Ecotoxicology and Environmental Safety 108 (2014) 294–301

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Assessment of the chemical, microbiological and toxicological aspects of post-processing water from underground coal gasification Magdalena Pankiewicz-Sperka a,n, Krzysztof Stańczyk a, Grażyna A. Płaza b, Jolanta Kwaśniewska c, Grzegorz Nałęcz-Jawecki d a

Department of Energy Saving and Air Protection, Główny Instytut Górnictwa (Central Mining Institute), Plac Gwarków 1, 40-166 Katowice, Poland Department of Environmental Microbiology, Institute for Ecology of Industrial Areas, 6 Kossutha, 40-844 Katowice, Poland c Department of Plant Anatomy and Cytology, University of Silesia, 28 Jagiellońska, 40-032 Katowice, Poland d Department of Environmental Health Sciences, Medical University of Warsaw, 1 Banacha, 02-097 Warsaw, Poland b

art ic l e i nf o

a b s t r a c t

Article history: Received 10 October 2013 Received in revised form 26 June 2014 Accepted 27 June 2014

The purpose of this paper is to provide a comprehensive characterisation (including chemical, microbiological and toxicological parameters) of water after the underground coal gasification (UCG) process. This is the first report in which these parameters were analysed together to assess the environmental risk of the water generated during the simulation of the underground coal gasification (UCG) process performed by the Central Mining Institute (Poland). Chemical analysis of the water indicated many hazardous chemical compounds, including benzene, toluene, ethylbenzene, xylene, phenols and polycyclic aromatic hydrocarbons (PAHs). Additionally, large quantities of inorganic compounds from the coal and ashes produced during the volatilisation process were noted. Due to the presence of refractory and inhibitory compounds in the post-processing water samples, the microbiological and toxicological analyses revealed the high toxicity of the UCG post-processing water. Among the tested microorganisms, mesophilic, thermophilic, psychrophilic, spore-forming, anaerobic and S-oxidizing bacteria were identified. However, the number of detected microorganisms was very low. The psychrophilic bacteria dominated among tested bacteria. There were no fungi or Actinomycetes in any of the water samples. Preliminary study revealed that hydrocarbon-oxidizing bacteria were metabolically active in the water samples. The samples were very toxic to the biotests, with the TU50 reaching 262. None of biotests was the most sensitive to all samples. Cytotoxicity and genotoxicity testing of the water samples in Vicia uncovered strong cytotoxic and clastogenic effects. Furthermore, TUNEL indicated that all of the water samples caused sporadic DNA fragmentation in the nuclei of the roots. & 2014 Elsevier Inc. All rights reserved.

Keywords: Underground coal gasification (UCG) Post-processing water Toxicological assays Environmental risk

1. Introduction Underground coal gasification (UCG) is currently regarded as a promising alternative method of obtaining energy from coal (Chen et al., 2011; Eftekhari et al., 2012; Liu et al., 2011; Prabu and Jayanti, 2012; Stańczyk et al., 2012). Intensified research on the UCG process began in the 1930s in the former Soviet Union. Largescale studies were also carried out in the United States in the 1970s and 1980s (Shafirovich and Varma, 2009). In Europe, UCG

Abbreviations: UCG, underground coal gasification; PAHs, polycyclic aromatic hydrocarbons; TU, toxic units; BTEX, benzene, toluene, ethylbenzene, xylene; EC, effective concentration; BOD-5, five days biochemical oxygen demand; COD, chemical oxygen demand; EC20, threshold toxicity; EC50, median toxicity; LC, lethal dose n Corresponding author. Fax: þ48 32 259 22 67. E-mail address: [email protected] (M. Pankiewicz-Sperka). http://dx.doi.org/10.1016/j.ecoenv.2014.06.036 0147-6513/& 2014 Elsevier Inc. All rights reserved.

process research was conducted from 1982 to 1999 (Wiatowski et al., 2012). At present, wide research in the field of UCG processing continues in China and Australia (Kapusta and Stańczyk, 2011; Liu et al., 2007, 2009; Yang, 2007; Yang et al., 2008). In 2007, studies were resumed in Europe, through the HUGE Project (Hydrogen Oriented Underground Coal Gasification for Europe, 2007–2010), coordinated by the Central Mining Institute in Poland. The UCG process is based on the direct injection of a gasifying agent to the ignited coal seam followed by collection of a gas product at the surface (Kapusta and Stańczyk, 2011). Postprocessing water is generated both during the UCG process and after its completion. During the reactor operation time, postprocessing water mainly derives from condensates separated during gas collection and treatment. After the termination of the process, condensates are mostly separated from the warm and humid gas coming out of the post-reaction zone and mine water flowing into the post-reaction zone. During UCG, a number of

