Assessing toluene biodegradation under temporally varying redox conditions in a fractured bedrock aquifer using stable isotope methods

Water Research 165 (2019) 114986

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Assessing toluene biodegradation under temporally varying redox conditions in a fractured bedrock aquifer using stable isotope methods Philipp Wanner a, *, 1, Ramon Aravena a, b, Jeremy Fernandes a, Michael BenIsrael c, Elizabeth A. Haack d, David T. Tsao e, Kari E. Dunfield c, Beth L. Parker a a

G360 Institute for Groundwater Research, College of Engineering and Physical Sciences, University of Guelph, 50 Stone Road East, Guelph, Ontario, N1G 2W1, Canada Department of Earth and Environmental Sciences, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, N2L 3G1, Canada c School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, Ontario, N1G 2W1, Canada d EcoMetrix Inc., 6800 Campobello Road, Mississauga, Ontario, L5N 2L8, Canada e BP Corporation North America Inc, Naperville, USA b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 May 2019 Received in revised form 9 August 2019 Accepted 13 August 2019 Available online 13 August 2019

In complex hydrogeological settings little is known about the extent of temporally varying redox conditions and their effect on aromatic hydrocarbon biodegradation. This study aims to assess the impact of changing redox conditions over time on aromatic hydrocarbon biodegradation in a fractured bedrock aquifer using stable isotope methods. To that end, four snapshots of highly spatio-temporally resolved contaminant and redox sensitive species concentrations, as well as stable isotope ratio profiles, were determined over a two-years time period in summer 2016, spring 2017, fall 2017 and summer 2018 in a toluene contaminated fractured bedrock aquifer. The concentration profiles of redox sensitive species and stable isotope ratio profiles for dissolved inorganic carbon (DIC) and sulfate (d13CDIC, d34SSO4, d18OSO4) revealed that the aquifer alternates between oxidising (spring 2017/summer 2018) and reducing conditions (summer 2016/fall 2017). This alternation was attributed to a stronger aquifer recharge with oxygen-rich meltwater in spring 2017/summer 2018 compared to summer 2016/fall 2017. The temporally varying redox conditions coincided with various extents of toluene biodegradation revealed by the different magnitude of heavy carbon (13C) and hydrogen (2H) isotope enrichment in toluene. This indicated that the extent of toluene biodegradation and its contribution to plume attenuation was controlled by the temporally changing redox conditions. The highest toluene biodegradation was observed in summer 2016, followed by spring 2017 and fall 2017, whereby these temporal changes in biodegradation occurred throughout the whole plume. Thus, under temporally varying recharge conditions both the core and the fringe of a contaminant plume can be replenished with terminal electron acceptors causing biodegradation in the whole plume and not only at its distal end as previously suggested by the plume fringe concept. Overall, this study highlights the importance of highly temporally resolved groundwater monitoring to capture temporally varying biodegradation rates and to accurately predict biodegradationinduced contaminant attenuation in fractured bedrock aquifers. © 2019 Elsevier Ltd. All rights reserved.

Keywords: Fractured sedimentary rock Aromatic hydrocarbons Stable isotope methods Redox conditions Biodegradation Plume attenuation

1. Introduction Aromatic hydrocarbons such as benzene, toluene, ethylbenzene, and xylene (BTEX) are major constituents of gasoline, jet fuel and

* Corresponding author. E-mail address: [email protected] (P. Wanner). 1 Current address: Institute of Geological Sciences, University of Bern, Baltzerstrasse 1e3, 3012, Bern, Switzerland. https://doi.org/10.1016/j.watres.2019.114986 0043-1354/© 2019 Elsevier Ltd. All rights reserved.

other oil products (Farhadian et al., 2008; Seagren and Becker, 2002). BTEX are often stored as light non-aqueous phase liquids (LNAPLs) in above or underground tanks for residential and industrial purposes (Bedient et al., 1999; Holliger et al., 1997). Due to the frequent leakage of the storage tanks and their transfer lines, BTEX compounds have become common subsurface contaminants (Christophersen et al., 2005; Tabani et al., 2016). When BTEX compounds are released as LNAPLs, they migrate through the vadose zone and are partially volatilized forming a vapour plume (BenIsrael et al., 2019; Rivett et al., 2011). If larger quantities are

