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Ikeda, Shiro, Kostova, Irena, Sekine, Hideaki, Sekine, Yoshika, 2016, Effect of Coal Fly Ash Leachate on the Bioluminescence Intensity of Vibrio fischeri. Coal Combustion and Gasification Products 8, 60-67, doi: 10.4177/CCGP-D-16-00001.1

Effect of Coal Fly Ash Leachate on the Bioluminescence Intensity of Vibrio fischeri

Shiro Ikeda1, Irena Kostova2,*, Hideaki Sekine3, Yoshika Sekine1,3

1Graduate School of Earth and Environmental Sciences, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan2Department of Geology, Paleontology, and Fossil Fuels, Faculty of Geology and Geography, Sofia University “St. Kl. Ohridski,” 15 Tzar Osvoboditel Boulevard, 1000Sofia, Bulgaria

3Graduate School of Science, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan

A B S T R A C T

Coal fly ash is a residue of coal-fired thermoelectric power plants (TPPs) and is mostly dumped in ash ponds or landfill sites, eventhough it potentially contains significant amounts of water-soluble hazardous contaminants. Bioassay using the bioluminescentbacterium Vibrio fischeri is known to be applicable for assessing the short-term and sublethal toxicity of complex mixtureswithout the need for precise chemical characterization. However, this type of bioassay is potentially adversely influenced by thepH-induced protein denaturation of cells. Because coal fly ash leachates often have alkaline or acidic properties, when applyingthe V. fischeri–based bioassay to the samples, we need to know potential effect of the leachates on the bioluminescence of thebacteria. This study accordingly aimed to investigate the feasibility of applying the V. fischeri bioassay to coal fly ash leachateas a screening method. Fly ash samples were collected from 12 TPPs located in three East European countries: Bulgaria, Greece,and Serbia. The fly ash samples were prepared in sterilized distilled water by ultrasonic extraction and filtration using 0.45-mmΦmembrane filters. The filtrates were then mixed with a solution of the test bacterium. The bioluminescence intensity wasmeasured using a luminometer. The results showed the ostensible influence of pH on bioluminescence intensity pronouncedwhen following the typical protocol using a 5.0‐g/L solid:liquid ratio. Accordingly, the pH of water extracts should be adjustedto within a range of 6 to 9 by dilution to observe the inhibition of bioluminescence by coal fly ash leachate as the objective endpoint.

– 2016 The University of Kentucky Center for Applied Energy Research and the American Coal Ash AssociationAll rights reserved.

A R T I C L E I N F O

Article history: Received 31 March 2016; Received in revised form 29 August 2016; Accepted 29 August 2016

Keywords: coal fly ash; bioassay; Vibrio fischeri; bioluminescence intensity; thermoelectric power plant

1. Introduction1. Introduction

Coal combustion supplies 48% of the electricity generated in Bul-garia and 25% of that in Japan. In Bulgaria, the combustion of 35.2Mt of coal produces approximately 10.4 Mt of solid combustionwastes annually (U.S. Department of Energy, Energy InformationAdministration, 2004). However, if not properly controlled, the efflu-ents produced can potentially cause serious environmental andhuman health problems. The Bulgarian thermoelectric power plants(TPPs) are among the largest sources of atmospheric pollution in Eur-ope, emitting sulfur dioxide (SO2) and other harmful gaseous and

particulate matters. These emissions lead to serious environmentalpollution not only of air, but also of waters, soils, and plants. Expo-sure to such chemicals through the environmental medium and foodsposes adverse health risks.Coal fly ash, which is collected by dust collectors, is a by-product

of coal-fired electric power plants and is mostly dumped in ash pondsor landfill sites, even though it potentially contains significantamounts of toxic elements (Hjelmar, 1996; Mester et al., 1999). Oneof the environmental concerns regarding these wastes is the magni-tude of the involved products. There are many reports on means toreduce the amount of fly ash waste that must necessarily be disposedof, including use as construction materials (taking advantage of itscementitious or binding characteristics) (Van der Sloot and De Groot,* Corresponding author. E-mail: irenko@gea.uni-sofia.bg

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doi: 10.4177/CCGP-D-16-00001.1– 2016 The University of Kentucky Center for Applied Energy Research and the American Coal Ash Association. All rights reserved.

