**Isolation and Characterization of a Novel Bacterium from the Marine Environment for Trichloroacetic Acid Bioremediation**

#### **Mahshid Heidarrezaei 1,2,\* , Hoofar Shokravi <sup>3</sup> , Fahrul Huyop 4,5 , Seyed Saeid Rahimian Koloor <sup>6</sup> and Michal Petr ˚u <sup>6</sup>**


Received: 23 May 2020; Accepted: 30 June 2020; Published: 2 July 2020

**Abstract:** Halogenated compounds are an important class of environmental pollutants that are widely used in industrial chemicals such as solvents, herbicides, and pesticides. Many studies have been carried out to explore the biodegradation of these chemicals. Trichloroacetic acid (TCA) is one of the main halogenated compounds that are carcinogenic to humans and animals. The bacterium was isolated from the northern coastline of Johor Strait. In this study, the ability of strain MH2 to biodegrade TCA was evaluated by a growth experiment and dehalogenase enzyme assay. The growth profile of the isolated strain was examined. The doubling time for *L. boronitolerans* MH2 was found to be 32 h. The release of chloride ion in the degradation process was measured at 0.33 <sup>×</sup> <sup>10</sup>−<sup>3</sup> <sup>±</sup> 0.03 mol·<sup>L</sup> <sup>−</sup><sup>1</sup> after 96 h when the growth curve had reached its maximum within the late bacterial exponential phase. The results showed that the strain had a promising ability to degrade TCA by producing dehalogenase enzyme when cell-free extracts were prepared from growth on TCA as the sole carbon source with enzyme-specific activity, 1.1 <sup>±</sup> 0.05 <sup>µ</sup>molCl−min−<sup>1</sup> ·mg−<sup>1</sup> protein. Furthermore, the morphological, and biochemical aspects of the isolated bacterium were studied to identify and characterize the strain. The morphological observation of the isolated bacterium was seen to be a rod-shaped, Gram-positive, motile, heterotrophic, and spore-forming bacterium. The amplification of the 16S rRNA and gene analysis results indicated that the isolated bacterium had 98% similarity to *Lysinibacillus boronitolerans*. The morphological and biochemical tests supported the 16S rRNA gene amplification. To the best of the authors' knowledge, this is the first reported case of this genus of bacteria to degrade this type of halogenated compound.

**Keywords:** biodegradation; dehalogenase-producing bacteria; haloalkanoic acids; trichloroacetic acid; 16S rRNA; *Lysinibacillus*

#### **1. Introduction**

Xenobiotic compounds are synthetic molecules which include halogenated hydrocarbon, polyaromatic hydrocarbons, polycyclic biphenyls, and lignin [1,2]. They are biologically active compounds that are widely used in several drug and pesticide industries [3,4]. Hence, xenobiotic compounds often appear in industrial wastewaters and aquatic ecosystems [5,6]. Xenobiotic compounds can be considered stable, in the thermodynamic sense. Moreover, they are fairly resistant to biodegradation by the native microorganisms, and persistent in the environment [7]. Damage caused by xenobiotic compounds poses significant health and ecological risks in developing countries [8]. The parent xenobiotic compounds are not directly toxic but they can be transformed into harmful oxy-radicals or carbon-centered radicals that attack the double bonds of cellular macromolecules generating oxidative damage [9]. Xenobiotic compounds have the potential to cause toxic effects on humans and animals with consequent acute carcinogenic, teratogenicity, and mutagenic effects [7]. Nonetheless, a diverse group of aerobic and anaerobic groups of bacteria often found in various habitats are capable of degrading xenobiotic compounds [10]. Several researches exist about biodegradation of xenobiotic compounds using dehalogenase-producing bacteria such as organofluorine [11,12], organochlorine [13,14], and organobromine [15,16].

Halogenated hydrocarbons are organic compounds that have many significant industrial applications such as multipurpose solvents, plasticizers, pesticides, fuel additives, flame retardants, and anesthetics [17–19]. Several halogenated hydrocarbon like polychlorinated biphenyl (PCB) residues and chlorinated hydrocarbon pesticide residues were detected in human adipose tissue, milk, and blood serum. Transfer and accumulation of these chemicals in the human body will consequently cause serious health problems [20]. One of the most well-known applications of halogenated aliphatics in the industry is related to the mixed substituted chlorofluorocarbons, CFC [21]. Millions of tons of halogenated aliphatics are produced annually and mainly have been used in a variety of manufacturing processes of solvents and cleaning agents [22]. These chemical compounds are common pollutants of aquatic habitats, as they are relatively water-soluble and can migrate to underground water-supplies and therefore threaten groundwater quality [23].

Haloacetic acids (HAAs) are recognized as oxidation products of airborne C2-halocarbons which have been demonstrated to be fast in volatilization [24]. They are carcinogenic in humans even at low concentrations [25]. Several studies show that haloacetic acids are biodegradable in anaerobic environments including soil and, wastewater, [26]. HAAs come either from brominated and/or chlorinated organic halogen compounds. These organic halogenated compounds are important by-products of the reaction between organic compounds present in water and chlorine in water-treatment plants [27]. There are five contaminating by-products of halogenated organic compounds, which exceed the maximum levels established by the US Environmental Protection Agency (60 µg·L −1 ). These by-products include monochloroacetic acid (MCA), dichloroacetic acid (DCA), trichloroacetic acid (TCA), monobromoacetic acid (MBA), and dibromoacetic acid (DBA). As a result, the literature reporting on the analysis of HAAs mostly is focused on these five species [28]. TCA is a member of a very small group of "moderately strong" acids with ionization constants in the range 0.1–10 that is the most commonly used agent for chemical peeling [29,30]. TCA is one of the main contaminants of the environment that are carcinogenic to humans and animals [31]. The DCA and TCA are the most abundant HAAs detected in water resources containing nearly 80% of existing HAAs [32]. Hence, the decontamination of water resources from these compound is one of the major challenges for the preservation of the aquatic ecosystem and wastewater treatment [33].

Biodegradation is one of the main natural processes for the removal of xenobiotics such as chloroaliphatic compounds from the environment using microorganisms [34,35]. The major chemical processes for the metabolism of xenobiotic compounds are oxygenation, oxidation, reduction, hydrolysis, and conjugation, which are catalyzed by enzymes [36]. Dehalogenases are known as enzymes that catalyze the degradation reaction of halogen atoms from halogenated organic compounds. Dehalogenase-producing bacteria can detoxify harmful halogenated compounds from their substrates

by cleaving carbon–halogen bonds that link halogens in aliphatic compounds [37,38]. Based on cleavage nature, dehalogenase enzymes can be classified as haloalkane, dichloromethane, halohydrin, and L- and D-haloalkanoic acid dehalogenases [39]. Each specific group of these enzymes has its enantioselectivity and product inhibition characteristics [40]. Dehalogenase enzyme is present in bacterial systems and plays an important role in the biodegradation of environmental pollution caused by halogenated hydrocarbons [41].

Several research projects have been conducted to study the degradation mechanism of dehalogenase-producing bacteria for bioremediation of halogenated hydrocarbons and related hazardous compounds in soil and water ecosystems. Muslem et al. [42] characterized *Bacillus cereus* WH2 strain for its ability to metabolize β-haloalkanoic acids as carbon and energy sources. Alrawas and Huyop [35] isolated *Ralstonia* sp. TCAA-2 native to Danga Bay coast, Malaysia, to biodegrade TCA. Hamid et al. [43] further characterized dehalogenase enzyme functions of *Rhizobium* sp. RC1 bacteria. It was reported that RC1 can grow in α-haloalkanoic acid as the sole carbon source. α-haloalkanoic acids are halogenated compounds that are widely liberated into the ecosystem through the use of weed herbicides in the agricultural sector. Adamu et al. [44] studied the catalytic properties and mechanistic analysis of the microbial dehalogenases specific in *Rhizobium* sp. RC1. The isolated strain was reported to have outstanding performance in detoxifying L-2-haloacid. Selvamani et al. [45] characterized deploying *Trichoderma asperellum* SD1 to degrade 3-chloropropionic acid in the terrestrial ecosystem. Bagherbaigi et al. [46] used *Arthrobacter* sp. S1 for degradation of α and β-haloalkanoic acids.

As such, most of the research to date has focused on bioremediation of aquatic and terrestrial habitats. Nonetheless, there is a very little study to decontaminate TCA using dehalogenase- producing bacteria and their focus is on terrestrial bacteria or the identified bacteria are non-native to the coastal region of Johor strait. To the best of the authors' knowledge, this is the first reported case that shows *Lysinibacillus* bacteria could degrade halogenated compounds such as TCA. Because of the issues described above, this study intends to characterize a native bacterium with the capability of degrading TCA. To this end, several bacterial strains capable of aerobically degrading TCA were isolated from the northern coastline of Johor Strait in Malaysia and evaluated for their morphological and biochemical characteristics to degradation capabilities of TCA. The phylogenetic tree of the isolated strain was constructed using 16S-rRNA sequencing and it was recognized that the characterized bacterium was a new subspecies of the genus *Lysinibacillus*. The new bacterium was given the scientific name of *Lysinibacillus boronitolerans* MH2.

#### **2. Materials and Methods**

#### *2.1. Sampling, Enrichments, and Growth*

The seawater sample was collected from the northern coastline of Johor Strait in Malaysia. The water sample (1 mL) was added into 250 mL flasks containing 100 mL of minimal medium and TCA as a carbon source. Minimal medium contained basal salts solution, trace metals solution; and distilled water. The composition and proportion of the constituent of the minimal medium are shown in Table 1. The medium was adjusted to pH 7 before autoclaving. Then, the bacterial culture was incubated aerobically at 30 ◦C conditions in an orbital shaker set at 200 rpm. In order to prepare a solidified medium, two flasks (labeled Flask 1 and Flask 2) were prepared. Flask 1 contained 1.6 g oxide agar added into 48 mL distilled water (DW). Flask 2 was filled with 10 mL of trace metal salts and 10 mL of basal salts added into 30 mL DW. Flask 1 and flask 2 were autoclaved separately at 121 ◦C, for 15 min at 101.3 kPa. After autoclaving, solution in both flasks 1 and 2 were mixed and 1 mL of 1 M concentration TCA was added to the mixture, and the obtained medium was poured on agar plates. After the solidification of the agar, bacterial dilution was streaked onto the plates. Among the isolated strains, three different bacterial colonies were detected that the colony with the highest growth rate was retained for further purification. No further study was carried out for the identification of the two other isolated bacteria strains. The selected colony was picked and sub-cultured by repeated

streaking on a fresh agar plate to purify for further analysis. The selected colony was diluted in a drop of distilled water and transferred into minimal medium containing the ingredients shown in Table 1.


**Table 1.** Composition and proportion of the constituents in trichloroacetic acid (TCA)-enriched minimal medium.

The bacteria were grown in minimal medium containing TCA as the sole carbon source. Chlorine atoms are eliminated by hydrolytic dehalogenation from the compound [48]. Chloride ion liberation had been monitored by the halide ion assay to prove the degradation of TCA [49]. The concentration of chloride ions was determined by converting absorbance value to concentration in mmol·L <sup>−</sup><sup>1</sup> based on the standard curve constructed using sodium chloride as the standard measurement of soluble chloride [50,51]. The suspension of the selected colony was used to evaluate the growth profile and TCA degradation. Several direct and indirect methods can be used for the evaluation of the bacterial growth profile. A colony-forming unit (CFU) is a direct method to evaluate the actual colony forming units per volume of culture in given bacterial incubation. The enumeration of CFU is one of the most accurate methods to find out the number of viable bacteria strains growing in the presence of harsh chemicals [52]. On the other hand, the spectrophotometric method is a widely used laboratory technique to indirectly estimate the bacterial concentration and quantitative analysis of the released chloride ions [44,53]. Spectrophotometry methods are based on the absorption and scattering of light beams passing through the culture medium. These methods are sensitive and selective; however, enumeration of CFU by plate count might be a disadvantage due to the fact that cells grown in clusters into a single colony and assumption that each colony arises from one cell is not accurate. In addition, commercial TCA may contain impurities and colony count may due to the growth of impurities rather than on TCA. In this study, absorbance A680nm and A460nm are used for analysis of the bacterial growth and released chloride ions, respectively. Chloride was liberated during the growth of TCA. The absorbance of the released chloride ions was recorded every 6 h using Jenaway spectrophotometer at A460nm. Growth experiment and chloride ion released analysis were carried out in triplicate.

#### *2.2. Preparation of Cell-Free Extracts and Dehalogenase Enzyme Assay*

Apart from measuring cell growth (A680nm) and chloride ions released (A460nm) in the growth medium, the presence of dehalogenase was assessed as the liberation of halide in cell-free extracts in vitro. Cells from a 100 mL culture grown in 10 mm TCA and nutrient broth were harvested by centrifugation at 20,000× *g* for 10 min at 4 ◦C during the late-growth phase, between 72–96 h (Figure 1). The pellets were resuspended in 10 mL of 0.1 M tris-acetate, 1 mm ethylenediaminetetraacetic acid (EDTA), 10% (mass·vol−<sup>1</sup> .) glycerol, pH 7.5, and washed three times with 0.1 M tris-acetate buffer by centrifugation at 20,000× *g* for 10 min at 4 ◦C. Finally, the cells were then resuspended in 3 mL of the same buffer and maintained at 0 ◦C for sonication in an MSE Soniprep 150 W ultrasonic disintegrator at

a peak amplitude (λ = 10 microns) for 30 s. Any unbroken cells and cell wall materials were removed by centrifugation at 25,000× *g* for 20 min at 4 ◦C. The cell-free extracts activity was assayed immediately after the preparation as previously described by Mesri et al. [54].

**Figure 1.** The growth curve of the MH2 bacterium measured at A680nm and chloride ions released measured at A460nm. The control culture without strain MH2 was incubated under the same growth conditions. All readings were based on triplicate analysis.

#### *2.3. Biochemical and Morphological Characterization*

Bergey's manual

Morphological tests, namely Gram staining, spore staining, and motility tests as well as biochemical tests including catalase, oxidase, urease, gelatin liquefaction, citrate, MacConkey agar, casein hydrolysis, starch, indole, and triple sugar iron (TSI) tests, were carried out for basic identification of bacteria generics and inferring their properties following the standard outline in Bergey's manual [55]. Table 2 shows the morphological and biochemical characteristics of the selected bacteria.


#### **Table 2.**Morphological and biochemical characterization of*L. boronitolerans*MH2.


**Table 2.** *Cont.*

#### *2.4. Molecular Identification*

Genomic DNA from bacterial cells of strain MH2 was isolated using the Wizard® Genomic Purification Kit (Promega). Universal polymerase chain reaction (PCR) forward and reverse primers Fd1 (5'-aga gtt tga tcc tgg ctc ag-3') and Rp1 (5'-acg gtc ata cct tgt tac gac tt-3'), respectively, were synthesized by 1st Base® Laboratory Malaysia Sdn. Bhd., 43300 Seri Kembangan, Selangor, Malaysia [58]. The amplified 16S rRNA gene has to be purified before sequencing. QIAquick polymerase chain reaction (PCR) purification kit (Qiagen) was used to purify the DNA. The sequencing was carried out by ABI PRISM® 377 DNA sequencer (1st Base® Laboratory Malaysia Sdn. Bhd., 43300 Seri Kembangan, Selangor, Malaysia). The thermocycling conditions for PCR amplification are presented in Table 3.