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released, BTEX compounds migrate to greater depths reaching the groundwater on which they float due to the lower density of LNAPLs compared to water (Wiedemeier et al., 1999). While floating on the groundwater, BTEX compounds are slowly dissolved forming a persistent shallow groundwater contaminant plume (Kim and Corapcioglu, 2003). Under fluctuating water table conditions, BTEX compounds are distributed over a “smear zone”, whereby the contaminants adsorb to the aquifer material in the capillary fringe and are subsequently volatilized contributing to the vapour plume formation in the unsaturated zone (Lahvis et al., 1999). It has been demonstrated that conventional remediation approaches such as pump and treat or excavation techniques often do not lead to a full clean-up of BTEX contaminated sites (Wiedemeier et al., 1999). Alternatively, monitored natural attenuation (MNA) is accepted to manage contaminated BTEX sites (Scow and Hicks, 2005). An important prerequisite for MNA is the natural depletion of the source zone and the occurrence of appropriate microbes and redox conditions allowing contaminant biodegradation, which creates steady-state conditions of the organic contaminant plume (Chapelle, 1999; Parker et al., 2019). Biodegradation of BTEX compounds mainly occurs through microbial oxidation, whereby microorganisms use several terminal electron acceptors including oxygen (O2), nitrate (NO
3 ), manganese (Mn4þ), ferric iron (Fe3þ), sulfate (SO2
4 ) and CO2, which differ in their redox potential (energy yield per unit of oxidised organic carbon). Previous conceptual models assumed that in BTEX plumes, a spatial redox zonation occurs with methanogenic conditions close to the source zone and progressively less reducing conditions in horizontal and vertical transverse and in longitudinal direction: sulfate reducing, followed by manganese, ferric iron, nitrate reducing and eventually aerobic conditions (Christensen et al., 2000; Lovley, 2003; Wiedemeier et al., 1999). More recently it became evident that the redox zonation concept is not adequate and that the plume fringe conceptual model is superior to describe redox conditions and biodegradation in BTEX plumes (Anneser et al., 2010; Meckenstock et al., 2015). The plume fringe conceptual model suggests that terminal electron acceptors are depleted in the BTEX plume core and that BTEX biodegradation under aerobic nitrate, iron, manganese, and sulfate reducing conditions only occurs at the plume fringe, where the terminal electron acceptors are replenished due to mixing processes between contaminated and uncontaminated groundwater. However, despite of the improved conceptual knowledge about the spatial evolution of redox conditions, the extent of temporal variations of redox conditions and their effect on BTEX biodegradation in groundwater is still poorly understood mainly due to the lack of field data at adequate temporal resolution. It remains unclear whether changing groundwater redox conditions can cause terminal electron acceptor replenishment in the plume core and thus, influencing BTEX biodegradation in the entire plume and not only at the at the distal end as postulated by the plume fringe concept. Furthermore, it is unknown how temporal redox variability affects the BTEX plume behaviour, such as steady state conditions and thus, if MNA is appropriate for managing BTEX contaminated sites under these conditions. Stable isotope methods have been established as an effective tool to track reactive transformation processes affecting organic and inorganic species (Aelion et al., 2010; Basu et al., 2014; Caschetto et al., 2017; Hunkeler et al., 2008; Wanner et al., 2016, 2018). The method makes use of the preferential cleavage of bonds between light compared to heavy isotopes, leading to an enrichment of heavy isotopes in the parent compared to the daughter compound. For organic contaminants such as BTEX, stable isotope methods were commonly applied to a single element (e.g. C). However, recent studies revealed the benefit of a multi-element

approach (e.g. C, H) to identify different biodegradation conditions and pathways (Badin et al., 2014; Palau et al., 2014, 2017; Wanner et al., 2017). This study aims to a) assess temporally varying redox conditions in a complex fractured rock aquifer and b) evaluate whether multielement stable isotope measurements on BTEX (d13C, d2H) combined with stable isotope analysis on dissolved inorganic carbon (d13CDIC) and sulfate (d34SSO4, d18OSO4) can be used to assess the impact of changing redox conditions over time on BTEX biodegradation. To that end, a site was selected where a fractured bedrock aquifer was contaminated by toluene decades ago, sustaining a shallow groundwater plume. Almost 30 years after the first contamination occurred, multiple high-resolution redox sensitive species concentration and d13CDIC, d34SSO4 and d18OSO4 profiles as well as toluene concentration and toluene-specific multi-element stable isotope ratios (d13CToluene, d2HToluene) profiles were measured in the fractured bedrock aquifer. The profiles were determined in four snapshots at different distances from the toluene contamination source over a time period of two years (June 2016 to June 2018). The highly resolved temporal groundwater measurements provide novel insight into varying redox conditions in a shallow fractured rock aquifer and their effect on toluene biodegradation. These investigations are important for evaluating whether contaminant plumes also reach steady state conditions in fractured rock aquifers when redox conditions change over time and if MNA can be used as a remediation strategy under such conditions for managing contaminated sites. 2. Material and methods 2.1. Study site The investigated site is a historical manufacturing facility located in southwestern Ontario, Canada, which operated between 1952 and 1990. For the manufacturing processes, toluene was used as a solvent and was stored in several above ground tanks connected to the facility by buried supply lines (Fig. 1A). The geology of the site consists of a 2.2 m thick overburden overlying an approximately 55 m thick sequence of Silurian-era, fractured dolostone aquifer (Fig. 1B). The fractured dolostone bedrock can be divided into three hydrogeological units (HGU) based on their vertical hydraulic conductivity (Fernandes, 2017). The shallowest unit (HGU 1) has a thickness of 4.8 m and shows a poor vertical conductivity to the underlying HGUs, whereas the middle and bottom HGUs (HGU 2 and 3) span larger vertical intervals (9.0 and 8.0 m) and show moderate vertical hydraulic conductivities (Fig. 1B). During the site investigation period, the groundwater table was shallowest in spring (~2.1 m below ground surface, mbgs) and became gradually deeper during summer and fall (~3.2 mbgs) indicating that the fractured bedrock aquifer was mainly recharged in spring during snowmelt (Fig. 2). Groundwater flows in the northern direction with a velocity ranging between 0.01 and 3.0 m/day (Fig. 1A) (Fernandes, 2017). The fractured bedrock aquifer was contaminated by toluene in the early stages of facility operation due to the leakage of the supply lines connecting the above ground storage tanks to the facility. In 2015 the toluene contamination of the site was further characterized in detail using the discrete fracture network (DFN) approach developed by Parker et al. (2012). These investigations identified the primary contamination source in the north-eastern corner of the site, which consists of toluene as residual LNAPL in the shallow bedrock in the zone of water table fluctuations (Fig. 1A). Dissolution from the residual toluene LNAPL sustains a shallow groundwater toluene plume in the main direction of groundwater flow to the north. To monitor the temporal evolution of the toluene groundwater plume, 11 groundwater

