.

KEYWORDS: Biochemical Oxygen Demand; Biodegradability; Deep Eutectic Solvents; Ionic Liquids; Sequential Injection Analysis; Saccharomyces cerevisiae.


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Introduction Biochemical oxygen demand (BOD) is one of the most important and widely used parameters to assess water quality, estimating the organic pollution of industrial wastewater or natural waters. BOD measures the amount of oxygen required by aerobic microorganisms to decompose organic matters in wastewater.1 Although the conventional 5-day (BOD5) method is well-established and provides reliable results, there are drawbacks in terms of applicability in laboratories with a large number of samples to analyze and it is not suitable for active intervention, such as environmental monitoring or process control. In fact, it is a laborious and time-consuming procedure that requires considerable experience and skill of operators to get reproducible results. Some alternative techniques have been developed to overcome the disadvantages of the conventional method. For example, sensors based on dissolved oxygen (DO) probes and biological recognized elements. The first biosensor for rapid BOD estimation was developed by Karube and coworkers in 19972 through the combination of a DO electrode and immobilized microorganisms in a membrane. Thenceforth a variety of other biosensors have been employed to determine BOD, most of them also incorporating immobilized microbial cells. Some of the microbial cells applied were Bacillus subtilis,3-4 Escherichia coli,5 Pseudomonas sp. ,6 Photobacterium phosphonium 5 and Trichosporon cutaneum.4, 7 In general, these systems required only a few minutes to obtain a reasonable signal and provided a good relationship between the output signal and BOD concentration.8 However, most of these systems have complex requirements of maintenance and the stability of some microbial cultures seems to be influenced by heavy metals and other toxic substances.9 Nakamura et al.10 published an alternative method to rapidly determine BOD, which based on a chemiluminescent reaction between luminol and oxygen species resulting from redox reaction between quinone, yeast Saccharomyces cerevisiae and potential pollutants, under the


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catalytic action of ferricyanide.10 Previous studies showed that ferricyanide is an efficient hydrophilic mediator for shuttle electrons from redox center of reduced bacterial enzymes to the electrode in the presence of organic compounds and so it has been widely used in the recent works involving redox reactions.11 Eukaryotes have some advantages as viable microbial in biosensors, as they are easy of handle, widely available, with a high rate of survival. Therefore, yeasts are one of the preferable biomaterials for the development of BOD biosensors.9, 11-12 Seo and coauthors8 were one of the few investigators to apply flow techniques, namely flow injection analysis (FIA), in the determination of BOD. The developed bioreactor with encapsulated S. cerevisiae and an oxygen electrode exhibited reasonable results in terms of sensitivity and reproducibility. Recently, Oota13 and Liu14 research groups also implemented automatic mediated BOD biosensors systems with good performance. FIA system operates as continuous circulation which demands the use of considerable amounts of carrier and sample, hence we opted for this work by another automatic approach, sequential injection analysis (SIA).15 SIA ensures an effective computer control of the relevant analytical parameters and hence a precise control of the reaction conditions. It is also an asset for the development of methods displaying great reproducibility, reasonable sampling rate and reduced consumption of reagents.16-17 Moreover, SIA has already proved to be a robust and accurate solution to bioassays using living microorganisms.18-19 In this work, we describe the development and optimization of a novel high-throughput method to determine BOD as well as an application of the method to the study of ionic liquids (ILs) and deep eutectic solvents (DESs), to estimate their impact in the aquatic habitats. ILs have noteworthy physical and chemical properties, such as negligible vapour pressure, nonflammability, chemical and thermal stability and a large range of solvation20-21 which resorted in


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an increasing interest of scientific21-26 and industrial22, 27 communities in this family of heterogeneous compounds and its potentialities. The large range of solvation, chemical, and thermal stability raises concerns about ILs potential environmental fate and risk of persistence in the environment, particularly in the aquatic ecosystem. DESs have been described as a promising “greener” alternative to conventional ILs, but maintaining similar physical properties and phase behavior. These solvents are easy to prepare, cheaper and use more environmentally-friendly ingredients than ILs. Therefore, they have been portrayed as less toxic and more biodegradable than traditional ILs.28-29However, more studies are needed to confirm the real environmental impact of these solvents and also their comparison with classic ILs. In this context, the aim of the current work was to develop a new automatic chemiluminescent method associated to a double-mediator system (ferricyanide and quinone) and Baker’s yeast for rapid determination of BOD and evaluate ILs and DESs biodegradability. It is expected that the developed methodology can be applied as an alternative screening approach to determine BOD and predict biodegradability.