**Table 3.** Thermocycling conditions for polymerase chain reaction (PCR) amplification.


The phylogram of unidentified bacteria was rebuilt using Mega5 Molecular software and compared with the sequences from the 16S rRNA gene stored in the Gene Bank by the National Center for Biotechnology (NCBI) using the Basic Local Alignment Search Tool (BLASTn) for nucleotides [59]. Sequences were aligned using Bioedit Sequence Alignment Editor X and a neighbor-joining tree was constructed [60]. Bootstrap consensus tree was inferred from 500 replicates.

#### **3. Results**

A seawater sample was taken from the marine environment and three strains of bacteria were isolated using minimal media. Once the bacterial strains were purified, only one isolate (MH2) was selected from three bacterial samples due to its distinctively superior growth and survival abilities in solid minimal media compared to the other two bacteria for further analysis. The doubling time for MH2 strain was found 32 h and the maximum released chloride ion due to dehalogenase enzyme activity was approximately 0.33 <sup>×</sup> <sup>10</sup>−<sup>3</sup> <sup>±</sup> 0.03 mol·<sup>L</sup> −1 . 16S rRNA phylogenetic analysis was conducted to determine the phylogenetic relatedness and the species affiliation of the MH2 strain.

#### *3.1. Growth of* L. boronitolerans *MH2*

The growth profile of the isolated *L. boronitolerans* MH2 strain bacteria was examined in fixed time intervals. The growth profile exhibiting the lag, exponential, stationary, and decline phase as it was expected. The growth of the bacteria by measuring through light absorbance of the solutions measured at 680 nm (A680nm) by using a JENWAY, 6300 ultraviolet (UV)-visible spectrophotometer at every 6 h. The measured values for the growth of MH2 were recorded based on 10 mm of the TCA-enriched medium. In the lag phase, the bacterium growth was very little because the bacterium does not immediately adapt to the growth condition and the carbon source which was toxic to the bacteria. In the exponential phase, the growth rate of the MH2 bacterium increased. At the end of the exponential phase, the growth curve reached its maximum rate at 96 h. After a long exponential phase, the growth curve reached a stationary phase and then the bacterium enters the decline phase. The growth profile shows that MH2 was able to grow in minimal medium containing 10 mm of TCA. According to Slater et al. [51], the bacterium absorbs TCA from the compound as the sole carbon source and releases chloride ions. The liberated chloride ion during the metabolism of MH2 strain was monitored by a halide ion assay order as an indicator of TCA degradation in the medium [51]. The results of chloride ion released and the growth curve of the bacterium was plotted as shown in Figure 1.

Figure 1 shows that both the halide ion assay and growth curves of the bacterium in minimal medium exhibited identical trends indicating that the concentration of the released chloride ion and the growth are directly related to each other. The curves for halide ion assay and growth of MH2 were in contrast with the control experiment. The concentration of the chloride ion in the cultivation solution was determined by converting absorbance corresponding to the concentration using the standard curve. The standard curve was constructed using NaCl as a typical standard for measuring soluble chloride concentration [61]. Doubling time of the bacteria in 10 mm TCA-enriched minimal medium was estimated to be 32 h.

#### *3.2. Enzyme Activity in Cell-Free Extracts from Cell Growth in Trichloroacetic Acid (TCA) Medium and Nutrient Broth*

Dehalogenase activity in cell-free extracts was assessed by the release of free halide in an enzyme-substrate reaction in vitro. Cell-free extracts from strain MH2 grown on 10 mm TCA medium as the sole carbon source was prepared. Halide liberation was measured from extracts of cells grown in TCA using TCA as a substrate. However, no halide was liberated by extracts of the same bacteria grown in nutrient broth. This suggests that in nutrient broth no expression of the bacterial dehalogenase gene takes place. The enzyme specific activity of the dehalogenase for TCA was 1.1 ± 0.05 µmolCl −min −1 ·mg <sup>−</sup><sup>1</sup> protein. −1 −1

#### *3.3. Morphology and Biochemical Characteristics*

The result of the bacteria characterization based on morphological and biochemical analysis shows that the isolated bacterium grew well at aerobic conditions and can readily metabolize TCA and produce chloride ions by dechlorination treatment. The morphological observations for Gram-staining and spore staining using light microscopy 1000× magnification are shown in Figures 2 and 3, respectively. The result of Gram-staining analysis indicated that the MH2 strain is a Gram-positive bacterium and produced creamy to pink colonies on solid minimal medium. *L. boronitolerans* MH2 was further examined for biochemical characteristics.

**Figure 2.** The Gram-staining of strain MH2 observation under light microscope (×1000 magnification). Purple colour bacteria appeared as the result of Gram staining which indicated MH2 was a Gram-positive bacterium.

A spore staining test was performed to determine if MH2 is an endospore-forming bacterium. Spores were observed during the microscopic examination that proves that bacterium MH2 was capable of producing spores. Microscopic observation of the spore staining is shown in Figure 3.

**Figure 3.** Spore staining for strain MH2 observed under a light microscope (×1000 magnification). Spores were seen as black spots in each cell.

negative results. Morphological and biochemical tests were conducted based on Bergey's manual Tables 4 and 5 showed the summary of morphological characteristics of MH2 and the results of biochemical tests, respectively. The oxidase, urease, casein, and starch hydrolysis tests showed positive results and catalase, gelatin, citrate, MacConkey agar, indole, and TSI demonstrated negative results. Morphological and biochemical tests were conducted based on Bergey's manual systematic bacteriology [56].


**Table 4.** Morphological character of MH2.



Positive (+); Negative (−).

#### *3.4. Polymerase Chain Reaction (PCR) Amplification of 16s rRNA Gene Analysis*

Colony PCR (cPCR) is performed to verify the construct of the DNA-sequence. 16S rRNA genes were amplified using universal primers Fd1 and Rp1, respectively. The amplified genes were monitored with agarose gel electrophoresis. In Figure 4, the unique amplified band (1500 bp) was observed and compared with the 1 Kb DNA ladder.

− − − − − −

**−**

**Figure 4.** Amplification of the 16S rRNA gene of strain MH2 showing 1500 bp DNA fragment on an agarose gel (1%) (Lane 2). Lane 1: 1kb DNA ladder (Promega); Lanes 3 and 4, negative controls, polymerase chain reaction (PCR) mixture without forward (Fd1) and reverse (Rp1) primer respectively, showing no amplification.

The continuous stretch (1274 bp) of 16S rRNA gene was investigated to determine the closest phylogenetic neighbors. The BLAST program was employed and it was revealed that the MH2 has 98% identity matches in sequence with *L. boronitolerans*. Amplification and direct sequence analysis of partial length of 16S rRNA indicate that bacterium MH2 is phylogenetically related to *L. boronitolerans*. The results of the 16S rRNA analysis supported the biochemical characteristics of the bacterium belong to the same genus and species.

#### *3.5. Phylogeny Tree Analysis*

The phylogenetic tree was constructed using the neighbor-joining method and the maximum composite likelihood method. The bootstrap consensus tree offered from 500 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are excluded. The percentage of replicate trees is shown next to the branches. Evolutionary analyses were conducted in MEGA5 [59]. Figure 5 presented the phylogeny tree of the MH2 strain.

The maximum composite likelihood method was used to estimate the evolutionary distances. The units of evolutionary distances were based on the number of base substitutions per site; 11 nucleotide sequences were involved in the analysis. All gaps and missing data were excluded for tree building before analysis. Only 1238 positions were considered in the final dataset.

**Figure 5.** Neighbor-joining tree of strains M2 based on 16S rRNA gene sequences. Scale Bar 0.001 indicates sequence divergence.

#### **4. Discussion**

Biodegradation is an effective biological process to clean up polluted environments through the isolation of efficacious halo-organic compound biodegraders. Notably, the BLASTn search on the 16S rRNA gene sequence revealed that MH2 has the 98% identity match with *Lysinibacillus boronitolerans* (accession number: KC59351.1). Hence, to identify the capability of MH2 to degrade the sample pollutants, this study grew the MH2 bacterial isolate in TCA-enriched minimal media as the sole carbon source (pollutants).

The initial composition of the culture medium and the concentration of the substrate (TCA) are two important parameters that can affect the degrading ability of the bacteria. Using artificial seawater or sterilized seawater as a medium for cultivation of sea-isolated bacterium was refrained from due to the potential interference of natural chloride ions in seawater with those liberated during the growth experiment. Minimal medium contained basal salts solution, trace metals solution, and distilled water was used in this study to provide the required growth culture for the bacteria. The existing nitrilotriacetic acid (N(CH2COOH)3) in the trace metals solution is low enough not to interfere significantly with the results of the experiment and to be consistent with the hypothesis of utilizing TCA as the sole carbon and energy source in the bacteria growth process. The optimal concentration of the TCA pollutant sample was selected 10 mm in the study of the growth profile and the ion liberation experiments. Using TCA in concentrations higher than 20 mm can stifle the cell growth due to toxic effects while the lower concentration of pollutants was not enough to observe the induced catalytic reaction of the dehalogenase-producing bacteria. Growth was strictly monitored by measuring the cells' turbidity and the amount of chloride ions released at appropriate time intervals. An uninoculated flask treated in the same way was used as a control. This is important to make sure the chloride measured in the growth medium was due to the cells using the TCA rather than the auto-degradation of the substrate in the growth medium.

MH2 strain was capable of degrading hydrocarbon compounds. The nature and type of carbon sources are among the most important factor to determine bacterial growth. Haloacetates (i.e., MCA, DCA, and TCA) are common classes of water chlorination by-products. Several bacteria are available that can grow on MCA. *Burkholderia* sp. DehCL1 [62], *Bacillus* sp. TW1 [63], *Xantobacter autotrophicus*

GJ10 [64,65], and *Pseudomonas* sp. R1 [66] are examples of bacteria that can degrade MCA haloacetates. Additionally, Meusel and Rehm [67] described *Xanthobacter autotrophicus* GJ10 that can degrade DCA. To the best of the authors' knowledge, this is the first report on *Lysinibacillus* sp. isolated from seawater adapted in metabolizing TCA.

The results showed that the highest growth in *L. boronitolerans* MH2 bacterium was achieved at 96 h. Low absorbance was observed for the lag phase due to the low intensity and slow growth of the bacterial biomass produced. In broth, the doubling time of the *L. boronitolerans* MH2 was 32 h. Chloride ion concentration during the biodegradation process was monitored by a chloride ion assay [61,68]. Based on the standard curve of the NaCl, the released chloride ion by *L. boronitolerans* MH2 strain after 96 h was 0.33 <sup>×</sup> <sup>10</sup>−<sup>3</sup> mol·<sup>L</sup> −1 . The study of the growth curve shows that the *L. boronitolerans* MH2 was capable of growing in a minimal medium having TCA as the sole source of carbon and energy. Growth on TCA was further analyzed by preparing the cell-free extracts from growth on TCA as described in Figure 1 and Section 3.2. The presence of dehalogenase was detected by measuring the enzyme-specific activity. The *L. boronitolerans* MH2 dehalogenase described herein appears to be inducible because MH2 cells grown in nutrient broth lacking TCA exhibited no dehalogenase activity.

Hydrolytic dehalogenation of TCA produces oxalate as a final product where oxalate serves as the carbon source and it can be immediately converted to CO<sup>2</sup> [69]. The number of bacteria capable of using oxalate as the sole source of carbon is very limited [70]. Biochemical and morphological experiments were carried out according to Bergey's manual [56] aimed at verifying the obtained result of 16S rRNA gene analysis. The results of biochemical characteristics supported the findings of the bacteria suggested by the 16S rRNA analysis. The growth profile of the isolated strain was examined and the results showed that *L. boronitolerans* MH2 bacterium has a promising ability as a dehalogenase-producing bacterium.

#### **5. Conclusions**

Using dehalogenase enzymes to detoxify chlorinated organic compounds was envisaged as a promising biological control method. The applicability of *L. boronitolerans* MH2 is an important outcome discovered in current analysis suggesting dehalogenase-producing bacteria is important for the future to the exploitation of the bacterium for in situ efforts to detoxify halogen-contaminated environments. More importantly, these findings further add to the limited body of knowledge with regards to the degrading of halogenated compounds by the bacteria.

**Author Contributions:** Resources, M.H., H.S., F.H., S.S.R.K., and M.P.; investigation, M.H.; writing—original draft preparation, M.H., H.S.; writing—review and editing, M.H., H.S., F.H.; visualization, M.H., H.S., F.H., M.P. and S.S.R.K.; supervision, F.H.; project administration, M.H., H.S., F.H., M.P., and S.S.R.K.; funding acquisition, F.H., M.P. and S.S.R.K.; All authors have read and agreed to the published version of the manuscript.

**Funding:** The APC is founded by Ministry of Education, Youth, and Sports of the Czech Republic and the European Union (European Structural and Investment Funds Operational Program Research, Development, and Education) in the framework of the project "Modular platform for autonomous chassis of specialized electric vehicles for freight and equipment transportation", Reg. No. CZ.02.1.01/0.0/0.0/16\_025/0007293.

**Acknowledgments:** The authors would like to thank the Department of Biosciences, Faculty of Science, Universiti Teknologi Malaysia, for facilities and services given and acknowledged Ministry of Education, Youth, and Sports of the Czech Republic and the European Union (European Structural and Investment Funds Operational Program Research, Development, and Education) for founding APC in the framework of the project "Modular platform for autonomous chassis of specialized electric vehicles for freight and equipment transportation", Reg. No. CZ.02.1.01/0.0/0.0/16\_025/0007293.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **The Use of MgO Obtained from Serpentinite in the Synthesis of a Magnesium Potassium Phosphate Matrix for Radioactive Waste Immobilization**

**Svetlana A. Kulikova 1,\* , Sergey E. Vinokurov <sup>1</sup> , Ruslan K. Khamizov <sup>1</sup> , Natal'ya S. Vlasovskikh <sup>2</sup> , Kseniya Y. Belova <sup>1</sup> , Rustam K. Dzhenloda <sup>1</sup> , Magomet A. Konov <sup>2</sup> and Boris F. Myasoedov <sup>1</sup>**


**Featured Application: MgO is used for the synthesis of a magnesium potassium phosphate matrix as a material for immobilization of radioactive waste in order to ensure radiation safety during storage or disposal of waste.**

**Abstract:** Magnesium oxide is a necessary binding agent for the synthesis of a magnesium potassium phosphate (MPP) matrix based on MgKPO<sup>4</sup> × 6H2O, which is promising for the solidification of radioactive waste (RW) on an industrial scale. The performed research is devoted to finding a cost-effective approach to the synthesis of MPP matrix by using MgO with an optimal ratio of the quality of the binding agent and the cost of its production. A method for obtaining MgO from the widely available natural mineral serpentinite was proposed. The phase composition, particle morphology, and granulometric composition of MgO were studied. It was found that the obtained MgO sample, in addition to the target periclase phase, also contains impurities of brucite and hydromagnesite; however, after calcining at 1300 ◦C for 3 h, MgO transforms into a monophase state with a periclase structure with an average crystallite size of 62 nm. The aggregate size of the calcined MgO powder in an aqueous medium was about 55 µm (about 30 µm after ultrasonic dispersion), and the specific surface area was 5.4 m2/g. This powder was used to prepare samples of the MPP matrix, the compressive strength of which was about 6 MPa. The high hydrolytic stability of the MPP matrix was shown: the differential leaching rate of magnesium, potassium, and phosphorus from the sample on the 91st day of its contact with water does not exceed 1.6 <sup>×</sup> <sup>10</sup>−<sup>5</sup> , 4.7 <sup>×</sup> <sup>10</sup>−<sup>4</sup> 8.9 <sup>×</sup> <sup>10</sup>−<sup>5</sup> g/(cm<sup>2</sup> ·day), respectively. Thus, it was confirmed that the obtained MPP matrix possesses the necessary quality indicators for RW immobilization.