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Fig. 1. A) Map of the toluene contaminated site including the locations of the multilevel wells along the two transects (A-A0 and BeB’; black circles and dark red line), the former above ground storage tank locations (grey shaded rectangles), the supply lines of the storage tanks (black lines), range in groundwater flow directions (blue arrows) and the toluene LNAPL source zone (red shaded area). B) Cross-section along transect A-A0 , which is also representative for transect BeB0 , showing the site subsurface hydrogeology including the three different hydrogeological units (HGU 1e3) and completion depths for the multilevel systems used for the high spatio-temporal resolution sampling. The continuous blue line indicates the highest water table (spring 2017/summer 2018) and the dashed blue line represents the lowest water table (summer 2016/fall 2017) during the sampling period. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2. Temporal evolution of precipitation (blue bars: rain, black bars: snow) and water table (open blue squares) at the investigated site before, during and after the conducted sampling campaigns that were carried out in June 2016, April 2017, September 2017 and June 2018 (green shaded areas). The precipitation data was obtained from a weather station located closely (~35 km) to the investigated site (Government of Canada, 2019). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

multilevel monitoring systems (MLS) were installed at different distances from the toluene source along two transects, A-A0 and BeB’ (Fig. 1A). Each of the multilevel wells contains 6 to 8 depthdiscrete groundwater sampling ports over a depth interval of 2.44e21.49 mbgs resulting in 73 ports total. 2.2. Groundwater sampling A detailed description of the groundwater sampling procedure can be found in section 1.1. of the Supporting Information (SI) In brief, groundwater was sampled from all 73 ports in June (summer) 2016, April (spring) 2017, September (fall) 2017 and June (summer) 2018 (Table S1, SI) using a Geopump® peristaltic pump with dedicated 0.64 cm OD diameter Teflon® tubing. After measuring the field parameters (pH, dissolved oxygen (DO), and temperature (T)), groundwater samples for concentration analysis of redox

sensitive species (Fe, Mn, SO2
4 , NO3 , CH4), HCO3 and dissolved inorganic carbon (DIC) were collected in 60 mL plastic containers.

Subsequently, groundwater samples for stable oxygen and sulfur isotope analysis of sulfate (d18OSO4 and d34SSO4) and for stable carbon isotope analysis of DIC (d13CDIC) were taken in 1 L plastic bottles. Furthermore, groundwater samples for toluene concentration and compound-specific carbon and hydrogen isotope analyses (d13CToluene, d2HToluene) were collected in 40 mL (VOA) glass vials without headspace. 2.3. Concentration and stable isotope analysis Detailed descriptions of analytical methods are available in section 1.1. of the SI. Briefly, the concentrations of redox sensitive

species (Fe, Mn, SO2
4 , NO3 ) and bicarbonate (HCO3 ) were measured in groundwater samples from all sampling campaigns (Table S1, SI) using a liquid ion chromatograph coupled to a mass spectrometer (IC-MS). Methane concentrations were analysed in groundwater samples from spring 2017, fall 2017 and summer 2018 (Table S1, SI) using a Varian 3800 gas chromatograph (GC) equipped

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with a flame ionization detector at 250  C. Toluene concentration analysis was conducted on groundwater samples from all sampling campaigns (Table S1, SI) using an Agilent 7890A GC equipped with a DB-VRX column (20 m; 0.18 mm ID; 1 mm film thickness). Toluenespecific carbon and hydrogen isotope analysis (d13CToluene, d2HToluene) was carried out on the groundwater samples taken in summer 2016, spring 2017 and fall 2017 (Table S1, SI) using a TRACETM GC coupled to a DeltaPlus XP isotope ratio mass spectrometer (IRMS) via a combustion III interface (Thermo Finnigan; Germany). Stable sulfur and oxygen isotope ratio analysis of sulfate (d18OSO4 and d34SSO4) was performed on the groundwater samples that were obtained in summer 2016 and fall 2017 (Table S1, SI). The sulfur isotope ratios of sulphate (d34SSO4) were analysed using a Carlo Erba Elemental Analyser (EA) coupled to a Finnigan Delta IRMS and the oxygen isotope ratios of sulfate (d18OSO4) were determined with an Isochrom IRMS Micromass coupled to the EA through a combustion interface. The stable carbon isotope ratios of DIC (d13CDIC) were also determined in the groundwater samples taken in summer 2016 and fall 2017 (Table S1, SI) and analysed with an Agilent 6990 GC coupled to an Isochrom (Micromass UK) IRMS. 3. Results and discussion 3.1. Toluene concentration data The organic contaminant concentration measurements in the multilevel wells revealed that toluene is the only groundwater contaminant at the site (Fig. 3). The highest toluene concentration