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heterogeneous and homogenous reactions take place (Kapusta and Stańczyk, 2011; Yang et al., 2008) that contaminate UCG postprocessing water with hazardous chemicals, such as organic aromatic compounds, including benzene, toluene, ethylbenzene, xylene, phenols and polycyclic aromatic hydrocarbons (PAHs). Additionally, large quantities of heavy metals can be released from coal tars and ashes produced in the volatilisation process, which is favoured by the high temperature of the process and the presence of numerous chemicals (Humenick and Fletcher Matox, 1978; Liu et al., 2006, 2007; Stuermer et al., 1982; Yang, 2009). UCG post-processing water resembles coke wastewater, in terms of their physico-chemical compositions (Kim et al., 2008), and both are subject to legal regulations related to their removal and treatment. Water Framework Directive 2000/60/EC, article 16 (Directive 2000/60/EC, 2000) contains a list of priority hazardous substances, some of which were identified in the UCG postprocessing water (benzene, naphthalene, PAHs and heavy metals). Although the problem of water pollution generated during UCG has been described, no full analysis of post-processing water has been performed, not only in terms of its physico-chemical characteristics but also in terms of the toxicological and microbiological characteristics. Microbiological and toxicological studies are very relevant for environmental risk assessment and developing effective methods to treat the contaminated water. The only attempt at toxicity analysis was described by DeGraeve (1980). The authors described bioassays which were used to determine the toxicity of the untreated condenser water from the Hanna-3 UCG experiment (Wyoming, USA) and its major toxic constituents (phenol, ammonia, and phenol and ammonia mixture) to fathead minnows (Pimephales promelas), rainbow trout (Salmo gairdneri) and Daphnia pulicaria (DeGraeve, 1980). Their study results indicated a very high toxicity of the samples that ranged from LC50 ¼0.1 to 0.18 percent, depending on the assay. Whole-sample toxicity programmes have been promoted since the 1970s in the USA. With the implementation of the Water Framework Directive, the use of biotests in Europe is expected to increase significantly (Wahdia and Thompson, 2007). There are no EU regulations on the application of the bioassays for monitoring effluents. However, in some countries, bioassays are utilised for effluent control (Direct Toxicity Assessment and Special Waste Regulations in UK; Wahdia and Thompson, 2007). Due to the complexity of effluents, a battery of bioassays should be applied for toxicity screening. Organisms from different taxonomic groups should be chosen for the battery. According to Pessala et al. (2004), the combination of tests should be selected individually for each sample type. Pollumaa et al. (2004) proposed bioassays with luminescent bacteria, protozoa, crustaceans and algae for evaluating the toxicity of oil-shale industry solid wastes polluted with heavy metals and hydrocarbons. In our project, a battery of six bioassays was applied, comprising bacterial, protozoan, crustacean and plant assays. No single bioassay was found to be most sensitive for all of the samples. Luminescent bacteria are very sensitive to simple organics with EC50 values in the range of 0.1 to 10 mg/L (Kaiser and Palabrica, 1991). However, the toxicity effects of nonpolar (e.g., hydrocarbons and their hydrophobic derivatives) and polar toxins (e.g., phenols), are additive and depend on the lipophilicity of the compounds. Further, Spirotox with Spirostomum ambiguum is much more sensitive than the bacteria to heavy metals (Nałęcz-Jawecki and Sawicki, 1998), but is less sensitive to organics. Of note, the effluents contain thousands of compounds and only a small fraction are identified chemically. Blum et al. (2011) suggested monitoring heterocyclic compounds, as they are typical constituents of coal tars, their aqueous solubilities are several times higher than those of PAH and BTEX, and their toxicity is high.

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Nakajima et al. (2013) monitored the toxicity of effluents obtained from hydrothermally treated coals. Their results suggested that the increased toxicity in Daphnia magna assays was caused by lower molecular weight compounds (phenols) formed during the treatment. Borrely et al. (2004) applied biotests with daphnia and luminescent bacteria to study the detoxification of effluents with an electron beam accelerator and found that in some cases the toxicity increased due to the formation of hydrogen peroxide. The aim of this study was to carry out a comprehensive characterisation of post-processing water samples, which were collected during a UCG experiment performed by the Central Mining Institute in Poland. The assays included chemical, microbiological and toxicological analyses.

2. Materials and methods 2.1. Design of the UCG experiment The experiment was carried out on a UCG pilot plant. The experimental installation enables simulation of the UCG process in ex situ conditions. Black coal from the Wieczorek coal mine was used. Selected parameters of used coal are shown in Table S1. The UCG process experiment was conducted for a period of 96 h. During the first 48 h of the process, oxygen was used as a gasifying agent. Oxygen flow to the reaction zone was constant and was 4 m3/h. Between 48 and 82 h after the initiation of the process, a significant deterioration occurred in the quality of the produced gas (stream flow ratio 6 m3/h). Due to this deterioration, after 82 h, the process was carried out with air enriched by oxygen (oxygen content 44 vol%). A simplified scheme of the experiment installation is presented in Fig. S1.

2.2. Post-processing water sampling The post-processing water samples were collected at three steps of the UCG simulation experiment, e.g., after 12 h (Sample I), 48 h (Sample II) and 72 h (Sample III). The purpose of this sampling procedure was to determine if the relevant phase of the process has a significant influence on the wastewater composition. For the microbiological analysis, the water samples were collected in sterilised 1-L polyethylene flasks. The samples were transported to the laboratory for chemical, toxicological and microbiological assays, which were performed immediately.