**Keywords:** serpentinite; magnesium oxide; calcination; particle size distribution; specific surface area; magnesium potassium phosphate matrix; radioactive waste; immobilization; hydrolytic stability; strength

#### **1. Introduction**

Industrial activities associated with the production and use of materials containing radioactive substances inevitably lead to the generation of radioactive waste (RW) of various activity levels. The largest amount of RW is generated during the operation of nuclear fuel cycle enterprises and exploitation of nuclear power reactors of various purposes. In some countries, including the USA, Sweden, and Finland, spent nuclear fuel (SNF) of nuclear power reactors is classified as RW and is stored without reprocessing. In other countries, including Russia, France, and Japan, SNF is subject to reprocessing for the purpose of

**Citation:** Kulikova, S.A.; Vinokurov, S.E.; Khamizov, R.K.; Vlasovskikh, N.S.; Belova, K.Y.; Dzhenloda, R.K.; Konov, M.A.; Myasoedov, B.F. The Use of MgO Obtained from Serpentinite in the Synthesis of a Magnesium Potassium Phosphate Matrix for Radioactive Waste Immobilization. *Appl. Sci.* **2021**, *11*, 220. https://doi.org/10.3390/ app11010220

Received: 2 December 2020 Accepted: 23 December 2020 Published: 28 December 2020

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**Copyright:** © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/).

extraction of uranium and plutonium for reuse, as well as a number of valuable components from fission products and actinides, and the residue part is considered to be RW. In case of uncontrolled spread of radioactive substances, they have a negative impact on humans and environmental objects. Therefore, solving the problem of RW management in order to ensure their reliable isolation from the human environment is key for the further development of nuclear energy and industry. For controlled storage and/or final disposal of RW, it should be converted to a stable solidified form using stable matrices.

Earlier, we showed in [1–5] that magnesium potassium phosphate (MPP) matrix MgKPO<sup>4</sup> × 6H2O is an effective and multipurpose mineral-like material for immobilization of different RW, and it possesses the number of benefits over cement and glass-like compounds. Therefore, MPP matrix was investigated for solidification of liquid intermediate level waste (ILW) [1], high level waste (HLW) [2,3], and also RW containing radiocarbon <sup>14</sup>C [4] and spent electrolyte [5], obtained as a result of pyrochemical reprocessing of mixed nitride uranium-plutonium SNF. This matrix is obtained by the acid-base reaction (1) of magnesium oxide (MgO) with potassium dihydrogen phosphate (KH2PO4) in an aqueous solution at room temperature, and it is an analog of the natural mineral K-struvite [6].

$$\rm MgO + \rm KH\_2PO\_4 + 5H\_2O \to MgKPO\_4 \times 6H\_2O \tag{1}$$

Magnesium oxide is a necessary binding agent for reaction (1) for the synthesis of MPP matrix; it is usually produced by calcination of carbonate minerals—magnesite (MgCO3) [7] and dolomite (CaMg(CO3)2) [8]. It is widely used to obtain the refractories in the production of steel and cement (70–80% of world consumption) in electrical engineering, agriculture, for wastewater treatment, and gas absorption. Magnesium oxide on the market with a purity of at least 99 wt% has a high cost—up to \$5000 per ton. At the same time, it is obvious that for the competitiveness of the technology of RW solidification using the MPP matrix, cheaper raw materials should be used; for example, so that the cost of MgO is at the level of the cost of Portland cement (\$300–400 per ton). For this reason, the aim of the study was to find a cost-effective approach to synthesizing the MPP matrix through the use of MgO with low production cost. In this regard, a natural mineral, serpentinite (Mg3Si2O5(OH)4), which contains 32–38% of MgO [9], is of special interest. There are various technologies for reprocessing of serpentinite, primarily methods using mineral acids: sulfuric acid [10], nitric acid [9], and hydrochloric acid [11].

Earlier in [12], while testing several commercial MgO samples obtained from magnesite, we recommended the use of MgO powder with an average particle size of about 50 µm, which has a high degree of crystallinity (the average crystallite size is not less than 40 nm), without impure readily soluble magnesium phases (first of all, magnesium hydroxide) to obtain a homogeneous compound based on MPP matrix with a high compressive strength (up to 15 MPa). The specific surface of the conditioned MgO powder was no higher than 7 m2/g [1]. It was also noted that the impurities in MgO of metal compounds, primarily silicon, calcium, and iron, do not significantly influence the synthesis conditions and the mechanical strength of the compound.

This article presents the results of studying the characteristics of MgO powder obtained from serpentinite, as well as determination of composition, mechanical strength, and hydrolytic stability of the prepared MPP matrix samples.

#### **2. Materials and Methods**

#### *2.1. Obtaining MgO from Serpentinite*

The flow chart for obtaining MgO from serpentinite is presented in Figure 1. In this work, an average sample of serpentinite from the "Bedenskoye" deposit, having a composition presented in Table 1, was used as a source of magnesium oxide.

*Схема получения из серпентинита добавлена в Хамизов Р Х*

привести блок схему здесь

**Figure 1.** Flow chart for obtaining MgO from serpentinite.

**Table 1.** Chemical composition of serpentinite.


\* Loss on Ignition.

45 μm), obtained in a ball mill with ceramic balls, were – – — — 2− — Several repeated experiments were carried out to decompose serpentinite and remove magnesium hydroxide. In each of them, 100 g of finely ground serpentinite powder (with a grain boundary size d < 45 µm), obtained in a ball mill with ceramic balls, were placed in a laboratory autoclave with a mechanical stirrer, and 650 g (520 mL) of circulating solution containing 35% NaHSO4, up to 7% MgSO<sup>4</sup> and up to 10% (NH4)2SO<sup>4</sup> were added. The mixture was stirred for 4 h at 120 ◦C. The obtained hot suspension was filtered under reduced pressure created by a water-jet pump on a Buchner filter with a thermostatic shirt at 95 ◦C to obtain filtrate No. 1, and then at the same temperature, the filter precipitate was washed by 250 mL of hot deionized water. Filtrate No. 1 and wash water were combined, and 912–925 g (730–740 mL) of mixed solution, containing no less than 3.4% of Mg(II) and no more than 0.08% of Fe(II, III), were obtained in different experiments that corresponded to the degree of magnesium removal—no less than 80%, and iron—up to 11.5%. The solution was placed in a cold-storage, where it was cooled to +4 ◦C and kept there for 2 h. As a result, a suspension was obtained, which was filtered on a thermostatted Buechner filter at 5 ◦C. Filtrate No. 2 (no more than 490 g with up to 0.5% magnesium and 0.1% iron) and a glassy precipitate with a pale yellowish-pink color were obtained. Chemical analysis of such precipitate (no less than 370 g), dried at 105 ◦C for 1 h until the loss of more than 10% moisture, showed a magnesium content of 6.5% and a molar ratio of Mg 2+ :NH<sup>4</sup> + :SO<sup>4</sup> <sup>2</sup><sup>−</sup> = 1:2:2 that corresponded to the double salt—magnesium ammonium sulfate (Boussingaultite). The iron content in the dry sediment did not exceed 0.06%.

The precipitate was dissolved in 650 mL of hot deionized water (95 ◦C), and a 25% ammonia solution (purity qualification "analytical grade") was added to the obtained solution up to pH of 8, and 2 g of ammonium carbonate, (NH4)2CO<sup>3</sup> ("reagent grade"), was also added. A hot suspension of light green color was obtained, which was placed in an autoclave, in which the temperature was maintained at 95 ◦C. Air was passed through the suspension, adjusting the outlet of gases from the autoclave so that an excess pressure is created in it. The mixture was kept for 4 h with stirring. A light brown suspension was discharged from the autoclave, which was filtered through hot thermostatted Buchner filter with a water-jet pump using a filter with pore size 1–2.5 nm. The resulting filtrate No. 3 was again placed in an autoclave, previously washed with deionized water, and then filtrate No. 3 was cooled to room temperature. 80 mL of the solution of 25% ammonia were poured into the contents of the autoclave, and 45 g of ammonium carbonate were added. The autoclave was closed, the temperature was raised up to 65 ◦C, and the mixture was stirred under excess pressure for 4 h. A suspension of white color with a bluish sheen was obtained, which was filtered through a thermostatted Buchner filter using a paper filter with pore size 1–2.5 nm. Filtrate No. 4 and precipitate of magnesium hydroxide-carbonate were obtained. The precipitate was dried at 120 ◦C and then subjected to preliminary calcination at a temperature of 650 ◦C for 3 h.

Filtrate No. 2 was heated up to 50 ◦C, a 25% ammonia solution was added to it to pH 8, and the mixture was stirred for 5 h on a magnetic stirrer with heating. A finely dispersed light brown suspension was formed, which was separated in a laboratory centrifuge to obtain a supernatant.

The supernatant and filtrate No. 4 were mixed, and the resulting solution was subjected to vacuum evaporation on a Buchi R-124 rotary evaporator (Bunker Lake Blvd., Ramsey, MN, USA) at 85 ◦C and a pressure of 0.15 bar until a wet crystalline mass was obtained. This mass was placed in a muffle furnace and first dried at 120 ◦C for 1 h; then the temperature was raised to 310 ◦C and decomposition was carried out for another 7 h. In repeated experiments, at least 335 g of precipitate were obtained, to which 320 mL of deionized water and 5 g of ammonium sulfate were added after cooling. A recycled solution was obtained with a content of at least 35% NaHSO4, up to 7% MgSO4, and up to 10% (NH4)2SO4, which was used to decompose the next portion of serpentinite.

In each of the consecutive carried out experiments, at least 30 g of the preproduct was obtained. The samples of magnesium oxide obtained in the course of six successive experiments were combined, and an average composition of precalcined magnesium oxide was obtained, in which the content of iron and manganese oxides did not exceed 0.01 wt%.

#### *2.2. Preparation of the MPP Matrix*

The synthesis of the MPP matrix was carried out according to reaction (1) at the MgO: H2O:KH2PO<sup>4</sup> weight ratio of 1:2:3. Previously in studies [1,13–16], it was shown that to reduce the rate of reaction (1) and, accordingly, to ensure a technologically acceptable setting time of the mixture for the purpose of high-quality mixing and tight packing of the resulting mixture into containers for subsequent storage, MgO powder should be used after preliminary heat treatment at 1300–1500 ◦C. Thus, MgO obtained from serpentinite in accordance with the method in Section 2.1 and precalcined at temperatures of 1300 ◦C for 3 h (hereinafter referred to as calcined MgO) in a muffle furnace (SNOL 30/1300, AB UMEGA GROUP, Utena, Lithuania) and KH2PO<sup>4</sup> ("Rushim" LLC, Moscow, Russia) crushed to a particle size of 0.15–0.25 mm were used for synthesis of MPP matrix. The excess of MgO in relation to the stoichiometry of reaction (1) was 10 wt% [1]. To decrease the rate of reaction (1), boric acid was added to the initial mixture in an amount corresponding to its 1.5 wt% content in the sample. The obtained mixture was placed in PTFE molds.

Cubic samples of the MPP matrix with dimensions of 2 <sup>×</sup> <sup>2</sup> <sup>×</sup> 2 cm<sup>3</sup> were prepared and kept for at least 15 days to cure at ambient atmospheric conditions.

#### *2.3. Investigation of MgO and MPP Matrix Samples*

The phase composition of MgO and MPP matrix samples was determined by X-ray diffraction (XRD) method using an Ultima-IV X-ray diffractometer (Rigaku, Tokyo, Japan). The XRD data were interpreted using the Jade 6.5 software package (MDI, Livermore, CA, USA) with PDF-2 powder database. The average crystallite size of the studied MgO samples was calculated by the Scherrer [12]. The composition of MgO was determined using the Rietveld method [17], with a PROFEX GUI software package for BGMN [18].

The microstructure of MgO and MPP matrix samples was investigated by the scanning electron microscopy (SEM) using a JSM-6700F (Jeol, Tokyo, Japan) and Vega 3 (Tescan, Brno, Czech Republic) microscopes; the electron probe microanalysis of the samples was performed by energy-dispersive X-ray spectroscopy (EDS) using an X-ACT analyzer (Oxford Inst., High Wycombe, UK).

The elemental composition of MgO powder was studied using an Axious Advanced PW 4400/04 X-ray fluorescence (XRF) spectrometer (PANalytical B.V., Almelo, Netherlands).

Adsorption measurements of MgO powder samples were carried out in an ASAP 2000 automatic sorbtometer (Micromeritics, Norcross, GA, USA); the specific surface area was calculated using the Micromeritics software package.

The particle size distribution of MgO samples was determined using a SALD-7500 nano laser diffraction granulometer (Shimadzu, Kyoto, Japan), including the use of 60 W ultrasound for 5 min.

The compressive strength of MPP matrix samples was determined using a Cybertronic 500/50 kN test machine (Testing Bluhm & Feuerherdt GmbH, Germany). At least three compound samples were used in experiment.

The hydrolytic stability of MPP matrix samples was determined in accordance with the semi-dynamic standard test GOST R 52126-2003 [19]. Before leaching, monolithic cubic samples were immersed in ethanol for 5–7 s, then the samples were dried in air for 30 min. Next, the samples were placed in a PTFE container, and double-distilled water was poured in as a leaching agent (pH 6.6 ± 0.1, volume 100 mL), which was replaced at regular time intervals. The samples were removed from the container at the set time, washed with double-distilled water (volume 100 mL), and combined with the leachate, and the content of the matrix components in the solutions after leaching was determined by ICP–AES (iCAP-6500 Duo, Thermo Scientific, Waltham, MA, USA). The calculations of the differential leaching rate LRdif (g/(cm<sup>2</sup> ·day)) of the matrix components from samples were made according to Equation (2).

$$\text{LR} = \frac{\mathbf{c} \cdot \mathbf{V}}{\mathbf{S} \cdot \mathbf{f} \cdot \mathbf{t}} \text{ }\tag{2}$$

where c—element concentration in solution after leaching, g/L; V—the volume of leaching agent, L; S—the open geometric surface area of the monolithic samples, cm<sup>2</sup> ; f—element content in matrix, g/g; and t—leaching time, days (for calculating the differential leaching rate t—duration of the n-th leaching period between shifts of contact solution).

