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Biotic and abiotic degradation behaviour of ethylene glycol monomethyl ether (EGME)
ARTICLE IN PRESS
Water Research 39 (2005) 2002–2007 www.elsevier.com/locate/watres
Biotic and abiotic degradation behaviour of ethylene glycol monomethyl ether (EGME) A. Fischer, C. Hahn Institute of Waste Management and Contaminated Site Treatment, Dresden University of Technology, Pratzschwitzer Str. 15, D-01796 Pirna, Germany Received 24 June 2004; received in revised form 30 November 2004; accepted 2 March 2005 Available online 5 May 2005
Abstract Glycol ethers are widely used in many processes in the chemical industry. Their high water solubility means they are used as solvents for different purposes (e.g. lacquers and varnishes). Since glycol ethers are known to produce toxic metabolites such as the teratogenic methoxyacetic acid during biodegradation, the biological treatment of glycol ethers can be hazardous. However, using oxidizing agents like hydrogen peroxide could be a feasible option for treating wastewater containing glycol ether. In this study, both-, biodegradation and abiotic oxidation experiments with ethylene glycol monomethyl ether (EGME) as contaminant were performed. The biodegradation experiments were conducted with a synthetic model wastewater containing 15 wt% NaCl and 5000 mg l1 of EGME. While experiments with the fungus Aspergillus versicolor resulted in the exhaustive biotic degradation of EGME, the toxic metabolite methoxyacetic acid (MAA) was produced as a ‘dead end’ product. Sodium hydroxide was added to adjust the decreasing pH caused by the production of MAA. In abiotic degradation experiments with EGME, other degradation products—organic acids and toxic aldehydes, e.g. methoxy acetaldehyde (MALD)—were detected. It must be taken into account that EGME and its biotic and abiotic degradation products are usually not analysed in routine wastewater measurements owing to their physical properties. r 2005 Elsevier Ltd. All rights reserved. Keywords: Ethylene glycol monomethyl ether; Methoxyacetic acid; Methoxy acetaldehyde; Biotic degradation; Abiotic degradation
1. Introduction Glycol ethers are produced in large quantities worldwide. In the United States, the annual production of EGME alone is estimated at more than 35,000 tonnes (Shih et al., 2001). Glycol ethers can be designated as ‘unusual contaminants’. Although they are highly water-soluble, they are Corresponding author. Tel.: +49 3501 530039;
fax: +49 3501 530022. E-mail address: [email protected] (C. Hahn).
not normally determined in ground and surface water because of their physical properties. Since many glycol ethers are even miscible with water, detection after the clean-up of groundwater samples often fails because they are difficult to separate. In contrast to other ethers, most glycol ethers have high boiling points and low vapour pressures, resulting in very poor detection limits for analytical methods such as GC/FID with headspace injection. For the analytical detection of ethers, extraction techniques like SPME methods (e.g. Bensoam et al., 1999) or direct aqueous injection (DAI) (e.g. Zenker et al., 2003) could be more suitable (see Section 2.5). However, these methods are not applied for the routine
0043-1354/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2005.03.032
ARTICLE IN PRESS A. Fischer, C. Hahn / Water Research 39 (2005) 2002–2007
detection of conventional environmental contaminants. Therefore, glycol ethers are likely to remain undetected in waterworks or during wastewater treatment. The biodegradation of EGME has been addressed in several scientific investigations (Groeseneken et al., 1989; Miller et al., 1983; Moss et al., 1985; Shih et al., 2001). The main metabolite found is the teratogenic methoxyacetic acid (MAA). Research into the toxicity of this substance has been conducted since the mid-1980s (Foster et al., 1983; Yonemoto et al., 1984). This study examined the biotic and abiotic degradation of EGME. For biotic experiments a synthetic model wastewater (derived from cellulose industry) with an extremely high salt concentration (15%) was used. As high salt concentrations in wastewater are well known to reduce the biodegradation of diethylene glycol dimethyl ether (Mangelsdorf et al., 2002), the degradation behavior of EGME under saline conditions is of high interest. During wastewater treatment, oxidants are commonly used in order to reduce the chemical oxygen demand (COD). A method based on hydrogen peroxide is often used in this respect, such as the FENTOX procedure (Wurdack et al., 2001), which is known to be effective for treating recalcitrant compounds. Fenton’s Reagent is frequently the basis used for these methods. The purpose of this study was to investigate the biotic and abiotic degradation pathways of EGME in order to better assess the potential risk of EGME in treated and untreated wastewater. A matter of particular interest was to determine the quantity of intermediate products, such as organic acids (formic acid, acetic acid, glycolic acid, glyoxylic acid, oxalic acid or methoxyacetic acid) and aldehydes (glycolaldehyde, formaldehyde, acetaldehyde and methoxyacetaldehyde (MALD)). To our knowledge, the effect of saline conditions on biotic degradation of EGME was not investigated before. Literature about the oxidation of EGME was also not available. Aschmann et al. (2001) investigated the kinetics of the reactions of several glycol ethers (not EGME) with OH radicals. The authors found that a multitude of reaction pathways are possible, whereas aldehydes as intermediates are generated predominantly. Partial oxidation of EGME with different reagents may be applied sometimes in laboratories in order to produce related aldehydes or acids.