1986; Singh et al., 1993; Chi et al., 2015) or soil amendment(Ram et al., 2006; Karmakar et al., 2010). Different alternatives arecurrently under consideration for a recycling method for coal flyash as an aluminum resource, which has substantial economicand environmental value (Li et al., 2014; Shemi et al., 2015; Zhanget al., 2015). Utilization of coal fly ash as a collection, a fixation,or both types of regent for accidental environmental contaminantsis also a novel aspect, e.g., in the use in the solidification ofliquid radioactive waste (Osmanlioglu, 2014; Roy et al., 2015) andspilled oil (Shishkin et al., 2014).However, fly ash contains elevated soluble major and trace con-

taminants that could adversely affect plants and soil (Georgakopouloset al., 2002); thus, rain and other sources of water (wet dumpingprevents the fly ash from becoming airborne) (Chakraborty andMukherjee, 2009) provide pathways for potentially toxic trace con-taminants to enter ecosystems. There are a number of reports onsoil pollution (Love et al., 2013) or ground-water contamination(Rai and Szelmeczka, 1990) caused by coal fly ash. To examine thehazardous nature of such fly ash, a freshwater fish (Ali et al., 2004,2007) Daphnia magna (Tsiridis et al., 2012), strains of the bacteriumSalmonella Typhimurium (Ames test), and earthworms (Manerikaret al., 2008) are used as bioassay test organisms. According to Spearsand Lee (2004), approximately 70% of coal fly ash produces a neutralor alkaline leachate. Alkaline fly ash samples derived from subbitu-minous Montana coal ranged from pH 10.5 to 11.5 when mixedwith distilled water, whereas a pH value of 4.1 was recorded for a dis-tilled water leachate of fly ash resulting from the combustion of bitu-minous coal in Illinois (Talbot at al., 1978).Bioassays are known to provide a means for assessing the toxicity

of complex mixtures without the need for precise chemical charac-terization. The marine luminescent bacterium Vibrio fischeri isone of the test organisms widely used for bioassay. V. fischeri ordi-narily emits light blue luminescence (λ 5 490 nm) caused by tran-scription induced by quorum sensing (Fuqua et al., 1994).However, the intensity of bioluminescence produced by V. fischeridecreases rapidly if the bacteria are exposed to toxic componentsbecause of the inhibition of quorum sensing (Miyamoto et al.,2000). The suppression of bioluminescence intensity caused by theinhibition of quorum sensing can be used an index of short-termsublethal toxicity. Kaiser demonstrated the significant relationshipbetween luminescent bacterial test data and acute toxicity data formany aquatic and land species, including fishes (Kaiser, 1998).The Water Framework Directive established by the European Unionin 2000 requires an integrated approach for assessing water qualityin a river basin, so the V. fischeri-based bioassay has been appliedextensively for the monitoring or control of river water quality,particularly in Europe (Reineke et al., 2002; Zhang et al., 2012;Serpa et al., 2014). There are also a number of reports of its applica-tion in other aqueous environments, including industrial wastewater (Reemtsma et al., 1999; Wang et al., 2002; Eilersen et al., 2004;Dries at al., 2014; Carbajo et al., 2015). However, use of this type ofbioassay is potentially hindered by the ostensible positive influenceof pH-induced protein denaturation in cells, which causes a reduc-tion in bioluminescence under test conditions. Because coal fly ashleachates are often alkaline or acidic in nature, we need to knowthe potential effect of leachates on the bioluminescence of bacteriawhen applying the V. fischeri–based bioassay to samples.This study accordingly aimed to investigate the feasibility of