Leaching mechanism of MPP matrix components was assessed according to a de Groot and van der Sloot model [20], which can be presented as Equation (3), where values of the coefficient A (slope of the line) correspond to the following mechanisms: <0.35—surface wash-off (or a depletion if it is found in the middle or at the end of the test); 0.35–0.65 diffusion transport; and >0.65—surface dissolution [21]. The calculation of B<sup>i</sup> was carried out according to Equation (4).

$$\log(\mathbf{B}\_{\mathbf{i}}) = \mathbf{A}\,\log(\mathbf{t}\_{\mathbf{n}}) + \text{const} \tag{3}$$

$$\mathbf{B}\_{\mathbf{i}} = \mathbf{A}\_{\mathbf{n}} \cdot \frac{\mathbf{V}}{\mathbf{S}} \cdot \frac{\sqrt{\mathbf{t}\_{\mathbf{n}}}}{\left(\sqrt{\mathbf{t}\_{\mathbf{n}}} - \sqrt{\mathbf{t}\_{\mathbf{n}-1}}\right)} \tag{4}$$

#### **3. Results and Discussion**

#### *3.1. Features of the Obtaining Process of MgO from Serpentinite*

The advantage of the bisulfate method of serpentinite reprocessing provides the possibility of creating a closed-loop technological process, where all reagents are recovered. This process was first proposed in [22]. In contrast to the complex chemical formulas used in this work, we introduced simplifications that make it possible to better understand the discussed cyclic process. If we present the formula of serpentinite in the form of a ratio of the main macrocomponents, oxides of magnesium, silicon, and iron, in accordance with the composition presented in Table 1, then the process of decomposition of serpentinite with ammonium bisulfate can be presented as reaction (5).

$$\begin{array}{c} \text{3 MgO} \times 2\text{SiO}\_2 \times 0.04\text{ Fe}\_3\text{O}\_4 + 6.4\text{ NH}\_4\text{HSO}\_4 \rightarrow 2\text{SiO}\_2 \times \text{H}\_2\text{O} + 3\text{ Mg(NH}\_4\text{)}\_2\text{(SO}\_4)\_2 + \\\ \text{0.08 FeNH}\_4\text{(SO}\_4\text{)}\_2 + 0.08\text{ Fe(NH}\_4\text{)}\_2\text{(SO}\_4)\_2 + \text{H}\_2\text{O} + 0.08\text{ (NH}\_4\text{)}\_2\text{SO}\_4 \end{array} \tag{5}$$

In reaction (5), in contrast to [22], it was taken into account that in concentrated solutions containing sulfate and ammonium, Mg (II) sulfate and Fe (II) and Fe (III) sulfates form double salts with ammonium sulfate [23,24]. From the mixture obtained in accordance with reaction (5), it is possible to separate the silicic acid phase (as well as the undecomposed part of serpentinite, which takes place under the usually used real conditions), but this separation is possible only at elevated temperature, since the solubility of double magnesium sulfate and ammonium decreases sharply with temperature decreasing. Therefore, the separation in the work presented by us is carried out at temperatures above 90 ◦C. In this case, a simple cooling operation allows most of the magnesium to be removed from the system in the form of Boussingaultite, reaction (6).

$$3\text{ Mg(NH\_4)\_2(SO\_4)\_2} + 18\text{ H}\_2\text{O} \rightarrow 3\text{ Mg(NH\_4)\_2(SO\_4)\_2} \times 6\text{H}\_2\text{O} \tag{6}$$

This process, similar to recrystallization, significantly reduces the problems of further purification of the magnesium product. However, it should be taken into account that all Tutton salts (double sulfates formed by two and singly charged cations) with a monoclinic crystal structure are capable of cocrystallization; therefore, Mohr's salt Fe (Fe(NH4)2(SO4)<sup>2</sup> × 6H2O), like a similar manganese salt, can pollute the Boussingaultite. That is why at the stage of purification of a magnesium compound not an ammonia solution is used, but a mixture of an ammonia solution and ammonium carbonate, since it is impossible to achieve quantitative precipitation of manganese hydroxide at pH 8. Within the framework of the processes presented for macrocomponents, the purification of double magnesium and ammonium sulfate from iron occurs in the following simple way, reaction (7).

$$0.08\,\text{Fe(NH}\_4\text{)}\_2\text{(SO}\_4\text{)}\_2 + 0.16\,\text{NH}\_4\text{OH} + 0.04\,\text{H}\_2\text{O} + 0.02\,\text{O}\_2 \to 0.08\,\text{Fe(OH)}\_3\downarrow + 0.16\,\text{(NH}\_4\text{)}\_2\text{SO}\_4 \tag{7}$$

The Fe (III) double salt (ferric ammonium alum) predominantly remains in the filtrate after the Boussingaultite is separated. The filtrate is purified from iron by the reaction (8).

$$0.08\text{ FeNH}\_4\text{(SO}\_4\text{)}\_2 + 0.24\text{ NH}\_4\text{OH} \rightarrow 0.08\text{ Fe(OH)}\_3\downarrow + 0.16\text{ (NH}\_4\text{)}\_2\text{SO}\_4\tag{8}$$

A very important problem is the problem of magnesium precipitation from double ammonium magnesium sulfate. Magnesium hydroxide is practically impossible to quantitatively precipitate with just ammonia from solutions containing excessive amounts of ammonium sulfate. We used the process of precipitation of a hydroxide-carbonate complex, similar to the analogous process in [22], according to reaction (9).

$$1.3\text{ Mg(NH}\_4\text{)}\_2\text{(SO}\_4\text{)}\_2 + 3\text{ NH}\_4\text{OH} + 1.5\text{ (NH}\_4\text{)}\_2\text{CO}\_3 \rightarrow 1.5\text{ (MgOH)}\_2\text{CO}\_3\downarrow + 6\text{ (NH}\_4\text{)}\_2\text{SO}\_4\tag{9}$$

Reactions (5), (7)–(9) give a total of 6.4 mol of ammonium sulfate. Removal in solid form by evaporation and subsequent heat treatment of ammonium sulfate leads to the production of all reagents necessary for the implementation of the cyclic process, reaction (10).

$$6.64 \text{ (NH}\_4\text{)}\_2\text{SO}\_4 \to 6.4 \text{ NH}\_4\text{HSO}\_4 + 6.4 \text{ NH}\_3 \tag{10}$$

In particular, when this amount of ammonia is dissolved, 6.4 mol of its aqueous solution can be obtained, of which 3.4 mol isfor carrying out reactions (7)–(9), as well as 3 mol for obtaining 1.5 mol of (NH4)2CO<sup>3</sup> for reaction (9). A real technological process can also be closed in terms of carbon dioxide, which is released during the calcination of magnesium hydroxide-carbonate obtained in accordance with reaction (9).

While carrying out sequentially repeated laboratory experiments, as can be seen from the description in Section 2.1, a closed process for ammonium bisulfate was carried out. To standardize the conditions for laboratory experiments, the collection of gas components and recuperation of ammonia solution on a small scale were not carried out.

According to our estimates, the cost of the enlarged proposed process for the production of MgO from serpentinite should not exceed \$400 per ton, which corresponds to the cost of Portland cement.

#### *3.2. Effect of Calcination of MgO Powder*

The obtained X-ray diffraction patterns of MgO powder samples are shown in Figure 2. It was established that the dominant phase in the studied samples is the target phase with the periclase structure, which is identified by the reflexes 2.43, 2.11, and 1.49 Å (Figure 2). The average crystallite size of MgO and calcined MgO was 40 and 62 nm, respectively, which corresponds to the requirements [12]. It should be noted that MgO samples prepared by high-temperature processing do not contain an impurity (Figure 2b) of Mg(OH)<sup>2</sup> (brucite) and Mg5(CO3)4(OH)<sup>2</sup> × 4H2O (hydromagnesite), which present in amounts about 21 and about 7 wt%, respectively, in the initial MgO sample (Figure 2a). It was previously noted that the presence of such phases in MgO during the synthesis of the MPP matrix is extremely undesirable, because it leads to an unacceptable increase in the rate of reaction (1) and produces an inhomogeneous compound with low strength.

**Figure 2.** X-ray diffraction patterns of MgO (**a**) and calcined MgO (**b**). 1—MgO (periclase); 2— Mg(OH)<sup>2</sup> (brucite), 3—Mg<sup>5</sup> (CO<sup>3</sup> )4 (OH)<sup>2</sup> × 4H2O (hydromagnesite).

According to XRF analysis, the calcined sample of magnesium oxide contains 0.22 wt% impurities (Table 2), that corresponds to the chemical purity of reagent "analytical grade" in accordance with Russian standard [25].


**Table 2.** Chemical composition of calcined MgO.

When studying the morphology of MgO powder particles, it was found that the initial sample consists of particles of irregular shape with a size of several to tens of µm (Figure 3a), and the surface structure of this powder presents staggered flake layer, which is typical of Mg(OH)<sup>2</sup> (Figure 3b). The morphology of MgO changed from a flake appearance into cubic crystals (Figure 3c) after its calcination; the discovered effect was previously also observed in [16].

**Figure 3.** Scanning electron microscope (SEM) images of MgO (**a**,**b**) and calcined MgO (**c**).

The obtained data on the granulometric composition of MgO powders are presented in Figure 4 and in Table 3. The distribution of particle agglomerates by size of the initial and calcined powder can be characterized as monomodal with a value of about 55 µm and a complication in the region of small sizes with values less than 1 µm (Figure 4a) and less than 3 µm (Figure 4c), respectively. The effect of ultrasound on MgO samples leads to the destruction of large agglomerates. For example, as a result of the influence of ultrasound on the initial powder, about 56% of the particles acquire a size of less than 8 µm (Figure 4a,b), and in the case of a calcined powder, about 79%—less than 31 µm (Figure 4c,d). It is noted

that, in general, calcination leads to partial agglomeration of MgO particles, for example, to an increase of the number of aggregates with a size of about 100 µm (Figure 4d).

**Figure 4.** Size distribution of MgO (**a**,**b**) and calcined MgO (**c**,**d**) (dashed curve—size distribution after the effects of ultrasound on powders).


**Table 3.** The results of granulometric analysis of MgO.

\* After exposure by ultrasound on powders.

It was found that the initial MgO powder has a large specific surface area (64.5 m2/g), apparently due to the flake layer structure. In this case, as a result of calcining MgO powder at 1300 ◦C for 3 h, the specific surface area of magnesium oxide decreases to 5.4 m2/g, which corresponds to the previously obtained data [1].

#### *3.3. Study of the Obtained Samples of the MPP Matrix*

For the synthesis of the MPP matrix, we used a precalcined MgO powder, the characteristics of which are given in the previous Section. When studying the phase composition of the synthesized samples of the MPP matrix, it was found that their main crystalline

phase is the target phase MgKPO<sup>4</sup> × 6H2O, which is an analog of K-struvite [6] with main reflections at 5.86; 5.56; 5.40; 4.25; 4.13 Å etc.) (Figure 5). The samples also contain phase of MgO (periclase), which is associated with the excess of the used MgO in relation to the stoichiometry of reaction (1).

**Figure 5.** X-ray diffraction pattern of the magnesium potassium phosphate (MPP) matrix. 1— MgKPO<sup>4</sup> × 6H2O (K-struvite); 2—MgO (periclase).

The SEM micrograph of the surface of the MPP compound is shown in Figure 5. The elemental composition of the predominant phases in the compound sample includes matrix components of the basic composition MgKPO<sup>4</sup> × 6H2O with insignificant variations in the Mg/K ratio, as we noted earlier in [1]. Open pores with a linear size of about 100 µm are also observed (Figure 6).

**Figure 6.** SEM image of the MPP matrix.

The compressive strength of the MPP matrix obtained using MgO after calcining at 1300 ◦C for 3 h was 6.19 ± 0.45 MPa, which meets the regulatory requirements for a cement compound: no less than 4.9 MPa [26].

The determination results of hydrolytic stability of MPP compound to the leaching of matrix-forming components are shown in Figure 7a,b. Data in Figure 7a shows that the differential leaching rate of magnesium, potassium, and phosphorus from the compound on the 91st day of contact of the sample with water is 1.6 × 10 −5 , 4.7 × 10 <sup>−</sup><sup>4</sup> 8.9 <sup>×</sup> <sup>10</sup> <sup>−</sup><sup>5</sup> g/(cm<sup>2</sup> day),

respectively. It was found that the leaching of the matrix-forming elements for 91 days of contact of the sample with water is controlled by various mechanisms. Leaching of potassium and phosphorus from the MPP matrix in the first seven days occurs due to its wash-off of from the surface of the compound, followed by depletion of the surface layer (Figure 7b, coefficients A in Equation (3) are for potassium −0.43 and 0.17, and for phosphorus −0.50, 0.04, and −0.66). Leaching of magnesium in the first 10 days occurs due to its wash-off of from the surface of the compound, and in the next 81 days due to diffusion from its inner layers (Figure 7b, −0.81 and 0.48). The obtained results on the hydrolytic stability (rate and mechanism of leaching) of the MPP matrix obtained using MgO obtained from serpentinite are consistent with the previously obtained data for the MPP matrix obtained from commercial MgO [1].

**Figure 7.** Kinetic curve of the leaching rate (**a**) and logarithmic dependence of the release (**b**) of the matrix components from the MPP matrix.

#### **4. Conclusions**

The applicability of MgO obtained during the reprocessing of widely available mineral raw materials—serpentinite by almost waste-free and economically profitable way for the synthesis of the MPP matrix was established. The characteristics of the obtained matrix correspond to the requirements for the material for RW immobilization and for preventing of release of highly toxic radionuclides into the environment.

**Author Contributions:** Conceptualization: S.A.K., S.E.V., and R.K.K.; methodology: S.A.K., S.E.V., N.S.V., and R.K.K.; validation: S.A.K., S.E.V., and N.S.V.; formal analysis: S.A.K., K.Y.B., and S.E.V.; investigation: S.A.K., N.S.V., K.Y.B., and R.K.D.; resources: R.K.D.; writing—original draft preparation: S.A.K., S.E.V., and R.K.K.; writing—review and editing: M.A.K. and B.F.M.; supervision: S.E.V., R.K.K., M.A.K., and B.F.M.; project administration: S.E.V.; funding acquisition: S.E.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the GEOKHI RAS state assignment (0137-2019-0022).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data sharing not applicable.

**Acknowledgments:** The authors thank V.V. Krupskaya and I.A. Morozov (Lomonosov Moscow State University; Institute of Geology of Ore Deposits, Petrography, Mineralogy, and Geochemistry of Russian Academy of Sciences) for the opportunity provided to use Ultima-IV X-ray diffractometer (Rigaku) and I.N. Gromyak (Laboratory of Methods for Investigation and Analysis of Substances and Materials, GEOKHI RAS) for performing the ICP-AES analysis.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

#### **References**


## *Article* **Electrochemical Determination of Lead Using A Composite Sensor Obtained from Low-Cost Green Materials:Graphite/Cork**

**Iasmin B. Silva <sup>1</sup> , Danyelle Medeiros de Araújo <sup>2</sup> , Marco Vocciante <sup>3</sup> , Sergio Ferro 4,\* , Carlos A. Martínez-Huitle 1,\* and Elisama V. Dos Santos 1,\***


**Abstract:** The purpose of this study was to develop an inexpensive, simple, and highly selective cork-modified carbon paste electrode for the determination of Pb(II) by differential pulse anodic stripping voltammetry (DPASV) and square-wave anodic stripping voltammetry (SWASV). Among the cork–graphite electrodes investigated, the one containing 70% w/w carbon showed the highest sensitivity for the determination of Pb(II) in aqueous solutions. Under SWASV conditions, its linear range and relative standard deviation are equal to 1–25 µM and 1.4%, respectively; the limit of detection complies with the value recommended by the World Health Organization. To optimize the operating conditions, the selectivity and accuracy of the analysis were further investigated by SWASV in acidic media. Finally, the electrode was successfully applied for the determination of Pb(II) in natural water samples, proving to be a sensitive electrochemical sensor that meets the stringent environmental control requirements.

**Keywords:** cork–graphite electrode; electrochemistry; lead; environmental application

#### **1. Introduction**

Lead is a highly toxic heavy metal that causes serious environmental problems due to its non-biodegradability. It is commonly released into the environment because of mining activities, natural processes, and the development of new technological devices [1,2], being frequently used by the automotive, plastics, paints, and ceramics industries for its corrosion resistance [3].