2. Materials and methods 2.1. Biotic degradation experiments In order to treat a high contaminated and high saline model wastewater, several halotolerante microorganism strains with a wide degradation spectra under salt stress were sought and investigated by project partners (Beck
2003
Fig. 1. Cultivation of Aspergillus versicolor in Fernbach Flasks.
et al., 2004). Thirty-five strains were isolated from soil samples originating from contaminated sites or saline environments. The strains were investigated regarding their biodegradation potential, the fungus Aspergillus versicolor tiraboschi showed the best results. As the metabolism of fungi can be inhibited because of motions like stirring or shaking, the fungus was cultivated in static cultures. Fernbach flasks (Fig. 1) are ideal culture vessels since the surface of the culture has a high extension. Oxygen supply is provided and filigrane hyphae can grow without being damaged as a result of motions. The flasks were filled with 400 ml culture medium (Glasgow media (FGSC) Aspergillus buffered minimal) containing 16.14 mmol l1 EGME and 150 g l1 salt (sodium chloride). As auxiliary substrate 0.5 wt% glucose was added. The pH was 5.8 and the temperature varied between 20 and 25 1C. The experiments were carried out twice at room temperature. For the weekly sampling, the flasks were opened and 6 ml of solution were taken. The pH of the culture was measured and in case of deviation it was adjusted with 0.5 M NaOH. The samples were sterilized by filtration through a 0.2 mm filter and stored at 17 1C. EGME and DOC were determined at once, analyses of aldehydes, organic acids and glucose were carried out for all samples at the end of the degradation experiments. 2.2. Abiotic degradation experiments The abiotic experiments were conducted without the addition of salt. Chloride is known to be able to react with oxidizing agents to a certain extent, albeit mainly with stronger oxidizers such as ozone. The main product is chlorine (e.g. Oum et al., 1998). However, high levels of salt ought only to result in lower yields and not fundamentally alter reaction pathways. The formation of a different range of products during oxidation in comparison to experiments without salt is unlikely. Furthermore, the high salt levels sometimes interfered with the analytical determinations of some intermediates (aldehydes). Therefore, for the abiotic experiments it
ARTICLE IN PRESS A. Fischer, C. Hahn / Water Research 39 (2005) 2002–2007
2004
and time-consuming purification of the crude product was not made.
was decided not to quench the impact of Fenton’s Reagent by using high concentrations of salt. The abiotic degradation experiments were performed in accordance with Turan-Ertas and Gural (2002) by stepwise addition of modified Fenton’s Reagent as oxidizing agent, in order to find out the minimal amount of oxidizing agent. The reaction vessel was a 500 ml Erlenmeyer flask filled with 250 ml of EGME solution. The initial concentration of EGME was 1 g l1. For each step of oxidation, 250 ml Fe(III) chloride solution (120 g l1) and 250 ml in 10-fold molar excess hydrogen peroxide solution (wt%) was added, resulting in a Fe(III) concentration in each flask of 0.47 mmol l1. The experiments were carried out twice at room temperature. Samples were stored at 12 1C over night before being measured, because preliminary tests had shown, that the reaction lasted about 12 h after having added the oxidizing agent.
2.4. Determination of aldehydes as PFBHA derivatives The derivatization of aldehydes and ketones with O(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA) reagent is a specific, very sensitive method for GC/ECD analysis. The detection limit is approximately 1 mg l1. The derivatization procedure was adopted from APHA-AWWA-WEF (1995) and Yamada and Somiya (1989). For GC/MS control analysis, a Hewlett-Packard HP 5890 with split/splitless injector and a capillary column (Hewlett Packard, PONA, 50 m, 0.2 mm i.d., 0.5 mm film thickness) was used with helium as carrier gas (1.0 ml min1). Two microliter of aldehyde derivatives solutions were injected (split ration 1:10) and analysed (scan modus). The temperature settings were: injector temperature 250 1C, oven temperature program: 50 1C (6 min) -20 1C/min -280 1C (10 min), and detector temperature 290 1C. Fig. 2 shows the mass spectrum of MALD after derivatization with PFBHA. The PFBHA derivatives of all the aldehydes investigated have a base peak at m/z 181, which is the pentafluorotropylium ion C6F5CH+ 2 . The ion with m/z 239 (molecule mass 30) results from the loss of NO, which is often observed for aldehyde derivatives (Yu et al., 1995). Ions with m/z ¼ molecule mass were not observed in all cases.
2.3. Synthesis of methoxy acetaldehyde (MALD) As MALD is not commercially available, it had to be synthesized. Aldehydes are frequently synthesized from the corresponding alcohol by a common oxidant. However, aldehydes are often further oxidized to the corresponding acids. The purification of MALD by distillation is difficult due to its boiling point (92 1C) being close to that of other by-products (e.g. water). Moreover, detecting aldehydes is rather difficult. The aldehydes examined showed decomposition during heating within the GC injector and it was not possible to analyse them with GC/MS. Therefore, MALD was synthesized by a method first described by Meerwein et al. (1958). The formation of MALD is confirmed by the simultaneous formation of chlorobenzene (reaction product from 4-chloroaniline), which can be determined by GC/MS. The required starting substance (diazonium salt of 4-chloroaniline) was prepared according to instructions from Beyer and Walter (1981). A difficult
2.5. Determination of EGME by DAI For the analytical detection of glycol ethers, SPME methods can be useful (e.g. Bensoam et al., 1999). However, for routine analysis EGME had to be determined in a mixture of hazardous compounds which had very different physicochemical properties (project HALOMIK, see point 5). It was not possible to find an 181
Abundance
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117 131 120
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252 268 281 297 240
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m/z Fig. 2. Mass spectrum of methoxy acetaldehyde (MALD) after derivatization with PFBHA.