2.3. Chemical analyses Chemical analyses of the water samples were conducted in the Laboratory of Water and Sewage Analysis of Główny Instytut Górnictwa (Central Mining Institute). The samples were filtered to remove coal tars and other undissolved residues. The inorganic contaminants (such an ammonia, nitrogen, free and bound cyanides, sulphates, B, Cr, Ti, Pb, Fe, Cd, Cu) and organic pollutants (phenols, BTEX and PAHs) were measured. The chemical analyses were carried out according to standard analytical methods. To determine pH and conductivity, potentiometry methods were used according to PN-90/C-04540.01 and PN-EN 27888:1999 standards. Ammonia nitrogen was determined by Flow Injection Analysis (FIA) with gaseous diffusion and spectrophotometric detection (according to PN-EN ISO 11732:2007). The sulphates were determined by a gravimetric method after precipitation with barium. Free- and bound- cyanides and phenolics were determined by Segment Flow Analysis (SFA) with spectrophotometric detection (PN-EN ISO 14403:2004; PN-EN ISO 14402:2004). Boron and other metals (Cr, Zn, Cd, Cu, Mo, Ni, Pb, and Ti) were determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES) (PB-07.22v.2.0). The BOD-5 and COD indices were determined by electrochemical and spectrophotometric methods (PN-EN 18991:2002; PN-EN 1899-2:2002; PB-07.26v.1.10). For the determination of BTEX compounds, gas chromatography coupled with a mass spectrometer (GC–MS) was used (Agilent Technologies 7890A). To determine the levels of 15 polycyclic aromatic hydrocarbons, solid phase extraction (SPE) high-performance liquid chromatography (HPLC) on Supelclean ENVI-C18 cartridges was carried out using the Agilent Technologies HPLC 1200 Series system.

2.4. Toxicological analysis Organisms differ in their sensitivity to various groups of chemicals. Thus the battery of six bioassays, testing for both acute toxicity and chronic toxicity, comprising bacterial, protozoan, crustacean and plant assays were chosen. The following commercially available tests were used for the ecotoxicological assays:

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Microtoxs, Spirotox, Thamnotoxkit F™, Protoxkit F™ and MARA. TUNEL was employed for the cytotoxicity and genotoxicity assays. 2.4.1. Ecotoxicological tests Microtoxs is based on the analysis of light emission changes in the luminescent bacteria Vibrio fischeri. All materials were purchased from SDI Europe. The test was carried out in the Microtox M500 (Modern Water, Inc.) analyser according to standard ISO 11348-3. The Spirotox test, with the ciliated protozoan S. ambiguum, was carried out according to the procedure detailed by Nałęcz-Jawecki (2005). The Thamnotoxkit F™ assay, with larvae of the crustacean Thamnocephalus platyurus hatched from dormant eggs, was carried out according to the manufacturer’s instructions (Thamnotoxkit, 1995). Both assays were performed in disposable, polystyrene multi-well plates (24 wells). After introducing the organisms into the wells, the wells were tightly closed with SealPlates film (Sigma Aldrich) to prevent crosscontamination. Protoxkit F™ is a multigenerational protozoa growth inhibition assay with a ciliate Tetrahymena thermophila. The test is based on optical density measurements. The assays were carried out in disposable, polystyrene spectrophotometric cells with lids (Protoxkit, 1998). The D. magna assay was performed in glass beakers according to standard ISO 6341. The microbial assay for risk assessment (MARA) is a multispecies, growth inhibition toxicity test with 11 microorganisms individually lyophilised in microplate wells. The MARA assays and all of the necessary reagents needed for the test were purchased from NCIMB Ltd. The toxicity tests were performed according to the standard operational procedure (Nałęcz-Jawecki et al., 2010) with the modification. The multiwall plates were tightly closed with SealPlates film (Sigma Aldrich) to prevent cross-contamination between the wells of the microplate. All toxicity tests were performed on a 2-fold dilution series of the samples. The endpoints specific for each test were expressed as a percent of the samples. Two endpoints were calculated in the toxicity tests: median effective concentration (EC50) and threshold effective concentration (EC20). In the MARA biotests, the microbial toxicity concentration (MTC) for each strain of microorganism was calculated with the special software supplied by the manufacturer (NCIMB Ltd.) (Nałęcz-Jawecki et al., 2010). Additionally, in this assay, the EC50 and EC20 values were calculated on the basis of the raw data on the inhibition of growth. The results obtained from each microbial test were transformed into toxic units according to the following formula: TU50 ¼

1  100 EC50

To examine the relative response of a specific strain in the MARA array versus the most sensitive strain, relative sensitivity (RS) values were determined (NałęczJawecki et al., 2010). The RS values were calculated for each microbial strain using the following formula: RSi ¼

MTCmin  100 MTCi

where RSi is a RS value for the “i” microorganism, MTCmin is the MTC value of the most sensitive strain, and MTCi is the MTC value of the “i” microorganism. 2.4.2. Analysis of cytogenetic effect Vicia faba (cv. White Windsor broad bean) was selected as the test plant species. The V. faba assay, which combines two test targets, cytotoxicity and genotoxicity, was performed according to the protocol detailed by Kanaya et al. (1994), with small modifications. The seeds were soaked for 12 h in tap water at room temperature and then the seed coats were carefully removed. The naked beans were placed on sterile moist cotton for 4 days at 22 1C until the primary roots had grown. The primary root tips were cut off to allow the development of lateral roots to 1–2 cm. The secondary roots were exposed to the tested water solutions for 2 h or 24 h at 22 1C in the dark. Each water sample was tested in three concentrations: undiluted and diluted with tap water in 1:3 and 1:10 proportions (to find the concentration that would allow the cyto- and geno-toxicity tests to be performed). After treatment, the seedlings were washed and half of the secondary roots were immediately fixed or the seedlings were left for a recovery period of 24 h and then fixed. Three seedlings per treatment (including concentrations and recovery time) were used. Tap water was used as a negative control, and 0.1 mM MH (maleic acid hydrazide, Sigma, CAS 123-3301) was used as a positive control (Kanaya et al., 1994). Each treatment experiment was conducted 3 times. The roots were fixed in ethanol:acetic acid (3:1 v/v) for 4 h at room temperature and then stained using the Feulgen method. Cytogenetic slides were prepared using the squash technique. Five slides, each from one root meristem, were prepared for each experimental group. The cytotoxicity of the samples was estimated from the mitotic activity of the root meristem cells and was quantified as a mitotic index (percent). To analyse the mitotic index (MI) 10,000 cells (2000 per slide) were observed for each sample. Genotoxicity was assessed with the frequency of aberrant cells in root meristems. For each treatment group, chromosomal aberrations (bridges, fragments and laggard chromosomes) were counted in 250 cells during anaphase and early telophase (50 per slide). The frequency of