Since the nitrate and chloride salts of lead show excellent solubility in water [4,5], lead is normally present in soil and aquatic ecosystems in ionic form, as Pb(II). According to the World Health Organization (WHO), a Pb(II) concentration as low as 0.24 µmol L−<sup>1</sup> can cause decreased intelligence in children, behavioral difficulties, and learning problems. For this reason, the concentration of lead in water and soils should always be below the WHO limit and, consequently, must be monitored.

Nowadays, several analytical methods are employed for lead detection, such as spectroscopy [6], optical colorimetry [7], inductively coupled plasma mass spectrometry (ICP-MS) [8], atomic absorption spectrometry (AAS) [9], and fluorescence spectrometry [10]. However, these analytical methods are expensive (they require trained operators, complex equipment, solvents or gases, and so on) and, in some cases, sample preparation procedures are required. In this context, electrochemical techniques have been investigated because of their significant advantages such as simplicity of operation, high sensitivity, low cost, and

**Citation:** Silva, I.B.; de Araújo, D.M.; Vocciante, M.; Ferro, S.; Martínez-Huitle, C.A.; Dos Santos, E.V. Electrochemical Determination of Lead Using A Composite Sensor Obtained from Low-Cost Green Materials:Graphite/Cork. *Appl. Sci.* **2021**, *11*, 2355. https://doi.org/ 10.3390/app11052355

Academic Editor: Fethi Bedioui

Received: 13 January 2021 Accepted: 1 March 2021 Published: 6 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

easy handling [11–15]. In general, electrochemical sensors are rapid, portable, inexpensive, and highly sensitive and offer a low limit of detection, good reproducibility, good signal-tonoise ratio, and selective detection [12]. Consequently, electrochemical sensors have been applied for the determination of heavy metals in the environment, industrial products, food matrices, electronic waste, and clinical materials [16–18].

Among the electrochemical sensors used, graphite-modified electrodes have been extensively developed due to their higher selectivity, sensitivity, high specific area, unique electrical conductivity, self-assembly behavior, mechanical flexibility, extreme resistance to oxidation, natural origin, and low cost [19–22]. However, these properties can be improved by including other modifiers in their composition.

Recently, cork has emerged as a promising low-cost and efficient green material for various environmental applications (e.g., compound detection [13,23,24], soil and water remediation [25,26]). Cork is a natural organic polymeric material, which has modest electrical, magnetic, and optical properties and exhibits self-cleaning behavior and antibacterial activity. For raw cork (RAC), electrical conductivity (σ) values of approximately 1.2 <sup>×</sup> <sup>10</sup>−<sup>10</sup> and 1.67 <sup>×</sup> <sup>10</sup>−<sup>13</sup> S m−<sup>1</sup> were registered at 25 and 50 ◦C, respectively [27]. Based on the existing literature, two types of cork are often used: raw cork (RAC) and regranulated cork (RGC). Their differences are mainly due to their composition, which depends on the thermal pretreatment applied to RAC to produce RGC.

In the present communication, cork–graphite composite electrodes to be used as electrochemical sensors for the detection of lead ions are discussed. The effects of the cork composition, the cork–graphite ratio, and the supporting electrolyte for detecting Pb(II) were investigated. The performance in Pb(II) detection of two voltammetric techniques (differential pulse adsorptive stripping voltammetry (DPASV) and square-wave adsorptive stripping voltammetry (SWASV)) was also evaluated. Finally, the applicability of the cork–graphite voltammetric device was successfully demonstrated by detecting Pb(II) in real water matrixes (groundwater, tap water, and "produced water") as well as verifying the selectivity, repeatability, reproducibility, and stability of the sensors.

#### **2. Materials and Methods**

The highest quality commercially available chemicals were used. Graphite powder and Pb(NO3)<sup>2</sup> were sourced from Sigma (Brazil); the former was used without further purification. Acetate buffer, NaNO3, CdCl2, H2SO4, NaCl, FeCl2, KCl, CaCl2, MgSO4, ZnCl2, AlCl3, and MnSO<sup>4</sup> were sourced from Merck (Brazil). The raw cork (RAC) used in the experimental studies was provided by Corticeira Amorim S.G.P.S., S.A. (Portugal); the granules were washed twice with distilled water in cycles of 2 h at 60 ◦C to remove impurities and other water-extractable components that could interfere with the electrochemical analysis. Before use, the RAC was dried at 60 ◦C in an oven for 24 h [23]. Aqueous solutions were prepared using ultrapure water obtained from a Millipore Milli-Q direct-0.3 purification system.

#### *2.1. Preparation of Cork-Modified Electrodes*

The RAC granules were reduced in size using a ball mill and sieved to obtain the finest fractions. The fraction below 150 µm (designated as RAC powder) was selected for use in this work. The cork–graphite composite sensor (working electrode) was prepared by mechanical homogenization of RAC and graphite (Gr) in different proportions (10:90, 70:30, and 90:10 %w/w), using 0.3 mL of paraffin oil as a binder and mixing everything in an agate mortar for about 30 minutes, as previously reported [23]. The paste was packed in a polypropylene nozzle (model K31-200Y) used as a support, and the sensor surface was smoothed over a tissue paper. Before use, the sensor was electroactivated by cyclic voltammetry between <sup>−</sup>1.1 and 0 V (scan rate: 100 mV s−<sup>1</sup> ) in 0.5 M H2SO4. The different sensors are referred to as GrRAC-X, where X is the amount of cork (RAC) expressed as %w/w. The unmodified graphite sensor (Gr) was prepared as described for the GrRAC-X

sensors, but in the absence of RAC powder. Electrode stability was also determined by repetitive determinations of Pb (25 µM) in 0.5 M H2SO4.

#### *2.2. Electrochemical Measurements*

The electrochemical tests were performed using an Autolab PGSTAT302N (Metrohm) controlled with NOVA 1.8 software, and a three-electrode cell including an Ag/AgCl (3.0 M KCl) reference electrode, a Pt wire auxiliary electrode, and one of the cork–graphite sensors (GrRAC) as the working electrode (geometrical area of approximately 0.45 mm<sup>2</sup> ). Differential pulse anodic stripping voltammetry (DPASV) measurements were performed with different concentrations of Pb(II) ions in acetate buffer solutions (pH 4.5), 0.5 M NaNO3, and 0.5 M H2SO4. The accumulation of Pb(II) ions on the surface of the composite sensor was achieved by applying a potential of −1.2 V (vs. Ag/AgCl) for different preconcentration times (40, 70, 100, 130, and 160 s), during which the stirring conditions were kept constant for 30, 60, 90, 120, and 150 s; the remaining 10 s were considered as an equilibration time, without stirring. Subsequently, the anodic stripping scan was performed at 50 mV s−<sup>1</sup> , with a modulation amplitude of +0.05 V, and a modulation time of 0.04 s. Square-wave anodic stripping voltammetry (SWASV) measurements were performed in 0.5 M H2SO4. In this case, a preconcentration potential of −1.2 V was applied to the working electrode for 120 s under continuous magnetic stirring, with a scanning frequency of 80 Hz, an amplitude of 50 mV, and a step potential of 5 mV. All electrochemical studies were conducted without deaerating and performed at room temperature (25 ± 2 ◦C). Each measurement was performed in triplicate, and obtained data were subjected to statistical analysis and reported as mean ± standard deviation (SD). For the determination of Pb(II) in different water matrices (tap water, groundwater, and produced water), the water samples were spiked with a known quantity of a standard solution of Pb(II) and the determination of Pb(II) was performed using the standard addition method.

#### **3. Results and Discussion**

#### *3.1. Effect of the Supporting Electrolyte*

In order to evaluate the voltammetric response of the proposed modified sensor, the quantification of Pb(II) was carried out in different supporting electrolytes. Figure 1a–c show the DPASV response for the determination of Pb(II) using a GrRAC-70% sensor (this composition was selected for preliminary analysis based on the results reported in the literature) from solutions of 0.1 M acetate buffer (pH 4.5), 0.5 M NaNO3, and 0.5 M H2SO4, respectively, using a preconcentration time of 30 s. The sulfuric acid solution proved to be the most suitable electrolytic solution because it provided a well-defined voltammetric signal and the response increased linearly without significant deviations (Figure 1). The limit of detection (LOD), for each of the supporting electrolytes used, was estimated by the equation *LOD = 3* × *Sy/x/b*, where *Sy/x* is the residual standard deviation and *b* is the slope of the calibration plot, in accordance with IUPAC recommendations of the mean value for samples analyzed in triplicate. This approach allows the control of both false positive and negative errors (α = β = 0.05), as recommended by IUPAC [28,29], and has been confirmed and recommended by experts in the field [30,31]. For the 0.1 M acetate buffer solution, no significant current response was obtained, resulting in an LOD of 4.8 µM; a similar outcome was obtained in 0.01 M acetate buffer solution, where an LOD of approximately 3.2 µM confirmed the poor performance of the sensor in acetate buffer solutions. Conversely, the analytical approach significantly improved when 0.5 M NaNO<sup>3</sup> and 0.5 M H2SO<sup>4</sup> solutions were used, which resulted in LOD values of 2.8 (Figure 1b) and 1.6 µM (Figure 1c). Comparing the values obtained with an unmodified graphite electrode and with the modified cork–graphite electrodes in H2SO4, the LOD is 3 times higher on graphite (≈4.8 µM) than that obtained with the GrRAC-70% electrode. The best result obtained using the composite material shows that the cork–graphite mixture is able to influence the intensity of the current signals.

**Figure 1.** DPASV curves recorded for different concentrations of Pb(II) in (**a**) 0.1 M acetate buffer (pH 4.5), (**b**) 0.5 M NaNO<sup>3</sup> , and (**c**) 0.5 M H2SO<sup>4</sup> . Lead concentrations: (a) 0, (b) 1.2, (c) 2.4, (d) 4.8, (e) 7.1, (f) 9.5, (g) 11.8, (h) 14.0, (i) 16.3, (j) 18.6, (k) 20.8, (l) 25 µM. Inserts: plots of the electrochemical response, in terms of current, as a function of the lead concentration.

According to the literature [32], cork has a great adsorption capacity, which is assumed to occur through a so-called biosorption mechanism consisting of an initial physical adsorption (rapid metal uptake) and then a slower chemisorption. The biosorption mechanism is the result of several kinds of interactions, such as complexation, coordination, chelation, ion exchange, inorganic microprecipitation, and hydrolysis products of metal ions; in the case of metal ion biosorption, ion exchange is usually the main mechanism. Hence, the type of cork, the pH conditions, and the contact time determine the interactions that can occur between the target compound and the cork surface. Depending on the cork used, specific active sites may predominate in its surface composition (phenolic, carboxylic, sulfonic, phosphate, and amino groups as well as coordination sites), in addition to the cork surface charge depending on pH conditions [33]. It is also important to consider that carbonaceous materials have micropores and mesopores, the accessibility of which will be increased following the inclusion of cork as a surface modifier. Thus, an improvement in voltammetric current signals can be achieved. Another important feature to consider is that the surface morphology of GrRAC-70% is more homogeneous, as evidenced by the SEM micrographs, which can positively influence its current response [34].

In the case of the acetate buffer as the supporting electrolyte, the formation of Pb(CH3COO)<sup>2</sup> can decrease the availability of Pb(II) in solution; in addition, the acetate ions can compete with Pb(II) ions for the active sites available on the graphite–cork surface. As a result, a poor current response is achieved, with limitations on the selectivity and sensitivity of the modified electrode. Indeed, lead concentrations below 10 µM (Figure 1a), which affect the LOD, cannot be efficiently detected. In the case of NaNO3, the lack of complexing activity by nitrate anions and the possibility of preferential interactions between the composite material (cork–graphite) and Pb(II) ions in solution allow significant improvements in the current response, with consequent benefits in terms of the linearity of the response, although superficial adsorption phenomena may be highlighted for lead concentrations below 10 µM (Figure 1b). Finally, well-defined voltammetric signals were observed at the GrRAC-70% electrode when H2SO<sup>4</sup> was used (Figure 1c). Due to the acidic conditions, lead is present in solution in its cationic form, Pb2+, and the cork surface is also completely protonated and positively charged. When the working electrode is negatively polarized, the lead ions compete with protons for surface sites; however, the surface accumulation of Pb ions is favored due to ion exchange mechanisms with active sites. In fact, the current response increased linearly without any significant deviations. Therefore, H2SO<sup>4</sup> was selected as the supporting electrolyte for the subsequent experiments.

#### *3.2. Influence of the Preconcentration Time*

The effect of the preconcentration time (40, 70, 100, 130, and 160 s) on the voltammetric response for Pb(II) detection was studied in 15 mL of 0.5 M H2SO<sup>4</sup> by using the GrRAC-70% sensor. The results indicate that the peak current increased with the preconcentration time, from 40 to 160 s, as illustrated in Figure 2. For all tests, the initial potential was held constant at −1.2 V under stirring conditions for different times (30, 60, 90, 120, and 150 s), with an additional resting time of 10 s without stirring; subsequently, the stripping voltammetry was carried out at 50 mV s−<sup>1</sup> . As seen in Figure 2, a decrease in current was achieved when the preconcentration time was extended to 160 s; thus, 130 s was chosen as the most suitable preconcentration time for further analysis. Ten replicates were considered in order to study the effect of preconcentration. According to Student's *t*-test at a confidence level of 95% (parameter denominated as *p*), there were no significant differences between the experimental value (23.5 µM of Pb) and the theoretical value (25 µM of Pb). The observed trend can be motivated by the high adsorption rate due to the porous structure of the cork [33]; for preconcentration times greater than 130 s (120 s under stirring conditions and 10 s in rest conditions), the GrRAC-70% sensor plausibly reached the maximum adsorption capacity on its surface.

− <sup>−</sup> − **Figure 2.** Effect of the preconcentration time on the quantification of Pb(II) ions by DPASV, using the GrRAC-70% composite sensor. Experimental conditions: 25 µM of Pb(II) (n = 10; R<sup>2</sup> = 0.98; *p* = 95%). Reduction potential: <sup>−</sup>1.2 V (vs. Ag/AgCl); scan rate: 50 mV s−<sup>1</sup> ; potential range: −1.0 to 0 V. Supporting electrolyte: 0.5 M H2SO<sup>4</sup> .