ARTICLE IN PRESS A. Fischer, C. Hahn / Water Research 39 (2005) 2002–2007
2.6. Determination of organic acids, dissolved organic carbon (DOC) and glucose The organic acids (MAA, formic acid, acetic acid) were analysed by high-performance ion chromatography (HPIC) using a Metrohm ion chromatograph with a Metrosep Organic Acids IC exclusion column. DOC was detected with a Shimadzu TOC analyser equipped with an autosampler. The samples were filtered before analysing (0.2 mm cellulose acetate filter). Glucose was determined photometrically by the modified Anthrone method (Jermyn, 1975). The photometer used was a Spectrocord 50 manufactured by Analytik Jena.
3. Results and discussion 3.1. Biotic degradation The biodegradation experiment (Fig. 3) was carried out over 2 months. After 4 weeks the consumption of glucose was almost completed and the degradation of EGME started. Simultaneously, MAA began to be formed. The measured DOC values fit well with the calculated DOC sum of all the analysed compounds. Other metabolites (e.g. aldehydes and acids) were not found; evidently MAA was the only metabolite of this biodegradation. The biodegradation of MAA was not observed, thus the acid can be considered as ‘‘dead end’’ product. At the beginning of the investigations, the degradation of EGME stopped after a few days as a result of the acidic effect of the produced MAA. Therefore, the pH of the solution had to be adjusted to pH 7 (with sodium hydroxide) to compensate for the acidic effect of MAA
3000 DOC glucose EGME MAA
2500 DOC [mgl-1]
adsorption material which could adsorb all these compounds to a higher extent. The most suitable method for the analytical determination of these compounds was DAI. Therefore, this method was also used for EGME analyses. For GC-FID analysis, a Perkin Elmer autosystem gas chromatograph with split/splitless injector and FID detector and a capillary column (DB-Wax, 30 m, 0.45 mm i.d., 0.85 mm film thickness) was employed with nitrogen as carrier gas (1.0 ml min1) as well as hydrogen (45 ml min1) and oxygen (ml min1) as detector gases. One micorliter of water samples were injected (split ratio: 1:10) into the GC/FID and analysed. The injector liners were filled with silylated glass wool. The liners had to be often changed due to precipitation and incrustation of salt. The temperature settings were: injector temperature 250 1C, oven temperature program: 50 1C (2 min) -15 1C/min -230 1C (1 min) and detector temperature 270 1C.
2005
2000 1500 1000 500 0 0
10
20
30 40 time [d]
50
60
70
Fig. 3. Biotic EGME degradation (all compounds calculated as organic carbon (DOC)).
(pKa ¼ 3.48), which is slightly stronger acidic than formic acid (pKa ¼ 3.7). The amount of MAA produced and the related consumption of the base were in good agreement. The simultaneous neutralization led to the complete biodegradation of EGME. Under environmental conditions this acidification effect can be neglected because dilution effects will prevent a lower pH. Only under unfavourable conditions (e.g. high amounts of EGME in the soil) the biodegradation of EGME can lead to the acidification of the environment and finally to the repression of biodegradation. The high concentrations of salt can be responsible for the resistance of MAA to degradation. However, MAA has often been observed as a metabolite of glycol ethers (e.g. Harada and Nagashima, 1975). Normally metabolites are only found if their biodegradation is slower than the biodegradation of the starting molecule. A precondition for the biodegradation of an organic compound is good water solubility, and MAA is even miscible with water. Therefore, MAA has to be considered as a recalcitrant compound. However, given the toxic properties of MAA, the biodegradation of EGME and the simultaneous production of MAA during wastewater treatment should be carefully monitored. The investigations proved a high salt tolerance of the fungus, though this strain is not halophilic. The glucose was an essential auxiliary substrate, degradation experiments without any other substrate than EGME showed poor degradation results, as it is typical for the so-called diauxie. Experiments with lower concentrations of salt led to faster biodegradation, with MAA as metabolite (data not shown). The biodegradation of EGME under high saline conditions was accomplished in the presented experiments, but no mineralization was observed. The production of the toxic MAA in high amounts could
ARTICLE IN PRESS A. Fischer, C. Hahn / Water Research 39 (2005) 2002–2007
2006
not be prevented, therefore, the abiotic pretreatment of EGME containing wastewaters could be a solution.
3.2. Abiotic degradation In contrast to the biological degradation experiments, no production of MAA was observed in the abiotic oxidation experiments. Fig. 4 shows the progress of the experiment. After the addition of 0.94 mmol l1 Fe3+ and 10 mmol l1 hydrogen peroxide, more than 99% of the initial EGME concentration was degraded, although the DOC loss was merely 57%. It was proven, that formic acid was the main product of the abiotic degradation of EGME. After EGME was oxidized, oxidation of formic acid began. The other products were determined as acetic acid, formaldehyde and glycolaldehyde. After the first addition step of Fenton’s Reagent, the calculated and measured DOC values correlated well. However, after the second and third addition of the reagent, a deviation appeared between the two parameters, indicating that not all the substances produced by this experiment were determined. At the end of the experiment, the measured and calculated DOC coincided well again. The apparent deficit in the mass balance during oxidation was somewhat caused by MALD, which was not to quantify (see Section 2.3). The overestimation of DOC at the beginning of the experiment may have been caused by the slight evaporation of EGME. Furthermore, some aldehydes were detected (albeit in tiny amounts) which could not be identified.