Experimental Section Reagents Ten millimolar luminol was dissolved in NaOH 0.1 M and stored for 2-3 days for stabilization in a light-protected bottle. Then, the stock solution was diluted to 12.5 µM and the obtained working solution was used in the BOD assays. Potassium hexacyanoferrate (III) 20 mM, 1,2naphthoquinone-4-sulfonic acid (NQS) 5 mM, hydrogen peroxide 20 mM and purchased Baker’s yeast S. cerevisiae 1.6 mg mL-1, from McCormick, were daily prepared in water. The carrier


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solution phosphate buffer was prepared by dissolution of 2.4 g KH2PO4, 1.82 g Na2HPO4.2H2O, 80 g NaCl and 2.0 g KCl in water in a final volume of 1 L, being the pH adjusted to 7.0 with HCl. The reagents applied in the Winkler method were prepared according to established procedure.30 PolySeed® was prepared according to the protocol of InterLab31 and the obtained suspension was after decanted and used in the BOD5 assays. A BOD standard solution containing 150 mg L-1 of glucose and glutamic acid (GGA) in equal quantities was employed as a positive control in both procedures. The tested compounds (Table 1) were kept in a carefully controlled anhydrous environment.

Table 1. List of the ILs and DESs tested in this study.

Ionic Liquids (ILs)

1 2 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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Name

Abbreviation (molar ratio)

1-ethyl-3-methylimidazolium acetate

emim [Ac]

1-ethyl-3-methylimidazolium trifluoromethanesulfonate

emim [TfMs]

1-ethyl-3-methylimidazolium methanesulfonate

emim [Ms]

1-butyl-3-methylimidazolium chloride

bmim [Cl]

1-butyl-3-methylimidazolium acetate

bmim [Ac]

1-butyl-3-methylimidazolium tetrafluoroborate

bmim [BF4]

1-hexyl-3-methylimidazolium chloride

hmim [Cl]

1-butyl-1-methylpyrrolidinium chloride

bmpyr [Cl]

1-butyl-1-methylpyrrolidinium tetrafluoroborate

bmpyr [BF4]


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Deep Eutectic Solvents (DESs)

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Tetrabutylammonium chloride

N4,4,4,4 [Cl]

Tetrabutylammonium acetate

N4,4,4,4 [Ac]

Tetrabutylphosphonium methanesulfonate

tbph [Ms]

Choline chloride:Urea

chol [Cl]:U (1:2)

Choline chloride:Thiourea

chol [Cl]:TU (1:2)

Choline chloride:D-(-)-Fructose

chol [Cl]:Fru (2:1)

Choline chloride:Acetamide

chol [Cl]:A (1:2)

Choline chloride:Citric acid

chol [Cl]:CA (1:1)

Choline chloride:Lactic acid

chol [Cl]:LA (1:2)

Choline chloride:Malonic acid

chol [Cl]:MA (1:1)

Choline chloride:L-(+)-Tartaric acid

chol [Cl]:TA (2:1)

Choline chloride:Glycerol

chol [Cl]:Gly (1:2)

Choline chloride:Zinc chloride

chol [Cl]:ZnCl2 (1:2)

Preparation of DESs In this work, DESs were prepared following standard procedures32-34 in accordance with the molar ratios presented in Table 1. Prior to being used, choline chloride was dried under vacuum at 60 ºC and all other reagents used for the synthesis of DESs were vacuum dried over silica gel and phosphorus pentoxide until they reached a constant weight. Briefly, the eutectic mixtures were obtained by heating the two components to 80/100 ºC and stirring until a homogeneous liquid was formed. The synthesized DESs were vacuum dried prior to the biodegradability evaluation.