#### *3.3. Influence of the Cork Concentration*

The performance of the composite electrode is affected by the amount of cork that is mixed with the graphite. In a previous work, we showed that the quantity of cork influences the electroactive area of the sensor as well as the electron transfer during the oxidation of caffeine [13]. In order to evaluate the effect of the quantity of cork on the detection of Pb(II) by DPASV, 0.5 M H2SO<sup>4</sup> was used as the supporting electrolyte. As can be observed in Figure 3a–c, the peak current recorded on GrRAC depends on the amount of cork present in the composite sensor. In particular, a linear relationship between the peak current and the Pb(II) concentration was obtained in the Pb(II) concentration range from 1 to 25 µM in 0.5 M H2SO<sup>4</sup> (inserts in Figure 3a–c), considering at least 11 different concentrations of the analyte. Pb(II) calibration curves were obtained for each of the prepared sensors. From the analytical curves obtained using GrRAC-10%, GrRAC-70%, and GrRAC-90% by DPASV, it can be seen that the stripping peak currents (Ip) increased linearly with the concentration of Pb(II) (inserts in Figure 3). The calculated correlation equations are

GrRAC-10%: I<sup>p</sup> (µA) = (0.05 <sup>±</sup> 0.03)×C - (0.1 <sup>±</sup> 0.05); R<sup>2</sup> = 0.97

GrRAC-70%: I<sup>p</sup> (µA) = (0.08 <sup>±</sup> 0.04)×C - (0.3 <sup>±</sup> 0.1); R<sup>2</sup> = 0.98

GrRAC-90%: Ip (µA) = (0.11 <sup>±</sup> 0.09)×C - (0.2 <sup>±</sup> 0.1); R<sup>2</sup> = 0.95

According to Figure 3, the best performing GrRAC sensor in terms of sensitivity, capable of providing an LOD for Pb(II) of only about 0.8 µM, was GrRAC-70%. In contrast, the GrRAC-10% and GrRAC-90% sensors provided higher LODs of approximately 1.5 and 1.2 µM, respectively. This difference in the LODs is attributable to the different degrees of dispersion of the surface active sites, as reported in our previous study on the determination of caffeine [23]. The highest peaks were obtained with GrRAC-70% and this cork–graphite ratio was used for detecting Pb(II). The results show that Pb(II) stripping signals with GrRAC-70% are superior to those obtained with the GrRAC-10% and GrRAC-90% electrodes. This can be attributed to the honeycomb macroporous structure of the cork granules, which favor the presence of propagation paths through the cork cells, forming macroporosity with textural properties. Based on our previous results, the surface morphology of GrRAC-70% appears particularly homogeneous because the graphite sheets are arranged in close contact within the porosities of the cork [23].

**Figure 3.** DPASV curves recorded for different concentrations of Pb(II) with (**a**) GrRAC-10%, (**b**) GrRAC-70%, and (**c**) GrRAC-90%. Lead concentrations: (a) 0, (b) 1.2, (c) 2.4, (d) 4.8, (e) 7.1, (f) 9.5, (g) 11.8, (h) 14.0, (i) 16.3, (j) 18.6, (k) 20.8, (l) 25 µM. Inserts: plots of the electrochemical response, in terms of current, as a function of the lead concentration.

#### *3.4. SWASV Analysis*

The GrRAC-70% composite sensor produced the best DPASV results for Pb(II) detection and was therefore chosen as the working electrode for evaluating Pb(II) traces by SWASV. Figure 4 shows the SWASV voltammetric responses of Pb(II) under pre-selected experimental conditions: 0.5 M H2SO<sup>4</sup> as the supporting electrolyte, −1.2 V as the preconcentration potential, 120 s of preconcentration time plus 10 s of resting time. The stripping voltammetric peaks of Pb(II) ions appeared at −0.44 V for the GrRAC-70% sensor. The peak current (Ip) increased linearly with the concentration of Pb(II) in the range from 1 to 25 µM; the linear regression equation (I<sup>p</sup> vs. C) was obtained as

$$\mathbf{I}\_{\mathbf{P}} \text{ (}\mu\text{A)} = (0.4 \pm 0.1) \times \text{C} - (0.8 \pm 0.2) \text{; R}^2 = 0.98$$

**Figure 4.** SWASV curves of GrRAC-70% recorded for different concentrations of Pb(II) in 0.5 M H2SO<sup>4</sup> : (a) 0, (b) 1.2, (c) 2.4, (d) 4.8, (e) 7.1, (f) 9.5, (g) 11.8, (h) 14.0, (i) 16.3, (j) 18.6, (k) 20.8, (l) 25 µM. Inserts: plots of the electrochemical response, in terms of current, as a function of the lead concentration.

The LOD was found to be 0.3 µM. Compared with DPASV, SWASV produced much better results: the regression residuals are randomly distributed around zero and the linearity is practically perfect. Another important point is that no noticeable alterations were noted in the calibration curves recorded on different days, confirming the stability of the GrRAC-70% composite sensor. The GrRAC-70% sensor used in this work remained stable for at least two months of intensive use.

− Table 1 collects the results available in the literature and relating to the analysis of Pb(II) with different electrodes and allows a comparison with the results obtained in this study. The ease of sensor preparation and the analytical protocol suggested here offer advantages over the other methods reported.

**Table 1.** Comparison of the analytical parameters of the sensors reported in the literature for the determination of Pb(II).


<sup>1</sup> Mercaptothiazoline-disulfide-bridged periodic mesoporous organosilica. <sup>2</sup> 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol modified multiwalled carbon nanotube electrode. <sup>3</sup> Nanocomposite of polypyrrole (PPy) and carbon nanofibers (CNFs)-modified carbon paste electrode (CPE). <sup>4</sup> Silver ring electrode. <sup>5</sup> Electrochemically reduced graphene oxide–multiwalled carbon nanotubes–L-cysteine. <sup>6</sup> Carbon paste electrode (CPE) modified with ion-imprinted polymer nanoparticles and multiwalled carbon nanotubes. <sup>7</sup> Inkjet-printed multiwalled carbon nanotubes.

#### *3.5. Study of the Interferences*

In order to evaluate the specificity of the suggested approach, the sensor response in the presence of several potentially interfering species was investigated. In particular, the response to Pb(II) was evaluated in solutions containing 10 µmol/L of the following cations: Fe2+, Na<sup>+</sup> , K<sup>+</sup> , Ca2+, Mg2+, Zn2+, Al3+, Mn2+, Cu2+, and Cd2+. No additional signals were recorded when Fe2+, Na<sup>+</sup> , K<sup>+</sup> , Ca2+, Mg2+, Zn2+, Al3+, Mn2+, or Cu2+ ions were present in solution during the determination of Pb at different concentrations (Figure 5). Conversely, a well-defined peak signal for Cd2+ was observed at <sup>−</sup>0.8 V. However, no changes in the Pb current peak were observed for the GrRAC-70% composite sensor in the presence of Cd2+ ions in solution (Figure 5). Therefore, the GrRAC-70% sensor can be used to detect Pb(II) even in the presence of other metals. −

**Figure 5.** SWASV curves of GrRAC-70% recorded for different concentrations of Pb(II) in 0.5 M H2SO<sup>4</sup> in the presence of a fixed concentration of interfering metal ions (10 µmol/L).

#### *3.6. Stability*

The stability of GrRAC was previously examined by determining caffeine, obtaining results of good consistency with a relative standard deviation (RSD) of 1.41% (*n* = 3); this outcome suggested that the cork–graphite composite sensor can be reused. A similar stability assessment was carried out in the case of Pb(II) by recording a series of voltammetric analyses over 10 days. No significant changes in the Pb peak current were observed after five measurements over that time period (Pb(II) = 15 µM; RSD = 1.52%, *n* = 5). The cork–graphite composite sensor was washed and stored at 25 ◦C after each experiment.

#### *3.7. Analytical Applications*

The effectiveness of the proposed method, for the detection of Pb(II) in real samples, was also tested by analyzing tap water, groundwater, and "produced water" (a brackish water that is extracted as a by-product from underground during the process of oil and natural gas extraction). The electrochemical determination of Pb(II) was based on SWASV in acidic medium. For this, a quantity of Pb(II) was added to the water samples to obtain a well-known concentration between 10 and 50 µM for each sample. Then, the prepared samples were analyzed using the GrRAC-70% composite sensor under the optimized experimental conditions reported in Section 3.4. The validity of the proposed method for the determination of Pb(II) was evaluated using the standard addition method and recovery studies were conducted on the samples. As shown in Table 2, the recoveries ranged from 89 to 115 (*n* = 3), indicating that the proposed method can be efficiently applied for the

detection of Pb(II) in real water samples. In all cases, the relative standard deviation (RSD) values ranged from 0.3 to 2.1%, which confirms that the developed detection approach is potentially applicable.

**Table 2.** Pb(II) content in real water samples, measured with SWASV using the GrRAC-70% composite sensor.


<sup>1</sup> Mean of three determinations <sup>±</sup> standard deviation.

#### **4. Conclusions**

Cork–graphite-based sensors offer a fast, reliable, cost-effective, and simple way to determine Pb(II) in real samples. The composite sensor exhibits higher sensitivity and reproducibility than conventional unmodified graphite sensors, and the low LOD allows for reduced matrix effects in dilute solutions. As for the materials tested, the affinity of the cork with the analyte allowed a substantial improvement in sensitivity. According to the results reported in this work, the sensor obtained by mixing 70% w/w of cork with 30% w/w of graphite allowed obtaining higher voltammetric responses and a rapid detection of Pb(II). The proposed approach is precise, with a limit of quantification of 0.3 µM, reproducible, and less expensive, both in terms of time and materials, compared to other analytical methods. The composite electrode can be applied effectively for the determination of Pb(II) in acidic media. As for the physical and chemical properties, which favor the interactions with the analytes to be detected or/and quantified, more experiments are needed to better understand the chemical and electrochemical processes that occur on the cork–graphite surface when the current is applied or when cork participates as a mediator.

Finally, even if the LOD reported in this work (0.3 µmol L−<sup>1</sup> ) is slightly above the limit established by the WHO (Pb: 0.24 µmol L−<sup>1</sup> ), there is room for improvement; for example, the size of the electrochemical sensor could be reduced, in order to approximate a micro-electrode, while the use of other carbon-based modifiers could allow improving the sensitivity.

**Author Contributions:** Conceptualization, E.V.D.S. and C.A.M.-H.; methodology, M.V.; formal analysis, I.B.S., S.F., and C.A.M.-H.; investigation, I.B.S. and C.A.M.-H.; resources, C.A.M.-H.; data curation, I.B.S., E.V.D.S., and C.A.M.-H.; writing—original draft preparation, I.B.S. and D.M.d.A.; writing—review and editing, M.V., S.F., and C.A.M.-H.; funding acquisition, D.M.d.A. and C.A.M.-H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (Brazil), grant number 306323/2018-4, and Fundação de Amparo à Pesquisa do Estado de São Paulo (Brazil), grant numbers 2014/50945-4 and 2019/13113-4.

**Acknowledgments:** I.B. Silva acknowledges the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior for her fellowship. Financial supports from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq–306323/2018-4) and from Fundação de Amparo à Pesquisa do Estado de São Paulo (Brazil) (FAPESP 2014/50945-4 and 2019/13113-4) are gratefully acknowledged. The authors also thank V.J.P. Vilar from the University of Porto (Portugal) for providing the cork samples used in this study.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Economic Valuation of Reducing Submerged Marine Debris in South Korea**

#### **Se-Jun Jin <sup>1</sup> , Young-Ju Kwon <sup>1</sup> and Seung-Hoon Yoo 2,\***


Received: 27 July 2020; Accepted: 31 August 2020; Published: 2 September 2020

**Abstract:** Submerged marine debris (SMD) scattered between sea level and the bottom of the sea damages the habitats of marine life and threatens its growth in South Korea. The collection of SMD is more difficult and expensive than that of coastal and floating debris. The government is trying to achieve a 33% reduction in SMD by 2023 by expanding its collection, which requires huge additional investments and additional information about the economic value or benefits of the reduction. This article seeks to conduct an economic valuation of the reduction by employing contingent valuation (CV), which asks people to indicate their willingness to pay (WTP) for the reduction. A dichotomous choice CV survey was undertaken with 1000 households by a professional survey firm through person-to-person interviews during July 2019. Overall, people understood the CV questions well and reported the WTP responses for a hypothetical market successfully created with CV. Although 37.9% of interviewees stated zero WTP, the average of the yearly household WTP was estimated as 5523 Korean won (KRW) (USD 4.92). This value ensures statistical significance. The population's WTP for the reduction would be KRW 110.30 billion (USD 99.75 million) per year over the next five years. It was found that the reduction is socially beneficial since the value was greater than the costs involved in the reduction.

**Keywords:** submerged marine debris; economic valuation; contingent valuation; economic benefit; willingness to pay

#### **1. Introduction**

The "plastic-free" movement is taking place around the world with plastic waste emerging as a serious environmental problem. In particular, the ocean often becomes the final destination for plastic waste, resulting in problems such as the creation of an island made up of plastic waste [1]. Waste containing plastics that flows into the ocean is called marine debris. The marine debris containing plastic is being pointed out as a global pollution problem [2], and various discussions and international agreements to strengthen regulations to reduce marine debris in the future are under way [3–7].

In line with this international situation, South Korea has tried to reduce marine debris. Marine debris in the past was mostly composed of decomposable materials, but postindustrial marine debris consisting of synthetic materials such as plastics cannot be decomposed. Due to its high buoyancy, plastic goods move thousands of miles into the ocean current and threaten marine ecosystems and wildlife [2]. Marine debris also causes damage to the economy and the marine environment [8]. Thus, in order for the marine ecosystem to provide sustainable services, it is urgent that the marine debris be proactively managed [9,10].

Three sides of the Korean Peninsula are surrounded by the sea, with the result that the country actively engages in fishing and trading activities. There are numerous ports and fishing ports on the East Sea, the South Sea, and the West Sea (the Yellow Sea). Moreover, rivers are connected to the sea. Inevitably, the country's geographical situation provides a route for land-based waste to flow into the sea. The inflow of waste through the rivers flows into the stream due to increased flow rates caused by a rainy season or typhoon. Currently, the country is actively carrying out policies and projects to mitigate floating marine debris on the coast [11], which is one type of marine debris.

It is difficult to identify the distribution and inflow path of the other type of marine debris, known as submerged marine debris (SMD), because it is located at the bottom of the sea. It is relatively easy and cheap to collect floating marine debris, but collection of SMD is expensive because it requires divers and special equipment. In addition, the disposal cost of marine waste in South Korea, approximately KRW 2.24 million (USD 1995) per ton in the case of sunken fishing net, is about eight times higher than that of land waste, which is approximately 270,000 Korean won (KRW) (USD 240) per ton. The SMD from the ocean as well as SMD flowing from land into the ocean has a negative impact on the marine environment. For instance, destruction of habitats of marine life, deterioration of the quality of marine products, threat to maritime safety, and damage to marine resources can arise [12,13]. In summary, although SMD is not classified as special waste, the collection of SMD is more difficult and expensive than that of coastal and floating debris because it is submerged at the bottom of the sea and requires special equipment and diving personnel to collect.

The South Korean government has established a legal basis for marine debris management at the national level and has pursued various polices to reduce marine debris, such as prevention, collection, and publicity. In particular, the government is trying to achieve a 33% reduction in SMD by 2023 by expanding its collection. The government's intent is to develop a collection system that considers the effects of SMD on the marine ecosystem and to pursue the collection of SMD in a way that reflects the use of space and ecological characteristics. Since the reduction requires a considerable amount of investment, the government is interested in the value people place on reducing SMD [1,14]. From an economics' point of view, people's willingness to pay (WTP) for the reduction is interpreted as the economic value or benefits ensuing from the reduction [15,16]. Whether or not the reduction is socially beneficial can be determined through comparing the economic benefits with the costs involved in the reduction.

This paper attempts to determine the economic value or benefits of the reduction of SMD by collecting and exploring people's WTP for the reduction. For this purpose, a survey-based economic technique called a contingent valuation (CV) method was adopted, and the results from a CV survey of 1000 interviewees are reported.

In South Korea, SMD occurs mainly through three channels. First, waste from the land or riverside flows into the river when heavy rain falls, and then into the sea through the estuary. If the nature of these wastes is investigated, more than 90% consists of trees and grass, although some of it is household waste such as waste appliances, waste plastics, and waste vinyl. It is impossible to identify the polluters in these cases. Therefore, it is difficult to find the polluters and make them pay for the cost of collecting SMD. In fact, the polluters may be ordinary people.

