SUSTAINABILITY OF NATURAL ATTENUATION OF AROMATICS (BTEX) Final report

Transcription

1 SUSTAINABILITY OF NATURAL ATTENUATION OF AROMATICS (BTEX) Final report Client: NICOLE, Port of Rotterdam, Shell Global Solutions Project code: Date: 20 July 2007

2 Client: Project title: NICOLE, Port of Rotterdam, Shell Global Solutions Sustainability of Natural Attenuation of aromatics (BTEX) Project code: Document type: final report, version 2 Publication date: 20 July 2007 Project manager: N.J.P. van Ras Author(s): N.J.P. van Ras, R.O. Winters, S.H. Lieten, J.E. Dijkhuis and M.J.C. Henssen (Bioclear), W.A. van Hattem (Havenbedrijf Rotterdam), G. Lethbridge (Shell Global Solutions) Keywords: natural attenuation, BTEX, anaerobic, sustainability, protocol Post address: P.O. Box 2262, 9704 CG Groningen The Netherlands Visit address: Rozenburglaan 13C, Groningen The Netherlands Telephone: 31 (0) Telefax: 31 (0) info@bioclear.nl Website: The cover of this report is made of recycled polypropylene All rights reserved. No part of this material may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopy, recording or by any information storage and retrieval system, without written permission from Bioclear. Bioclear supports industry, authorities, engineering companies and institutes in the area of environmental technology For all orders to Bioclear the General Conditions for research orders to Bioclear, as registered at the Chamber of Commerce at Groningen, are applicable.

3 SUMMARY i SUMMARY Introduction The NICOLE project Monitored Natural Attenuation: demonstration & review of the applicability of MNA at 8 field sites has shown that industry, regulators and scientists recognise Natural Attenuation (NA) as a useful risk management tool that results in loss of contamination and prevention of plume migration. However, when NA is used to prevent plume migration in the future, the sustainability of NA processes is important. None of the existing protocols considers this element, but this information is essential to give a reliable extrapolation of plume behaviour in the future and to determine the applicability of NA at a site. Background information Due to the naturally occurring degradation processes, active remediation measures might not be necessary or can be reduced and a more cost-effective remediation strategy can be implemented. Most protocols that are available to determine the occurrence of NA mainly focus on monitoring of processes that indicate whether degradation has occurred in the past. The information obtained is then extrapolated to predict plume development in the future, for instance using modelling. However, when predicting plume behaviour in the future, the sustainability of NA is important. Will the required NA processes also occur in the future? Are estimations and expectations used in modelling still valid in, for instance, 5 or 10 years from now? Together with Dutch governmental and industrial parties a systematic protocol to determine the sustainability of NA of chlorinated ethenes was developed. The developed protocol helps industry, governments and consultants to get a reliable picture of the NA processes and the sustainability of NA and is used as a management tool to make decisions in the case of NA processes for chlorinated ethenes and to optimise monitoring activities. Besides chlorinated solvents, the aromatic hydrocarbons (BTEX) are an important group of contaminants in industry and are frequently detected in groundwater plume areas for which natural attenuation is considered. The objective of this NICOLE project was to develop a protocol to evaluate the occurrence and to determine the sustainability of NA of BTEX. Activities and results Recent knowledge about (anaerobic) BTEX degradation was collected and recent research results were integrated. Specific measurements and/or tests with samples from sites of the participants and other samples were performed (anaerobic bench scale feasibility tests, isotopic analyses, bioavailable iron(iii) tests, molecular analyses) and confirmed BTEX degradation under various conditions. Together with the knowledge and experiences gained with the protocol for chlorinated ethenes, a new protocol to determine the sustainability of NA of BTEX was developed. This protocol provides information about the mechanisms of and prerequisites for NA of BTEX. It includes (analytical) tools to determine the occurrence of BTEX degradation at a site and a systematic step by step approach to determine the sustainability of the process in the future. In this report, the protocol is described in more detail.

4 SUMMARY ii Conclusions The recently developed protocols provide the required structured approach to determine the feasibility of MNA as stand-alone option or cost-effective addition to active measures and help to take away possible doubts of site owners and regulatory authorities. The protocols consider the most important groups of mobile contaminants in the groundwater (chlorinated ethenes and BTEX). The protocol for chlorinated ethenes is available via the website of SKB ( The protocol for BTEX is available via the website of NICOLE (

5 TABLE OF CONTENTS SUMMARY I 1. INTRODUCTION General introduction Background information and objectives Approach and activities Structure of the report 2 2. NATURAL ATTENUATION OF BTEX Definition of natural attenuation BTEX degradation under aerobic conditions Degradation under anaerobic conditions Nitrate-reducing conditions Manganese-reducing conditions Iron-reducing conditions Sulphate-reducing conditions Methanogenic conditions Aspects that might influence anaerobic BTEX degradation Resume Typical situation at BTEX contaminated sites 8 3. TOOLS TO DETERMINE THE OCCURRENCE OF NATURAL ATTENUATION Lines of evidence for natural attenuation of BTEX First line of evidence Second line of evidence Shift in redox conditions Detection of degradation products Compound specific stable isotope analyses (CSIA) Third line of evidence Laboratory microcosms Molecular detection of specific benzene degrading micro-organisms In-situ microcosms (Bac-trap) General approach NA feasibility study PROTOCOL TO DETERMINE THE SUSTAINABILITY OF NA OF BTEX Introduction Sustainability of NA of BTEX Step 1: determining degradation of BTEX Step 2: calculation of the acceptor-donor ratio in individual monitoring wells Step 3: Assess prerequisites for sustainable NA at site scale Does sustainable natural attenuation of BTEX result in an acceptable situation? Conceptual model Quantification of the conceptual model Use of conceptual model to define remediation target active measures CHECKLIST REFERENCE LIST 27 APPENDIX: A. Literature review BTEX degradation, ongoing research B. Sampling strategy C. Calculations

6 INTRODUCTION 1 1. INTRODUCTION 1.1. General introduction The NICOLE project Monitored Natural Attenuation: demonstration & review of the applicability of MNA at 8 field sites [1] has shown that industry, regulators and scientists recognise Natural Attenuation (NA) as a useful risk management tool that results in loss of contamination and prevention of plume migration. However, if NA is used to prevent plume migration in the future, the sustainability of the NA processes is important. The sustainability of NA is seen as the potential for NA processes to continue in the future. Will the degradation processes that have occurred in the past or occur now also occur in the next years or decades, at the same rates? None of the existing protocols to evaluate NA considers this element, although this information is essential in order to reliably extrapolate the plume behaviour to the future situation and thus determine the applicability of NA at a given site. Within the NICOLE project Sustainability of natural attenuation of aromatics (BTEX), a protocol to determine the sustainability of the NA of BTEX was developed Background information and objectives Due to naturally occurring degradation processes, active remediation measures at a contaminated site might not be necessary or to a reduced extent. In this way a more cost-effective remediation strategy can be implemented. Most protocols that are available to determine the occurrence of NA mainly focus on monitoring of processes that indicate whether degradation has occurred in the past. The information obtained is then extrapolated to predict plume development in the future, for instance using modelling programs. However, when predicting plume behaviour in the future, the sustainability of NA is important. Will the required NA processes also occur in the future, at the same degradation rates? Will the estimations and expectations used in the modelling still be valid in, for instance, 5 or 10 years from now? These uncertainties are mainly caused by a lack of understanding and detailed information about the long-term sustainability of the NA potential of the aquifer. A structured argumentation with a conceptual model and a comprehensive data set is necessary to get MNA approved and to take away possible doubts of site owners and regulatory authorities [1]. A new protocol to determine the sustainability of NA of chlorinated ethenes was developed in 2002, together with Dutch governmental and industrial parties [2]. The developed protocol helps industry, governments and consultants to obtain a reliable picture of the NA processes and the sustainability of NA and is used as a (management) tool to make decisions in the case of NA processes for chlorinated ethenes and to optimise monitoring activities. Besides chlorinated solvents, aromatic hydrocarbons (BTEX) are important contaminants that are frequently detected in groundwater plumes in which natural attenuation is likely. The objective of this NICOLE project was to develop a protocol to evaluate the occurrence and determine the sustainability of NA of BTEX.

7 INTRODUCTION Approach and activities Recent knowledge on BTEX degradation was collected by means of a literature study with articles published from If relevant, earlier published results were evaluated as well. Research groups in Europe and the US were contacted and recent research results were integrated. Specific measurements and/or tests with samples from BTEX contaminated sites were performed (intrinsic bench scale feasibility tests, isotopic analyses, bioavailable iron(iii) tests, molecular analyses). The site-specific results of these measurements and tests are not included in this report, but these tests confirmed BTEX degradation under various conditions and the knowledge gained was used for the sustainability protocol for BTEX. Together with the knowledge and experiences gained through the protocol for chlorinated ethenes [2], a new protocol to determine the sustainability of NA of BTEX was developed. This protocol includes information about the mechanisms of and prerequisites for NA of BTEX and the (analytical) tools to determine the occurrence and the sustainability of BTEX degradation at a site. The protocol provides the required structural approach to determine the feasibility of MNA as stand-alone option or cost-effective addition to active measures and helps to eliminate possible doubts of site owners and regulatory authorities Structure of the report In chapter 2 of this report, recent knowledge about the degradation of BTEX in the subsurface is included. In chapter 3, tools to determine the occurrence of natural attenuation of BTEX are presented and explained. Chapter 4 describes the developed protocol to determine the sustainability of BTEX degradation. In chapter 5 a checklist is included that draws the attention of users to aspects that are of importance for the sustainability of natural attenuation of BTEX components. The reference list is presented in chapter 6.

8 NATURAL ATTENUATION OF BTEX 3 2. NATURAL ATTENUATION OF BTEX It is widely known and accepted that benzene and other BTEX compounds can be degraded readily under aerobic conditions. Recent studies have shown that these compounds can also be degraded under anaerobic conditions. These degradation processes play an important role in the natural attenuation of BTEX components at contaminated sites Definition of natural attenuation Various definitions of the term natural attenuation exist. In general, natural attenuation refers to a variety of processes that reduce the mass, toxicity, volume, mobility and/or concentrations of hazardous compounds in soil and groundwater. These processes can be classified as either: physical (dispersion, volatilisation etc.); chemical (chemical degradation); biological (biodegradation). Natural attenuation processes that reduce the concentrations and/or the mobility of a hazardous compound but not the total mass are referred to as nondestructive mechanisms. Examples of these mechanisms are physical dispersion and sorption. Natural attenuation processes that result in a reduction of the total mass are referred to as destructive mechanisms and include chemical and biological degradation processes. In this report, NA is defined as the sum of nondestructive and destructive processes that result in a loss of the total mass of BTEX and consequently reduce concentrations, volume and migration of these contaminants. An overview is presented in table 1. Table 1. Overview of natural attenuation processes in the soil process type destructive or non-destructive dispersion physical non-destructive diffusion physical non-destructive dilution physical non-destructive volatilisation physical non-destructive sorption physical non-destructive chemical oxidation chemical destructive chemical reduction chemical destructive biological oxidation biological destructive biological reduction biological destructive For BTEX components, destructive mechanisms due to biological degradation processes are far more important than chemical destructive mechanisms or nondestructive mechanisms. This protocol therefore focuses on the sustainability of biological degradation processes.