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Apparatus The analytical flow system (Fig. 1) applied to determine BOD incorporated a buret Bu1S from Crison Instruments S.A. (Allela, Barcelona, Spain) and a 10-port multiposition CheminertTM selection valve. A glass syringe of 5 mL total dispense volume (Hamilton Bonaduz AG, Switzerland) was coupled to the device and driven by a stepper motor. Solenoid head-valves enabled the commutation of the syringe either to the manifold or to the carrier. The synchronization between the devices was performed by computer resorting to visual basic software and the communication with the instruments was accomplished by means of RS-232C asynchronous protocols, using embedded dynamic libraries. A sequential output of the commands and evaluation of the equipment status was performed through the implementation of the control algorithm based on the use of a set of interdependent timers. The applied software enabled the control of flow rate, flow direction, valve position, stop flow duration, sample, and reagents volumes, as well as data acquisition and processing. During the assays, the analytical signals were recorded on strip chart recorder (Kipp & Zonen BD 111) or acquired via computer. Manifold components were connected by means of 0.8 mm i.d. PTFE tubing, which was also used for the holding coil (2 m). A mixing chamber with an internal volume of ca. 400 µL, containing a magnet was used to guarantee adequate mixing conditions. Chemiluminescence measurements were made in a Camspec CL-2 equipped with a 60 µL flow cell and 5 mm of the optic path, which was positioned in front of a photomultiplier. The detector was composed of two input ports and one output port, a configuration that promoted the reagents mixture in the cell and ensured that the reaction between luminol and the active oxygen species


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occurred inside the cell. The temperature of the assay was monitored with a heating-stirring plate (IKA C-MAG HS7) and maintained around 23 ± 3 ºC. Yeast suspension was maintained in continuous stirring during the assays.

Figure 1. Schematic representation of the SIA system applied in the determination of BOD values. C: carrier (phosphate buffer, pH 7.0); S: syringe (5 mL); HC: holding coil (2 m); SV: selection valve; H: hydrogen peroxide; L: luminol; Fe: potassium hexacyanoferrate (III); NQS: 1,2-naphthoquinone-4-sulfonic acid; Y: yeast S. cerevisiae; SS: standard solution (GGA); MC: mixing chamber; D: detector; W: waste.

Sequential injection procedure The analytical cycle established, in the SIA system, to determine BOD is schematized in Table 2. In each cycle, were sequentially aspirated 12.5 µL of NQS, 31.3 µL of Fe (III), 18.8 µL of yeast and 43.8 µL of sample which composed the mixture 1. The flow was reversed and the reaction zone was propelled by the carrier solution to the mixing chamber, where remained for 300 seconds. After the stop period, mixture 1 was propelled until the entrance of detector cell. Next,


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aliquots of hydrogen peroxide (25 µL), luminol (12.5 µL) and Fe (III) (31.3 µL) were aspirated and the obtained reaction zone (mixture 2) was propelled in direction of second entrance of cell detector for 3.4 seconds. After, it was sequentially propelled mixture 1 and mixture 2 to chemiluminescence cell positioned in front of a photomultiplier. Then, it was acquired the increase on chemiluminescence signal proportional to luminol oxidation in the presence of reactive oxygen species. Blank assays were performed for each tested condition in the course of optimization. This enabled the evaluation not only of the obtained chemiluminescence signal in the absence of sample but also the resulting increment when substituted by GGA standard solution 150 mg L-1. Each condition was evaluated in triplicate.

Table 2. Analytical cycle applied in SIA system to determine BOD.

Step

Solution

Volume (µL)

Time (sec)

Flow rate (mL min-1)

Action

1

NQS

12.5

1.5

0.5

Aspiration of NQS

2

Fe (III)

31.3

1.9

1

Aspiration of K3[Fe(CN)6]

3

Yeast

12.5

1.5

0.5

Aspiration of S. cerevisiae

4

GGA

43.8

2.6

1

Aspiration of GGA

5

Mixture 1

200

12

1

Propulsion to mixing chamber

6

---

---

300

0

Stop period

7

Mixture 1

180

11

1

Aspiration from mixing chamber

8

Mixture 1

150

9

1

Propulsion to detector cell

9

H 2O 2

25

1.5

1

Aspiration of H2O2

10

Luminol

12.5

1.5

0.5

Aspiration of luminol

11

Fe (III)

31.3

1.9

1

Aspiration of K3[Fe(CN)6]