The second SMD channel consists of fishing-related waste that is intentionally or accidentally thrown into the ocean. Fishermen's fishing gear, fishing nets, waste nets, Styrofoam for buoys, and feed bins for cultivating aquatic products are flowing into the ocean. In the case of fishing-related waste, fishermen could be charged with SMD collection costs since they are clearly the polluters. However, not all fishermen discharge waste into the ocean. It is not easy to accurately identify the polluters. In addition, in South Korea, fishermen are exempt from various taxes and are given subsidies because their income levels are lower than those of other occupations. Therefore, imposing a financial burden on fishermen is not a very feasible alternative.

Third, waste intentionally or accidentally dumped by coastal inhabitants or islanders also flows into the ocean. Likewise, it is not easy to figure out who among these residents has leaked waste, and their income levels are low, making it difficult for them to bear new burdens even if they are polluters.

In summary, identifying the causal provider of SMD is not easy and, even if it is possible, imposing collection costs is not socially acceptable. In the end, the central government has no choice but to collect and utilize SMD through the funds raised from taxes paid by the general public. Thus, the framework of this study, which randomly selected 1000 households nationwide and asked WTP questions, was reasonable. This is because the polluters are not economic players such as certain companies, but an unspecified majority of the general public.

The effects of SMD belong to the category of negative externalities, which have many other economic effects. The economic effects of SMD are summarized in three ways. First, there is a decrease in tourism income owing to marine environment pollution by SMD. When SMD flows onto the beach, the number of visitors decreases, which, in turn, reduces the income of accommodations and shops near the beach. Second, there is economic damage that occurs to fishermen. SMD caught in a net causes damage to fish catches or fishing nets. In addition, SMD caught in a ship screw can lead to a ship accident, resulting in huge economic losses. The third effect of SMD is a national economic loss due to transboundary pollution. When SMD crosses the border along the current, transboundary pollution occurs, which can cause conflicts between countries.

These various economic effects of SMD can affect the public's WTP for reducing SMD. Therefore, in order to control these effects, it was assumed in the study that everything remained in its current state except for the change in the goods to be assessed. The assessed goods presented to respondents in this study reflected a 33% reduction of SMD by 2023 as compared to the business-as-usual (BAU) state, assuming that there are no economic effects on SMD.

There are three sections in the subsequent content of the paper. Section 2 reports materials and methods. Section 3 shows the main results of the analysis. Conclusions are presented in Section 4.

#### **2. Materials and Methods**

#### *2.1. Survey Implementation and Data Collection*

In order to collect CV data, the method of deriving the WTP from respondents; the payment vehicle, unit, and period; the method of survey; and the sample size had to be determined [17]. First, out of four methods of open-ended questions, bidding game questions, payment card questions, and dichotomous choice (DC) questions, which have been used in the literature as methods of eliciting WTP, this study adopted the DC question method. This was because the DC question method has been most frequently employed in the literature and possesses various merits, such as incentive compatibleness and mitigation of the respondents' cognitive burden [18]. In addition, Korea Development Institute [19] and Arrow et al. [20] present methodological guidance to be followed in applied CV research. For example, the survey correctly explained to respondents that there exist substitutes for the good, that the respondents' income is limited, and that consumption of other goods should be reduced to pay the WTP they have reported. In addition, this survey evaluated one of the many projects that the government should undertake. As will be explained below, this study tried to follow most of these guidelines.

Six important points had to be determined in order to conduct an actual field survey with a well-made CV questionnaire. First, the method of survey should be determined. This study adopted a person-to-person interview, which can facilitate the delivery of information rather than utilizing a relatively low-cost telephone, mail, or Internet method. In addition, the survey was conducted by experienced interviewers belonging to a professional opinion research institute.

Second, the size of the sample had to be determined. In this study, the population was all households in South Korea, and the population size is 19,971,359. The appropriate sample size had to be determined from this, with a 95% confidence level usually considered. In addition, a sample error of 5% is widely applied in South Korea, but a sample error of 3.1% was adopted in this study for more rigidity. The appropriate size of the sample was thus derived as approximately 1000, and 1000 observations were collected for the final analysis. Although a larger sample is better, it is important to size the sample at an appropriate level because the cost of the survey increases accordingly. In this regard, the size of the sample was set at 1000 following the suggestion of Korea Development Institute [19] and Arrow et al. [20]. In particular, since the costliest method of a person-to-person individual interview was conducted in this study, the sample size of 1000 was considered large enough and appropriate.

Third, the unit of the survey had to be determined. In this study, households were chosen out of individuals and households. This was because Korea Development Institute [19] proposed the use of households as a unit of the CV survey. In addition, conducting surveys of individuals may cause an issue as to what to do with the population when expanding the sample value to the population value. In other words, whether to include people under 20 or over 65 years of age who may lack economic ability can have a significant impact on the analysis results. On the other hand, conducting a survey of households is free of this issue. To improve the reliability of the data, the participants from households were limited to the household owner and his/her spouse, who have the actual burden of tax payment.

Fourth, the payment period must be determined. Naturally, the longer the payment period, the larger the total WTP, and the shorter the payment period, the smaller the total WTP. Therefore, it is important to reasonably determine the payment period. In this survey, payment was due for the next 10 years. This was because this period has been used in most applied CV works conducted in South Korea.

Fifth, the payment vehicle had to be fixed. The payment vehicle was decided as the yearly household income tax. Income tax is the most widely applied payment vehicle in empirical CV research for South Korea as it has the advantage of being relatively familiar to interviewees and not tied to everyday spending. In addition, Korea Development Institute [19] suggests as a guideline for applied CV studies that yearly household income tax should be used as a means of payment.

Sixth, the method of eliciting WTP responses had to be determined. Instead of the direct open-ended question method, the close-ended question method most widely applied in the literature was adopted. Of several close-ended questions, a DC question asking if an interviewee is willing to pay a specific amount was employed. The main part of the survey questionnaire in this study is given in Appendix A.

Various versions of the DC question are actually found in the literature. This study tried to apply a one-and-one-half-bounded (1.5B) model. Cooper et al. [21] proposed the model, which has several advantages [22–26]. The procedure of applying the model can be explained in the following manner. First, two bid amounts, *D<sup>L</sup>* and *D<sup>H</sup>* (*D<sup>L</sup>* < *DH*), should be determined through preliminary investigation. Half of all respondents were asked to agree on payment after presenting the smaller (*D<sup>L</sup>* ) of the two amounts first. If "yes" was responded, the higher bid (*DH*) was presented and an additional question about its payment was asked. If "no" was stated to *D<sup>L</sup>* , an additional question was not needed. The remaining respondents were given a higher amount (*DH*) first. If "yes" was reported, an additional question was not asked. However, if "no" was answered, the lower amount (*D<sup>L</sup>* ) was presented to the respondent. Thus, six responses were possible: "no," "yes-no," "yes-yes," "no-no," "no-yes," and "yes."

This study sought to take a closer look at the cases of "no" and "no-no" responses among these. Responses that indicated no intention of paying a lower amount (*D<sup>L</sup>* ) were further classified into zero WTP and WTP greater than zero and less than a lower amount (*D<sup>L</sup>* ). Therefore, a further question was asked to identify to which of the two classifications the "no" and "no-no" responses belonged. To this

end, a further question was presented about whether the interviewee had no intention of paying a dime, i.e., to check for a zero WTP. The final number of cases was, therefore, eight:

$$\begin{cases} \begin{aligned} I\_q^{YY} &= \mathcal{K}\{\mathcal{D}\_q^H < \mathcal{E}\_q\} = \mathcal{K}(\text{qh answer is "yes-yes"})\\ I\_q^{YY} &= \mathcal{K}\{\mathcal{D}\_q^L < \mathcal{E}\_q \le \mathcal{D}\_q^H\} = \mathcal{K}(\text{qh answer is "yes-no"})\\ I\_q^{LY} &= \mathcal{K}\{\mathcal{E}\_q \le \mathcal{D}\_q^L\} = \mathcal{K}(\text{qh answer is "no-yes"})\\ I\_q^{YN} &= \mathcal{K}\{\mathcal{E}\_q = 0\} = \mathcal{K}(\text{qh answer is "no-no"})\\ I\_q^Y &= \mathcal{K}(\mathcal{D}\_q^H < \mathcal{E}\_q) = \mathcal{K}(\text{qh answer is "yes"})\\ I\_q^{NY} &= \mathcal{K}\{\mathcal{D}\_q^L < \mathcal{E}\_q \le \mathcal{D}\_q^H\} = \mathcal{K}(\text{qh answer is "no-yes"})\\ I\_q^{NYY} &= \mathcal{K}\{\mathcal{E}\_q \le \mathcal{D}\_q^L\} = \mathcal{K}(\text{qh answer is "no-no-yes"})\\ I\_q^{NNN} &= \mathcal{K}\{\mathcal{E}\_q = 0\} = \mathcal{K}(\text{qh answer is "no-no-no"})\end{aligned} \tag{1}$$

where *I* and *J* are binary variables with zero or one, *q* indicates *q*th interviewee, and *K*(·) is an indicator function. If the proposition in parenthesis is true, the function has a value of one. Otherwise, the function has a value of zero.

The WTP data obtained from a CV survey conducted on 1000 households during July 2019 is summarized in Table 1.


**Table 1.** Willingness-to-pay data obtained and used in this study.

Notes: <sup>a</sup> Unit is Korean won (USD 1.0 = KRW 1122.8 at the time of the survey). <sup>b</sup> Numbers reported in parentheses mean the percentage of the number of observations.

#### *2.2. Method: CV*

The CV method has various advantages and disadvantages. Three advantages are as follows. First, unlike revealed preference approaches, such as the hedonic price technique and the travel cost technique, CV is a stated preference technique and can be used to estimate the economic value that explicitly includes non-use value. Second, from an economic point of view, the CV technique can theoretically provide an accurate estimate of the economic value or benefits from the supply of a certain good, while the revealed preference techniques have room for underestimation or overestimation. Third, since the validity and reliability of the CV approach is proven to some extent in the literature [22–26], the CV approach has been a widely applied one.

The CV method also has three disadvantages. First, the application of CV is more costly than that of other economic techniques because a survey of many respondents is essentially needed. For researchers facing budget constraints, the application of CV may be restrictive. Second, a valuation through CV based on the data collected using the questionnaire can be subject to various biases, as it can be influenced by the content of the questionnaire, the attitude of the interviewer, and the operation of the survey method. Third, since CV techniques are based on stated responses gathered from people instead of human behavior, people are less likely to believe in the value obtained by using CV.

The DC question method has several merits and demerits. There are two typical merits to the DC method. First, it is quite familiar to respondents. Even if a person has not experienced a referendum, the type of question is similar to deciding whether to buy a good on the market. Therefore, people can answer DC questions without much difficulty. Second, it is incentive compatible for people to respond. People buy a certain good if their utility from the purchase and consumption of it is greater than or equal to the price of the good; they do not otherwise buy the good. If a person's WTP is greater than the presented bid, she/he will answer "yes" and otherwise "no." There is no reason to take a strategic behavior when a person is faced with the DC question.

The DC method has two demerits. First, the use of the DC question results in discrete interval data rather than continuous point data. This makes an econometric analysis of the CV data less statistically efficient than other value elicitation methods such as open-ended questions. Thus, the use of the DC method requires the collection of a large number of observations and demands a large survey cost. Second, a pretest survey is required to determine a list of bid amounts to be presented to the respondents, resulting in costs associated with the pretest survey and longer application period than other methods.

Reducing SMD is a typical nonmarket good. A nonmarket good means that it cannot be traded in the usual market. A good traded in the market is easily valued, but a nonmarket good does not have a market and its value is not well observed [27]. Therefore, for the purpose of assessing the economic benefits ensuing from the reduction, it is necessary to create a hypothetical market for the reduction and hypothetically trade the reduction in the market. A CV method is a typical economic method that can be done in this way, and it has been widely utilized in the literature [28–34]. The CV technique uses a questionnaire to explain the good to be assessed to the randomly chosen potential consumers and then to make a hypothetical transaction, leading them to reveal their WTP for consuming the good [35–39]. Next, the researcher estimates the WTP model and calculates the average WTP by applying an econometric model to the WTP data collected from a survey.

Therefore, the first thing a researcher should do for the application of CV is to carefully make the questionnaire. CV questionnaires usually have three components. The first component addresses questions about potential consumer perceptions and experiences toward the good being assessed. The second component presents an explanation of the good to be evaluated and a question about the WTP. Questions about the individual characteristics of the consumers are shown in the third component.

The most important part of the CV questionnaire is the explanation of the good under investigation. The good should be identical for all respondents and should be accurately described in the questionnaire. The BAU state, which can be a reference for valuation, and the target state to be assessed should be clearly described. In other words, the object of valuation in CV is the amount of the WTP to obtain a change from the BAU state to the target state. Moreover, the policy measures associated with how to get the change should be fully explained. The policy measures presented in the CV survey were the establishment of a scientific forecasting system for early prediction of inflows and travel paths of marine debris, and research and development on how to collect and treat the SMD.

#### *2.3. Modeling the CV Data*

As mentioned earlier, this study attempted to explicitly deal with zero WTP in analyzing the DC CV data. To this end, the spike model given in Kriström [40], Habb and McConell [41] and Yoo and Kwak [42] was applied. Hanemann's [43] approach to modeling DC CV data was also used. Therefore, the CV data model used in this study was the 1.5B DC spike model. The 1.5B DC spike model can explicitly reflect zero WTP as well as positive WTP responses. The mean WTP estimate obtained from analyzing the 1.5B DC spike model using a total of 1000 observations was considered reasonable in its use for information about the benefits ensuing from the reduction. For the application of this model, the cumulative distribution function (cdf) of WTP had to first be defined. This study adopted a logistic function that is almost always applied in the spike model. Thus, the cdf, *ME*(·), can be specified as:

$$M\_E(\mathbb{E}; \tau\_0, \tau\_1) = \begin{cases} \left[1 + \exp\left(\tau\_0 - \tau\_1 E\right)\right]^{-1} & \text{if } E \ge 0\\ 0 & \text{if } E < 0 \end{cases} \tag{2}$$

where τ<sup>0</sup> and τ<sup>1</sup> are parameters of *ME*(·). If *E* = 0, the equation in the first line on the right side becomes the spike. Thus, the spike means Pr(*E* = 0). *E* is a bid presented to respondents and Pr(·) means a probability.