micronuclei (MN) was estimated for 2000 interphase cells on each slide. The slides were examined using bright-field microscopy (Olympus Provis AX). The photographs were taken using a Hamamatsu CCD camera. The results are presented as the means 7 S.D., and the statistical significance of the differences between the control and treated groups was estimated with Student’s t-test. The TUNEL (TdT-mediated dUTP nick-end labelling) test was performed in order to analyse the frequency of nuclei with DNA fragmentation in the cells of V. faba roots. The test was performed after a 2 h treatment with water samples, without a recovery period. The frequency of TUNEL-positive nuclei after 0.1 mM MH treatment was also estimated. 1–2 long roots were fixed with freshly prepared 4 percent paraformaldehyde (Fluka) for 1 h at room temperature. The in situ Cell Death Detection Kit, Fluorescein (Roche), was employed according to the procedure described by Juchimiuk and Maluszynska (2005). The positive control was a 50 mL DNAse solution (1U) applied to one slide of the control sample. For a negative control of the TUNEL reaction, a mixture without terminal transferase was used. Each slide held one root tip. The slides were stained with 2 mg/mL DAPI (40 ,6diamidino-2-phenylindole), air dried and then mounted in Citifluor. The slides were evaluated with a fluorescence microscope using a FITC filter (with an excitation filter of 495 nm and a barrier filter of 525 nm) and a DAPI filter (with an excitation filter of 355 nm and a barrier filter of 450 nm). The labelled nuclei were counted, and the total number of analysed nuclei was calculated. The frequency of labelled cells was calculated on the basis of 2000 cells analysed on 2 slides for two treatment experiments.

2.5. Microbiological analysis Culturable microorganisms were evaluated with serial 10-fold dilutions of water samples. 1 mL of each sample was dispersed in 10 mL of sterilised physiological saline (0.8 percent NaCl) by shaking for 2 min. After the dilutions (10  1–10  5), 1 mL of each dilutions was pipetted onto plates. The pour-plate method was used for the quantification and isolation of microorganisms. Aerobic bacteria were incubated on SMA medium (Standard Methods Agar, BioMérieux) containing 100 mg cycloheximide/l. Psychrophilic bacteria were incubated at 22 1C for 72 h; mesophilic bacteria were incubated at 37 1C for 24 h; thermophilic bacteria were incubated at 45 1C for 24 h. The terms “psychrophilic”, “mesophilic” and “thermophilic” are used to describe the incubation temperature. Fungi were incubated on MEA medium (Malt Extract Agar, BioMérieux) with 100 mg/L chloramphenicol at 22 1C for 7 days. After incubation, the bacterial and fungal colonies were counted and the colony forming units (CFUs) were calculated. Actinomycetes were isolated on a selective medium, which contained: 2.0 g/L sodium caseinate, 0.1 g/L asparagine; 4.0 g/L sodium propionate, 0.5 g/L dipotassium phosphate, 0.1 g/L magnesium sulphate, 0.001 g/L ferrous sulphate, 5.0 g/L glycerol, 20 g/L agar, and 500 mg/L cycloheximide. The plates were incubated at 22 1C for 7 days. A mixed culture of iron-oxidizing bacteria was grown in Winogradski nutrient medium: 0.5 g/L K2HPO4, 0.5 g/L NaNO3, 0.2 g/L CaCl2  6H2O, 0.5 g/L MgSO4  7H2O, 0.5 g/L NH4NO3, 6.0 g/L ammonium iron citrate, 20.0 g/L agar (pH 4.8) at 2571 1C for 5 days. The Mn-oxidation agar media consisted of: 0.001 g/L FeSO4  7H2O, 0.15 g/L MnSO4  H2O, 2 g/L peptone, 0.5 g/L yeast extract and 15 g/L agar, all in 10 mM HEPES buffer (pH 7.4). The sporeforming bacteria were isolated by the method described in the Manual of Methods for General Microbiology (Gerhardt, 1981). Anaerobic bacteria were evaluated as described in Manual of Environmental Microbiology (Hurst et al., 2007). Detection of hydrocarbon-oxidizing bacteria on solid media was performed according to Petrovic et al. (2008) with some modifications. Two solid media were used: Tauson (T) medium of the following composition: (NH4)2SO4 1.0 g/L; CaSO4 0.5 g/L; MgSO4  7H2O 0.3 g/L; FeSO4  7H2O 0.005 g/L; K2HPO4 0.15 g/L; KH2PO4 0.15 g/L; 20.0 g/L agar) and MSWY medium (MS) composed with: NaCl 5.0 g/L; KCl 0.75 g/L; MgSO4  7H2O 7.0 g/L; yeast extract 1.0 g/L; proteose peptone 1.0 g/L; K2HPO4 0.3 g/L; KH2PO4 0.3 g/L; 20.0 g/L agar). After the basic media was sterilised, a 1 percent TTC solution was added to one part each of the basic media, MST and TT. Then, 1 mL of each water sample, 100 mL of crude oil and all of the prepared media (T, MS, MST, TT) were put into Petri dishes. Each water sample was prepared in triplicate on MS, T, MST and TT media. The resultant colonies were enumerated after 20 days of incubation at 30 1C. All grown colonies on the MS and T media were counted, whereas only the red colonies on the MST and TT media were counted. The abundance of microorganisms was given in CFU ml-1. All microbiological analyses were performed in triplicate.