9 NATURAL ATTENUATION OF BTEX BTEX degradation under aerobic conditions BTEX is readily degraded under aerobic conditions. Under aerobic conditions, BTEX serves as an electron donor and oxygen is used as a terminal electron acceptor. Mono- and dioxygenases are involved in this degradation process. Several aerobic metabolic pathways for the degradation of benzene, toluene, ethylbenzene and xylene (BTEX), have been identified, and numerous bacteria are able to use BTEX as a carbon source and electron donor under aerobic conditions. Aerobic degradation can be important at the plume fringe in groundwater that is aerobic by nature. However, the groundwater in Europe is usually anaerobic and the consumption of oxygen during the degradation often exceeds the supply of oxygen, resulting in an anaerobic source and plume zone. In these cases, anaerobic degradation processes prevail. There are only a few specific sites where aerobic degradation of BTEX at the plume fringe can play a role in the natural attenuation process, for instance at sites in highly permeable soils with shallow contaminated groundwater. The overall reaction equation for BTEX components under aerobic conditions are shown in figure 1, for all BTEX components. C6H O2 6 CO2 + 3 H2O C7H8 + 9 O2 7 CO2 + 4 H2O C8H O2 8 CO2 + 5 H2O Figure 1. Benzene (C6H6), toluene (C7H8) and ethylbenzene/xylene (C8H10) degradation under aerobic conditions [3] Degradation under anaerobic conditions As mentioned at the start of this chapter, due to the prevalence of anaerobic groundwater conditions in Europe, anaerobic degradation of contaminants plays an important role in remediation of pollutants. In general, of the BTEX compounds, benzene is the most recalcitrant under anaerobic conditions and therefore often a distinction is made between benzene and the TEX components. In this paragraph a short summary of the possibilities for anaerobic degradation of benzene and other BTEX components is presented. Different micro-organisms are able to use different electron donors and electron acceptors for their energy balance. During the anaerobic degradation of BTEX, BTEX serves as an electron donor. Da Silva et al. [4] showed benzene degradation under laboratory conditions to occur under nitrate-, manganese-, iron- and sulphate-reducing conditions and under methanogenic conditions. Nitrate, manganese(iv), iron (III), sulphate and carbon dioxide can be used as terminal electron acceptors for the degradation of BTEX. Other compounds like humic substances and chlorate have also been mentioned in the literature as suitable terminal electron acceptors for the degradation of BTEX, but are only relevant in a few specific situations (humic substances) or as a stimulated bioremediation approach (chlorate). Degradation in the presence of humic substances and chlorate will thus not be discussed in this report.

10 NATURAL ATTENUATION OF BTEX 5 In the following paragraphs, the anaerobic degradation of BTEX with nitrate, manganese(iv), iron(iii), sulphate and carbon dioxide as electron acceptor will be shortly discussed and reaction equations are presented. More detailed information is included in the literature review (appendix A) Nitrate-reducing conditions Caldwell and Suflita (1999) [5] mention various references, stating that anaerobic benzene and TEX degradation has been shown conclusively under nitrate reducing conditions. As first intermediates of the degradation of benzene both toluene and phenol have been detected, this indicates that benzene degradation under nitrate reducing conditions can occur both via phenol and via toluene [6]. Phenol and toluene will degrade subsequently to, eventually, carbon dioxide, water and biomass. The reaction equations for BTEX components under nitrate reducing conditions are shown in figure 2, for all BTEX components. C6H6 + 6 NO H + 6 CO2 + 3 N2 + 6 H2O C7H NO H + 7 CO N H2O C8H NO H + 8 CO N H2O Figure 2. Benzene (C6H6), toluene (C7H8) and ethylbenzene/xylene (C8H10) degradation under nitrate reducing conditions [3] Manganese-reducing conditions Although manganese(iv) is not an electron acceptor that is ubiquitous in the environment, it can be of importance at specific sites. Anaerobic biodegradation of BTEX in general and benzene specifically with manganese(iv) has been reported in literature [7;8]. Research of Villatoro-Monzón et al. suggests that the degradation rate of benzene under manganese(iv) reducing conditions is significantly higher than under Fe(III) reducing conditions [9]. The reactions that occur under manganese(iv) reducing conditions are shown in figure 3, for all BTEX components. C6H MnO H + 6 CO Mn H2O C7H MnO H + 7 CO Mn H2O C8H MnO H + 8 CO Mn H2O Figure 3. Benzene (C6H6), toluene (C7H8) and ethylbenzene/xylene (C8H10) degradation under manganese reducing conditions [3].

11 NATURAL ATTENUATION OF BTEX Iron-reducing conditions Anaerobic benzene and TEX degradation has been shown conclusively to occur under Fe(III)-reducing conditions. In experiments conducted by Caldwell and Suflita [5] microbiological BTEX degradation was observed under iron-reducing conditions. The addition of iron(iii) -naturally present as immobilized iron in the soil matrix- to a methanogenic benzene contaminated soil stimulated the degradation of benzene [5]. Furthermore, Anderson and his team [10] observed benzene degradation in a petroleum-contaminated soil under iron-reducing conditions. The observation was done during laboratory experiments. The degradation occurred without a lag phase, indicating that degradation most likely also occurred in-situ. This petroleum-contaminated site was greatly enriched with Geobacteraceae [10]. Members of the Geobacteraceae have also been found at several other sites under iron(iii)-reducing conditions [11] and are known to completely oxidise aromatic compounds [10]. It is likely that these organisms play a role in the degradation of BTEX and/or intermediates. This process was also found to occur at various field locations. Results showed that when iron became depleted the degradation of benzene stopped [5]. Botton observed the production of phenol as an intermediate in the degradation of benzene under iron-reducing conditions [6]. For the degradation of BTEX under iron-reducing conditions bioavailable iron is needed, which is normally only a small percentage of the total iron present in the soil. The reactions that occur under iron reducing conditions are shown in figure 4, for all BTEX components. C6H Fe(OH) H + 6 CO Fe H2O C7H Fe(OH) H + 7 CO Fe H2O C8H Fe(OH) H + 8 CO Fe H2O Figure 4. Benzene (C6H6), toluene (C7H8) and ethylbenzene/xylene (C8H10) degradation under iron reducing conditions [3] Sulphate-reducing conditions Anaerobic degradation of BTEX has been demonstrated under sulphate-reducing conditions by Lovely and Caldwell [12] [5]. The addition of sulphate to a methanogenic benzene contaminated soil stimulated the degradation of BTEX, especially of benzene [13;14]. These observations are based on laboratory experiments using soil from a petroleum-contaminated aquifer. The results indicate that anaerobic benzene degradation may occur under field conditions under sulphate-reducing conditions. In three field studies performed within the Dutch soil remediation program SKB, sulphate injection was used to stimulate BTEX degradation. At all three sites evidence was found for anaerobic degradation of BTEX and at two of the sites a significant degradation (up to 90%) of BTEX was realised within 12 months [15].

12 NATURAL ATTENUATION OF BTEX 7 The reactions that occur under sulphate-reducing conditions are shown in figure 5, for all BTEX components. C6H SO H + 6 CO H2S + 3 H2O C7H SO H + 7 CO H2S + 87 H2O C8H SO H + 8 CO H2S + 5 H2O Figure 5. Benzene (C6H6), toluene (C7H8) and ethylbenzene/xylene (C8H10) degradation under sulphate reducing conditions [3] Methanogenic conditions Caldwell and Suflita [5] mention various references, stating that anaerobic benzene degradation has been demonstrated under methanogenic conditions. In soil from a petroleum-contaminated aquifer anaerobic degradation of benzene was recorded under methanogenic conditions without addition of any other electron acceptor [16]. However under methanogenic conditions BTEX degradation seems to occur at low degradation rates. The degradation reactions for the different BTEX compounds under methanogenic conditions are shown in figure 6. C6H H2O 2.25 CO CH4 C7H8 + 5 H2O 2.5 CO CH4 C8H H2O 2.75 CO CH4 Figure 6. Benzene (C6H6), toluene (C7H8) and ethylbenzene/xylene (C8H10) degradation under methanogenic conditions [3] Aspects that might influence anaerobic BTEX degradation Several researchers suggest that the presence of other electron donors like natural organic matter or co-contaminants like TPH, PAH or phenols can influence the degradation of BTEX by competition for electron acceptors or preferential degradation of these electron donors instead of BTEX. Also, the degradation of benzene can be limited by the preferential degradation of the other TEX components. Hunt et al. observed that benzene was degraded only after complete removal of xylenes and toluene [17]. Hunt et al., Edwards et al. and Langenhoff et al. mention the presence of TEX or naphtahlene as possible cause for the persistence of benzene in their research [18;17;19]. The extent to which benzene degradation is limited seems to be dependent on the prevailing redox conditions. Inhibition was found under nitrate- or sulphate reducing conditions, but not under iron(iii)-reducing conditions [20]. Besides inhibition of benzene degradation, the degradation of other organic contaminants will consume electron acceptors like nitrate or sulphate and will therefore influence the available oxidation capacity of groundwater for degradation of BTEX. This aspect is important for the sustainability and has to be taken into account.

13 NATURAL ATTENUATION OF BTEX Resume The above mentioned references indicate that anaerobic BTEX degradation occurs under various redox conditions. Degradation is unspecific with regard to the electron acceptor, although degradation rates vary under the different redox conditions and degradation of BTEX (and especially benzene) may be limited under methanogenic conditions. Also, the degradation rates for TEX components under methanogenic conditions are generally low compared to the degradation rates under nitrate-, iron- or sulphate-reducing conditions. Several micro-organisms that are involved in the anaerobic degradation of BTEX have been identified. Under different redox conditions different groups of microorganisms appear to be involved in the degradation of BTEX. Most likely more micro-organisms that are currently unknown can be involved in the degradation of BTEX components. Specific micro-organisms that can be used as indicator for BTEX or, more specifically, benzene degradation have not been identified yet. Natural attenuation of BTEX can be influenced by the presence of other compounds that can be used as electron donor by micro-organisms. These compounds will compete for available electron donors, therefore influencing the sustainability of the degradation of BTEX. Also, the degradation of benzene can be limited by the preferential degradation of the other BTEX components. When a NA approach for a BTEX contamination is foreseen, special emphasis has to be paid on the occurrence of benzene degradation Typical situation at BTEX contaminated sites As said, BTEX degradation can occur under different redox conditions. Degradation causes a depletion of electron acceptors. When degradation of BTEX occurs and the amount of BTEX and therefore the need for electron acceptors for degradation is high, various zones tend to arise (see figure 7). In the source zone with high concentrations of BTEX, all electron acceptors are depleted and methanogenic conditions are present (CH4). Downgradient of the source zone, conditions change gradually from methanogenic to the natural redox conditions. In the example in figure 7, the natural redox conditions are nitrate reducing and a transition zone with sulphate and iron-reducing conditions can be distinguished. Figure 7. Representation of redox conditions in a BTEX plume when nitrate reducing conditions prevail in the uncontaminated groundwater.