Two facts are significant: although no MAA was formed in the abiotic oxidation experiments, aldehydes were produced which were stable over a considerable long period without being rapidly oxidized. The aldehydes generated are hazardous; MALD in particular is known to have genotoxic and mutagenic effects (Kitagawa et al., 2000; Chiewchanwit and Au, 1994; Ma et al., 1993). The abiotic degradation of the aldehydes and acids was completed by additional doses of Fenton’s Reagent.
4. Conclusions The results indicate that EGME can be degraded by the fungus Aspergillus versicolor. However, no mineralization occurs during biotic degradation because the metabolite MAA is stable (‘dead end’ product). As the degradation is based on diauxie, glucose was essential as auxiliary substrate. In contrast to many hydrophilic organic compounds (e.g. ethanol), EGME is not easily biodegradable. Under unfavourable conditions the production of MAA can lead to acidification, causing the biodegradation of EGME to stop unexpectedly. Furthermore MAA is known to be teratogenic. The abiotic degradation of EGME by Fenton’s Reagent is fast and complete. Unlike biodegradation, the abiotic oxidation products are mainly formic acid and acetic acid, along with small amounts of formaldehyde and acetaldehyde. Although not all degradation products were identified, broad evidence was found for the formation of the mutagenic MALD. It must be considered, that biotic and abiotic wastewater treatment of EGME could generate harmful byproducts which should be monitored.
500 EGME formic acid 400
acetaldehyde
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We would like to thank the German Ministry of Education and Research for kindly funding this work (project HALOMIK, ref. no. 02wa0133). We also own many thanks to D. Beck, S. Ho¨rnig and W. Meusel from UAS Anhalt in Koethen, Germany, for the support in the cultivation of the fungus.
100
References
formaldehyde DOC [mgl-1]
Acknowledgements
300
aceticacid DOC
0 0
0.47 0.94 1.41 1.88 2.35 2.82 Fe3+ [mmol/L]
Fig. 4. Abiotic EGME degradation and corresponding oxidation products (all compounds calculated as organic carbon).
APHA-AWWA-WEF, 1995. PFBHA liquid–liquid Extraction Gas Chromatographic Method. In: Greenberg, A.E., Trussel, R.R., Clesceri, L.S. (Eds.), Standard Methods for the Examination of Water and Wastewater, 16th ed. American Public Health Association, Washington, DC. Aschmann, S.M., Martin, P., Tuazon, E.C., Arey, J., Atkinson, R., 2001. Kinetic and product studies of the reactions of
ARTICLE IN PRESS A. Fischer, C. Hahn / Water Research 39 (2005) 2002–2007 selected glycol ethers with OH radicals. Environ. Sci. Technol. 35 (20), 4080–4088. Beck, D., Meusel, W., Ho¨rnig, S., Strech, D., Gerloff, A., Nitschke, S., 2004. Kultivierung und Abbauleistung von Aspergillus versicolor in Ru¨hrreaktoren, Blasensa¨ulen und Festbettreaktoren unter Salzstress. Poster presentation. 22. DECHEMA-Jahrestagung der Biotechnologen, 4.-6. Mai 2004, Wiesbaden. Bensoam, J., Cicolella, A., Dujardin, R., 1999. Improved extraction of glycol ethers from water by solid-phase micro extraction by carboxen polydimethylsiloxane-coated fiber. Chromatographia 50 (3/4), 155–159. Beyer, H., Walter, W., 1981. Aromatic diazo-compounds, textbook of organic chemistry (Aromatische Diazoverbindungen, Lehrbuch der Organischen Chemie). S. Hirzel Verlag, Stuttgart, Germany, p. 543. Chiewchanwit, T., Au, W.W., 1994. Cytogenetic effects of 2methoxyethanol and its metabolite, methoxyacetaldehyde, in mammalian cells in vitro. Mutat. Res. 320, 125–132. Foster, P.M.D., Creasy, D.M., Foster, J.R., Thomas, L.V., Cook, M.V., Gangolli, S.D., 1983. Testicular toxicity of ethylene glycol monomethyl and monoethyl ethers in the rat. Toxicol. Appl. Pharmacol. 69, 385–399. Groeseneken, D., Veulemas, H., Masschelein, R., Van Vlem, E., 1989. Experimental human exposure to ethylene glycol monomethyl ether. Int. Arch. Occup. Environ. Health 61, 243–247. Harada, T., Nagashima, Y., 1975. Utilization of alkylether compounds by soil bacteria. J. Ferment. Technol. 53, 218–222. Jermyn, M.A., 1975. Increasing the sensitivity of the anthrone method for carbohydrate. Anal. Biochem. 68, 332–335. Kitagawa, K., Kawamoto, T., Kunugita, N., Tsukiyama, T., Okamoto, K., Yoshida, A., Nakayama, K., 2000. Aldehyde dehydrogenase (ALDH) 2 associates with oxidation of methoxyacetaldehyde; in vitro analysis with liver subcellular fraction derived from human and Aldh2 gene targeting mouse. FEBS. Lett. 476, 306–311. Ma, H., An, J., Hsie, A.W., Au, W.W., 1993. Mutagenicity and cytotoxicity of 2-methoxyethanol and its metabolites in Chinese hamster cells (the CHO/HPRT and AS52/GPT assays). Mutat. Res. 298, 219–225. Mangelsdorf, I., Boehncke, A., Koennecker, G., 2002. Diethylene glycol dimethyl ether, concise international chemical assessment document 41. Fraunhofer Institute of Toxicology and Aerosol Research, Hannover, Germany, World Health Organization, Geneva, published under the sponsorhip of UNEP, ILO and WHO. Meerwein, H., Allendo¨rfer, H., Beekmann, P., Kunert, F., Morschel, H., Paweller, F., Wunderlich, K., 1958. Reduc-