3. Results 3.1. The chemical characterisation of UCG post-processing water The chemical analysis of the post-processing water that was obtained during the experimental simulation of the UCG process for hard coal is shown in Tables S2 and S3. UCG post-processing water contains eighteen toxic organic pollutants. High concentrations of BTEX (benzene, toluene, ethylbenzene, xylene) and polycyclic aromatic hydrocarbons (PAHs) were detected. The BTEX concentrations

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ranged from 1890–2350 mg/L, and the PAH concentrations ranged from 1660 to 3150 mg/L. Benzene (995–1770 mg/L) made up more than 50 percent of the total BTEX concentration, while naphthalene (2855–1473 mg/L) made up over 85 percent of the PAHs in each sample. These organic compounds are highly toxic to the environment and to living organisms. Among the inorganic contaminants, high concentrations of nitrogen (1500–2300 mg/L) and sulphates (up to 1330 mg/L) were detected. The ammonia and cyanide present in the samples are also hazardous inorganic contaminants. High temperatures during the UCG process promote increased solubility of the contaminants, and facilitate their migration. High concentrations of toxic chemical compounds present in the samples impinge on high values of BOD-5 (biochemical oxygen demand) and COD (chemical oxygen demand) parameters. For comparison, moderately polluted rivers may have a BOD-5 value in the range of 2 to 8 mg/L. BOD-5 values for municipal waste water are usually in the range of 20 mg/L or less. As is shown in Table S3, BOD-5 values for the samples are more than one hundred times larger. As Tables S2 and S3 show, the parameters of each sample are substantially similar to each other and no significant difference was observed between them. This suggests that the composition of the received post-processing water changed little during the course of the experiment. As might be expected in the case of aromatic compounds, their concentrations were the highest at the beginning of the experiment, decreasing slightly over the duration of the experiment. The inorganic contaminant and heavy metal concentrations increased during the experiment. This situation occurs because aromatic hydrocarbons and other organic compounds form reactions during the coal gasification reaction. The process temperature is then the highest which favours the formation of a number of PAHs and other aromatics. With time, these reactions tend to disappear, and the main source of contaminant generation is the ashes formed during the gasification process. Heavy metals present in the ashes migrate into the water, resulting in their increased concentration in the post-processing water.

3.2. Toxicological characterisation of UCG post-processing water 3.2.1. Bioassay results In the current study, the toxicity of post-processing water from underground coal gasification was evaluated with a battery of six bioassays comprising two bacterial, two protozoan and two crustacean tests. In the preliminary Spirotox and Thamnotoxkit F™ tests that were performed according to standard procedures, toxic effects were observed in the control wells. It was assumed that the effects were caused by volatile compounds present in the test samples. Thus, in the basic tests all microplates were tightly closed with SealPlates film to prevent cross contamination between the wells in the microplates.

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The samples were very toxic, with the TU50 reaching 262 (Table 1). None of biotests was the most sensitive for all of the samples. Two crustacean biotests were comparably sensitive to all three samples. The toxicity of sample III was similar in both protozoan assays, while samples I and II were 5 times more toxic to Spirotox than the Protoxkit F™. From the bacterial assays, Microtoxs was much more sensitive than MARA, with TU50 values 10- to 20-fold higher. The testing revealed that samples I and II had very high threshold toxicity (EC20), especially in the Microtoxs assay. The TU20/TU50 ratio illustrates the increased effect with increasing concentrations. The TU20/TU50 ratio was as high as 4.1 and 6.9 (for sample I and sample II). In the other biotests, the ratios were much lower, between 1.2 and 3.4. The EC20 value indicates the concentration that causes the first symptoms of toxicity in the tested population. The MTC value is the endpoint calculated by the MARA software, on the basis of raw data about the inhibition of each microorganism’s growth. Additionally, in this project, we decided to calculate TU50 and TU20 values to unify the endpoints of all of the bioassays and to allow calculation of the TU20/TU50 ratios. As seen in Table 1, the TUMTC lies between the TU50 and TU20 values, and the TU20/TU50 ratio is similar to the other tests. Despite the fact that MARA is the least sensitive bioassay in this study, it has one important feature. Eleven MTC values were obtained for each sample giving a unique “toxic fingerprint” profile of the sample (Fig. 1). MARA is the first assay that generates a fingerprint, allowing comparison of the substances, if they contain similar toxicants (toxicants that similarly affecting organisms with different sensitivities) (Nałęcz-Jawecki et al., 2010). A similar spectrum of toxic effects on microbial strains was observed for the first and second samples. Three bacterial strains (1, 7 and 8) were the most sensitive to the samples, while strain 5 was the least sensitive. In contrast, strain 11 was the second-most sensitive to sample III, with a TU equal to 50 percent of the highest value.