was detected in the multilevel wells located closest down gradient to the source zone (M29, M21, M28, M22; Fig. 1) during all four sampling campaigns (summer 2016, spring 2017, fall 2017, summer 2018). In these wells, the peak toluene concentrations occurred at shallow depth (~312.5 m above sea level, masl) close to the bedrock-overburden interface with concentrations ranging from 74,000 (M21) up to 571,200 mg/L (M29) (Fig. 3) being close to the toluene solubility limit of 632,000 mg/L (Polak and Lu, 1973). Closer to the surface (~313.0 masl) and with increasing depth, the toluene concentrations strongly decreased to below 15,000 mg/L (Fig. 3). During low water table conditions in summer 2016/fall 2017 the high toluene concentration zone at ~312.5 masl was partly exposed to the unsaturated zone. This led to toluene volatilization and to an increased toluene concentration in the unsaturated zone in summer 2016 and fall 2017 (BenIsrael et al., 2019). The observations of the highest toluene concentration at ~312.5 masl and the steep vertical concentration gradients are consistent with the low vertical hydraulic conductivity of the fractured bedrock aquifer and the conceptual assumption that the toluene LNAPL in the source zone floats on the water table continuously dissolving and sustaining a shallow groundwater plume. In addition to the vertical variability, a temporal change of toluene concentration was observed. In the most contaminated wells, the peak toluene concentration at shallow depth was three times lower in fall 2017 compared to the other sampling campaigns (summer 2016, spring 2017, summer 2018; Fig. 3). At greater depth (<309 masl) a stronger temporal toluene concentration variation, by several orders of magnitude, was detected from 0.19 to 1410 mg/L during the different sampling

Fig. 3. Toluene concentrations with depth on a logarithmic scale in the most contaminated multilevel wells (M29, M21, M28, M22) along cross section A-A0 and BeB0 in summer 2016 (blue squares), spring 2017 (green triangles), fall 2017 (black circles) and summer 2018 (red crosses). Continuous blue lines indicate highest water table (spring 2017/summer 2018) and dashed blue lines represent lowest water table (summer 2016/fall 2017) during the sampling period. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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campaigns. However, the toluene concentrations at greater depth were not consistently lower or higher during a particular sampling campaign (Fig. 3). With increasing horizontal transverse distance (5e22 m) from the most contaminated wells (M21, M22, M28, M29), the peak toluene concentrations decreased by five orders of magnitude to below 5 mg/L in M24, M25, and M26 and below detection limit in M20 and M30 during all four sampling campaigns (Fernandes, 2017). A large toluene decrease was also observed in the direction of groundwater flow, whereby the toluene concentrations in M23, located at 40 m distance from the source zone, also declined by five orders of magnitude to below 5 mg/L (Fernandes, 2017). Based on the spatial plume delineation, the locations of the multilevel monitoring wells can be divided into plume core (M21, M22, M28 M29), plume fringe (M23, M24, M25, M27) and non-impacted wells (M20, M30; Table S2, SI). 3.2. Redox sensitive species and bicarbonate concentration data The rapidly decreasing toluene concentrations within a few tens of meters in the horizontal and vertical transverse and longitudinal directions from the source zone indicate that the migration of the plume is strongly attenuated, likely due to a combination of physical (e.g sorption and matrix diffusion) and reactive processes. To evaluate to what extent reactive processes contribute to the strong attenuation of the toluene plume, it was investigated whether redox conditions were favourable for toluene biodegradation. Moreover, it was examined whether HCO
3 , representing the toluene biodegradation product under various redox conditions (aerobic, nitrate, manganese, iron and sulfate reducing), is detected at high concentrations. Among the redox sensitive species, nitrate was only detected in a few ports of

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the multilevel monitoring systems (data not shown), while O2, Fe,
Mn, SO2
4 , CH4 and HCO3 were detected across the site (Figs. 4 and 5). Only small changes in concentrations of redox sensitive species were observed between the transects A-A0 and BeB’ in the horizontal and vertical transverse and longitudinal directions relative to groundwater flow in each of the four sampling campaigns (Figs. 4 and 5). This reveals the absence of a longitudinal and transverse redox zonation in the toluene plume being in agreement with the plume fringe conceptual model (Meckenstock et al., 2015). In contrast, a temporal variation of the redox conditions was observed, especially pronounced for the O2, Fe and Mn concentrations. In summer 2016/fall 2017, elevated Fe and Mn concentrations up to 7500 and 330 mg/L, respectively and generally low O2 concentrations (<1.57 mg/L) were measured (Fig. 4). The high Fe and Mn concentrations likely occurred due to the reduction of iron and manganese bearing minerals as reduced iron and manganese (Fe2þ, Mn2þ) are much more soluble compared to their oxidised equivalents (Fe3þ, Mn4þ). The peak Fe and Mn concentrations occurred at shallow depth (~312.5 masl) in the high toluene concentration zone, whereas with increasing depth, Fe and Mn concentrations declined to concentrations below 700 and 70 mg/L, respectively at 305.0 masl (Fig. 4B, C, E and F). The coincidence of the elevated Fe, Mn concentrations with the high toluene concentrations at shallow depth (~312.5 masl) suggests that Fe and Mn reduction is related with toluene biodegradation in summer 2016/fall 2017 at shallow depth. The supposed occurrence of toluene biodegradation at shallow depth is further substantiated by the high HCO
3 concentrations at ~312.5 masl in the plume core and fringe wells, reaching concentrations up to 510 mg/L, while with increasing depth, the HCO
3 concentrations dropped to below 320 mg/L in both transects (Fig. 5C and F). High