applying the widely used V. fischeri bioassay to coal fly ash leachateas a screening method, using fly ash samples collected at 12 TPPs

located in three East European countries (Bulgaria, Serbia, andGreece). The test was conducted by using the commercially availableROTASTM V. fischeri bioassay kit (Rapid Onsite Toxicity Audit Sys-tem, Cybersense Biosystems, Abingdon, U.K.). Water extracts ofcoal fly ash samples were initially tested with the kit following thetypical protocol using a 5.0‐g/L solid:liquid ratio, and the effects ofthe resulting changes in pH on the bioluminescence were investigat-ed. The major ionic components in leachates were also analyzed tocharacterize each fly ash sample and to infer the inhibitory or stimu-lating activity of the coal fly ash leachates.

2. Experimental2. Experimental

2.1. Sampling and preparation

The fly ash from 12 TPPs located in three East European countries(Bulgaria, Greece, and Serbia) was used as samples. The fly ash sampleswere collected from the electrostatic precipitators (ESPs) of sevenBulgarian TPPs (Republika, Bobov Dol, Dimitrovgrad, Varna, Ruse,Maritza East 2, and Maritza East 3), three Greek TPPs (Megalopolis,Agios Demetrios, and Meliti), and two Serbian TPPs (TENT A andTENT B). Information on the TPPs, source areas of coal, and coal rankare provided in Table 1. ESPs are particulate collection devices thattrap dust from flue gas by using the force of an induced electrostaticcharge (Figure 1). Each ESP is composed of one to five rows (first, sec-ond, third, fourth, and fifth rows). Most of the samples were collectedfrom individual rows of an ESP, although some were obtained as bulksamples (i.e., a mixture of fly ash collected from all the rows of anESP). All samples were stored in vinyl bags at room temperature untilanalyses.

2.2. Bioassay using the luminescent bacterium Vibrio fischeri

In this study, the V. fischeri–based bioassay system ROTASwas used for evaluation of bioluminescence inhibition. The packagesystem was originally developed by Cybersense Biosystems and dis-tributed by Hitachi Chemical (Tokyo, Japan) for the toxicity screen-ing of contaminated soils (Cybersense Biosystems, 2005).ROTAS has three extractionmodes: sterilized, distilled, and deionized

water (leachable); hydrochloric acid (metals), and methanol (organics).The leachable mode was applied in this study. Figure 2 shows a sche-matic view of the sample preparation and measurement of biolumines-cence intensity. Fly ash samples were extracted with 10 mL of sterilizeddistilled and deionized water by ultrasonic extraction for 15 min. Afterfiltration using 0.45-mmΦMilliporeHmembrane filters, 1 mL of filtratewas used as the sample solution. Rehydration medium (2.0% NaClaq,saline water for protection from osmotic damage) was added to afreeze-dried bacterial powder of V. fischeri, stored at 252 K, and thenallowed to stand for 50 min at 301¡1 K. This facilitated full reactiva-tion of bacterial luminescence. One milliliter of prepared sample solu-tion was then transferred to each well of a 24-well plate, and 1 mL ofthe bacterial solution was slowly added to the sample solution. The24-well plate was then placed into a luminometer (RT001, STL, Reading,U.K.), and time courses of luminescence intensity (L) were measured for15 min.Relative bioluminescence intensity (BLI) was calculated from the

measured L with time (t) using Eq. 1 to calibrate the initial biolumi-nescence intensity to 100%.

BLI~LtB0

L0 � Bt� 100ð%Þ (1)

Ikeda et al. / Coal Combustion and Gasification Products 8 (2016) 61

where L0 is the initial bioluminescence intensity (at t 5 0), Lt is thebioluminescence intensity (at t 5 t), B0 is the initial blank biolumi-nescence intensity, and Bt is the measured blank bioluminescenceintensity. To evaluate the effect of samples on bioluminescenceintensity, bioluminescence inhibition (INH) was calculated from theBLI (%) at 15 min using Eq. 2.