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tion of aromatic diazo-compounds with ethers, 1,3-dioxolanes and tertiary amines (U¨ber die Reduktion aromatischer Diazoverbindungen mit A¨thern, 1,3-Dioxolanen und tertia¨ren Aminen). Angew. Chem. 8, 211–215. Miller, R.R., Hermann, E.A., Langvardt, P.W., McKenna, M.J., Schwetz, B.A., 1983. Comparative metabolism and disposition of ethylene glycol monomethyl ether and propylene glycol monomethyl ether in male rats. Toxicol. Appl. Pharmacol. 67, 229–237. Moss, E.J., Thomas, L.V., Cook, M.W., Walters, D.G., Foster, P.M.D., Creasy, D.M., Bray, T.J.B., 1985. The role of metabolism in 2-methoxy ethanol induced testicular toxicity. Toxicol. Appl. Pharmacol. 79, 480–489. Oum, K.W., Lakin, M.J., DeHaan, D.O., Brauers, T., Finlayson-Pitts, B.J., 1998. Formation of molecular chlorine from the photolysis of ozone and aqueous sea-salt particles. Science 279, 74–77. Shih, T.-S., Liou, S.-H., Chen, C.-Y., 2001. Urinary 2-methoxy acetic acid accumulation in response to 2-methoxy ethanol exposure. Archives of Environmental Health January. Turan-Ertas, T., Gural, M.D., 2002. Oxidation of diethylene glycol with ozone and modified Fenton processes. Chemosphere 47 (3), 293–301. Wurdack, I., Specht, O., Renner, T., Goerlach, T., Wabner, D., 2001. BayFORREST research project F 187: Oxidative Degradation of Refractory COD with Fenton’s Reagent (BayFORREST-Forschungsvorhaben F 187: Oxidativer Abbau von refraktaerem CSB mit Fentons Reagenz). In: Wilderer, P.A., Wimmer, M. (Eds.), Booklet 12 of the Bavarian research network for waste research and waste material recycling, status report in December (Berichtsheft 12 des bayerischen Forschungsverbundes fu¨r Abfallforschung und Reststoffverwertung, Statusbericht im Dezember), Munich, pp. 211–227. Yamada, H., Somiya, I., 1989. The determination of carbonyl compounds in ozonated water by the PFBOA method. Ozone Sci. Eng. 11, 127–141. Yu, J., Jeffries, H.E., Le Lacheur, L.M., 1995. Identifying airborne carbonyl compounds in isoprene atmospheric photooxidation products by their PFBHA oximes using gas chromatography/ion trap mass spectrometry. Environ. Sci. Technol. 29, 1923–1932. Yonemoto, J., Brown, N.A., Webb, M., 1984. Effects of dimethoxyl phthalate, monomethoxyethyl phthalate, 2methoxyethanol and methoxyacetic acid on post-implantation rat embryos in culture. Toxicol. Lett. 21, 97–102. Zenker, M.J., Borden, R.C., Batlaz, M.A., 2003. Occurence and treatment of 1,4-dioxane in aqueous environments. Environ. Eng. Sci. 20 (5), 423–432.
Water Research 39 (2005) 2002–2007 www.elsevier.com/locate/watres
Biotic and abiotic degradation behaviour of ethylene glycol monomethyl ether (EGME) A. Fischer, C. Hahn Institute of Waste Management and Contaminated Site Treatment, Dresden University of Technology, Pratzschwitzer Str. 15, D-01796 Pirna, Germany Received 24 June 2004; received in revised form 30 November 2004; accepted 2 March 2005 Available online 5 May 2005
Abstract Glycol ethers are widely used in many processes in the chemical industry. Their high water solubility means they are used as solvents for different purposes (e.g. lacquers and varnishes). Since glycol ethers are known to produce toxic metabolites such as the teratogenic methoxyacetic acid during biodegradation, the biological treatment of glycol ethers can be hazardous. However, using oxidizing agents like hydrogen peroxide could be a feasible option for treating wastewater containing glycol ether. In this study, both-, biodegradation and abiotic oxidation experiments with ethylene glycol monomethyl ether (EGME) as contaminant were performed. The biodegradation experiments were conducted with a synthetic model wastewater containing 15 wt% NaCl and 5000 mg l1 of EGME. While experiments with the fungus Aspergillus versicolor resulted in the exhaustive biotic degradation of EGME, the toxic metabolite methoxyacetic acid (MAA) was produced as a ‘dead end’ product. Sodium hydroxide was added to adjust the decreasing pH caused by the production of MAA. In abiotic degradation experiments with EGME, other degradation products—organic acids and toxic aldehydes, e.g. methoxy acetaldehyde (MALD)—were detected. It must be taken into account that EGME and its biotic and abiotic degradation products are usually not analysed in routine wastewater measurements owing to their physical properties. r 2005 Elsevier Ltd. All rights reserved. Keywords: Ethylene glycol monomethyl ether; Methoxyacetic acid; Methoxy acetaldehyde; Biotic degradation; Abiotic degradation
1. Introduction Glycol ethers are produced in large quantities worldwide. In the United States, the annual production of EGME alone is estimated at more than 35,000 tonnes (Shih et al., 2001). Glycol ethers can be designated as ‘unusual contaminants’. Although they are highly water-soluble, they are Corresponding author. Tel.: +49 3501 530039;
fax: +49 3501 530022. E-mail address: [email protected] (C. Hahn).