Concerning the model, the log-likelihood function can be specified as:

$$\begin{array}{ll} \ln L = & \sum\_{q=1}^{1000} \left[ \left( I\_q^{YY} + I\_q^Y \right) \ln \left[ 1 - M\_E \left( E\_q^H; \tau\_0, \tau\_1 \right) \right] \right] \\ & + \left( I\_q^{YY} + I\_q^{YY} \right) \ln \left[ M\_E \left( E\_q^H; \tau\_0, \tau\_1 \right) - M\_E \left( E\_q^L; \tau\_0, \tau\_1 \right) \right] \\ & + \left( I\_q^{YY} + I\_q^{NNY} \right) \ln \left[ M\_E \left( E\_q^L; \tau\_0, \tau\_1 \right) - M\_E \left( 0; \tau\_0, \tau\_1 \right) \right] \\ & + \left( I\_q^{NN} + I\_q^{NNN} \right) \ln M\_E \left( 0; \tau\_0, \tau\_1 \right) \end{array} \tag{3}$$

Maximum likelihood (ML) estimation method relates to obtaining parameter estimates that maximize the log-likelihood function. This study employed the ML estimation method. Thus, the estimates for τ<sup>0</sup> and were obtained by maximizing Equation (3). In addition, the mean WTP was derived from Equation (2) as [44,45]:

$$\left[ (1/\tau\_1) \ln[1 + \exp(\tau\_0)] \right] \tag{4}$$

#### **3. Results and Discussion**

#### *3.1. Estimation Results*

The results obtained through an application of maximum likelihood estimation to the model, Equation (3), are given in Table 2. The dependent variable is the probability of responding "yes" to a suggested bid. As the value of the bid becomes greater, the probability should be reduced. The coefficient estimate for bid amount is negative and statistically significant at the 1% level. This is quite reasonable. Given that the sample proportion of "no-no" and "no-no-no" responses, that is, zero WTP was 37.9%, the estimated spike of 0.3859 implies that the data were well represented by the spike model. Moreover, the spike secures statistical significance at the 1% level.

**Table 2.** Estimation results of the model and the mean willingness to pay (WTP).


Notes: <sup>a</sup> The unit is 1000 Korean won (USD 1.0 = 1122.8 at the time of the survey). <sup>b</sup> CI indicates confidence interval. <sup>c</sup> The null hypothesis is that all the parameter estimates are jointly zero. The # means statistical significance at the 1% level.

The Wald test can be employed for the specification test of the model. The null hypothesis is that the estimated coefficients for bid amount as well as constant terms are not distinguishable from zero. In other words, the hypothesis implies the meaninglessness of the model. The statistic was 656.28. Since this value is sufficiently large, the hypothesis could be rejected without deficiency. In addition, the *p*-value for the statistic was 0.000. Thus, the model possessed statistical significance.

The yearly household average WTP for the reduction was obtained as KRW 5523 (USD 4.92). Uncertainties can be involved in the estimation of the average. In such cases, it may be a good idea to

report confidence intervals together rather than just point estimate. To this end, this study adopted a parametric estimation technique developed by Krinsky and Robb [46] to present a confidence interval for the average. This method assumed that the estimates of the constant term and coefficient for the bid amount, given in Table 2, followed a bivariate normal distribution and produced an empirical distribution of the mean WTP by extracting the coefficients from this distribution and calculating the mean WTP 5000 times. Cutting the appropriate proportion from the left and right sides of this empirical distribution can find 95% and 99% confidence intervals. In other words, to find 95% and 99% confidence intervals, 2.5% and 0.5% are cut from the left and right sides of the distribution, respectively. Table 2 also reports them.

The results presented in Table 2 do not contain other covariates related to the interviewee's characteristics. However, other factors could influence the likelihood of reporting "yes" to an offered bid. For instance, some variables concerning the interviewee, such as gender, household income, and education level, can be introduced. A model including some covariates can be considered for investigating the possible effects of such variables. For this purpose, four variables were reflected in the model with covariates. Basic information about the covariates is described in Table 3.


**Table 3.** Information about some variables considered in this study.

Table 4 shows the estimation results of the model containing the variables described in Table 3. The Wald statistic for the specification test of the model was 607.79. This indicates that the null hypothesis of the model's being meaningless was rejected, considering that its *p*-value became 0.000. One of the purposes of estimating the model with covariates is to check for internal consistency or theoretical validity. Except for the estimated coefficient for Head variable, all the coefficient estimates were statistically significant. Thus, it seems that the model employed in this paper secured internal consistency. As explained above, the sign of the coefficient means the direction of the effect of the variable on the likelihood of responding "yes" to a provided bid. The coefficient estimates for Education, Age, and Income variables had statistical significance. Respondents with higher levels of education had a higher possibility than respondents with lower levels of education. The age of the respondents was negatively correlated with the possibility. An interviewee with a higher income was more likely to answer "yes" to a proposed bid than an interviewee with a lower income. Contrary to our prior expectations, the estimates of the coefficient for the Head variable was not statistically significant at a level of 10%. Whether or not the respondent was the head of the household did not affect the respondent's determination of WTP for the reduction. This was quite an interesting finding because it is often thought in the country that this variable will affect the respondent's decision on WTP.

The important purpose of estimating a model containing covariates is to verify the theoretical validity or internal consistency of the model. The model used in this study appeared to be meeting these since the estimation results were overall significant. Table 4 also presents the yearly household average WTP estimate and its confidence intervals. The mean WTP was KRW 5412 (USD 4.82), which is not much different from the results given in Table 2 (KRW 5523 or USD 4.92). In addition, the confidence intervals were much the same as those given in Table 2.


**Table 4.** Estimation results of the model containing the variables described in Table 3.

Notes: <sup>a</sup> The unit is 1000 Korean won (USD 1.0 = 1122.8 at the time of the survey). <sup>b</sup> CI indicates confidence interval. <sup>c</sup> The null hypothesis is that all the parameter estimates are jointly zero. The \* and \*\* indicate statistical significance at the 10% and 5% levels, respectively.

#### *3.2. Discussion of the Results*

This study investigated people's WTP for reducing SMD in South Korea by 33% by 2023 by means of expanding SMD collection. It is possible to compare three sample characteristics with three population characteristics given in Statistics Korea [47]. First, the sample proportion of female persons can be compared with the population proportion of female persons. The first (50.0%) is not different from the second (49.9%). Second, three areas with a large number of households can be investigated. The sample proportions of Gyeonggi, Seoul, and Busan respondents were 23.9%, 20.1%, and 7.2%, while the population proportions of Gyeonggi, Seoul, and Busan respondents were 23.7%, 19.4%, and 7.0%, making no significant difference. Third, the average monthly income of households can be examined. The sample value was KRW 4.86 million (USD 4136) and the population value was KRW 4.92 million (USD 4187), almost the same. The authors also think that the population could be reasonably represented by the sample because sampling was entrusted to a specialized survey institute. Therefore, extending the results for the previously presented sample to the population would not be a problem.

One of the most important purposes of the applied CV study was to expand the location value of WTP obtained from the sample to the population. In this regard, we should have determined whether to use the location values for the sample. Usually, mean, median, and mode are used for location value. The median WTP obtained in this study was zero and the mean WTP was estimated to be positive. The mode WTP could not be computed because we used DC WTP question. It was necessary to determine which, of mean or median, to use. Median is known to be all the more robust than mean because mean is vulnerable to outliers but median is not. Median can be useful for identifying the central tendency of the sample, but it is not used for expanding a sample value to a population value because it can cause underestimation to overestimation in the expansion. Therefore, the mean WTP has been almost always employed in the literature to estimate population value using sample value.

As explained earlier, we conducted stratified random sampling using 16 strata. The sample size allocated to each stratum was decided based on the Census implemented by Statistics Korea in 2015. The sample size of each stratum was, thus, consistent with the population. In this study, the total value was calculated by multiplying the mean WTP by the number of households in the population instead of using a mean formula applied for stratified sampling.

The average of the household's yearly WTP for the reduction was computed as KRW 5523 (USD 4.92). This sample value can be extended to the population. The population's total WTP was derived as the multiplication of the relevant number of households by the average WTP. Since the CV survey was conducted throughout the country, the relevant population became the entire country. There were 19,971,359 households when the survey was implemented [47]. The yearly population value would be KRW 110.30 billion (USD 99.75 million). Comparison of this value with the costs involved in the reduction is an interesting task.

An economic feasibility analysis of the reduction was tried as a final exercise. To this end, some prerequisites needed to be examined and determined. First, the period for the analysis had to be set. It was determined as five years, beginning from 2019 when the survey was implemented and when the reduction begins in 2023. Second, a social discount rate should be set. Concerning this, the government-run Korea Development Institute announced the suggested use of 4.5% as a social discount rate. This study adopted the value. Third, the time of "present" as a baseline for calculating the present value had to be set. In this study, this was set to be 2019, the time when the survey was conducted.

The next important information that was needed was benefits and costs arising from the reduction. As presented earlier, the economic benefits ensuing from the reduction would occur annually for 10 years, from 2019 to 2023. The costs largely relate to collection and treatment of SMD and were taken from "The Third Marine Debris Master Plan (2019–2023)" contained in Korea Ministry of Oceans and Fisheries [48]. The costs amounted to about KRW 6.69 billion (USD 0.60 million), KRW 10.20 billion (USD 0.91 million), KRW 11.22 billion (USD 1.00 million), KRW 12.35 billion (USD 1.10 million), and KRW 12.66 billion (USD 1.13 million) over the period 2019-2023, respectively. All benefits and costs are expressed in a 2019 constant price. The ratio of benefit over cost (B/C), which is one of the indicators for cost-benefit analysis (CBA), can be calculated from the constant values of benefits and costs. It is a simplification of the real CBA.

The present value of the benefits arising from the reduction was computed as KRW 1103.02 billion (USD 98.24 million) and that of the costs arising from the reduction became KRW 48.16 billion (USD 4.29 million). Thus, the net present value of the reduction became KRW 863.89 billion (USD 76.94 million), which is larger than zero and thus implies that the reduction passed the economic feasibility analysis. Furthermore, the ratio of benefit over cost was computed to be 18.93, which is larger than one, confirming the finding that the reduction secures economic feasibility. In conclusion, the reduction of SMD in South Korea is socially beneficial. Therefore, a continuous and stable reduction must be conducted.

#### **4. Conclusions**

South Korea is trying to reduce SMD by 33% by 2023 through expanding the collection of it. The implication from this study is all the more interesting since no research that has evaluated people's WTP for reducing SMD is found in the literature. Therefore, this study can be a useful contribution to the literature. In particular, the information about the value people place on the reduction is widely demanded to determine whether the reduction has sufficient public support. For the purpose of providing this information to policymakers, this article empirically looked into people's WTP for the reduction, employing data collected through a survey of 1000 households through person-to-person interviews during July 2019. In addition, the spike model was estimated not only using the entire sample data, including observations with zero WTP, but also only using observations with positive WTP. In this regard, the mean WTP estimate obtained for the sample can be extended over the population.

Judging from the comments of the supervisor and interviewers, the survey was implemented without difficulty and successfully enough to collect opinions representative of the population. Overall, people understood the CV questions well and reported the WTP responses in a hypothetical market successfully created with CV. Respondents stated a significant amount of WTP for the reduction. This study can provide three important policy implications. First, people's WTP for reducing SMD was quantitatively assessed. Although 37.9% of interviewees stated zero WTP, the average of the yearly household WTP was calculated to be KRW 5523 (USD 4.92), which is not big compared to the average household's annual income (KRW 58.9 million or USD 52.5 thousand). However, it has statistical

significance and can be utilized as a logical basis for the government to continue to push for reduction of SMD. In fact, the various CV empirical studies conducted in South Korea have often encountered too many zero WTP responses [22–24,26]. For example, Lim and Yoo [22], Kim et al. [23], Kim et al. [24], and Kim and Yoo [26] reported that the proportion of zero WTP responses was 56.5%, 46.5%, 61.7%, and 63.6%, respectively. The zero WTP response rate in this study is fairly small compared with the preceding studies. Therefore, it can be judged that the public is giving considerable value to the reduction.

Second, according to microeconomics, WTP means the economic benefits that arise from the reduction. The study evaluated the economic benefits that ensue from a 33% reduction in SMD by 2023 through an expansion of its collection and found that the population's total WTP was KRW 110.30 billion (USD 99.75 million).

Third, the economic benefits can be compared with the costs caused by the reduction. In this regard, a cost-benefit analysis of the reduction indicated that the reduction is socially beneficial and, therefore, the investment on the reduction can be economically justified. This is because the net present value was larger than zero. Moreover, the benefit/cost ratio was greater than one. Thus, the reduction of SMD should be stably and continuously performed.

As addressed above, the CV approach has some limitations due to using a survey of respondents. For example, in a hypothetical market created with CV, payments of a certain amount are not actually made but are made hypothetically. Therefore, it would be useful to conduct a test for the respondents' sincerity that adopts some statistical methods. One method is to include questions which gather ordinal data on a Likert-type scale in the CV questionnaire and then to compute a Cronbach's alpha to test for internal consistency. Unfortunately, this study did not contain the questions in the CV questionnaire. This point needs to be appropriately handled in future CV studies.

To the best of the authors' knowledge, there have not been many cases studies that applied CV to reducing marine debris, specifically SMD. Thus, one purpose of this study was to add a case study of South Korea to the literature. In particular, the implications of this study are all the more useful because there have been no related studies for the country. Nevertheless, this study needs to be improved in several respects to ensure that it is distinct from previous studies. First, if more observations are obtained through additional budgeting, the respondents can be segmented and the analysis could be made according to various criteria, such as geolocation of the respondent, whether the respondent had knowledge of campaigns around the issue prior to the survey, and relevance of the local issue, so as to obtain differentiated implications for each segmented group. Second, because the presented results are preliminary or partial, they can be supplemented by including enterprises in the survey. Since households consume products produced by businesses, there is a view that businesses are the ultimate polluting sources of the seas and oceans. Therefore, a follow-up study can be carried out by including enterprises as interviewees and using taxes paid by entrepreneurs and companies as the payment vehicle. In addition, future research should be conducted on companies that can respond to the polluters pay principle. Only then will this be helpful in establishing a more detailed policy on SMD.

**Author Contributions:** This paper was written through the collaboration of the three authors. S.-J.J. collected data, analyzed the collected data statistically, and compiled the results; Y.-J.K. compiled the data and wrote a significant portion of the paper; and S.-H.Y. proposed research ideas, secured the necessary budget for the survey, and supervised the entire process of the study. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was a part of the project titled A Study on the Integrated Management of Marine Space (PE99843), funded by the Korea Institute of Ocean Science and Technology (KIOST).

**Acknowledgments:** The authors are grateful to the three anonymous reviewers for their valuable comments and suggestions for improving this manuscript. This research was supported by the project titled A Study on the Integrated Management of Marine Space (PE99843), awarded by the Korea Institute of Ocean Science and Technology (KIOST).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Appendix A. Main Part of the Survey Questionnaire**

#### *Part 1. Questions about Socio-Economic Characteristics*

The interviewees were asked to respond to their socio-economic characteristics, such as the gender of the individual, the number of family members, the level of education, and the monthly income per household (before tax deduction). Questions about the number of family and income were open-ended questions, while the question about the level of education was as follows:

**Table A1.** Please check with √ your education level in years.


*Part 2. Questions about Willingness to Pay for Reducing the Submerged Marine Debris (SMD) in South Korea*

Type A. Q1. Is your household willing to pay additional income tax of 1000 Korean won (lower bid amount) annually for the next 10 years for reducing SMD in South Korea, supposing that the protection is certain to succeed?


Type A. Q2. Is your household willing to pay additional income tax of about 3000 Korean won (upper bid amount) annually for the next 10 years for reducing SMD in South Korea, supposing that the protection is certain to succeed?


Type B. Q1. Is your household willing to pay additional income tax of about 3000 Korean won (upper bid amount) annually for the next 10 years for reducing SMD in South Korea, supposing that the protection is certain to succeed?


Type B. Q2. Is your household willing to pay additional income tax of about 1000 Korean won (lower bid amount) annually for the next 10 years for reducing SMD in South Korea, supposing that the protection is certain to succeed?


Q3. Then, is your household not willing to pay anything for reducing SMD in South Korea?


#### **References**


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*Article*