INH~100� BLI (2)

To normalize the different weights (W, mg) of each fly ash sample,we used inhibition per weight, INH/W (%/mg), which was calculatedusing Eq. 3 as an inhibitory index.

INH=W~100� BLI

W(3)

In this bioassay method, different lots of bacterial solutions wereused at each measurement. The reproducibility of each measurementis important for quality assurance and quality control. To determinereproducibility, three lots of bacterial solutions were separately appliedto the same water extracts of Republika fly ash collected from the first

row of the ESP. The measured coefficient of variation (CV) was 4.0%.Because the BLI of samples in the 24 wells were measured simulta-neously by each detector in the luminometer, the accuracy of eachdetector was evaluated by using 0.050 g of Republika fly ash. One mil-liliter of water extracts of fly ashwas added to each of the 24 wells con-taining 1 mL of bacterial solution; subsequently, the time course of BLIwas measured. The average value of BLI (%) at 15 min was 26.0¡0.39(mean¡ SD), whereas the CV was only 1.5% (n 5 24). These valuesindicated the potential applicability of this system to the present study.

2.3. Dose–response test for extracted fly ash samples

To determine the 50% effective concentration (EC50) (Tisler andZagorc-Koncan, 2002; Flladosa et al., 2005) of fly ash samples,a dose–response test was conducted using ROTAS (leachable model)for eight typical Bulgarian fly ash samples collected from the first, sec-ond, and third rows of Republika and Bobov Dol TPPs, and from thetwo bulk samples collected from Maritza East 2 and 3 TPPs. Fly ashsamples weighing 0.1 and 0.05 g were prepared separately. All sampleswere subsequently extracted with 10 mL of sterilized distilled water byultrasonic separation for 15 min. After filtration using 0.45-mmΦpore‐size membrane filters, each solution was diluted with sterilizeddistilled water to prepare a dilution series of 1 in 10 (1/10) to 1 in1000 (1/1000). Time courses of BLI were measured by a luminometer,and the BLI values at 15min were converted to INH values using Eq. 2.

2.4. Chemical analyses of water soluble components

Anions in the water extracts of fly ash samples were quantified byion chromatography (IC). The ion chromatograph system consists of aDionex ICS-90 system and DS5 detector (Dionex Corp., Sunnyvale,CA). The following conditions were used: separation column, 4 6250 mm, IonPack AS-9HC analytical (Dionex); guard column, Ion-Pack AG4 4 mm (Dionex); suppressor, AMMS III (Dionex); injectionvolume, 25 mL; eluent, 5 mM sodium hydrogen carbonate at1.0 mL/min; regen, 15mM sulfuric acid. To prepare a 1‐mg/L standardwater solution containing a mixture of seven anions (F−, CH3COO

−,HCOO−, Cl−, NO�

2 , NO�3 , SO

2�4 ), a 1000‐mg/L solution was initially

prepared from reagent-grade NaF, CH3COONa, HCOONa, NaCl,

Table 1Table 1Description of coal fly ash samples collected from electrostatic precipitators (ESPs) of thermoelectric power plants (TPPs) located in East European counties (Bulgaria,Serbia, and Greece) used in this study

Row position for sample collection

n TPP (abbreviation) Country Source area of coal Coal rank 1 2 3 4 5

1 Republika (RP) Bulgaria Pernik coal basin (Bulgaria) Subbituminous X X X2 Bobov Dol (BD) Bulgaria Bobov Dol, Pernik, Oranovo-Simitli,

Chukurovo, Stanjanci, Beli Breg,and Bourgas coal basins (Bulgaria)