14 NATURAL ATTENUATION OF BTEX 9 If sustainable natural attenuation occurs, the life cycle of a BTEX plume can be separated in four different stages: initial expansion of the plume when leaching from the source zone and transport in the aquifer is greater than the biodegradation; a steady state in which the leaching from the source zone and transport is more or less equal to the biodegradation and further migration of the plume is prevented; a collapse of the plume when biodegradation exceeds the leaching of the source zone and the transport in the aquifer; exhaustion, when the leaching of the source zone to the plume in the aquifer stops and the plume contamination is degraded. For sustainable natural attenuation to occur it is important that the amount of electron acceptor available for the BTEX degrading micro-organisms exceeds their requirement. Furthermore, the use of electron acceptors for other processes like degradation of other contaminants has to be taken into account. In other words, the balance between electron donor (BTEX and possibly other contaminants) and electron acceptor determines whether the degradation process is sustainable. The supply of electron acceptor has to be sufficient to degrade the total load of contaminants and/or to meet the required amount necessary for the degradation of BTEX that may leach from a source zone and the degradation of the competing electron donors. In chapter 4, this aspect is explained and quantified.

15 TOOLS TO DETERMINE THE OCCURRENCE OF NATURAL ATTENTUATION TOOLS TO DETERMINE THE OCCURRENCE OF NATURAL ATTENUATION This chapter provides a brief overview of possible approaches to investigate the occurrence of natural attenuation of BTEX. The processes that are responsible for this natural attenuation and the monitoring techniques and tools that are available are explained. A more detailed description of these processes and tools can be found in the report of the NICOLE project Monitored Natural Attenuation: demonstration & review of the applicability of MNA at 8 field sites [1]. The occurrence of BTEX degradation can be determined by following three lines of evidence. These will be discussed separately in paragraphs 3.1, 3.2 and 3.4. Using these lines of evidence, the occurrence of natural attenuation of BTEX contamination can be shown Lines of evidence for natural attenuation of BTEX To evaluate the feasibility of MNA (Monitored natural attenuation) for a contaminated site several protocols and guidelines have been published. Although these protocols differ slightly, there is consensus on the general approach, the required data and the evaluation process. It is generally accepted that for implementation of MNA, the site owner needs to show conclusively that attenuation (degradation) occurs at rates sufficient to protect human health and the environment. The three lines of evidence (defined by the US EPA) can be applied to establish this: primary lines of evidence are data from historical groundwater and/or soil chemistry samples that demonstrate a clear and meaningful trend of decreasing contaminant mass and/or concentrations at appropriate monitoring or sampling points. Primary lines of evidence are used to determine whether plumes are shrinking, stable or expanding; secondary lines of evidence include data from the site characterization that indirectly demonstrate the type of natural attenuation processes active at the site and determine the rate at which such processes can reduce contaminant concentrations to required/acceptable levels. For example, the occurrence of biodegradation can be determined by measuring the levels of dissolved oxygen, nitrate, iron (II), sulphate, methane, carbon dioxide and other parameters and establishing the correlation between the BTEX concentrations and the electron acceptors and reduced products. Analyses for specific degradation products or compound-specific stable isotopes analyses are also considered as a second line of evidence; tertiary lines of evidence include data from field or laboratory microcosm studies (conducted in or with actual contaminated site samples) that directly demonstrate microbial activity in the soil or aquifer material and its ability to degrade the contaminants of concern. Also, detection of specific microorganisms involved in the degradation of contaminants can be used as a third line of evidence. In the following paragraphs the tools that can be used to obtain and interpret the data necessary for these three lines of evidence are explained in more detail, specifically regarding the BTEX components.

16 TOOLS TO DETERMINE THE OCCURRENCE OF NATURAL ATTENTUATION First line of evidence The first line of evidence for NA of BTEX is the evaluation of site-specific data of the BTEX concentrations over time. This requires a set of data from on site monitoring of BTEX concentrations in a time frame of at least several years. In general, a time series of 4 or more monitoring rounds with intervals of at least one year is preferred. The first line of evidence can be assessed at two different levels: overall interpretation of time series to demonstrate concentration trends (entire site); detailed analysis of spatial trends throughout the plume. In certain large contaminated areas various individual sources of contamination are present. These sources and their plumes cannot be assessed and presented at an individual basis therefore an overall assessment is conducted that is representative for the entire site and reflects the differences that exist between the various plumes. For an objective evaluation of the data several statistical tests are available. Examples of these tests are the Mann-Kendall and Mann-Whitney statistical test, developed by the State of Wisconsin, department of Natural Resources ( These tests identify the contaminant concentration trend in monitoring wells at the site as decreasing, stable, increasing or inconclusive Second line of evidence The second line of evidence consists of monitoring shift in redox conditions, detection of degradation products and isotope analyses of the contamination and its degradation products Shift in redox conditions For the microbial degradation of BTEX components micro-organisms use an electron acceptor. During degradation this electron acceptor is consumed and transformed into other chemical components. Degradation therefore results in a decrease in electron acceptor (like nitrate and sulphate) and an increase in metabolic products (like iron(ii), sulphide and methane). Mapping the redox conditions at a contaminated site and comparing the redox composition of uncontaminated groundwater with contaminated groundwater can be used as an evidence for the occurrence of degradation Detection of degradation products Detection of intermediates like (alkyl)phenols, benzoates and benzylsuccinate indicates the occurrence of an anaerobic BTEX degradation. At several sites the results of analyses of these degradation products indicate that the occurrence of specific (alkyl)phenols can be related to the composition of the BTEX contamination. For instance, the presence of toluene results in an increase in methylphenol concentrations and the presence of xylenes leads to an increase in dimethylphenol concentrations. Also, the anaerobic degradation of benzene leads to elevated levels of phenol at several contaminated sites. These intermediates however do not specifically indicate degradation of BTEX if (alkyl)phenols are also present as contamination at, for example, former gaswork sites or sites of the petrochemical industry.

17 TOOLS TO DETERMINE THE OCCURRENCE OF NATURAL ATTENTUATION 12 At one site in the Netherlands, the presence of (alkyl)phenols seemed to be indicative for limited BTEX degradation under methanogenic conditions. Under these conditions degradation of benzene occurred, but the degradation rate of intermediates (phenol) to finally CO2 was low leading to an accumulation of phenol to measurable concentrations. After addition of sulphate as a more suitable electron acceptor for the degradation of benzene, the degradation rate of benzene and phenol increased and phenol as intermediate product could not be measured under sulphate reducing conditions [15] Compound specific stable isotope analyses (CSIA) Organic compounds have an isotope ratio that is characteristic for that specific contamination. BTEX contaminants have a specific 13 C/ 12 C ratio (or 13 C) and a specific 2 H/ 1 H ratio (or 2 H). This ratio changes during biological degradation, because micro-organisms preferentially use the light isotopes. Due to biological degradation, the fraction of the heavy isotopes increases. The 13 C enrichment is greater under anaerobic conditions than under aerobic conditions. Non-biological processes like adsorption and dilution have no significant influence on 13 C or 2 H. The enrichment of heavier isotopes, called fractionation, can therefore be used to demonstrate biodegradation at a contaminated site. Analytical techniques are available to measure both the carbon and hydrogen ratios separately for each BTEX component. The advantage of measuring the 2 H is that for hydrogen the fractionation is much higher and can already be shown when only 30% degradation has occurred (compared to 60% degradation for 13 C). The disadvantage of the analyses for hydrogen isotopes is the higher detection limit. Compound specific isotope analyses can be used to show degradation by measuring several samples collected along the flow path of a groundwater plume. An increase in 13 C or 2 H of a specific compound (for instance benzene) is evidence for its biodegradation. The advantage of this approach is that evidence for biodegradation can be obtained in one monitoring round, because this approach considers the biodegradation process that has occurred during the time that the plume arose from the original source. However, to interpret the data and to calculate degradation rates detailed information about the direction and the rate of the groundwater flow is required. The interpretation can be hampered when several source zones are present or when contamination has occurred over a longer period of time. An increase in 13 C or 2 H of a specific compound in a specific monitoring well over time can be used as well. The advantage is that this method is not (or less) dependant on the origin of the contamination and the direction and rate of the groundwater flow. The disadvantage however is the need to collect several samples over a certain period of time. When the degradation rates are low many years are needed to demonstrate the occurrence of degradation. For the use and interpretation of CSIA in the quantification of in-situ biodegradation processes there are certain aspects that need to be kept in mind. These conditions are stated below: the degree of fractionation must be significant (in ); sampling in time or space is possible with concentrations that are above the detection levels (several tenths of µg/l); a relation between the source and the plume is established; it is important that the source of the contamination is known and homogeneous of composition.

18 TOOLS TO DETERMINE THE OCCURRENCE OF NATURAL ATTENTUATION Third line of evidence The third line of evidence includes evidence of degradation from field samples or laboratory microcosms through the monitoring of microbial activity. The third line of evidence is usually conducted in case no conclusive statement on the degradation of BTEX is possible using solely the first two lines of evidence Laboratory microcosms Laboratory microcosms (batch experiments) are conducted using soil and groundwater samples from the contaminated site. The experiment can be conducted under field conditions (intrinsic) or optimised conditions (stimulated) for the degradation. In this manner it is possible to observe whether degradation under field conditions occurs or whether the degradation is limited by a lack of nutrients or suitable electron acceptors Molecular detection of specific benzene degrading micro-organisms Detection of specific micro-organisms is possible using molecular detection techniques that target universal intracellular components like DNA, RNA and proteins. Anderson and his team [10] observed benzene degradation in a petroleumcontaminated soil under iron-reducing conditions and a correspondently enrichment with Geobacteraceae [10]. Members of the Geobacteraceae have also been found at several other sites under iron(iii)-reducing conditions [11]. This way, detection of (groups of) organisms can be used to support the occurrence of degradation of organic compounds under the prevailing redox conditions. Results from other studies show that the micro-organisms present at BTEX contaminated sites differ from site to site. Apart from differences between sites, differences in microbial diversity were also noted between the source, reference and plume areas of contaminated sites. So far no micro-organism or group of organisms has been identified that is always present at a site where BTEX degradation occurs. In other words, no indicatororganism has been identified for the anaerobic degradation of benzene or BTEX, as is the case for reductive dechlorination of chlorinated ethenes. Ongoing research in this field focuses on the identification of one (or a few) micro-organisms that are present if degradation of BTEX occurs. When identified, these organisms could be used as indicator organisms for BTEX degradation although the ability to degrade BTEX is widespread and none of them are obligates In-situ microcosms (Bac-trap) In-situ microcosms can be applied to demonstrate biodegradation in-situ. The microcosms (so-called bacterial traps or Bac-Trap ) are composed of carrier material with a high specific surface, coated with a labelled compound of interest (for instance 13 C benzene). These microcosms are placed in monitoring wells and are retrieved after a certain period of time. If degradation of the labelled compounds occurs, 13 C is incorporated in cell material. By specific analyses performed on biomass that is washed form the carrier material, the incorporation of 13 C in the biomass and therefore the actual in-situ biodegradation can be determined.