Fig. 4. Concentration profiles of O2, Fe and Mn in multilevel wells M21, M27 and M30 along cross sections A-A’ (AeC) and in multilevel wells M28, M23 and M20 along cross section BeB0 (DeF) in summer 2016 (blue squares), spring 2017 (green triangles), fall 2017 (black circles) and summer 2018 (red crosses). The multilevel wells shown are representative for the plume core (M21, M28), plume fringe (M27, M23) and non-impacted groundwater (M20, M30). Note that Fe and Mn concentrations are plotted on a logarithmic scale. Continuous blue lines indicate highest water table (spring 2017/summer 2018) and dashed blue lines represent lowest water table (summer 2016/fall 2017) conditions during the sampling period. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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Fig. 5. Concentration profiles of SO2
4 , CH4 and HCO3 in the multilevel wells M21, M27 and M30 along cross sections A-A’ (AeC) and in multilevel wells M28, M23 and M20 along cross section BeB0 (DeF) in summer 2016 (blue squares), spring 2017 (green triangles), fall 2017 (black circles) and summer 2018 (red crosses). The multilevel wells shown are representative for the plume core (M21, M28), plume fringe (M27, M23) and non-impacted groundwater (M20, M30). Note that SO2
4 concentrations are plotted on a logarithmic scale. Continuous blue lines indicate highest (spring 2017/summer 2018) and dashed blue lines represent lowest water table (summer 2016/fall 2017) conditions during the sampling period. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fe and Mn and low O2 concentrations were, however, also detected in the non-impacted wells (M20, M30; Fig. 4). This indicates that iron and manganese reduction in summer 2016/fall 2017 is not only related with the toluene plume but also with the little precipitation in summer 2016/fall 2017 (Fig. 2), causing low recharge with oxygen rich water and generally more reducing groundwater conditions. In contrast to summer 2016/fall 2017, much lower Fe and Mn concentrations (below 0.13 and 0.35 mg/L, respectively) and higher O2 concentrations of up to 7.17 and 4.80 mg/L, respectively were measured in spring 2017/summer 2018 in the plume core, plume fringe and non-impacted wells (Fig. 4). The generally low Fe and Mn with high O2 concentrations in spring 2017/summer 2018 are likely caused by the strong recharge due to snowmelt following snow accumulation in winter 2017 and spring 2018 (Fig. 2). This led to a recharge of the aquifer with oxygen-rich water and the replenishment of terminal electron acceptor, which likely resulted in Mn and Fe oxidation and precipitation. In spring 2017/summer 2018 high HCO
3 concentrations were also detected in the high toluene concentration zone (~312.5 masl) indicating that toluene biodegradation also occurred in spring 2017/summer 2018, however, under more oxic conditions compared to summer 2016/fall 2017. As opposed to Fe, Mn and O2, no temporal concentration variation was observed for SO2
4 and CH4 between spring 2017/summer 2018 and summer 2016/fall 2017 (Fig. 5A, B, D and E). Sulfate showed generally low (<0.02e42 mg/L) concentration in the high toluene concentration zone (~312.5 masl) and was elevated (up to 120 mg/L) at depths greater than 305 masl and closer to the surface (~313.0 masl) where the toluene concentrations are lower (Fig. 5A and D). Conversely, CH4 exhibited an opposite vertical

concentration pattern showing generally high concentrations in the high toluene concentration zone (up to 11.8 mg/L), and low concentrations (<2 mg/L) closer to the surface (~313.0 masl) and at depths greater than 305 masl in spring 2017, fall 2017 and summer 2018 (Fig. 5B and E). The high CH4 and low SO2
4 concentrations in the high toluene concentration zone (~312.5 masl) suggest that toluene biodegradation also occurs under methanogenic and sulfate reducing conditions. This is plausible in summer 2016/fall 2017 as in addition to Fe and Mn reduction, sulfate reduction and methanogenesis can occur. In contrast, in spring 2017/summer 2018 the occurrence of sulfate reduction and especially methanogenesis is rather unlikely due to the more oxic conditions in the aquifer. It seems that in spring 2017/summer 2018, the SO2
4 concentrations were low due to the poor replenishment of SO2
4 during the recharge of the aquifer with oxygenated snowmelt water and not due to reactive processes. Furthermore, in spring 2017 methane oxidation was likely less favourable compared to Fe and Mn causing the persistence of CH4 but not of Fe and Mn in the aquifer. Despite the identification of temporally varying redox conditions caused by changing recharge conditions, the resulting effect on toluene biodegradation and resulting plume attenuation is difficult to evaluate based on toluene, redox species and HCO
3 concentrations alone. To gain more insight into relations between temporally varying redox conditions and the extent of toluene biodegradation, dual element toluene-specific isotope ratios (d13CToluene, d2HToluene) were measured in summer 2016, spring 2017 and fall 2017. Furthermore, isotope ratios of sulfate (d34SSO4, d18OSO4) and DIC (d13CDIC) were determined in summer 2016 and fall 2017 to evaluate to what extent toluene was biodegraded via sulfate reduction and methanogenesis at these times.