3.2.2. Cytogenetic effects The V. faba micronucleus and chromosomal aberration tests have been previously recommended as a screening test for the genotoxicity of polluted water (Degrassi and Rizzoni, 1982; Ma et al., 1995). The effect of treatment with the tested water samples was observed as changes in the mitotic activity of V. faba root meristem cells (Tables 2 and 3). Control cells were characterised by a mitotic index of 8.1 71.1 percent. The mitotic activity of the cells treated with MH (positive control) was 4.6 7 0.6 percent. A decrease in the frequency of dividing V. faba cells was observed after treatment with all of the water samples. The mitotic activity of cells strongly depended on the concentrations of the tested water samples. There were no dividing cells observed after treatment with all tested samples at 100 percent concentrations, both for 2 h and 24 h of treatment. Due to these results, other

Table 1 Toxicity results of tested UCG post-processing water samples. Biotest

Microtoxs MARA

Sample I

Sample II

TU50

TU20

TU20/TU50

TU50

109 4.3

444 14.5

4.1 3.3

185 9.7

TUMTC1 ¼ 8.4 Spirotox Protoxkit F™ Thamnotoxkit F™ Daphnia 1

177 25.6 135 79

Sample III TU20 1282 17.9

TU20/TU50 6.9 1.8

TUMTC1 ¼ 16.4 218 39.0 314 200

1.2 1.5 2.3 2.5

177 25.0 137 152

Microbial toxicity concentration of the most sensitive strain TUMTC ¼ (1/MTCmin)  100%.

TU50

TU20

TU20/TU50

119 14.1

204 47.6

1.7 3.4

189 150 400 400

1.5 1.3 1.5 2.8

TUMTC1 ¼22.3 218 49.1 340 283

1.2 2.0 2.5 1.9

125 118 262 141

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Table 3 Mitotic activity and frequencies of micronuclei induced by UCG post-processing water samples after a 24 h exposure. Sample

Table 2 Mitotic activity and frequencies of micronuclei induced by UCG post-processing water samples after a 2 h exposure. Sample

Mitotic activity (%)

Micronuclei (%)

No recovery

24 h Of recovery time

No recovery

24 h Of recovery time

Water samples—100% I II III

0 0 0

0 0 0

0 0 0

0 0 0

Water samples—1:3 I II III

0 1.87 0.2a 2.6 70.2a

1.17 0.1a 2.47 0.3a 3.17 0.1a

3.57 0.2a 3.87 0.2a 2.57 0.1a

3.17 0.1a 3.17 0.2a 2.17 0.1a

Water samples—1:10 I II III

1.27 0.1a 2.2 70.2a 6.8 70.2a

2.47 0.1a 3.67 0.2a 7.57 0.4

12.07 0.3a 11.17 0.3a 14.27 0.5a 12.47 0.2a 4.87 0.2a 4.17 0.2a

8.1 71.1

8.25 7 0.9

0

Negative control (tap water) MH a

4.6 70.6

a

5.77 0.8

a

9.87 0.3

0 a

6.37 0.3a

Differ from the tap water.

concentrations of water samples (1:3, 1:10) were applied to analyse their cytotoxicity. The diluted water samples at the 1:3 and 1:10 concentrations inhibited mitotic activity more strongly than treatment with MH, except for the lowest concentration of sample III. Drastically decreased mitotic activity was observed in the root cells treated with sample I, both for 2 h and 24 h of treatment. The highest mitotic index was observed for sample III (6.87 0.2 percent for 2 h treatment, 5.8 70.5 percent for 24 h treatment), however it was still lower than that of the control cells. These results indicate that 24 h of exposure to the water

Micronuclei (%)

No recovery

24 h Of recovery No time recovery

24 h Of recovery time

Water samples—100% I II III

0 0 0

0 0 0

0 0 0

0 0 0

Water samples—1:3 I II III

0 0 1.17 0.1a

0.6 7 0.1a 0.4 70.1a 1.97 0.1a

2.07 0.2a 2.87 0.2a 1.57 0.1a

1.17 0.1a 2.07 0.2a 1.17 0.1a

Water samples—1:10 I II III

0.87 0.1a 1.87 0.1a 5.87 0.5a

1.17 0.2a 2.3 70.4a 6.9 70.7a

13.77 0.7a 11.27 0.8a 16.87 1.2a 14.27 0.9a 5.87 0.5a 4.37 0.3a

Negative control (tap water)

8.17 1.1

8.25 7 0.9

0

0

MH (2 h treatment)

4.67 0.6a

5.7 70.8a

9.87 0.3a

6.37 0.3a

a

Fig. 1. Fingerprints of tested samples in the MARA assays, RS—relative sensitivity values calculated for each microbial strain.