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12

Mixture 2

113

3.4

2

Propulsion to detector cell

13

Mixture 1

188

5.6

2

Propulsion of mixture 1 into the cell

14

Mixture 2

125

7.5

1

Propulsion of mixture 2 into the cell

15

---

688

16.5

2.5

Washing of 9-port

16

---

688

16.5

2.5

Washing of 10-port

17

---

625

12.5

3

Washing of mixing chamber

18

---

1200

16

4.5

Elimination of washing solution

20

---

3600

29

7.5

Refilling of syringe

Winkler method DO concentrations were determined using the conventional Winkler’s method according to an established procedure.30 In each assay, 4 mL of bacteria solution and 1.25 mL of a 250mM IL solution were added to a capped BOD bottle with a capacity of around 300 mL, obtaining a final concentration of IL of 1 mM. Then, the bottles were completely filled with the prepared mineral medium that contained 42.5 mg L-1 KH2PO4, 22.5 mg L-1 MgSO4.7H2O, 27.5 mg L-1 CaCl2 and 0.25 mg L-1 FeCl3.6H2O. The same procedure was applied to the tested DESs, in a concentration of 5 mg L-1. It was also performed blank and positive control assays, without sample or with 15 mL of GGA standard solution, respectively. The bottles were kept in an incubator for 5 days at 20 ºC, protected from light. After that period, it was determined the DO of the selected compounds by the Winkled method. Firstly, a 2-mL pipette was filled with KI, placed on the bottom of the bottle and 1 mL was drained. The same procedure was performed with the manganese chloride solution. The obtained mixture was left to precipitate for 1 hour. Then, it was introduced 5 mL of steaming hydrochloric acid solution into the bottle and the obtained solution was titrated with sodium thiosulphate N/10 solution until it became straw yellow. At


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that point, starch solution was added to the mixture and titration continued until the disappearance of blue color. BOD and % degradation was calculated according to the formulas indicated in the OECD test guideline 301 D.35

Results and Discussion In this work, it is described the development and implementation of an automated methodology to rapidly determine BOD and its application in the evaluation of biodegradability of a set of ILs and DESs. The method based on the redox reaction between quinone derivative and S. cerevisiae in the presence of organic substances that result in the formation of quinol or semiquinone radicals, which originate active oxygen species, such as superoxide anions and hydrogen peroxide. The active oxygen species react with luminol under the catalysis of ferricyanide, and it is registered as an increase in the chemiluminescence transient signal. Therefore, the production rate of active oxygen species was determined by luminol-dependent chemiluminescence reaction which is proportional to a rise in the microbial metabolic activity when in the presence of organic substances.10, 36

Optimization of the SIA assay The physical and chemical parameters eligible to influence the assay performance were assessed by the univariate approach. The results were analyzed in terms of chemiluminescence signal in


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the absence and presence of positive control (GGA) and differences between both. The tested parameters and respective selected final conditions are listed in Table 3.

Table 3. Results of the optimization of BOD assay in the flow system.

Parameter

Range

Selected

Volume of luminol (µL)

7.5 - 20

12.5

Volume of potassium hexacyanoferrate (III) (µL)

17.5 - 37.5

21.9

Volume of 1,2-naphthoquinone-4-sulfonic acid (µL)

10 - 18.75

12.5

Volume of H2O2 (µL)

18.75 - 31.25

25

Volume of S. cerevisiae (µL)

10 - 25

12.5

Luminol concentration (µM)

12.5 - 112.5

12.5

H2O2 concentration (mM)

1 - 20

20

Potassium hexacyanoferrate (III) concentration (mM)

5 - 30

20

1,2-naphthoquinone-4-sulfonic acid concentration (mM)

1 - 20

5

S. cerevisiae concentration (mg mL-1)

0.5 - 10

1.6

0.5 - 2

2

Propulsion of mixture 2 to detector (mL min )

0.5 - 2

1

Stop period in mixing chamber (min)

1.5 - 7.5

5

Propulsion of mixture 1 to detector (mL min-1) -1

A flow rate of 1 mL min-1 was adopted for the aspiration of potassium hexacyanoferrate (III), samples and H2O2. Luminol, 1,2-naphthoquinone-4-sulfonic acid, and S. cerevisiae were aspirated at 0.5 mL min-1 ensuring a good repeatability. The propulsion flow rate to detector cell was studied between 0.5 and 2 mL min-1 for mixture 1 and mixture 2 and it was selected 2 and 1 mL min-1, respectively.