19 TOOLS TO DETERMINE THE OCCURRENCE OF NATURAL ATTENTUATION General approach NA feasibility study To evaluate NA at a contaminated site, a tiered approached is most common. The aim is to obtain a good estimation of the probability that NA is occurring, preferably as soon as possible and with as little expense as necessary [21]. Typically, the first line of evidence is the starting point. Site-specific data concerning the history of activities (possible accidents, spillages), contaminants (time series), and (geo)hydrology is collected and evaluated. Data gaps can be identified and a plan for additional monitoring activities, if necessary, can be developed. Usually, the second line of evidence is followed as well. Analyses for redox conditions and degradation products are common. CSIA can be considered when the degradation of the separate BTEX components has to be confirmed or when degradation of BTEX is expected to be slow. This can be the case if methanogenic conditions prevail or if the degradation of benzene can be influenced by the presence of other organic compounds (TEX, co-contaminants). The measurements and experiments mentioned in the third line of evidence can be used to obtain additional information. The laboratory microcosms are a good alternative for CSIA if the prerequisites for the performance and interpretation of CSIA (data) are not met. These indicate that the potential for biodegradation exist in the aquifer. Detection of specific benzene degrading micro-organisms is, however, until now not specific enough to be used as a line of evidence for the degradation of the individual BTEX components. This general approach complies with most protocols for the evaluation of NA [21] and the results of an SKB-project in which several measurements and experiments were tested and evaluated as possible lines of evidence for the degradation of benzene [22].

20 PROTOCOL TO DETERMINE THE SUSTAINABILITY OF NA OF BTEX PROTOCOL TO DETERMINE THE SUSTAINABILITY OF NA OF BTEX 4.1. Introduction Laboratory and field observations show that anaerobic degradation of BTEX can occur under all redox conditions (chapter 2). Site specific measurements, laboratory experiments and accompanying protocols are available to determine whether NA of BTEX has occurred or occurs at this moment (chapter 3). However, for implementation of a remediation approach that uses natural degradation processes, it is important that the degradation processes are sustainable, in other words also occur in the future. The rate of the degradation processes is the second important factor that determines the feasibility of NA as a remediation process. In general, NA rates have to be high(er) at high groundwater flow velocities or high (residual) )concentrations compared to sites with low groundwater flow velocities or low (residual) concentrations. In paragraph 4.2., a protocol to determine the sustainability of NA of BTEX components is presented. Also, in paragraph 4.3. a conceptual model is presented to determine if NA results in an acceptable situation (no further migration of the plume, no impact on vulnerable receptors) Sustainability of NA of BTEX The step by step protocol to determine the sustainability of natural attenuation processes for BTEX is based on the assumption that natural attenuation is sustainable if sufficient oxygen, nitrate, bioavailable manganese(iv), iron(iii) and/or sulphate is present or if the influx of oxygen, nitrate and/or sulphate is sufficient to compensate the consumption by biological degradation processes. If this is not the case, natural attenuation might still be possible (because degradation of BTEX is also possible under methanogenic conditions) but degradation rates are likely to be (much) lower and the risk profile of the application of NA is much higher. In such situations, a more intensive monitoring and a sound remediation plan with a contingency plan and well-defined decisionmaking process to implement the contingency plan is required.

21 PROTOCOL TO DETERMINE THE SUSTAINABILITY OF NA OF BTEX 16 In figure 8 a flowchart is shown in which the approach of this protocol is presented. yes Is NA occuring? use lines of evidence (chapter 3) no calculate acceptor:donor ratio in wells in source and plume and redox conditions in reference other remediation or risk management measures necessary scenario 1 reference: not methanogenic source: ratio > 1 plume: ratio >1 scenario 2 reference: not methanogenic source: ratio < 1 plume: ratio >1 scenario 3 reference: not methanogenic source: ratio < 1 plume: ratio <1 scenario 4 reference: methanogenic source: ratio < 1 plume: ratio <1 NA sustainable calculate indicative remediation time and/or plume lenght based on: influx of oxygen, nitrate and sulphate total load of BTEX bioavailable Mn(IV) and Fe(III) downstream NA critical use conceptual model to predict plume behaviour, determine if additional measures are necessary and to develop monitoring plan yes no use conceptual model to determine requirements met? which remediation target should be achieved with additional measures Figure 8. Flowchart to determine the sustainability of NA at a BTEX contaminated site. The protocol is based on step by step approach. In the first step the occurrence of NA is investigated using the lines of evidence. In the second step the results of the NA study are used to calculate the electron acceptor : electron donor ratio per well and to determine the redox conditions in the reference monitoring well. These data are usually already available from the first step and no additional field activities are necessary. Based on these calculations the site is allocated to one of the four scenarios mentioned in the flow chart. In the third step a monitoring plan is developed. Depending on which scenario represents the site, additional field activities or calculation may be necessary to determine the sustainability. More detailed information about the steps of the protocol and calculations that have to be performed is given in the subparagraphs below Step 1: determining degradation of BTEX In the first step of the sustainability protocol, the occurrence of BTEX degradation at the site is investigated using the lines of evidence explained in chapter 3. A detailed description of how to interpret the collected data is considered to be outside of the scope of this report, but can be found in several existing protocols [3].

22 PROTOCOL TO DETERMINE THE SUSTAINABILITY OF NA OF BTEX 17 If the evaluation with one or more lines of evidence provide sufficient data to prove the occurrence of natural attenuation of the BTEX contamination, proceed with step Step 2: calculation of the acceptor-donor ratio in individual monitoring wells The second step in the protocol to establish the sustainability of natural attenuation is to calculate the ratio between the amount of electron acceptor (oxygen, nitrate, sulphate, possibly co-contaminants) and electron donor (BTEX and possibly co-contaminants) in the groundwater of the individual monitoring wells in source and plume (appendix B). More detailed information concerning the role of co-contaminants and calculation methods is presented in appendix C. In this appendix, the monitoring results of an imaginary BTEX contaminated site without co-contaminants is used as illustration of the protocol. Natural organic matter (measured as TOC) is not taken into account. If oxygen, nitrate and/or sulphate are present simultaneously with TOC, the TOC present is not likely to be readily biodegradable and will therefore not compete with BTEX for available electron acceptors. Aerobic, nitrate-, manganese-, iron- or sulphate reducing conditions in the reference monitoring well (upstream of the source and plume) are considered as a first indication for sustainable natural attenuation, methanogenic conditions are not favourable. Because the information available in this stage is usually derived from groundwater analyses, the amount of iron(iii), which is also a suitable electron acceptor for biological degradation of BTEX, is not taken into account in this step yet. Iron(III) cannot be measured in the groundwater; to determine the amount of iron(iii) available for NA soil samples have to be collected and analysed. This can be necessary if the results of the initial screening are not favourable for sustainable natural attenuation (see paragraph ). BTEX in the groundwater is in equilibrium with BTEX adsorbed to the soil and a calculation based on the dissolved BTEX concentrations will result in an underestimation. However, for the first evaluation of the sustainability this calculation is considered sufficient. In table 2, the amount of oxygen, nitrate and/or sulphate (in mg) required to degrade 1 mg of the separate BTEX compounds is presented. These values are based on the reaction equations presented in figure 1, (degradation with oxygen), figure 2 (degradation with nitrate) and figure 5 (degradation with sulphate). Table 2. Amount of oxygen, nitrate and sulphate (mg or mole) required per mg or mole BTEX oxygen nitrate sulphate mg/mg mole/mole mg/mg mole/mole mg/mg mole/mole Benzene Toluene Ethylbenzene Xylene

23 PROTOCOL TO DETERMINE THE SUSTAINABILITY OF NA OF BTEX 18 The concentrations BTEX, possible co-contaminants, oxygen, nitrate and sulphate in each well are used to calculate the ratio between electron acceptor and electron donor in each contaminated monitoring well (see appendix C, also for cocontaminants that can serve as electron donor or -acceptor). For sustainable natural attenuation, an excess of electron acceptor has to be present. Therefore, a ratio of more than 1 is considered favourable, a ratio less than 1 is not favourable for sustainable natural attenuation (see table 3). Table 3. Score for the electron acceptor : electron donor ratio per well ratio electron acceptor : electron donor favourable for NA of BTEX? less than 1 more than 1 no yes Step 3: Assess prerequisites for sustainable NA at site scale In step 2, the ratio between the electron donor (BTEX) and the sum of the electron acceptors (oxygen, nitrate and sulphate) is calculated for each contaminated monitoring well. If co-contaminants that serve as electron donor or -acceptor are present, these have to be taken into account as well. The reference monitoring well is evaluated as favourable for NA when the conditions are not methanogenic. In step 3, these results are used to evaluate the spatial trends of this ratio at the site. The possible scenarios are presented in table 4. This table does not contain all possible combinations, but the combinations not mentioned in the table are not likely to occur at a site and are therefore not considered. If the results of monitoring wells in the reference area, the source zone or in the plume are not in accordance with each other, the score for these areas is made based on a majority agreement. Table 4. Different scenarios for at a given site scenario 1 scenario 2 scenario 3 scenario 4 reference area not methanogenic not methanogenic not methanogenic methanogenic source area more than 1 less than 1 less than 1 less than 1 plume area more than 1 more than 1 less than 1 less than 1 overall score favourable critical critical very critical Scenario 1 In scenario 1, the conditions in the reference area are aerobic to sulphate-reducing and the ratio between electron acceptor and electron donor is favourable in source and plume area. In this scenario, the amount of electron acceptors is sufficient to meet the demand for biological degradation of the BTEX components, and the conditions for sustainable natural attenuation are favourable. Whether NA actually results in an acceptable situation (no further migration of the plume, no impact on vulnerable objects) depends on degradation rates, groundwater flow rates and the vicinity of vulnerable objects. This can be determined using the conceptual model presented in paragraph 4.3.

24 PROTOCOL TO DETERMINE THE SUSTAINABILITY OF NA OF BTEX 19 Scenario 2 and 3 In scenario 2 and 3, the conditions in the uncontaminated groundwater are favourable for sustainable natural attenuation (everything but methanogenic) but the influx of these electron acceptors is not sufficient to meet the demand for degradation of the BTEX contamination. This results in an electron acceptor : electron donor ratio of less than 1 in the source zone (scenario 2) or in the source zone and plume zone (scenario 3). Usually, this results in methanogenic conditions in the source zone and (part of) the plume. In these scenarios three additional aspects are of importance: 1. the influx of oxygen, nitrate and sulphate with the uncontaminated groundwater (load per time unit) to calculate the load of BTEX degraded per year; 2. the total load of BTEX present at the site (calculated based on the dissolved and adsorbed concentrations) to calculate an indicative remediation time and related plume length; 3. the amount of bioavailable manganese (IV) and iron(iii) present in the uncontaminated area downstream of the plume. Influx of oxygen, nitrate and sulphate, amount of BTEX degraded per time unit The first aspect is of importance to determine what the amount of BTEX is that is degraded in the source zone per time unit. This calculation is based on the assumption that the load of nitrate and/or sulphate that is supplied to the source zone per time unit is completely used during the degradation of BTEX (and possible co-contaminants). This assumption is valid because in scenario 2 and 3 the conditions in the source zone are likely to be methanogenic. The influx of oxygen, nitrate and/or sulphate (kg/year) is calculated using the concentrations of these components in the reference monitoring well(s) in kg/l, the groundwater flow rate (m/year) and the area of the cross section of the source zone perpendicular to the groundwater flow direction. The amount of oxygen, nitrate or sulphate needed for degradation of 1 mg benzene, toluene or ethylbenzene/xylenes varies from 3.1 (oxygen) to 4.6 to 4.9 (nitrate or sulphate). Due to the limited solubility of oxygen in water (< 10 mg/l), the inflow of nitrate and sulphate usually forms the most important supply of electron acceptors and an ratio of 5 is considered safe and reasonable. A calculation with equation (1) and a ratio of 5 yields the load of BTEX degraded per time unit: (1) BTEX degraded (kg/year) = sum influx oxygen, nitrate and sulphate (kg/year) 5 Indicative remediation time and maximum expected plume length With the results of the calculation to determine the amount of BTEX degraded per time unit due to the influx of oxygen, nitrate and/or sulphate and the total load of BTEX present (aspect 2) an indication of the time needed to degrade the total load of BTEX present at the site can be calculated (equation 2). (2) total load of BTEX (kg) indicative remediation time (year) = load BTEX degraded (kg/year)