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3.3. Stable isotope data 3.3.1. Temporal variation of toluene biodegradation The most depleted d13CToluene and d2HToluene values that were measured closest to the toluene source zone (shallowest port, multilevel M29), during the two years sampling period were
30.8‰ and
158‰, respectively in fall 2017. These values can be used as an indication for the isotopic composition of the toluene LNAPL given its proximity to the contamination source. Compared to the toluene source zone, temporal trends of heavy carbon (13C) and hydrogen (2H) isotope enrichment in toluene were observed (Fig. 6). In summer 2016 and spring 2017, shifts of d13CToluene values of ~2.3‰ were observed, whereas a smaller enrichment of heavy carbon isotopes (13C) was observed in fall 2017 (~1.5‰) (Fig. 6A and C). Similar trends but larger shifts were observed for the d2HToluene values with respect to the source zone. The largest shift of d2HToluene values was detected in summer 2016 (~80‰) and spring 2017 (~50‰) and the smallest in fall 2017 (~15‰). The temporal shifts of toluene-specific isotope ratios were generally larger, especially for the d2HToluene values, compared to those attributable to physical processes such as diffusion and sorption (Wanner and Hunkeler, 2015; Wanner et al., 2017), unequivocally identifying toluene biodegradation. The various enrichments of heavy carbon (13C) and hydrogen (2H) isotopes in toluene over time represent different extents of toluene biodegradation and become clearly evident in a dual isotope plot, in which the x- and y-axis represent the shifts of d13CToluene and d2HToluene values compared to the source zone (Dd2H vs. Dd13C; Fig. 6E). The highest extent of toluene biodegradation occurred in summer 2016 followed by spring 2017 and fall 2017. The temporal variability of the extent of toluene biodegradation is likely related to the changing redox conditions over time (Figs. 4 and 5), whereby the

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slopes (L ¼ Dd2H/Dd13C) in the dual isotope plot can be used, in addition to the redox species concentrations (Figs. 4 and 5), as an indication for the redox conditions under which toluene biodegradation occurred (Fig. 6E). For toluene biodegradation under aerobic and iron reducing conditions previous studies determined L values in a range between 1-68 and 24e62, respectively, while for nitrate (11e23), manganese (12e18) and sulfate (15e33), reduction, smaller ranges of L values were observed (Dorer et al., 2016; Herrmann et al., 2009; Mancini et al., 2006; Tobler et al., 2008; Vogt et al., 2008). In contrast to aerobic, iron, sulfate and nitrate reducing conditions, L values for toluene biodegradation under methanogenic conditions are unknown and thus, d13CToluene and d2HToluene values in a dual isotope provide no information whether methane production was linked to toluene biodegradation.

3.3.2. Toluene biodegradation in summer 2016 The highest extent of toluene biodegradation during summer 2016, revealed by the largest enrichment of heavy carbon (13C) and hydrogen (2H) isotopes in toluene (Fig. 6), coincided with iron, manganese and sulfate reducing as well as methanogenic conditions as indicated by the redox species concentrations (Figs. 4 and 5). The d13CToluene and d2HToluene values in the dual isotope plot reinforce the occurrence of toluene biodegradation via sulfate and iron reduction but not via manganese reduction (Fig. 6E). Hence, although elevated manganese concentrations were measured, toluene biodegradation via manganese reduction is likely not the main toluene biodegradation mechanism occurring in summer 2016. The occurrence of toluene biodegradation via sulfate reduction in summer 2016 can be further evaluated by considering the d34SSO4 and d18OSO4 profiles (Fig. 7A and B). The most depleted d34SSO4 and d18OSO4 values (
2.7‰ to 8.4‰ and 0.9‰ to 5.4‰, respectively) were found close to the surface (~313.5 masl) in

Fig. 6. Toluene-specific d13CToluene and d2HToluene profiles in the plume core (M21 and M29) and plume fringe (M27) multilevel wells in cross section A-A’ (A and B) and in the plume core multilevel wells (M22, M28) in cross section BeB’ (C and D). In figures AeD continuous blue lines indicate highest water table (spring 2017/summer 2018) and dashed blue lines represent lowest water table (summer 2016/fall 2017) conditions during the sampling period. The shallow ports (>308 masl) and the deep (<308 masl) ports are represented by the closed and open symbols, respectively, in summer 2016 (blue squares), spring 2017 (green triangles) and fall 2017 (black circles). (E) Dual isotope plot of d13CToluene and d2HToluene values, in which the x- and y-axis represent the shifts of d13CToluene and d2HToluene values compared to the toluene source zone (Dd2H vs. Dd13C). The dashed lines in the dual isotope plot (E) represent the range of slopes (L ¼ Dd2H/Dd13C) being indicative for different redox conditions. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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Figure 7. d34SSO4 (A) and d18OSO4 (B) profiles and d34SSO4 versus SO2
4 concentrations (C) in the plume core (M21, M22, M28, M29) and plume fringe (M26, M27) multilevel wells in both transects (A-A0 and BeB0 ). In figures A and B continuous blue lines indicate highest water table (spring 2017/summer 2018) and dashed blue lines represent lowest water table (summer 2016/fall 2017) during the sampling period. The measurements in summer 2016 and fall 2018 are indicated with blue squares and black circles, respectively, whereby the shallow (>308 masl) and deep (<308 masl) ports are indicated with closed and open symbols, respectively.