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The luminol reaction occurs in seconds, whereby we opted for the division of the reagents aspiration in two stages. First, we aspirated potassium ferricyanide, 1,2-naphthoquinone-4sulfonic acid, yeast and the sample, making mixture 1 that was propelled to a mixing chamber to promote the reaction between reagents. After the stop period and the propulsion of mixture 1 to the detector, the remaining solutions were aspirated and sent straight to the detector where the luminol chemiluminescent reaction was immediately registered by a photomultiplier positioned in front of the cell. The volume of luminol was studied between 7.5 and 20 µL and a volume of 12.5 µL was established in a compromise between repeatability and sensitivity, providing an amplified range for the study of samples requiring high microbial metabolic activity to be decomposed. A range of 17.5 to 37.5 µL in volume was tested for potassium ferricyanide and a volume of 21.9 µL was selected, since insignificant differences (< 5%) were observed for higher volumes. The volume of 12.5 µL of 1,2-naphthoquinone-4-sulfonic acid exhibited the better results among the evaluated range, hence, it was selected for future assays. H2O2 volume was studied between 18.8 and 31.3 µL, and it was adopted 25 µL in the final conditions, minimizing the chemiluminescence signal of blank assays, without impairing the differences observed between blank and GGA assays. Relatively to yeast, it was observed an increase in sensitivity up to 12.5 µL, expressed as an increase of more than four times in the difference between blank and GGA assays. Then, it was verified a decrease in the percent of increment of the chemiluminescence signal for volumes greater than 12.5 µL of S. cerevisiae, so this volume was used in the following studies. In general, it was selected small volumes of reagents, which promoted their mixture, without compromising the method sensitivity. The selection of small volumes also promoted the final mixture of all reagents in the chemiluminescence cell, as well as the


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configuration of the detector with the two input ports. Additionally, we used a mixing chamber with aim of increasing the contact between sample, yeast and quinine derivative. Relatively to reagents concentration the tested conditions are presented in figure 2. The concentration of luminol was fixed at 12.5 µM as a compromise between sensitivity and capacity to evaluate samples with high BOD. For concentrations of luminol below 22.5 µM the increase in the chemiluminescence signal between blank and control was only of 27%. Therefore, it was selected a lower concentration, guaranteeing a decrease in the blank signal, without significantly affecting the sensitivity of the assay. A reasonable blank chemiluminescence signal was observed for 20 mM of H2O2 without impairing the assay sensitivity. Potassium hexacyanoferrate (III) concentration was tested between 5 and 30 mM. The results suggested a maximum of catalytic activity for 20 mM, not justifying the use of superior concentrations in the following assays. The concentration of 1,2-naphthoquinone-4-sulfonic acid was tested between 1 and 20 mM, with an increase in chemiluminescence signal related to an increase in quinone concentration. However, it was fixed 5 mM for the upcoming studies due to solubility restraints. Regarding S. cerevisiae concentration, it was studied up to 10 mg mL-1 and opted for 1.6 mg mL1

in order to obtain a maximum of sensitivity.

140 120 100 Hydrogen peroxide % ∆CL

1 2 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


80 60

1,2-naphthoquinone-4sulfonic acid

40

Potassium hexacyanof errate (III)

20 0 0

10

20

30

40

Reagent concentration mmol L-1


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Figure 2. Representation of the effect of reagents concentration in the percentage of increase of chemiluminescence intensity resulting from differences between blank and GGA assays.

A stop period of 5 min in the mixing chamber was implemented with the objective of increasing the contact between sample, yeast, and quinone. The results showed that the difference in chemiluminescence between blank and control assays increased up to 5 min and slightly decreased for longer periods of time. A linear response was observed from 10 to 315 mg L-1 for GGA, described by the equation %∆ = 0.2761C (mg L-1) + 13.848 (R2 = 0.9944). The limits of detection and quantification were 9.53 and 31.75 mg L-1, respectively. The repeatability of the developed method was reasonable with a relative standard deviation of 7.1% (n = 10). The acquired data for studied ILs were interpolated on the obtained calibration curve and then converted to mg L-1 of O2 through the recognized relation 150 mg L-1 of GGA corresponds to 198 ± 30 mg L-1 of O2.31 After it was calculated BOD (mg O2/mg sample) and % degradation according to formulas indicated in OECD 301 D.35 The developed method can perform 8 cycles per hour while producing only 3.6 mL of effluent on each assay.