25 PROTOCOL TO DETERMINE THE SUSTAINABILITY OF NA OF BTEX 20 During this calculated indicative remediation time it is likely that the plume will extend due to the natural groundwater flow. If the groundwater flow rate is known, a first indication of the plume length can be estimated (equation 3): (3) indication of plume length (m) = groundwater flow rate (m/year) / R (-) indicative remediation time (year) with R the retardation factor (-) of the most mobile contaminant (benzene). Role of bioavailable manganese (IV) and iron(iii) downstream of the plume These initial calculations result an a first indication of plume length and remediation time to be expected. This is considered a worst-case calculation, because degradation in the source zone under methanogenic conditions (if applicable) is not taken into account. Also, contaminants will migrate to an area downstream of the current plume where bioavailable manganese (IV) and/or iron(iii) might be available. Manganese (IV) and/or iron(iii) are suitable electron acceptor for degradation of BTEX, and degradation of BTEX with manganese (IV) and/or iron(iii) will further reduce remediation time and plume length (the third aspect). The amount of bioavailable iron(iii) can be measured using soil samples from the uncontaminated area downstream of the plume and the Bioavailable Ferric Iron Assay (New Horizons Diagnostics, USA). This assay measures the amount of ferric iron (Fe 3+ ) in soil or sediment that can be reduced to ferrous iron (Fe 2+ ) by iron-reducing bacteria. The bioavailable Fe(III) content of soil or sediment samples can also be determined by means of chemical extraction. In one such technique, the sediment sample is homogenised and seperate samples tested for extractable Fe(II) (using 0.5M HCl), and extractable Fe(II) + Fe(III) (using 0.25M HCl M hydroxylamine hydrochloride). The bioavailable Fe(III) content is then calculated as the difference between the two extractions [23]. Research shows that the amount of bioavailable iron(iii) is typically 10-30% of the total amount of iron in a soil sample and this assumption can be used when the described assay is not available. Currently, no kit is available for the determination of bioavailable manganese (IV) and an assumption should be made based on the concentration of total manganese in a soil sample. The fraction of 10-30% mentioned for bioavailable iron(iii) should also be used to calculate the amount of bioavailable manganese (IV).

26 PROTOCOL TO DETERMINE THE SUSTAINABILITY OF NA OF BTEX 21 Summary scenario 2 and 3 The described additional measurements and calculations that are required for scenario 2 and 3 will result in a go/no go decision to continue with the conceptual model (paragraph 4.3) to determine the feasibility of natural attenuation as a remediation option. If the calculated indicative remediation time and/or maximum expected plume length are not acceptable or do not meet the requirements, natural attenuation is not feasible and active measures are necessary. Possible reasons for a no go decision are: - a vulnerable receptor is within the expected plume length; - the plume will migrate off-site; - time needed to reach remediation targets or a stable plume is too long. If this is not the case it is advised to continue with the conceptual model described in paragraph 4.3. Scenario 4 In the fourth scenario, the ratio between electron acceptor and electron donor is less than 1 in the source and plume area and the conditions in the reference monitoring well are methanogenic. This can occur in soils that are already strongly reduced. At these sites, there are no electron acceptors (nitrate, iron(iii) or sulphate) present that are favourable for degradation of BTEX. However, this does not mean that natural attenuation does not occur. Natural attenuation might still be possible (because degradation of BTEX is also possible under methanogenic conditions) but degradation rates are likely to be (much) lower and the risk profile of the application of NA is much higher. In such situations, a thorough NA investigation (see chapter 3) is required to determine the occurrence of BTEX degradation, with specific attention to the degradation of benzene. If degradation does occur and the rates are sufficient to reach an acceptable situation (see paragraph 4.3), natural attenuation under methanogenic conditions is expected to be sustainable because CO2 (the electron acceptor under methanogenic conditions) is most likely present in sufficient quantities. An intensive monitoring and a sound remediation plan with a contingency plan and well-defined decision-making process to implement the contingency plan is required Does sustainable natural attenuation of BTEX result in an acceptable situation? Conceptual model The first prerequisite to apply natural attenuation as a risk management approach is that degradation occurs. the second prerequisite is that the degradation is sustainable, in other words that it continues in the future. These aspects are explained in chapter 3 and paragraph 4.2, respectively. To determine if natural attenuation results in an acceptable situation (no further migration of the plume, no impact on vulnerable receptors) degradation rates, groundwater flow rates and the vicinity of vulnerable receptors have to be taken into account. In this paragraph a conceptual model to determine this aspect is presented.

27 PROTOCOL TO DETERMINE THE SUSTAINABILITY OF NA OF BTEX 22 The feasibility of NA of a BTEX contamination as a remediation approach depends on three aspects: the BTEX concentration present at a site; the BTEX degradation rate; the migration rate of the BTEX contamination. These three aspects, which can be quantified using field- and laboratory measurements, literature data and groundwater modelling, are closely related. This relationship between these aspects can be visualised using a three-axis graph, as shown in figure 9. Figure 9. Model illustrating the interaction between BTEX concentration, groundwater flow and degradation rate. The graph shows that at high BTEX concentrations, a high degradation rate (low half life time) and/or a low groundwater flow (and resulting slow migration rate) is needed to obtain an acceptable situation. At low BTEX concentrations, a longer half life time and/or a higher migration rate can still result in an acceptable situation. This interpretation can also be used if the migration rate or the half life time is taken as a starting point. For example: if the migration rate is high, the half life time has to be short and/or the BTEX concentration has to be low. At low migration rates, the BTEX concentration can be higher and/or the half life time longer. The same reasoning goes for the half life time as starting point.

28 PROTOCOL TO DETERMINE THE SUSTAINABILITY OF NA OF BTEX 23 As indicated, the three aspects can be quantified relatively easily. The concentrations of BTEX are determined using laboratory analyses on groundwater samples. The groundwater flow rate is calculated based on the permeability and porosity of the soil and the difference in groundwater table heights over the site (hydraulic gradient). Using the groundwater flow rate and the results of site specific organic carbon measurements and the resulting retardation factor, the migration rate of the BTEX contamination is calculated. If benzene is present, it is advised to use the characteristics of benzene to calculate the retardation factor because the retardation factor of benzene is low compared to the other BTEX components and benzene will therefore determine the migration rate of the contamination. The degradation rate of the different compounds can be derived from field data (trend evaluation, modelling) or can be quantified using the results of CSIA or bench scale feasibility studies (see chapter 3), although care should be taken when extrapolating laboratory degradation rates to field conditions. Site specific data are preferred, if these data are not available literature data or experiences at other (comparable) sites can be used as initial assumption. The prevailing redox conditions at the different areas at a contaminated site have to be taken into account, because degradation rates vary under different redox conditions. Degradation rates derived from trend analyses or CSIA are usually an average value over the plume. Bench scale feasibility studies with samples from the different redox zones in the plume (see figure 7) can be used to derive sitespecific data at the different conditions, but care has to be taken when translating the results to field conditions as temperature, mixing etc will influence the results. At least, a temperature correction should be used when the microcosms are incubated at room temperature. This correction can be made when assuming that degradation rates decrease with a factor 2 when the temperature decreases with 10 C. In table 5, degradation rates are presented for the degradation of the various BTEX compounds under different redox conditions (obtained from field and laboratory studies described in literature, collected by Suarez and Rifai [24]). Although these data are collected from publications before 1999, this review article is, to our best knowledge, the most comprehensive review to date. Table 5. First order degradation rate constants (day -1 ) for BTEX components under different redox conditions Suarez and Rifai [24] degradation rate constant (day -1 ) aerobic nitrate reducing iron reducing sulphate reducing methanogenic benzene n.c n.c. 1 toluene ethylbenzene n.r m-xylene o-xylene p-xylene : not calculated. Often recalcitrant under these conditions 2 : not reported, insufficient data

29 PROTOCOL TO DETERMINE THE SUSTAINABILITY OF NA OF BTEX Quantification of the conceptual model To quantify the combined effect of natural attenuation rate, migration rate and the concentrations of the contaminants that are present on plume development, a modelling exercise is necessary. In a model, these parameters are used as input and a plume prediction over time can be calculated and visualised. For relatively simple situations (scenario 1 and possibly scenario 4), indicative calculations or 2-dimensional models like Bioscreen ( or Webplume ( can be used. These models consider the degradation rate and migration rate constant over the plume. For more complex scenarios (scenario 2 and 3), a 3-dimensional model like Visual Modflow with Rt3D is advised. In these scenarios, different redox zones can be distinguished upstream, in the source and plume area and downstream, with resulting differences in degradation rates. These differences have to be incorporated in the model to obtain a reliable plume prediction, and 3-dimensinal models with multi-species reactive flow and transport simulation are necessary Use of conceptual model to define remediation target active measures The conceptual model can be used to determine the overall result of natural attenuation, contaminant migration and concentration of the contaminants (paragraph and ). Two results are possible: the current combination of the three aspects results in an acceptable situation, active measures are not necessary and a natural attenuation approach is feasible; the current combination of the three aspects does not result in an acceptable situation, for instance because the plume will migrate off-site, will reach a vulnerable receptor or the time needed for a natural attenuation approach is too long. If natural attenuation is not sufficient, active measures are needed. These measures are aimed at influencing the three aspects of the conceptual model. For instance, source excavation will decrease the concentrations of BTEX at the site, resulting in a favourable combination of the three aspects for natural attenuation of the residual contamination. Also, a stimulation of the biological degradation (for instance by injection of nitrate and/or sulphate) decreases the half life time which results in a favourable combination. A third option is to reduce the migration rate by redirecting uncontaminated groundwater or (geo)hydrological isolation measures. The conceptual model can be used to define the remediation targets for these active measures. If natural attenuation occurs, but the rate is not sufficient or the concentrations of BTEX are too high to reach an acceptable situation, active measures are necessary. However, it might not be necessary to remediate to very low levels. By using the conceptual model, the necessary remediation target of the active measures to reach an acceptable situation can be determined. By attuning the active measures to the natural attenuation capacity, a significant reduction of remediation costs can be obtained [25].

30 CHECKLIST CHECKLIST In addition to the protocol to determine the sustainability of natural attenuation of BTEX (paragraph 4.2) and the overall effect of natural attenuation (paragraph 4.3), a number of aspects must be closely observed (also in the future) in order to ensure the uninterrupted progress of natural attenuation. For example, active measures like groundwater extraction, compressed air injection and excavation of the source zone can influence the natural attenuation process at a site. For BTEX contaminants, this effect will usually be positive because it will reduce the concentration of the BTEX components, resulting in a favourable combination of the three aspects mentioned in chapter 4.3 and figure 9. It may also be the case that there is insufficient time to give NA an adequate opportunity despite the fact that the natural attenuation process is sustainable or because the reaction reservoir is restricted (adjacent sites), making NA inapplicable. An check list was formulated in order to highlight these aspects which, together with the explanation can be used to ascertain whether account must be taken of additional aspects at specific sites. The check list is included in table 9.