Subbituminous Lignite X X X

3 Maritza 3 (Dimitrovgrad) (D) Bulgaria Maritza East coal basin (Bulgaria) Lignite X X X4 Varna (V) Bulgaria Donetski coal basin (Ukraine) Bituminous X X X X X5 Ruse (R) Bulgaria Kuznetzki coal basin (Russia) Bituminous X X X6 Maritza East 2 (ME2) Bulgaria Maritza East basin (Bulgaria) Lignite Bulk sample7 Maritza East 3 (ME3) Bulgaria Maritza East basin (Bulgaria) Lignite Bulk sample8 TENT A (TA) Serbia Kolubara coal basin (Serbia) Lignite Bulk sample9 TENT B (TB) Serbia Kolubara coal basin (Serbia) Lignite Bulk sample

10 Meliti (ML) Greece Ptolemais coal basin (Greece) Lignite X X11 Megalopolis, Unit 1 (M1) Greece Megalopolis coal basin (Greece) Lignite Bulk sample

Megalopolis, Unit 4 (M4) Greece Megalopolis coal basin (Greece) Lignite Bulk sample12 Agios Demetrios, Unit 2 (AD2) Greece Ptolemais coal basin (Greece) Lignite Bulk sample

Agios Demetrios, Unit 3 (AD3) Greece Ptolemais coal basin (Greece) Lignite Bulk sample

Fig. 1.Fig. 1. Schematic view of the electrostatic precipitators (ESPs) in a typicalthermoelectric power plant in East European countries. The filtration devicenormally has several rows to trap fine particles efficiently.

62 Ikeda et al. / Coal Combustion and Gasification Products 8 (2016)

NaNO2, NaNO3, and Na2SO4, respectively, by dissolution into ion-exchanged water. All chemicals were weighted after drying in anoven at 80uC. Finally, 1 mL of the standard solution was used forcalibration and determination.Water-soluble alkalis are normally present in fly ash (Dhadse

et al., 2008); hence, the concentrations of Na+, K+, Ca2+, and Mg2+

in the water extracts of fly ash samples were determined by polarizedZeeman atomic absorption spectrophotometry (AAS) using a HitachiZ-5300 spectrometer. Diluted series of reagent grade KCl, CaCl2,Mg(NO3)2, and NaCl in ion-exchanged water were used for calibra-tion and determination. The ionic contents in a unit mass (mg/g)of fly ash were calculated from the measured concentrations deter-mined by IC and AAS. The pH of extracts was also determined usinga pH meter (AS 211, AS ONE, Osaka, Japan).The limits of quantitation of chemical measurements by IC (anions)

and AAS (cations) of final solutions were as follows: F−, 0.10 mg/L(signal‐to‐noise ratio [S/N] 5 10); CH3COO

−, 0.093 mg/L (S/N 5 10);HCOO−, 0.072 mg/L (S/N 5 10); Cl−, 0.073 mg/L (10σ); NO�

2 , 0.19mg/L (S/N 5 10); NO�

3 , 0.077 mg/L (S/N 5 10); SO2�4 , 0.083 mg/L

(S/N 5 10); Na+, 0.017 mg/L (10σ); Ca2+, 0.052 mg/L (10σ); Mg2+,0.018 mg/L (10σ); and K+, 0.11 mg/L (10σ).

3. Results and Discussion3. Results and Discussion

3.1. Bioassay of fly ash with Vibrio fischeri

Figure 3 shows representative results for the time course ofBLI measurements based on the typical protocol of theV. fischeri bio-assay for soil samples. BLI values of the blank, which were derivedfrom tests of sterilized distilled water (the extraction solvent), werecalibrated to 100% using Eq. 1. Copper(II) sulfate at 40 mg/L stronglyreduced the BLI to less than 20% within 2 min and worked as a posi-tive control for the tests using this method. In the case of fly ash sam-ples, the following two types of result were observed: inhibition, e.g.,the BLI of 5.0 g/L for Republika (first row) was markedly reduced inonly 1 min; and BLI-activated, e.g., immediately after the beginningof measurement, a significant increase in the BLI of 5.0 g/L for Repub-lika (second row) was observed, and the BLI value reached 141% in