Applicability of the developed method to assess the BOD of selected ILs and DESs


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After the optimization of the luminol-dependent chemiluminescence method, ILs and DESs oxygen demand was determined. Test compounds representing various chemical structures, incorporating different alkyl chain length, cations, and anions were chosen. With this, we intended to collect data that can be useful to predict the biodegradability of different ILs and DESs. Additionally, the data obtained in the SIA system was compared with the traditional BOD5 method and the information was compiled in Table 4. The results demonstrated significant differences in BOD values and biodegradability between tested compounds. In general, DESs demonstrated higher consumption of oxygen than ILs, but at the same time higher percentages of biodegradation.

Table 4. Results of BOD and % degradation obtained in the automatic and standard methods for the selected compounds.

Conventional method (BOD5)

SIA method

Tested sample mg O2 L-1 ± SDa

% biodegradation

mg O2 L-1 ± SDa

% biodegradation

bmim [Cl]

160 ± 5.85

46.9

148 ± 19.2

46.3

emim [Ac]

115 ± 4.20

38.4

99.5 ± 11.2

34.6

bmim [BF4]

193 ± 7.02

15.8

158 ± 23.7

13.7

emim [TfMs]

183 ± 6.67

65.0

174 ± 10.2

65.8

emim [Ms]

186 ± 6.79

60.0

177 ± 6.53

61.3

bmpyr [Cl]

173 ± 6.32

39.0

216 ± 28.2

52.1

tbph [Ms]

234 ± 8.55

25.0

215 ± 22.8

23.9


17


a

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N4,4,4,4 [Cl]

231 ± 8.44

26.0

248 ± 28.6

29.8

bmpyr [BF4]

180 ± 6.56

40.0

186 ± 6.95

43.9

bmim [Ac]

141 ± 5.14

34.4

117 ± 12.1

30.4

hmim [Cl]

183 ± 6.67

41.3

196 ± 21.6

47.0

N4,4,4,4 [Ac]

186 ± 6.79

19.3

232 ± 22.0

26.0

chol [Cl]:U

389 ± 32.5

71.6

395 ± 33.0

72.7

chol [Cl]:TU

399 ± 33.3

69.4

398 ± 33.2

69.0

chol [Cl]:Fru

385 ± 32.1

50.1

377 ± 31.5

49.1

chol [Cl]:A

288 ± 24.0

52.9

291 ± 24.3

53.5

chol [Cl]:CA

273 ± 22.8

63.3

270 ± 22.6

62.6

chol [Cl]:LA

246 ± 20.5

51.3

257 ± 21.4

53.5

chol [Cl]:MA

173 ± 14.4

49.2

184 ± 15.3

52.2

chol [Cl]:TA

342 ± 28.5

52.2

350 ± 29.2

53.3

chol [Cl]:Gly

258 ± 21.5

50.4

259 ± 21.6

50.6

chol [Cl]:ZnCl2

156 ± 13.0

61.0

156 ± 13.0

61.0

The values presented are the mean and the standard deviation of the replicates.

Relatively to ILs, it was observed differences in BOD values between tested compounds, which ranged from 99.5 to 248 mg L-1 O2 for emim [Ac] and N4,4,4,4 [Cl], respectively. The compounds incorporating the anion acetate (emim [Ac] and bmim [Ac]) exhibited the lowest BOD values among the tested ILs. Moreover, the IL emim [Ac] (BOD = 99.5 mg L-1 O2) required lower consumption of oxygen by aerobic microorganisms than the ILs incorporating other anions, such as emim [TfMs] and emim [Ms] (BOD = 174 and 177 mg L-1 O2, respectively). The similarity in