31 CHECKLIST 26 Table 9. Check list Attention point Technological Change in degradation conditions as a consequence of changes AT the site Are there or will there be other remediation measures applied at the site that may be influential? Can changes be expected in the redox conditions as a consequence of remediation activities? Has or is the temperature or acidity (ph) of the soil been or being changed? Have changes in the use of the site occurred recently or can they be expected? Change in degradation conditions as a consequence of changes OUTSIDE the site Can changes be expected in the redox conditions as a consequence of application of manure in agriculture or the inflow of less reduced groundwater? Can changes in the groundwater flow situation (direction, rate) be expected? Can groundwater be extracted in the vicinity? Risks Are the current human and ecological risks reduced to a sufficient degree? Have temporary safety measures been taken in relation to exposure? Definition of decontamination result Does the remediation aim to be achieved leave sufficient margin for the uncertainties related to NA? Is the reaction reservoir for NA defined or are there agreements concerning boundaries that should not be reached by the contamination? Organisational Financial Is the financial responsibility for the contamination and the NA process clear? Are there financial obstructions to the application of NA throughout the entire lifetime of the decontamination? Organisational opportunities for natural attenuation Is sufficient time available to give NA a chance? Can any conflicts in the planning at the authorities involved obstruct NA? Is the responsibility for the contamination clear in all stages of the remediation? Has an acceptable situation been achieved or will it be achieved? Do any changes to the standards and/or risk evaluation constitute an obstruction for NA? Do any potentially threatened receptors constitute an obstruction to NA? Are a contingency plan and the criteria for its implementation available? Change of regulatory authority Will the regulatory authority change in the near future, for instance to a different government body? Legal Adjacent sites Can damage claims relating to restrictions for adjacent sites be expected when NA is implemented? Has the responsibility for the remaining contamination been assigned in the property transfer deed? Are the restrictions regarding use clear and checked in terms of the (future) use of the site?

32 REFERENCE LIST REFERENCE LIST 1 Slenders, H., Langenhoff, A., Ballerstedt, H., Ter Meer, J., and Sinke, A Monitored Natural Attenuation: Demonstration and review of the applicability of MNA at 8 fiels sites, part 1: main report (draft). 2 Dijkhuis, J. E., van Bemmel, J. B. M., Henssen, M. J. C., and van Lotringen, R Methodiek voor het opstellen van de duurzaamheid van natuurlijke afbraak (D-NA) van gechloreerde ethenen. SKB SV-513, 3 Wiedemeier, T. H., Swanson, M. A., Moutoux, D. E., Gordon, K. E., Wilson, J. T., Wilson, B. H., Kampbell, D. H., Haas, P. E., Miller, R. N., Hansen, J. E., and Chapelle, F. H Technical protocol for evaluating natural attenuation of chlorinated solvents in ground water. EPA/600/R-98/128, 4 Da Silva, M. L., Ruiz-Aguilar, G. M., and Alvarez, P. J Enhanced anaerobic biodegradation of BTEX-ethanol mixtures in aquifer columns amended with sulfate, chelated ferric iron or nitrate. Biodegradation. 16,2: Caldwell, M. E., Tanner, R. S., and Suflita, J. M Microbial Metabolism of Benzene and the Oxidation of Ferrous Iron under Anaerobic Conditions: Implications for Bioremediation. anaerobe. 5,9: Botton, S Natural attenuation of BTEX in a polluted aquifer, give bugs a chance! 7 Chakraborty, R. and Coates, J. D Anaerobic degradation of monoaromatic hydrocarbons. Appl Microbiol Biotechnol. 64,4: Langenhoff, A. A. M., Brouwers-ceiler, D. L., Engelberting, J. H. L, Quist, J. J., Wolkenfelt, J. G. P. N., Zehnder, A. J. B., and Schraa, G Manganese reduction coupled to toluene oxidation. FEMS Microbiol Ecol. 22, Villatoro-Monzon, W. R., Mesta-Howard, A. M., and Razo-Flores, E Anaerobic biodegradation of BTEX using Mn(IV) and Fe(III) as alternative electron acceptors. Water Sci Technol. 48,6: Anderson, R. T., Gaw, C. V., Lovley, D. R., and Rooney-Varga, J. N Anaerobic Benzene Oxidation in the Fe(III) Reduction Zone of Petroleum-Contaminated Aquifers. Environ Sci Technol. 32,9: Lin, B., Van Verseveld, H. W., and Roling, W. F Microbial aspects of anaerobic BTEX degradation. Biomed Environ Sci. 15,2: Lovley, D. R., Coates, J. D., Woodward, J. C., and Phillips, E Benzene Oxidation Coupled to Sulfate Reduction. Appl Environ Microbiol. 61,3: Weiner, J. M. and Lovley, D. R Anaerobic benzene degradation in petroleum-contaminated aquifer sediments after inoculation with a benzene-oxidizing enrichment. Appl Environ Microbiol. 64,2:

33 REFERENCE LIST Weiner, J. M., Lauck, T. S., and Lovely, D. R Enhanced anaerobic benzene degradation with the addition of sulphate. Bioremediation Journal. 2,3&4: van Ras, N. J. P., van Bemmel, J. B. M., Dijkhuis, J. E., and Henssen, M. J. C In-situ benzeenafbraak onder sulfaatreducerende omstandigheden. SKB-project SV-604, 16 Weiner, J. M. and Lovley, D. R Rapid Benzene Degradation in Methanogenic Sediments from a Petroleum-Contaminated Aquifer. Appl Environ Microbiol. 64,5: Hunt, M. J., Beckman, M. A., Barlaz, M. A., and Borden, R. C Anaerobic BTEX degradation in laboratory microcosms and in situ columns. Intrinsic bioremediation Papers 3rd International in-situ and on-site bioreclamation symposium Edwards, E. A, Wills, L. E., Reinhard, M, and Grbic-Galic, D Anaerobic degradation of toluene and xylene by aquifer microorganisms under sulfate-reducing conditions. Appl Environ Microbiol. 58,3: Langenhoff, A. A. M., Schraa, G., and Zehnder, A. J. B Behaviour of toluene, benzene and naphtalene under anaerobic conditions in sediment columns. Biodegradation. 7, Nales, M., Butler, B. J., and Edwards, E. A Anaerobic benzene biodegradation: a microcosm survey. Bioremediation Journal. 2, Sinke, A Monitored Natural Attenuation, moving forward to consensus. Land contamination and reclamation. 9,1: Langenhoff, A. A. M. and van Ras, N. J. P Anaërobe afbraak van benzeen: het ultieme bewijs. SKB-project PT4120, 23 Lovely, D. R. and Phillips, E. J. P Rapid assay for microbially reducible ferric iron in aquatic sediments. Appl Environ Microbiol. 53, Suarez, M. P. and Rifai, H. S biodegradation rates for fuel hydrocarbons and chlorinated solvents in groundwater. Bioremediation Journal. 3,4: Henssen, M. J. C. and koopmans, M Stabiele eindsituatie wel of geen black-box? Haalbaarheid stabiele eindsituatie onderbouwd bij in-situ sanering Marconiplein. Bodem. 5,

34 APPENDICES Appendix A Literature review BTEX degradation, ongoing research Benzene occurs frequently as a ground and groundwater contamination, often together with other BTEX-compounds. Fields studies have shown that e.g. toluene, ethylbenzene and xylenes are relatively easily biodegraded under anaerobic conditions, but that it is more difficult to prove the anaerobic degradation of benzene (Davis et al 1999). Of all BTEX compounds, benzene is the most toxic, most water soluble (and thus mobile) and least degradable. It is the most critical compound in BTEX contaminations with respect to health risks, risks of dispersion and when it comes to containing the contamination in the long term. The main focus of this literature review is therefore the anaerobic degradation of benzene. Benzene and other BTEX compounds can be degraded readily under aerobic conditions, but it has long been thought that at least benzene was recalcitrant to anaerobic degradation. Over the past years, first indirect evidence accumulated that suggested benzene could be degraded anaerobically, but rather recently, this has been proven directly. During the anaerobic degradation of benzene and other TEX compounds, these compounds serves as an electron donor in the energy balance of micro-organisms. The electrons that are formed can be transferred to several electron acceptors, i.e. nitrate, iron(iii), sulphate or CO2. However, proving in situ anaerobic degradation of especially benzene remains difficult, and additional tools are needed to judge the situation and allow a firm statement on whether natural attenuation is occurring at a given location. As part of the development of a protocol on sustainable natural attenuation of BTEX contaminations, this document provides an overview of the state of the art of anaerobic benzene degradation. This overview is based on a review of the (scientific) literature published mainly since Lovley (2000) provided an overview of the knowledge of the subject developed until that year. A mini-review deals mainly with the possible degradation mechanisms of benzene (Coates et al 2002). Another mini-review involving mainly the micro-organisms involved in anaerobic benzene and related topics is provided by Chakraborty and Coates (2004). Anaerobic degradation pathways of benzene This paragraph provides an overview of the actual degradation pathway of benzene. It discusses the types of electron acceptors other than oxygen that can be used and the micro-organisms that have been associated with those reactions. Electron acceptors Benzene and the other TEX compounds can be degraded under anaerobic conditions by organisms using a wide variety of electron acceptors. Nitrate, manganese(iv), iron(iii), sulphate and carbon dioxide have been reported as terminal electron acceptors, while also the use of humic substances and chlorate have been mentioned. Oxidation of benzene was shown to occur together with sulphate reduction (Lovley et al 1994). In a petroleum-contaminated soil, benzene degradation was observed under iron reducing conditions. Although the observation was made in a laboratory, the degradation occurred without a lag, indicating that it also occurs in situ (Anderson et al 1998). The anaerobic degradation of benzene can be stimulated by addition of sulphate to a methanogenic benzene contaminated soil. The addition of iron(iii) and humic substances had similar effects (Weiner et al 1998). These observations are based on laboratory experiments using soil from a petroleum-contaminated aquifer.