not normally determined in ground and surface water because of their physical properties. Since many glycol ethers are even miscible with water, detection after the clean-up of groundwater samples often fails because they are difficult to separate. In contrast to other ethers, most glycol ethers have high boiling points and low vapour pressures, resulting in very poor detection limits for analytical methods such as GC/FID with headspace injection. For the analytical detection of ethers, extraction techniques like SPME methods (e.g. Bensoam et al., 1999) or direct aqueous injection (DAI) (e.g. Zenker et al., 2003) could be more suitable (see Section 2.5). However, these methods are not applied for the routine
0043-1354/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2005.03.032
ARTICLE IN PRESS A. Fischer, C. Hahn / Water Research 39 (2005) 2002–2007
detection of conventional environmental contaminants. Therefore, glycol ethers are likely to remain undetected in waterworks or during wastewater treatment. The biodegradation of EGME has been addressed in several scientific investigations (Groeseneken et al., 1989; Miller et al., 1983; Moss et al., 1985; Shih et al., 2001). The main metabolite found is the teratogenic methoxyacetic acid (MAA). Research into the toxicity of this substance has been conducted since the mid-1980s (Foster et al., 1983; Yonemoto et al., 1984). This study examined the biotic and abiotic degradation of EGME. For biotic experiments a synthetic model wastewater (derived from cellulose industry) with an extremely high salt concentration (15%) was used. As high salt concentrations in wastewater are well known to reduce the biodegradation of diethylene glycol dimethyl ether (Mangelsdorf et al., 2002), the degradation behavior of EGME under saline conditions is of high interest. During wastewater treatment, oxidants are commonly used in order to reduce the chemical oxygen demand (COD). A method based on hydrogen peroxide is often used in this respect, such as the FENTOX procedure (Wurdack et al., 2001), which is known to be effective for treating recalcitrant compounds. Fenton’s Reagent is frequently the basis used for these methods. The purpose of this study was to investigate the biotic and abiotic degradation pathways of EGME in order to better assess the potential risk of EGME in treated and untreated wastewater. A matter of particular interest was to determine the quantity of intermediate products, such as organic acids (formic acid, acetic acid, glycolic acid, glyoxylic acid, oxalic acid or methoxyacetic acid) and aldehydes (glycolaldehyde, formaldehyde, acetaldehyde and methoxyacetaldehyde (MALD)). To our knowledge, the effect of saline conditions on biotic degradation of EGME was not investigated before. Literature about the oxidation of EGME was also not available. Aschmann et al. (2001) investigated the kinetics of the reactions of several glycol ethers (not EGME) with OH radicals. The authors found that a multitude of reaction pathways are possible, whereas aldehydes as intermediates are generated predominantly. Partial oxidation of EGME with different reagents may be applied sometimes in laboratories in order to produce related aldehydes or acids.
2. Materials and methods 2.1. Biotic degradation experiments In order to treat a high contaminated and high saline model wastewater, several halotolerante microorganism strains with a wide degradation spectra under salt stress were sought and investigated by project partners (Beck
2003
Fig. 1. Cultivation of Aspergillus versicolor in Fernbach Flasks.
et al., 2004). Thirty-five strains were isolated from soil samples originating from contaminated sites or saline environments. The strains were investigated regarding their biodegradation potential, the fungus Aspergillus versicolor tiraboschi showed the best results. As the metabolism of fungi can be inhibited because of motions like stirring or shaking, the fungus was cultivated in static cultures. Fernbach flasks (Fig. 1) are ideal culture vessels since the surface of the culture has a high extension. Oxygen supply is provided and filigrane hyphae can grow without being damaged as a result of motions. The flasks were filled with 400 ml culture medium (Glasgow media (FGSC) Aspergillus buffered minimal) containing 16.14 mmol l1 EGME and 150 g l1 salt (sodium chloride). As auxiliary substrate 0.5 wt% glucose was added. The pH was 5.8 and the temperature varied between 20 and 25 1C. The experiments were carried out twice at room temperature. For the weekly sampling, the flasks were opened and 6 ml of solution were taken. The pH of the culture was measured and in case of deviation it was adjusted with 0.5 M NaOH. The samples were sterilized by filtration through a 0.2 mm filter and stored at 17 1C. EGME and DOC were determined at once, analyses of aldehydes, organic acids and glucose were carried out for all samples at the end of the degradation experiments. 2.2. Abiotic degradation experiments The abiotic experiments were conducted without the addition of salt. Chloride is known to be able to react with oxidizing agents to a certain extent, albeit mainly with stronger oxidizers such as ozone. The main product is chlorine (e.g. Oum et al., 1998). However, high levels of salt ought only to result in lower yields and not fundamentally alter reaction pathways. The formation of a different range of products during oxidation in comparison to experiments without salt is unlikely. Furthermore, the high salt levels sometimes interfered with the analytical determinations of some intermediates (aldehydes). Therefore, for the abiotic experiments it
ARTICLE IN PRESS A. Fischer, C. Hahn / Water Research 39 (2005) 2002–2007
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and time-consuming purification of the crude product was not made.