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BOD values and biodegradability between emim [Ms] and emim [TfMs] suggests that the differences in the anionic group composition, namely the addition of three fluorine atoms, did not significantly influenced neither the oxygen consumption nor the biodegradability. These results are in disagreement with the “rules” published by Boethling et al.,37 which predict that halogens (especially chlorine and fluorine) increase the persistence of the molecule in the environment. The anionic pair chloride/tetrafluoroborate also demonstrated small differences in terms of BOD. Regarding the cationic core, in general, the tested cations seem to influence the requirement of oxygen by S. cerevisiae to decompose the compounds. Tbph [Ms] (BOD = 215 mg L-1 O2) showed a higher microbial metabolic activity comparatively to emim [Ms] (BOD = 177 mg L-1 O2). The ILs incorporating tetrabutylammonium as cation also exhibited higher BOD values than respective imidazolium derivatives. Pyrrolidinium-based ILs, on the other hand, presented BOD values resembling to ILs incorporating imidazolium core. In brief, the cation core appeared to influence the biodegradability in the following sequence: pyrrolidinium ≈ imidazolium ≥ ammonium ≈ phosphonium. A similar sequence was observed by Abrusci in the studies conducted with the bacterium Sphingomonas paucimobilis.38 The increase in the side chain from 4 to 6 carbons, for pair bmim [Cl] and hmim [Cl] (BOD = 148 and 196 mg L-1 O2, respectively) resulted in a slight increase in the consumption of oxygen. Relatively to the percentage of biodegradation, the differences were insignificant between these two compounds. It is necessary to perform more detailed studies to understand the influence of unsubstituted alkyl side chains in the biodegradability of ILs and to confirm if molecules containing long side chain are more biodegradable.39-40


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Summarily, tbph [Ms], N4,4,4,4 [Cl], N4,4,4,4 [Ac] and hmim [Cl] showed the highest BOD values among the tested ILs and also the lowest percentages of biodegradation, with the exception of hmim [Cl]. Ferlin and coworkers had also observed lower levels of biodegradability for tetrabutylammonium-based ILs in Closed Bottle test.41-42 Emim [TfMs] and emim [Ms], in turn, exhibited the highest biodegradability, 61% and 55%, respectively. According to ISO and OECD, emim [TfMs] and emim [Ms] are classifiable as readily biodegradable, since it reached the level of 60% of biodegradation. For the remaining compounds, to determine if they are classifiable as readily biodegradable it would be necessary to perform a 28-day test. DESs were prepared by thermal mixing of choline chloride, an ammonium salt, with varying hydrogen-bond donors, with both components being considered environmentally-friendly ingredients. Choline, vitamin B4, is an essential nutrient to living organisms, acting as a precursor in the synthesis of acetylcholine and phospholipids.43-44 Therefore, it has been widely used in the synthesis of DESs, as well as in the design of new ILs based on the idea that it is harmless and biodegradable.45 The results obtained in this work for DESs seems to corroborate this concept since most of the tested compounds exhibited percentages of degradation greater than 60% or close, under the tested conditions. The remaining DESs presented levels of degradability near to 60% indicating that probably these compounds will also reach the level within 28-days period. Few works have been conducted until now to evaluate the biodegradability of these compounds. Studies of Wen and coauthors resorting to Closed Bottle test showed that chol [Cl]:U and chol [Cl]:A reached near 80% of degradation.46 Juneidei and coworks observed likewise that chol [Cl]:Gly, chol [Cl]:U and chol [Cl]:MA were readily biodegradable in Closed Bottle test.47


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BOD values demonstrated differences between selected DESs, with chol [Cl]:ZnCl2 and chol [Cl]:MA (BOD = 156 and 184 mg L-1 O2, respectively) showing the lowest consumption of oxygen. On the opposite, chol [Cl]:TU, chol [Cl]:U and chol [Cl]:Fru (BOD = 399; 389 and 385 mg L-1 O2, respectively) presented the highest BOD values. Curiously, the urea derivatives although required greater amounts of oxygen by yeast to metabolize the molecules also exhibited high percentages of degradation compared to other tested compounds. Statistical analysis was applied to compare the results obtained in the developed method with the conventional 5-day BOD method. Pearson’s analysis verified a strong positive correlation (P > 0.976) between automatic and conventional method. The t-student test also showed that for a 2sided significance level of 0.05 (P =0.962) the H0 could not be rejected; hence, it is possible that the obtained BOD, for tested samples, from both methods is not significantly different from each other. The correlation coefficient for the tested compounds was 0.952 for both SIA method and BOD5 (figure 3). The statistical results demonstrated the potential applicability of the developed method to determine BOD.

450 y = 1.0089x - 0.8239 R² = 0.9522

400 350 BODSIA mg L -1

1 2 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


300 250 200 150 100 50 0 0

50

100

150

200 250 BOD5 mg L -1

300

350

400

450


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Figure 3. Correlation of BOD values obtained in the SIA method with conventional 5-day BOD method for tested samples.