35 APPENDICES They strongly indicate that anaerobic benzene degradation may occur under field conditions under iron-reducing and sulphate-reducing conditions. In soil from the same location, anaerobic degradation of benzene was also recorded under methanogenic conditions (Weiner and Lovley, 1998). Anaerobic biodegradation of BTEX in general and benzene specifically with manganese(iv) has been reported in literature (Coates et al, 2002, Villatoro-Monzón et al, 2003). Caldwell et al (1999) observed benzene degradation under iron-reducing conditions. They also found that, when iron became depleted, benzene degradation stopped. In these experiments, microbiological oxidation of Fe(II) to Fe(III) was observed, a process also found at various locations in the field. It is suggested that some reports of biodegradation of hydrocarbons under nitrate-reducing conditions may in fact be indirect through reoxidation of Fe(II). This is similar to the suggestion made elsewhere that humic substances may be used as an electron acceptor (Weiner and Lovley 1998) by a mechanism described a few years earlier (Lovley et al 1996). Caldwell et al (1999) mention various references, stating that anaerobic benzene degradation has been shown conclusively under nitrate-, Fe(III)- and sulphate-reducing conditions, as well as under methanogenic conditions. In an experimental in situ bioremediation of a BTEX contamination, the degradation of benzene was shown to occur when both nitrate and sulphate were injected as electron acceptors, but only towards the end of the experiment, indicating benzene was only degraded after all other electron donors had been consumed (Cunningham et al 2000, Cunningham et al 2001). In a study on sediment-free enrichment cultures that were capable of degrading benzene under anaerobic conditions, it was observed that these cultures could be maintained under nitrate-, Fe(III)- and sulphate-reducing conditions, as well as under methanogenic conditions. It was also observed that organisms could switch between electron acceptors, while maintaining benzene degradation (Ulrich and Edwards 2004). Furthermore, chlorate has been mentioned as a possible electron acceptor (Chakraborty and Coates 2004) and manganese(iv) was also shown to act as such (Villatoro-Monzón et al 2003). This indicates that anaerobic benzene degradation is very non-specific with regard to the electron acceptor. Da Silva et al (2005) also showed benzene degradation to occur under nitrate-, Fe(III)- and sulphate-reducing conditions in the laboratory. Degradation rates The rate of benzene degradation strongly depends on the terminal electron acceptor used. Most values reported concern depletion of benzene per volume and time or have not been related to biomass concentrations or initial benzene concentration. Laboratory values range from 1 to 75 µm/d. Burland and Edwards (1999) report benzene degradation rates in microcosms and transfer cultures ranging from 3.2 to 18.7 µm/day under nitrate reducing conditions. Chakraborty and Coates (2004) cite the degradation of benzene under nitrate-reducing conditions by Dechloromonas-strains RCB and JJ to amount to 160 µm in five days. The most comprehensive study was performed by Ulrich and Edwards (2004), who reported degradation rates in enrichment cultures to vary depending on the electron acceptor used between 1 and 75 µm/d. The highest degradation rates were reported for methanogenic conditions, the lowest for nitrate- and sulphate-reducing cultures. Davis et al (1999) reported half-lives for toluene under field conditions to be 110 to 260 days depending on the local circumstances, suggesting that in situ degradation rates of benzene are likely to be much lower than the laboratory values. Schreiber et al (2004) report first-order rate constants for benzene estimated form field trials and laboratory experiments to be negligible under nitrate-reducing conditions and highest for methanogenic conditions (0.009 day -1 ).

36 APPENDICES Micro-organisms Few anaerobic benzene degrading organisms have been identified or named. However, the number of unidentified or as yet unnamed organisms able to degrade benzene under anaerobic conditions is steadily increasing. In one study alone, thirteen different organisms were found, suggesting the ability to perform this conversion is not uncommon. A petroleum-contaminated site, where benzene degradation was observed under ironreducing conditions, was greatly enriched in Geobacteraceae (Anderson et al 1998). When it comes to species of bacteria able to degrade benzene, Geobacter is mentioned, as are the Desulfobacteriacea. Only two strains shown to be able to degrade benzene have been identified: the strains JJ and RCB of the genus Dechloromonas. In strain RCB, the degradation was coupled to the reduction of nitrate or chlorate (Chakraborty et al 2005, Coates et al 2002). Anaerobic benzene degrading sediment-free enrichment cultures have been obtained under iron reducing conditions (Jahn et al 2005). Specific groups of microorganisms appear to be involved in degradation under different redox conditions. Members of the Azoarcus/Thauera cluster perform BTEX degradation under denitrifying conditions, Geobacteraceae under Fe (III) reducing conditions and Desulfobacteriaceae under sulphate reducing conditions (Lin et al 2002). In a broad study on anaerobic sulphate-reducing micro-organisms, four clones of benzene degrading cultures clustered with Desulfobacter latus based on the dsrab gene. However, a 16S rrna-based phylogenetical analysis clustered three of these four with Desulfococcus (Pérez-Jiménez et al 2001). The micro-organisms used in the study by Ulrich and Edwards (2004) were not identified to species. Thirteen different clones were analysed. These clustered with various known Bacteria and Archea, depending on the electron acceptor used. Reaction mechanisms A few possible pathways have been suggested for the anaerobic degradation of benzene. These pathways involve initial carboxylation, hydroxylation of benzene to phenol or methylation to toluene with subsequent transformation to benzoate or benzoyl-coa. Benzoyl-CoA is further degraded and finally yields CO2 (Coates et al 2002, Chakraborty and Coates, 2004). In figure 1 the different pathways of benzene degradation are shown. As yet it is still unclear which pathway is preferred under different redox conditions. Figure 1. Possible pathways anaerobic benzene degradation (Chakraborty and Coates, 2004).

37 APPENDICES During degradation of benzene under methanogenic conditions, phenol, acetate and propionate were recorded as degradation intermediates (Weiner and Lovley 1998). In sulphate reducing enrichment cultures, obtained from a petroleum contaminated site, it was established that both phenol and benzoate are intermediates in the anaerobic degradation of benzene by using radio labelled [ 13 C-UL] benzene as a substrate. Benzoate was also found under iron-reducing and methanogenic conditions (Caldwell and Suflita 2000). Isotope fractionation studies suggest the anaerobic degradation of benzene involves an initial hydroxylation to phenol under both nitrate-reducing, sulphate-reducing and methanogenic conditions (Mancini et al 2003). Anaerobic benzene degradation by the nitrate-reducing Dechloromonas strain RCB was shown to involve an initial hydroxylation to phenol, followed by carboxylation and loss of the hydroxyl group to form benzoate. It is suggested this pathway is also used by other anaerobially benzene degrading organisms using other terminal electron acceptors (Chakraborty and Coates 2005), as phenol and benzoate, together with acetate and propionate have been found in several instances where anaerobic benzene degradation was proven or suspected to occur. Ulrich et al (2005) studied the formation of reaction intermediates of anaerobic benzene degradation in enrichment cultures. In both nitrate-reducing and methanogenic cultures, the results strongly suggest an initial methylation to toluene. Under methanogenic conditions phenol was transiently produced, indicating a pathway involving an initial hydroxylation to phenol. The further anaerobic mineralization of benzoate has been elucidated and is discussed by Carmona and Diaz (2005). Detection of anaerobic degradation of benzene To be able to monitor the anaerobic degradation of benzene, tools to detect this degradation are needed. These tools should be specific and reliable, i.e.: they should indicate anaerobic benzene degradation if it is present, and not if it is not present and should not detect anything but anaerobic benzene degradation. From what has become known about anaerobic benzene degradation over the past few years, several ways could be used for its in situ detection, although the actual in situ circumstances may complicate matters. Detection of anaerobic degradation of BTEX compounds can be done in several ways: 1. Direct monitoring of depletion of the BTEX-compounds; 2. Monitoring of metabolic intermediates of the degradation; 3. Monitoring of the compounds involved in the respiration (terminal electron acceptors (TEA) and reduced products); 4. Compound specific isotope analyses (CSIA); 5. Detection of micro-organisms involved in anaerobic degradation of BTEX compounds (indicative); 6. in-situ or laboratory microcosms. Direct monitoring of depletion of the BTEX-compounds It is usually not possible to judge the presence of anaerobic degradation in-situ. Processes like transport of the contamination through the soil often cause too large changes in the concentration of the compound of interest to be able to determine whether degradation is occurring or not (see e.g. Davis et al 1999). Monitoring of the depletion can be done in the laboratory on a field sample. This sample may or may not be amended with the compound of interest. In this case also the formation of metabolic end products and other compounds can be monitored in order to make a well founded judgement.

38 APPENDICES Monitoring of metabolic intermediates of the degradation The use of metabolic intermediates to detect biodegradation is also known as signature metabolites analysis (SMA). It is an elegant way to prove the occurrence of biodegradation, but needs knowledge of the metabolic intermediates occurring in the degradation pathways of a given compound. CSIA is a useful method for detecting biodegradation of benzene. Phenol may be detected as a reliable and specific indicator of anaerobic benzene degradation under certain circumstances, as may toluene, benzoate and several other compounds. No compound is available, however, that will under all circumstances prove anaerobic benzene degradation. Metabolic intermediates can provide valuable information if they fulfil a number of criteria. They should be (Young and Phelps 2005): formed during active biodegradation of the targeted compound; specific to the process being monitored; normally absent in unimpacted environments; water soluble for ease of sampling; biodegradable in order to discriminate between past and present biodegradation. This listing largely excludes intermediates that are made industrially. Griebler et al (2004) studied SMA for aromatic hydrocarbons in combination with compound-specific isotope analysis (CSIA). This latter technique uses the fact that biodegradation pathways may preferentially degrade one of two isotopes (C or H), although this preference is usually weak. If biodegradation occurs, a shift in the isotope fractions of the remaining contamination indicates the occurrence of biodegradation. With regard to the biodegradation of benzene, Griebler et al (2004) found CSIA useful. No intermediates for the degradation of benzene are mentioned. Mancini et al (2003) described CSIA for benzene degradation under anaerobic conditions. They found the differences for both carbon and hydrogen to be large. These differences may be used for the development of a detection protocol for the assessment of biodegradation. Beller (2000) considered the metabolic indicators for detecting anaerobic alkylbenzene degradation. He mentioned benzylsuccinates and E-phenylitaconates as detectable intermediates of the degradation of toluene. These compounds are specific for their source compound and are not manufactured, ruling out the possibility of an industrial source. Furthermore, a rapid analysis for the benzylsuccinates was developed (Beller 2002). Benzoate is also mentioned, but the industrial production of this compound restricts is use as a reliable indicator. The other compounds mentioned occur as intermediates in the toluene pathway and could also serve as indicators. However, toluene often occurs as a contaminant together with benzene. In these cases, the presence of neither toluene, nor benzoate can be used as an indication of benzene degradation as they are both compound occurring in the degradation route of toluene. In case toluene is not present as a contaminant, this compound and benzoate may be used as indicators. Under these circumstances, also benzylsuccinate may be used. Benzoate has been indicated as an intermediate in anaerobic benzene degradation (Caldwell and Suflita 2000). This was confirmed by Chakraborty and Coates (2005), who also indicated that the formation of phenol is the first step in anaerobic benzene degradation and that this compound occurs is sufficiently high concentrations to allow detection. These conclusions were also reached by Ulrich et al (2005) who indicated that phenol is only formed under methanogenic conditions. Benzoate is rather unspecific, as it is formed in both pathways. Phenol is more specific, as it does not as a rule occur as a contamination together with benzene and it is not formed as an intermediate in the degradation of TEX-compounds.