the first 2 min. The pH of the sample solutions was remarkably differ-ent: the pH values of first and second row samples were 10.5 and 8.9,respectively. The relationship between the pH and INH/W of all thetested samples with a 5.0‐g/L solid:liquid ratio is shown in Figure 4.According to the instructions of another commercially available V.fischeri test, Microtox Acute Toxicity Test (Azur Environmental,1998), a range of pH 6–8 is suggested to be suitable for applicationbecause higher acidity and basicity may cause bacterial protein dena-turation, potentially having an ostensible influence on biolumines-cence. Only seven of the coal fly ash samples examined in thepresent study plotted in the range of the bioassay, causing either inhi-bition or stimulation. However, samples causing activation were alsofound in the pH 8–9 range. The increase in the BLI of luminescentbacteria by the major cations contained in seawater (i.e., Na+, K+,Ca2+, and Mg2+) is well known (Bendt et al., 2001; Yang et al.,2015). In the present study, we found that INH/W tended to correlatewith the contents of cations (Mg2+: r 5 0.81; Ca2+: r 5 0.77;

Fig. 2.Fig. 2. Flow chart of the sample preparation by ultrasonic extraction with sterilized distilled water, and measurement of bioluminescence intensity using a luminometerbased on the leachable mode of the ROTAS Vibrio fischeri kit.

Fig. 3.Fig. 3. Typical time courses of bioluminescence intensity (BLI) in cases of theblank (sterilized distilled water), positive control (40 mg/L, CuSO4aq), and fly ashsamples (first and second rows of Republika [RP] thermoelectric power plant [TPP]).They showed both inhibition and activation toward the luminescent bacteria.

Ikeda et al. / Coal Combustion and Gasification Products 8 (2016) 63

Na+: r5 0.64). Therefore, the bioassay is applicable when the samplesolution of coal fly ash leachate ranges from pH 6 to 9.Figure 5 shows the dilution effect on INH of water leachates of

eight typical fly ash samples (the first, second, and third rows of

Republika, and the first, second, and third rows of Bobov Dol,Maritza East 2, and Maritza East 3 TPPs) at different dilution levels.The pH values of samples at each concentration level are alsoshown. Although both inhibition (first row of Republika TPP andall rows of Bobov Dol) and activation (second and third rows ofRepublika, Maritza East 2, and Maritza East 3) types were found,sigmoid curves were obtained for each sample. However, in twoeffluent samples (first of Republika), it was found that the inhibitionof bioluminescence was not negated, even when the samples werewell diluted to be in the range of pH 6–8. This can probably beattributed to the hazardous chemical contaminants contained inthe coal fly ash leachates, which adversely affect the quorum sens-ing of the bacteria.

3.2. Major ionic components in coal fly ash leachate

The concentrations of chemical components in water extractionsof fly ash samples are present in Figure 6. The content of chemicals(mg/g) was calculated from concentrations (mg/L) of the water-extracted solutions. Ca2+ was the most abundant among the sub-stances, and Mg2+ and SO2�

4 were relatively rich in the fly ashsample. These results are in accordance with the data reported byKumar et al. (2005). The authors reported that chemical compositionof fly ash is as follows: SiO2, 59.4%; Al2O3, 23.6%; Fe2O3, 6.1%;CaO, 1.9%; MgO, 0.97%; SO3, 0.76%; alkalis, 1.4%; unburned S

Fig. 4.Fig. 4. Relationship between the pH and inhibition per weight (INH/W) of all thetested samples with a 5.0‐g/L solid:liquid ratio.

Fig. 5.Fig. 5. Dose–response curve obtained from Vibrio fischeri bioassay for the samples from first, second, and third rows of Republika (RP) and the first, second, and third rowsof Bobov Dol (BD) and Maritza East 2 and 3 (ME2 and ME3). ○ 5 bioluminescence inhibition (INH); ◆ 5 pH values at each concentration level.