was decided not to quench the impact of Fenton’s Reagent by using high concentrations of salt. The abiotic degradation experiments were performed in accordance with Turan-Ertas and Gural (2002) by stepwise addition of modified Fenton’s Reagent as oxidizing agent, in order to find out the minimal amount of oxidizing agent. The reaction vessel was a 500 ml Erlenmeyer flask filled with 250 ml of EGME solution. The initial concentration of EGME was 1 g l1. For each step of oxidation, 250 ml Fe(III) chloride solution (120 g l1) and 250 ml in 10-fold molar excess hydrogen peroxide solution (wt%) was added, resulting in a Fe(III) concentration in each flask of 0.47 mmol l1. The experiments were carried out twice at room temperature. Samples were stored at 12 1C over night before being measured, because preliminary tests had shown, that the reaction lasted about 12 h after having added the oxidizing agent.
2.4. Determination of aldehydes as PFBHA derivatives The derivatization of aldehydes and ketones with O(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA) reagent is a specific, very sensitive method for GC/ECD analysis. The detection limit is approximately 1 mg l1. The derivatization procedure was adopted from APHA-AWWA-WEF (1995) and Yamada and Somiya (1989). For GC/MS control analysis, a Hewlett-Packard HP 5890 with split/splitless injector and a capillary column (Hewlett Packard, PONA, 50 m, 0.2 mm i.d., 0.5 mm film thickness) was used with helium as carrier gas (1.0 ml min1). Two microliter of aldehyde derivatives solutions were injected (split ration 1:10) and analysed (scan modus). The temperature settings were: injector temperature 250 1C, oven temperature program: 50 1C (6 min) -20 1C/min -280 1C (10 min), and detector temperature 290 1C. Fig. 2 shows the mass spectrum of MALD after derivatization with PFBHA. The PFBHA derivatives of all the aldehydes investigated have a base peak at m/z 181, which is the pentafluorotropylium ion C6F5CH+ 2 . The ion with m/z 239 (molecule mass 30) results from the loss of NO, which is often observed for aldehyde derivatives (Yu et al., 1995). Ions with m/z ¼ molecule mass were not observed in all cases.
2.3. Synthesis of methoxy acetaldehyde (MALD) As MALD is not commercially available, it had to be synthesized. Aldehydes are frequently synthesized from the corresponding alcohol by a common oxidant. However, aldehydes are often further oxidized to the corresponding acids. The purification of MALD by distillation is difficult due to its boiling point (92 1C) being close to that of other by-products (e.g. water). Moreover, detecting aldehydes is rather difficult. The aldehydes examined showed decomposition during heating within the GC injector and it was not possible to analyse them with GC/MS. Therefore, MALD was synthesized by a method first described by Meerwein et al. (1958). The formation of MALD is confirmed by the simultaneous formation of chlorobenzene (reaction product from 4-chloroaniline), which can be determined by GC/MS. The required starting substance (diazonium salt of 4-chloroaniline) was prepared according to instructions from Beyer and Walter (1981). A difficult
2.5. Determination of EGME by DAI For the analytical detection of glycol ethers, SPME methods can be useful (e.g. Bensoam et al., 1999). However, for routine analysis EGME had to be determined in a mixture of hazardous compounds which had very different physicochemical properties (project HALOMIK, see point 5). It was not possible to find an 181
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m/z Fig. 2. Mass spectrum of methoxy acetaldehyde (MALD) after derivatization with PFBHA.
ARTICLE IN PRESS A. Fischer, C. Hahn / Water Research 39 (2005) 2002–2007
2.6. Determination of organic acids, dissolved organic carbon (DOC) and glucose The organic acids (MAA, formic acid, acetic acid) were analysed by high-performance ion chromatography (HPIC) using a Metrohm ion chromatograph with a Metrosep Organic Acids IC exclusion column. DOC was detected with a Shimadzu TOC analyser equipped with an autosampler. The samples were filtered before analysing (0.2 mm cellulose acetate filter). Glucose was determined photometrically by the modified Anthrone method (Jermyn, 1975). The photometer used was a Spectrocord 50 manufactured by Analytik Jena.
3. Results and discussion 3.1. Biotic degradation The biodegradation experiment (Fig. 3) was carried out over 2 months. After 4 weeks the consumption of glucose was almost completed and the degradation of EGME started. Simultaneously, MAA began to be formed. The measured DOC values fit well with the calculated DOC sum of all the analysed compounds. Other metabolites (e.g. aldehydes and acids) were not found; evidently MAA was the only metabolite of this biodegradation. The biodegradation of MAA was not observed, thus the acid can be considered as ‘‘dead end’’ product. At the beginning of the investigations, the degradation of EGME stopped after a few days as a result of the acidic effect of the produced MAA. Therefore, the pH of the solution had to be adjusted to pH 7 (with sodium hydroxide) to compensate for the acidic effect of MAA
3000 DOC glucose EGME MAA
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adsorption material which could adsorb all these compounds to a higher extent. The most suitable method for the analytical determination of these compounds was DAI. Therefore, this method was also used for EGME analyses. For GC-FID analysis, a Perkin Elmer autosystem gas chromatograph with split/splitless injector and FID detector and a capillary column (DB-Wax, 30 m, 0.45 mm i.d., 0.85 mm film thickness) was employed with nitrogen as carrier gas (1.0 ml min1) as well as hydrogen (45 ml min1) and oxygen (ml min1) as detector gases. One micorliter of water samples were injected (split ratio: 1:10) into the GC/FID and analysed. The injector liners were filled with silylated glass wool. The liners had to be often changed due to precipitation and incrustation of salt. The temperature settings were: injector temperature 250 1C, oven temperature program: 50 1C (2 min) -15 1C/min -230 1C (1 min) and detector temperature 270 1C.