summer 2016 and at greater depth (<308 masl) in fall 2017 (Fig. 7A and B) where the sulfate concentrations were highest (80e100 mg/ L) (Fig. 5A and D). These d34SSO4 and d18OSO4 values can be considered as background values being unaffected by sulfate reduction. In summer 2016, enriched d34SSO4 and d18OSO4 values of up to 22.1‰ and 15.0‰, respectively compared to the background values were observed in the high toluene concentration zone (~312.5 masl) and at slightly greater depth (~311.0 masl). (Fig. 7A and B). This indicates, in combination with the low sulfate concentration, that toluene biodegradation occurred at least to some degree under sulfate reducing conditions in summer 2016 at these depths. To gain additional information about whether sulfate reduction is the main toluene biodegradation pathway in summer 2016, carbon isotope ratio profiles of DIC (d13CDIC) can be considered. Toluene biodegradation via sulfate, iron and manganese reduction leads to a depletion and methanogenesis to an enrichment of heavy carbon isotopes (13C) in DIC (Conrad et al., 1997; Landmeyer et al., 1996). It can be assumed that d13CDIC values between
8.5‰ and
13.5‰ correspond to the background values representing a mixture between biogenic CO2 produced in the overburden (d13CDIC ¼
22‰) and DIC from carbonate mineral dissolution (d13CDIC ¼
0.5‰) (Aelion et al., 2010; Cerling et al., 1991; Conrad et al., 1997). In summer 2016, at a depth greater than 308 masl, d13CDIC values showed background values indicating

that neither sulfate, iron, manganese reduction nor methanogenesis occurred (Fig. 8A). This is consistent with the d34SSO4 and d18OSO4 background values at <308 masl supporting the assumption that sulfate reduction was absent at this depth (Fig. 7A and B). In contrast, close to the surface (~313.0 masl), where the toluene concentrations are lower than in the high toluene concentration zone at ~312.5 masl, more depleted d13CDIC values (
14.9‰) compared to background values were detected suggesting that toluene biodegradation occurred via sulfate, iron and/or manganese reduction (Fig. 8A and B). However, since d34SSO4 and d18OSO4 values were in the background range at ~313.0 masl, sulfate reduction can be excluded. Moreover, manganese reduction is likely also not taking place as shown by the dual isotope plot (Fig. 6E) suggesting that toluene biodegradation mainly occurs via iron reduction at ~313.0 masl. At slightly greater depth, in the high toluene concentration zone (~312.5 masl), where high HCO
3 concentrations (500 mg/L) indicated high toluene biodegradation, the d13CDIC values were within or close to background (Fig. 8A and B). This suggest that in contrast to the shallower depth, methanogenesis occurred in addition to iron reduction in the high toluene concentration zone (~312.5 masl) shifting the d13CDIC to more positive values into the background range, which explains the d13CDIC background values despite high toluene biodegradation.

P. Wanner et al. / Water Research 165 (2019) 114986

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Figure 8. d13CDIC profiles (A) and d13CDIC versus HCO
3 concentrations (B) in summer 2016 (blue squares) and fall 2017 (black circles), in the plume core (M21, M22, M28, M29) and plume fringe (M26, M27) multilevel wells in both transects (A-A0 and BeB0 ). The shallow (>308 masl) and deep (<308 masl) ports are indicated with closed and open symbols, respectively. In figure A, the continuous blue line indicates highest water table (spring 2017/summer 2018) and dashed blue lines represent lowest water table (summer 2016/fall 2017) during the sampling period.

3.3.3. Toluene biodegradation in spring 2017 In spring 2017, the intermediate extent of toluene biodegradation, indicated by the intermediate enrichment of heavy carbon (13C) and hydrogen (2H) isotopes in toluene compared to summer 2016 (high biodegradation) and fall 2017 (low biodegradation) (Fig. 6E), took place under aerobic conditions due to the elevated O2 concentrations (Fig. 4A and D). The Dd13C and Dd2H values in spring 2017 in the dual isotope plot support this assumption as well (Fig. 6E).

3.3.4. Toluene biodegradation in fall 2017 The least extent of toluene biodegradation in fall 2017, identified by the lowest enrichment of heavy carbon (13C) and hydrogen (2H) isotopes in toluene, concurred with sulfate, iron, manganese reducing and methanogenic conditions as indicated by the redox species concentrations (Figs. 4 and 5). The shifts of the d13CToluene and d2HToluene values (Dd13C and Dd2H) in the dual isotope plot in fall 2017 were consistent with the slopes (L ¼ Dd2H/Dd13C) for sulfate, manganese and at least partly for iron reducing conditions reinforcing the occurrence of toluene biodegradation under these redox conditions (Fig. 6E). The sulfate isotope ratio profiles in fall 2017 showed enriched d34SSO4 and d18OSO4 values with respect to background values of up to 29.4‰ and 15.4‰, respectively at depths ranging between 308.5 and 311.0 masl (Fig. 7A and B). The enrichment of heavy sulfur (34S) and oxygen (18O) isotopes in sulfate at this depth range was larger than in summer 2016 revealing that toluene biodegradation occurred to a larger extent under sulfate reducing conditions in fall 2017 compared to summer 2016. At shallower depth, in the high toluene concentration zone (~312.5), the sulfate isotopes were not measured due to the low sulfate concentration. However, for evaluating if toluene biodegradation mainly occurs via sulfate reduction in the high toluene concentration zone in fall 2017, d13CDIC profiles were considered. The d13CDIC profiles showed a larger enrichment of 13C compared to summer 2016 showing d13CDIC values of up to 0.6‰ (Fig. 8A) in the high toluene concentration zone (~312.0) and at slightly greater depth (~311.0 masl), where the elevated HCO
3 concentrations (350e425 mg/L) indicated toluene biodegradation. This revealed that in the high toluene concentration zone toluene biodegradation occurred to a larger extent under methanogenic conditions in fall