Mitotic activity (%)

Differ from the tap water.

samples caused a stronger effect than 2 h of treatment. When the cells were allowed time to recover after the treatment, the mitotic index was slightly higher than the values obtained immediately after treatment. It is possible that the analysed water samples may cause cell death which appears as a decline in the MI (Chandra et al., 2005). The inhibition of the mitotic index can also be attributed to be the effect of environmental effluents on DNA/ protein synthesis (Chauhan et al., 1998). The inhibition of the MI induced by these water samples could directly affect root growth and elongation, and cause lethal effects on the organism (Antosiewicz, 1990). The mitotic activity of the V. faba root cells is a good indicator of the cytotoxicity of the water samples tested. The water samples were also tested for potential clastogenicity in V. faba. Micronuclei in interphase cells were observed after the 1:3 and 1:10 water sample treatments (Tables 2 and 3). No micronuclei were induced by the undiluted water samples. These results confirm the previous data, which concluded that micronucleus frequency is directly proportional to the mitotic index (Degrassi and Rizzoni, 1982). The increased frequency of micronuclei in comparison to the control was not high for the cells treated with the 1:3dilutions, only 2 70.2 percent for sample I, 2.8 70.2 percent for sample II, and 1.5 70.1 percent for sample III. The highest frequencies of micronuclei were observed in the cells treated with the 1:10 dilutions. The highest frequencies of micronuclei were observed in Vicia cells after 24 h of treatment with water sample II (16. 8 71.2 percent) and water sample I (13.7 70.7 percent). Sample III caused micronuclei at a lower frequencies (5.870.5 percent) than did treatment with samples I and II. The treatment with water samples for 2 h caused micronuclei with a frequency of approximately 12–14 percent for samples I and II and 5 percent for sample III. With a recovery period after treatment with the analysed water samples, only a slight increase of MI and decrease of the frequencies of micronuclei were observed. Among chromosomal aberrations (CA), the fragments and bridges in anaphase and telophase were observed, however their frequencies were very low (below 1 percent) for all of the samples. The low frequencies of CA can be due to strong cytotoxic effects caused by the samples. A total inhibition of MI immediately after treatment, combined with only a slight increase after 24 h of recovery, was observed. This finding could indicate the cell death of Vicia cells after treatment with the analysed water samples. The TUNEL test is used to visualise DNA breakage, which is a common feature of cell death, but was also adapted to plant

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299

Fig. 2. The results of TUNEL method in Vicia faba root interphase cells: (a, a0 )—control, not treated cells, individual nuclei showed green fluorescence; (b, b0 )—positive control, after treatment with DNAse, almost all nuclei are showing green fluorescence; (c, c0 )—after treatment with water samples I. Left column: DAPI staining, all nuclei are stained. Right column: nuclei with or without green fluorescence as a result of TUNEL reaction. Bars represent 10 μm.

mutagenesis studies (Juchimiuk and Maluszynska, 2005). In this study, the TUNEL test was used to detect the DNA fragmentation in V. faba root meristematic cells induced by the tested water samples (Fig. 2). TUNEL-specific fluorescence was observed in the positive control (DNAase solution was used prior to the TUNEL reaction), where approximately 80 percent of the nuclei were labelled (Fig. 2b). The frequency of FITC-labelled nuclei for the control, untreated cells was approximately 1 percent (Fig. 2a). All of the tested water samples caused sporadic DNA fragmentation in the nuclei of the roots; only approximately 4 percent of the nuclei were labelled (Fig. 2c). These results suggest that the cells did not undergo the cell death process after treatment with water samples. In this study, the TUNEL test enables visualization of the consequences on DNA levels immediately after treatment with the water samples. This study confirmed that the TUNEL test is an effective method to monitor DNA damage caused by the water samples and to distinguish the cells subjected to the cell death process. These results suggest that the water samples analysed could be classified as cytotoxic and genotoxic waste. These effects were

attributable to the complex mixture found in the water samples. Our work is the first report, to our knowledge, to show evidence of genetic damage in plants induced by the UCG post-processing water. However, other water environmental pollutants, like PAHs, BTEX, and heavy metals, have been shown to be cytotoxic and genotoxic in plant cells (Leme et al., 2008,2009; Sang and Li, 2004). Taking into account that the V. faba test has correlates with other plant tests, we conclude that post-processing water caused genotoxic effects in the plant genome. 3.3. Microbiological characterisation of UCG post-processing water The microbiological characterisation of post-processing water samples is presented in Table 4. Due to the presence of refractory and inhibitory compounds in the UCG post-processing water samples, the number of microorganisms was very low and only a few microorganisms were detected in the water samples. Among the tested microorganisms, mesophilic, thermophilic, psychrophilic, spore-forming, anaerobic and S-oxidizing bacteria were found. The microbiological data show that psychrophilic bacteria dominated

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Table 4 Microbiological characterization of UCG post-processing waters. Microorganisms

Mesophilic bacteria Termophilic bacteria Psychrophilic bacteria Spore-forming bacteria Anaerobic bacteria Fe-oxidizing bacteria S-oxidizing bacteria Mn-oxidizing bacteria Actinomycetes Fungi

Units

CFU mL  1 CFU mL  1 CFU mL  1 CFU mL  1 CFU mL  1 CFU mL  1 CFU mL  1 CFU mL  1 CFU mL  1 CFU mL  1

Post-processing water samples Sample I

Sample II

Sample III

807 12 207 3 465 7 24 217 8 467 7 0 187 5 0 0 0

557 8 457 11 2377 23 947 12 587 9 0 107 2 0 0 0

35 74 157 3 47 1 147 7 107 5 0 97 3 0 0 0

Mean value from three replicates 7standard deviation.