Conclusions A new chemiluminescence automatic method was developed for the first time to rapidly estimate BOD. A precise control of the reaction conditions was guaranteed by resorting to SIA system. Furthermore, the implemented system enabled a wide improvement in the sample rate from 5 days in the conventional method to only 7.5 min in the presented method. The costs of the assay were also improved through a decrease of more than 75-times in reagents consumption and use of apparatus able to perform different analytical measurements. The obtained results demonstrated differences in BOD and biodegradation between tested compounds, highlighting that the chemical structure of the molecule influences its biodegradability. In general, DESs exhibited not only higher BOD values than ILs but also higher biodegradability. Six of the tested compounds reached the criteria to be classified as readily biodegradable under the tested conditions. Some of the remaining tested compounds presented promising indications relatively to their biodegradability and possibility of being designated as readily biodegradable. In summary, it was developed a new simple, economic and reliable screening tool suitable to determine chemical compounds BOD and to predict their risk of persistence in the environment.


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ASSOCIATED CONTENT Supporting information includes one document with a figure and its caption.

AUTHOR INFORMATION Corresponding Author * E-mail address: [email protected]; Phone: +351 220428670; Fax: +351 226093483.

Present Addresses b

A3D - Association for Drug Discovery and Development, Rua do Baixeiro, nº 38 Aradas, 3810-

399 Aveiro, Portugal c

Unidade de Investigação para o Desenvolvimento do Interior, Instituto Politécnico da Guarda,

Av. Dr. Francisco de Sá Carneiro, nº 50, 6300-559 Guarda, Portugal

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources This work has been financially supported by Fundação para a Ciência e a Tecnologia through project UID/QUI/50006/2013 and FEDER through COMPETE. It also received support from


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Associated Laboratory of Requimte projects competition through the project “Exploring new Deep Eutectic Solvents with the help of Ionic Liquids’ tunability”. Susana P. F. Costa and Ana M. O. Azevedo received a Ph.D. grant from FCT (SFRH/BD/86381/2012 and SFRH/BD/118566/2016, respectively). Marieta L. C. Passos thanks FCT for the Pos-doc grant (SFRH/BPD/72378/2010).

ABBREVIATIONS 1-butyl-1-methylpyrrolidinium chloride, bmpyr [Cl]; 1-butyl-1-methylpyrrolidinium tetrafluoroborate, bmpyr [BF4]; 1-butyl-3-methylimidazolium acetate, bmim [Ac]; 1-butyl-3methylimidazolium chloride, bmim [Cl]; 1-butyl-3-methylimidazolium tetrafluoroborate, bmim [BF4]; 1-ethyl-3-methylimidazolium acetate, emim [Ac]; 1-ethyl-3-methylimidazolium methanesulfonate, emim [Ms]; 1-ethyl-3-methylimidazolium trifluoromethanesulfonate, emim [TfMs]; 1-hexyl-3-methylimidazolium chloride, hmim [Cl]; biochemical oxygen demand, BOD; Choline chloride:Acetamide, chol [Cl]:A; Choline chloride:Citric acid, chol [Cl]:CA; Choline chloride:D-(-)-Fructose, chol [Cl]:Fru; Choline chloride:Glycerol, chol [Cl]:Gly; Choline chloride:Lactic acid, chol [Cl]:LA; Choline chloride:Malonic acid, chol [Cl]:MA; Choline chloride:L-(+)-Tartaric acid, chol [Cl]:TA; Choline chloride:Thiourea, chol [Cl]:TU; Choline chloride:Urea, chol [Cl]:U; Choline chloride:Zinc chloride, chol [Cl]: ZnCl2; deep eutectic solvents, DESs; dissolved oxygen, DO; flow injection analysis, FIA; glucose and glutamic acid, GGA; ionic liquids, ILs; sequential injection analysis, SIA; tetrabutylammonium chloride, N4,4,4,4 [Cl]; tetrabutylphosphonium methanesulfonate, N4,4,4,4 [Ms].


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TOC/ABSTRACT GRAPHIC

SYNOPSIS It is presented an alternative eco-friendly, economic and faster method to assess compounds with the potential to pollute water and persistence in the environment.


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ACS Sustainable Chemistry & Engineering L HC S C 3 ILs and

Biodegradability

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1 10 9

Fe

4 8

5 6

7

ACS Paragon Plus EnvironmentN Y

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Microfluidic Chemiluminescence System with Yeast - ACS Publications

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