39 APPENDICES Monitoring of the compounds involved in the respiration (TEA and reduced products) Respiratory activity consumes and produces compounds, the nature of which is dependent on what type of electron acceptor is used. A major drawback to using this type of biomarkers is that there are various inorganic reactions that occur in the soil that may have a decisive influence on the conclusions that may be drawn from the measurement of the concentrations of certain compounds. Furthermore, they are very non-specific to the electron donor used. Accumulation of reduction products will reflect general biological activity, without specifying the electron donor consumed. Only if benzene is the sole electron donor present, then monitoring of electron acceptors may be useful to demonstrate benzene degradation. Maurer and Rittmann (2004a) provide a critical review of the use of biochemical footprints for the biodegradation of BTEX. They stress that several abiotic chemical reactions should be taken into consideration in order to track the footprints of biodegradation of BTEX. more in particular they mention five processes: 1. fermentation and methanogenesis should be considered separately: as the onset of methanogenesis needs products to have been produced. Fermentation may be running without methanogenesis being apparent; 2. precipitation and dissolution of calcite: these processes change the concentration of dissolved carbon dioxide, which is also formed as a result of biodegradation; 3. precipitation and dissolution of amorphous iron sulphide (FeS): iron(ii) may precipitate with sulfide, removing the evidence of iron reduction and/or sulphate reduction; 4. conversion of FeS into the thermodynamically more stable pyrite (FeS2) with loss of sulphide and abiotic formation of H2. These processes will influence the occurrence of dissolved sulphide and hydrogen gas, which may also result from biological degradation, and thus obscure their use as a footprint of biodegradation; 5. reductive dissolution of solid iron(iii) by oxidation of sulphide. This process produces reduced dissolved iron, which is also the product of biological iron reduction. The abiotic process thus complicating the use of iron(ii) as a biodegradation footprint. These processes are accounted for in a combined biological and chemical model (CBCmodel) for the biodegradation of BTEX (Maurer and Rittmann 2004b). This model uses many compounds, and acetate and hydrogen gas are included as footprints of biological degradation. However, more specific degradation products like benzoate or toluene are not included. Furthermore, no evaluation of the model has yet been published. It is thus not known how well it describes actual natural attenuation of BTEX. The processes mentioned do underline the caution with which certain footprints should be used. The rate of in situ biodegradation of BTEX is assumed to be dependent on the electron acceptor used. It is thus important to determine which terminal electron acceptor process (TEAP) is active at any given location. It may, however, be difficult to decide which TEAP is predominant, as two TEAPs may be active simultaneously (Schreiber et al 2004). Detection of micro-organisms involved in anaerobic degradation of BTEX compounds Various micro-organisms have been found to be able to degrade benzene under anaerobic conditions. Many of them have been characterized genetically. However, there is not one specific organism that is known to completely mineralise benzene, or that may otherwise be used as a universal indicator of anaerobic benzene degradation.

40 APPENDICES Geobacteraceae were greatly enriched in a iron reducing, benzene degrading soil, suggesting quantitative detection of this family may be a way to detect locations of anaerobic benzene degradation (Anderson et al 1998). Phylogenetic analyses of DNA sequences recovered from benzene-degrading sediments clustered with Geobactereacea known to be able to degrade aromatic compounds, while sequences from uncontaminated sediments did not. This could lead to the development of rapid assays indicating the presence of anaerobic benzene degrading potential of contaminated sites (Anderson et al 1998). Detection of the dissimilatory sulphite reductase (dsrab) genes has been performed among cultures feeding on various carbon sources including benzene (Pérez-Jiménez et al 2001). This gene is coupled to the presence of sulphate reducing micro-organisms. The gene was detected among various consortia degrading several substrates, including benzene. The bssa gene coding for the benzylsuccinate synthase was found to be most numerous in a aquifer column exhibiting the highest toluene degradation of a number of columns (Da Silva and Alvarez 2004). Given that toluene may occur as a degradation intermediate of benzene, this gene may possibly serve to establish in situ anaerobic benzene degradation. This, however, needs further study. The in situ microbial populations and population dynamics can be studied using a combination of a mesocosm and genetic detection techniques (Hendrickx et al 2005). Although this specific study did not relate the occurrence of specific organisms to the anaerobic degradation of benzene, this device described could very well be used to do so. References literature review Anderson, R T, Rooney-Varga, J N, Gaw, C V and Lovley, D R Anaerobic in the Fe(III) Reduction Zone of Petroleum-Contaminated Aquifers. Environmental Science and Technology 32: Anderson, R T, Rooney-Varga, J N, Gaw, C V and Lovley, D R Aromatic and Polyaromatic Hydrocarbon Degradation under Fe(III)-Reducing Conditions. Environmental Science and Technology 32: Beller, H R Metabolic indicators for detecting in situ anaerobic alkylbenzene degradation. Biodegradation 11: Beller, H R Analysis of Benzylsuccinates in Groundwater by Liquid Chromatography/Tandem Mass Spectrometry and Its Use for Monitoring In Situ BTEX Biodegradation. Environmental Science and Technology 36: Burland, S M and Edwards, E A Anaerobic Benzene Biodegradation Linked to Nitrate Reduction. Applied and Environmental Microbiology 65: Caldwell, M E, Tanner, R S and Suflita, J M Microbial metabolism of Benzene and the Oxidation of Ferrous Iron under Anaerobic Conditions: Implications for Bioremediation. Anaerobe 5: Caldwell, M E and Suflita, J M Detection of Phenol and Benzoate as Intermediates of Anaerobic Benzene Biodegradation under Different Terminal Electron-Accepting Conditions. Environmental Science and Technology 34: Carmona, M and Díaz, E MicroCommentary: Iron-reducing bacteria unravel novel strategies for the anaerobic catabolism of aromatic compounds. Molecular Microbiology 58: Chakraborty, R and Coates, J D Anaerobic degradation of monoaromatic hydrocarbons. Applied Microbiology and Biotechnology 64: Chakraborty, R and Coates, J D Hydroxylation and Carboxylation Two Crucial Steps of Anaerobic Benzene Degradation by Dechloromonas Strain RCB. Applied and Environmental Microbiology 71:

41 APPENDICES Chakraborty, R, O Connor, S M, Chan, E and Coates, J D Anaerobic Degradation of Benzene, Toluene, Ethylbenzene, and Xylene Compounds by Dechloromonas Strain RCB. Applied and Environmental Microbiology 71: Coates, J D, Chakraborty, R and McInerney, M J Anaerobic benzene degradation a new era. Research in Microbiology 153: Cunningham, J A, Hopkins, G D, Lebron, C and Reinhard, M Enhanced anaerobic bioremediation of groundwater contaminated by fuel hydrocarbons at Seal Beach, California. Biodegradation 11: Cunningham, J A, Rahme, H, Hopkins, G D, Lebron, C and Reinhard, M Enhanced In Situ Bioremediation of BTEX-Contaminated Groundwater by Combined Injection of Nitrate and Sulfate. Environmental Science and Technology 35: Da Silva, M L B and Alvarez, P J J Enhanced Anaerobic Biodegradation of Benzene- Toluene-Ethylbenzene-Xylene-Ethanol Mixtures in Bioaugmented Aquifer Columns. Applied and Environmental Microbiology 70: Da Silva, M L B, Ruiz-Aguilar, G M L and Alvarez, P J J Enhanced anaerobic biodegradation of BTEX-ethanol mixtures in aquifer columns amended with sulfate, chelated ferric iron, or nitrate. Biodegradation 16: Davis, G B, Barber, C, Power T R, Thierrin, J, Patterson, B M, Rayner, J L and Wu, Q The variability and intrinsic remediation of a BTEX plume in anaerobic sulphaterich groundwater. Journal of Contaminant Hydrology 36: Griebler, C, Safinowski, M, Vieth, A, Richnow, H H and Meckenstock, R U Combined Application of Stable Carbon Isotope Analysis and Specific Metabolites Determination for Assessing In Situ Degradation of Aromatic Hydrocarbons in a Tar Oil- Contaminated Aquifer. Environmental Science and Technology 38: Hendrickx, B, Dejonghe, W, Boënne, W, Brennerova, M, Cernik, M, Lederer, T, Bucheli- Witschel, M, Bastiaens, L, Verstraete, W, Top, E M, Diels, L and Sprinael, D Dynamics of an Oligotrophic Bacterial Aquifer Community during Contact with a Groundwater Plume Contaminated with Benzene, Toluene, Ethylbenzene, and Xylenes: an In Situ Mesocosm Study. Applied and Environmental Microbiology 71: Jahn, M K, Haderlein, S B and Meckenstock, R U Anaerobic degradation of Benzene, Toluene, Ethylbenzene, and o-xylene in Sediment-Free Iron-Reducing Enrichment Cultures. Applied and Environmental Microbiology 71: Lin, B, Van Verseveld, H W and Roling, W F Microbial aspects of anaerobic BTEX degradation. Biomedical and Environmental Science 15: Lovley, D R, Coates, J D, Woodward, J C and Phillips, E J P Benzene Oxidation Coupled to Sulfate Reduction. Applied and Environmental Microbiology 61: Lovley, D R, Coates, J D, Blunt-Harris, E L, Phillips, E J P and Woodward, J C Humic substances as electron acceptors for microbial respiration, Nature 382: Lovley, D R Anaerobic benzene degradation. Biodegradation 11: Mancini, S A, Ulrich, A C, Lacrampe-Couloume, G, Sleep, B, Edwards, E A and Sherwood Lollar, B Carbon and Hydrogen Isotopic Fractionation during Anaerobic Biodegradation of Benzene. Applied and Environmental Microbiology 69: Maurer, M and Rittmann, B E 2004a. Modelling intrinsic bioremediation for interpret observable biochemical footprints of BTEX biodegradation: the need for fermentation and abiotic chemical processes. Biodegradation 15: Maurer, M and Rittmann, B E 2004b. Formulation of the CBC-model for modelling the contaminants and footprints in natural attenuation of BTEX. Biodegradation 15: Pérez-Jiménez, J R, Young L Y, Kerkhof, L J Molecular characterization of sulfatereducing bacteria in anaerobic hydrocarbon-degrading consortia and pure cultures using the dissimilatory sulfite reductase (dsrab) genes. FEMS Microbiology Ecology 35:

42 APPENDICES Schreiber, M E, Carey, G R, Feinstein, D T and Bahr, J M Mechanisms of electron acceptor utilization: implications for stimulating anaerobic biodegradation. Journal of Contaminant Hydrology 73: Ulrich, A and Edwards, E A Physiological and molecular characterization of anaerobic benzene-degrading mixed cultures. Environmental Microbiology 5: Ulrich, A C, Beller, H R and Edwards, E A Metabolites Detected during Biodegradation of 13 C6-Benzene in Nitrate-Reducing and Methanogenic Enrichment Cultures. Environmental Science and Technology 39: Villatoro-Monzón, W R, Mesta-Howard, A M and Razo-Flores, E Anaerobic biodegradation of BTEX using Mn(IV) and Fe(III) as alternative electron acceptors. Water Science and Technology 48: Weiner, J M, Lauck, T S and Lovley, D R Enhanced Anaerobic Benzene Degradation with the Addition of Sulfate. Bioremediation Journal 2: Weiner, J M and Lovley, D R Rapid Benzene Degradation in Methanogenic Sediments from a Petroleum-Contaminated Aquifer. Applied and Environmental Microbiology 64: Young, L Y and Phelps, C D Metabolic Biomarkers for Monitoring in Situ Anaerobic Hydrocarbon Degradation. Environmental Health Perspectives 113:

43 APPENDICES Appendix B Sampling strategy and monitoring parameters Sampling points The sampling strategy to determine the occurrence and sustainability of NA of BTEX (and other contaminants) includes monitoring wells located upstream (reference), in the source, in the plume parallel and perpendicular to the direction of the groundwater flow (figure 1). A minimum of 6 to 8 monitoring wells need to be selected. The required number of monitoring wells is dependent on the soil structure, presence of one or more aquifers and the number of contamination sources. Figure 1. Schematic view of a plume and the monitoring strategy. Cross-section (above) and birds eye view (below); with monitoring wells in the source area (B), downstream in de plume area (P), in the plume area perpendicular to the flow direction (L) and upstream of the source area (R).