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2000 1500 1000 500 0 0
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30 40 time [d]
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Fig. 3. Biotic EGME degradation (all compounds calculated as organic carbon (DOC)).
(pKa ¼ 3.48), which is slightly stronger acidic than formic acid (pKa ¼ 3.7). The amount of MAA produced and the related consumption of the base were in good agreement. The simultaneous neutralization led to the complete biodegradation of EGME. Under environmental conditions this acidification effect can be neglected because dilution effects will prevent a lower pH. Only under unfavourable conditions (e.g. high amounts of EGME in the soil) the biodegradation of EGME can lead to the acidification of the environment and finally to the repression of biodegradation. The high concentrations of salt can be responsible for the resistance of MAA to degradation. However, MAA has often been observed as a metabolite of glycol ethers (e.g. Harada and Nagashima, 1975). Normally metabolites are only found if their biodegradation is slower than the biodegradation of the starting molecule. A precondition for the biodegradation of an organic compound is good water solubility, and MAA is even miscible with water. Therefore, MAA has to be considered as a recalcitrant compound. However, given the toxic properties of MAA, the biodegradation of EGME and the simultaneous production of MAA during wastewater treatment should be carefully monitored. The investigations proved a high salt tolerance of the fungus, though this strain is not halophilic. The glucose was an essential auxiliary substrate, degradation experiments without any other substrate than EGME showed poor degradation results, as it is typical for the so-called diauxie. Experiments with lower concentrations of salt led to faster biodegradation, with MAA as metabolite (data not shown). The biodegradation of EGME under high saline conditions was accomplished in the presented experiments, but no mineralization was observed. The production of the toxic MAA in high amounts could
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not be prevented, therefore, the abiotic pretreatment of EGME containing wastewaters could be a solution.
3.2. Abiotic degradation In contrast to the biological degradation experiments, no production of MAA was observed in the abiotic oxidation experiments. Fig. 4 shows the progress of the experiment. After the addition of 0.94 mmol l1 Fe3+ and 10 mmol l1 hydrogen peroxide, more than 99% of the initial EGME concentration was degraded, although the DOC loss was merely 57%. It was proven, that formic acid was the main product of the abiotic degradation of EGME. After EGME was oxidized, oxidation of formic acid began. The other products were determined as acetic acid, formaldehyde and glycolaldehyde. After the first addition step of Fenton’s Reagent, the calculated and measured DOC values correlated well. However, after the second and third addition of the reagent, a deviation appeared between the two parameters, indicating that not all the substances produced by this experiment were determined. At the end of the experiment, the measured and calculated DOC coincided well again. The apparent deficit in the mass balance during oxidation was somewhat caused by MALD, which was not to quantify (see Section 2.3). The overestimation of DOC at the beginning of the experiment may have been caused by the slight evaporation of EGME. Furthermore, some aldehydes were detected (albeit in tiny amounts) which could not be identified.
Two facts are significant: although no MAA was formed in the abiotic oxidation experiments, aldehydes were produced which were stable over a considerable long period without being rapidly oxidized. The aldehydes generated are hazardous; MALD in particular is known to have genotoxic and mutagenic effects (Kitagawa et al., 2000; Chiewchanwit and Au, 1994; Ma et al., 1993). The abiotic degradation of the aldehydes and acids was completed by additional doses of Fenton’s Reagent.
4. Conclusions The results indicate that EGME can be degraded by the fungus Aspergillus versicolor. However, no mineralization occurs during biotic degradation because the metabolite MAA is stable (‘dead end’ product). As the degradation is based on diauxie, glucose was essential as auxiliary substrate. In contrast to many hydrophilic organic compounds (e.g. ethanol), EGME is not easily biodegradable. Under unfavourable conditions the production of MAA can lead to acidification, causing the biodegradation of EGME to stop unexpectedly. Furthermore MAA is known to be teratogenic. The abiotic degradation of EGME by Fenton’s Reagent is fast and complete. Unlike biodegradation, the abiotic oxidation products are mainly formic acid and acetic acid, along with small amounts of formaldehyde and acetaldehyde. Although not all degradation products were identified, broad evidence was found for the formation of the mutagenic MALD. It must be considered, that biotic and abiotic wastewater treatment of EGME could generate harmful byproducts which should be monitored.
500 EGME formic acid 400
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We would like to thank the German Ministry of Education and Research for kindly funding this work (project HALOMIK, ref. no. 02wa0133). We also own many thanks to D. Beck, S. Ho¨rnig and W. Meusel from UAS Anhalt in Koethen, Germany, for the support in the cultivation of the fungus.
100
References
formaldehyde DOC [mgl-1]
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aceticacid DOC
0 0
0.47 0.94 1.41 1.88 2.35 2.82 Fe3+ [mmol/L]
Fig. 4. Abiotic EGME degradation and corresponding oxidation products (all compounds calculated as organic carbon).
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