among the bacteria. The psychrophilic bacteria counts varied between 465 CFU/mL in sample I and 4 CFU/mL in sample III. There were no fungi or Actinomycetes in any of the water samples. Generally, the lowest number of detected microorganisms was in sample III. The results of the analysis of the analysis of the cultivation and biochemical methods are presented in Table S4. The hydrocarbon-oxidizing bacteria grew only on the MS and MST media. There was no growth of bacteria on T and TT media. The presence of nutrients in the MS medium yielded the growth of bacteria. Olga et al. (2008) suggested that the majority of hydrocarbon-oxidizing bacteria grow on mineral media with hydrocarbons and with other organic compounds. Previous microbiological research revealed that hydrocarbon-oxidizing bacteria growing on MS medium with TTC were metabolically active. The number of active hydrocarbonoxidizing bacteria was 3 times lower in sample III than in the rest of the tested water samples. There is no information on microbiological characterisation of the post-processing water from UCG processing. Only a few studies have analysed the microbiology of polluted coke wastewater. Whiteley et al. (2001) cultured and examined the Pseudomonad species, while others (Whiteley and Bailey, 2000; Manefield et al., 2005) used culture-independent methods, which may give a more realistic view of the composition of the microbial community. Felföldi et al. (2010) combined culture-independent methods (sequence-aided T-RFLP, taxon-specific PCR) with strain isolation and biodegradation tests, revealing a simply structured microbial community dominated by easily culturable heterotrophic bacteria in coke wastewater effluent. The predominant phylotype was related to the floc-forming Comamonas badia, which presented a phenol-degrading ability, so far an undescribed feature for the species. In addition to C. badia, dominant bacterial groups included the highly phenol-tolerant and -degrader Pseudomonas-, the non-phenol-degrader Sphingomonas- and the phenol-degrader and possibly thiocyanate-removing Rhodanobacter. The presence of the members of the sulphur-oxidizing and thiocyanate-degrading Thiobacillus genus was also detected.

4. Discussion Based on the results of the chemical analyses the post-processing water samples can be expected to be highly toxic. To see how the chemical compounds in the post-processing water affect the environment, toxicological and microbiological analyses were carried out. The bacterial bioassays indicated a high level of toxicity in the tested water samples. All of the six biotests had a very high TU50 index. The analysis of the cytotoxicity and genotoxicity of the water samples in Vicia showed the strongest effects for samples I and II. Treatment with sample III also resulted in cytotoxic and clastogenic effects,

however, it was weaker than for samples I and II. These results are correlated with the concentrations of the main organic contaminants present in the water samples, especially benzene, naphthalene and the phenolics (Table S2), for which the largest concentrations are observed in the initial stage of the process. In all of these bioassays it was necessary to pre-dilute the tested samples. A potential environmental risk posed by UCG post-processing water is associated with the underground coal gasification process. In cases of improper technological operation this water could cause considerable pollution problems by contacting the soil and ground water. These results suggest that the analysed post-processing water samples could be classified as cytotoxic and genotoxic waste. The microbiological analyses indicated the presence of metabolic activity of hydrocarbon-oxidizing bacteria in the water. This result is very important, considering the possibility of using bioremediation technologies for post-processing water from UCG. Bioaugmentation with specialised microorganisms could be used as an efficient and effective method of improving the removal efficiency of recalcitrant organic compounds in the post-processing water. The chemical composition of the water from the UCG process is similar to that of coke plant effluents (Kim et al., 2008). However, to assess the degrading potentials and physiological properties of the most important microbes of the communities, an integrated study, linking the results of culture-dependent and culture-independent methods is needed. The assays and analyses conducted represent the first step in research toward finding an effective biological method for UCG post-processing water treatment. The next microbiological research will be undertaken to characterise the microbial community composition and the specific bacteria (vaccine consisting of selected strains of bacteria). This can provide the solution to the problem of UCG post-processing water toxicity, its purification and the high environmental risk it poses.

5. Conclusions UCG post-processing water contains many toxic organic and inorganic compounds. Due to the presence of these contaminants, the microbiological and toxicological analyses revealed the high toxicity of the tested water samples. Chemical analysis itself does not provide a clear assessment of the environmental risk posed by the tested samples. The current study shows that the environmental risk posed by the post-processing water should be evaluated with an integrated chemical, toxicological and microbiological monitoring program. The utilisation of organisms from different trophic levels in toxicity tests helps to improve the environmental impact evaluation of the water discharge into different ecosystems. The water quality monitoring has historically only relied on chemical analyses. Such measurements may identify a compound present in the environment, but they do not provide insight into the biological effects of the present toxicants and the joint effects of mixtures of (un)known compounds on the biota. Therefore, bioassays are deployed as a complementary tool, giving insight to the biological effects of a test compound. The increasing interest in the UCG process makes it is necessary to more closely analyse the problem of environmental risk associated with water pollution. In conclusion, only the combination of chemical, microbiological and toxicological approaches provides a complete picture of the risk posed by UCG post-processing water and will aid in further research into effective biological methods of UCG postprocessing water treatment.

Acknowledgments The research presented in this paper was performed as a part of the HUGE 2 project (Hydrogen Oriented Underground Coal

M. Pankiewicz-Sperka et al. / Ecotoxicology and Environmental Safety 108 (2014) 294–301

Gasification for Europe—Environmental and Safety Aspects) and supported by the RFCS under Contract no. RFCR-CT-2011-00002 and by the Polish Ministry of Science and Higher Education.

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Assessment of the chemical, microbiological and toxicological aspects of post-processing water from underground coal gasification.

The purpose of this paper is to provide a comprehensive characterisation (including chemical, microbiological and toxicological parameters) of water a...
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