2017 compared to summer 2016 and to a minor extent via sulfate reduction. Moreover, at greater depth (~311.0 masl) the enriched d13CDIC values showed that although sulfate reduction occurred to some degree as shown by the enriched d34SSO4 and d18OSO4 values (Fig. 7), toluene biodegradation also occurred predominantly under methanogenic conditions. At depths <308 masl, the d13CDIC values and HCO
3 concentrations (250e300 mg/L) were close to background values similarly to summer 2016 (Fig. 8). Therefore, in fall 2017 toluene biodegradation was not occurring under sulfate or methanogenic conditions at greater depth (<307 masl). This is consistent with the d34SSO4 and d18OSO4 values that also showed background values at a depth greater than 308 masl (Fig. 7).

3.3.5. Spatial variation of toluene biodegradation In contrast to the temporal variation, a less strong spatial variation of toluene-specific d13CToluene and d2HToluene values were observed (Fig. 6). In vertical direction, no clear trends of heavy carbon (13C) or hydrogen isotopes (2H) enrichment in toluene was observed at shallow depths (>308 masl), where most of the toluene resides (Fig. 6). This is inconsistent with the elevated HCO
3 concentrations (Fig. 5C and F) and the shifts of d34SSO4, d18OSO4 and d13CDIC values with respect to background at shallow compared to greater depths pointing to the occurrence of higher toluene biodegradation at shallow compared to deeper depths (Figs. 7 and 8). At shallow depth, the absence of an enrichment of heavy carbon and hydrogen isotopes in toluene can be attributed to the large toluene mass that resides at shallow depth. The large toluene mass weakens the biodegradation-induced carbon and hydrogen isotope effect as the ratio between isotopically enriched biodegraded and light non-degraded toluene remains low even if toluene biodegradation occurs. Similar to the vertical direction, carbon and hydrogen isotope ratios of toluene also showed relatively small shifts in the range of Dd13CToluene ¼ 2‰ and Dd2HToluene ¼ 20‰ in groundwater flow direction between the two transects (A-A0 and BeB’) and in the horizontal transverse direction during the different sampling campaigns (Fig. 6). This reveals that progressive toluene biodegradation occurred to a minor degree in the transverse direction across the plume width and along the groundwater flow path, which is consistent with the redox species concentrations showing no redox zonation in either of these directions. The

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P. Wanner et al. / Water Research 165 (2019) 114986

absence of a spatial redox zonation and progressive toluene biodegradation in the transverse and groundwater flow directions reveals that the redox milieu affecting toluene biodegradation is mainly controlled by changing recharge conditions during the different seasons and to a minor extent by the toluene mass distribution in the fractured bedrock aquifer.

4. Conclusions and environmental significance The present study shows that the redox conditions in a toluene contaminated fractured bedrock aquifer can change rapidly from oxic to anoxic conditions over months being unrelated with the toluene plume but caused by temporally varying recharge conditions that are seasonal. The high resolution stable isotope data including toluene-specific multi-element (d13CToluene, d2HToluene) isotope analyses as well as d34SSO4, d18OSO4 and d13CDIC measurements revealed that the temporally varying redox conditions influence the extent of toluene biodegradation in the entire toluene plume. The largest extent of toluene biodegradation was observed in summer 2016 mainly under iron reducing conditions. Intermediate toluene biodegradation occurred in spring 2017 under oxic conditions, while the least extent of toluene biodegradation was detected in fall 2017 via iron, sulfate, and manganese reduction and via methanogenesis. Consequently, our study results reveal that under temporally varying redox conditions both the plume core and the fringe can be replenished with terminal electron acceptors influencing BTEX biodegradation in the whole plume and not only at the distal boundaries of the plume as previously suggested by the plume fringe conceptual model. This observation highlights the importance of high temporally resolved groundwater monitoring as a single measurement in time could lead to false estimations of biodegradation rates and predictions of plume attenuation. Therefore, the improved comprehension of temporally varying contaminant biodegradation rates will advance the quantitative site conceptual model used to evaluate whether or not monitored natural attenuation (MNA) can be used as a site management strategy in complex fractured bedrock aquifers.

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement The authors acknowledge BP Canada (Alan Scheibner, Mike Early, Larry Stone), the University Consortium for Field-Focused Groundwater Contamination Research, and the Natural Sciences and Engineering Research Council of Canada (NSERC) through a Collaborative Research and Development Grant (CRD) to Prof. Beth L. Parker, Prof. Kari Dunfield and Prof. Ramon Aravena for financial support. The authors also thank, Nathan Glas, James Hommersen, Isaac Noyes, Leon Halwa, Marina Nunes, Carlos Maldaner, Jonathan Munn, Juliana Camillo, Andrea Roebuck and Kamini Khosla for field support and Maria Gorecka, Rashmi Jadeja and Richard Elgood for analytical support.

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.watres.2019.114986.

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