64 Ikeda et al. / Coal Combustion and Gasification Products 8 (2016)

and moisture, 3.7%. Among the above mentioned oxides, only CaO,MgO, SO3, and alkalis are water-soluble components. Although noclear relationships were found between the contents of Na+, K+,F−, Cl−, and NO�

3 and the quality of burned coals, the pH maydepend on the source area of coal, the coal rank, or both, as shownin Figure 7. The pH values of the water extracts of fly ash from bitu-minous Ukrainian coal used at Varna TPP in Bulgaria were 7.1 (firstrow), 7.1 (second row), 7.0 (third row), 7.3 (fourth row), and 6.9 (fifthrow)—close to neutral pH. Meanwhile, those from lignite Serbian

and Greek coals were in the range of pH 9.1–11.8. In addition to thebasicity mostly determined by the source area and coal rank, weshould address other trace contaminants to identify the potentialcause of inhibitory activity suggested in section 3.2 by furtherstudies.

3.3. Variation of bioluminescence inhibition property by rowposition of ESP

As shown in Figure 1, fly ash particles are trapped by an ESPbefore entering into the atmospheric environment. At Republika,Bobov Dol, Dimitrovgrad, Varna, and Ruse, the fly ash sampleswere separately collected from different rows, and the particle sizedistribution was assessed. The median diameters of the particles col-lected from Republika were 196 mm (first row), 28 mm (second row)and 15 mm (third row), respectively. Samples from Bobov Dolshowed similar sizes—213 mm in the first row, 140 mm in the second,and 121 mm in the third. This means that most of the coarse fly ashparticles were trapped by the first row of an ESP. Figure 8 showsthe INH of Republika and Bobov Dol at pH 8, derived from thedose–response relationships shown in Figure 5. This result suggeststhe coal fly ash collected from a closest row to a boiler is the mosteffective on the reduction of bioluminescence.

Fig. 6.Fig. 6. Major ionic components in the water extracts of fly ash samples analyzed by atomic absorbance spectrometry and ion chromatography. Samples of Republika (RP),Bobov Dol (BD), Maritza 3 Dimitrovgrad (D), Varna (V), and Ruse (R) thermoelectric power plants (TPPs) were collected from every row of the electrostatic precipitator (ESP).Samples of Meliti (ML), TENT (T), Maritza East (ME), Megalopolis (M), and Agios Demetrios (AD) TPPs were collected from two different units and mixed.

Fig. 7.Fig. 7. Comparison of the pH values of water extracts of all of the coal fly ashsamples. For explanation of abbreviations, see Table 1 and the legend to Figure 6.

Ikeda et al. / Coal Combustion and Gasification Products 8 (2016) 65

4. Conclusions4. Conclusions

In this study, we investigated the feasibility of applying theV. fischeri bioassay to coal fly ash leachate as a screening method.The water leachates of coal fly ashes collected from Bulgarian,Serbian, and Greek TPPs were tested based on marine bacterialbioluminescence. By applying the typical protocol originally devel-oped for soil samples, the ostensible positive influence of high pH onbioluminescence intensity was often observed. Consequently, waterextracts should be adjusted to within the pH 6–9 range by dilution toobserve the inhibitory effect of coal fly ash leachate on biolumines-cence as the objective endpoint. The results obtained in this studyclearly indicate that further research is necessary to identify thehazardous species contained in coal fly ash, and this will be anarea of focus in our future studies.

AcknowledgmentsAcknowledgments

The authors thank Mr. Kazuyoshi Kurihara and Dr. BakuMaekawa (Hitachi Chemical Co. Ltd.) for supplying ROTAS kitsand Mr. Masafumi Oikawa (Graduate School of Science, TokaiUniversity) for his great help in this study.

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