This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 1 Multi-method assessment of the intrinsic biodegradation 2 potential of an aquifer contaminated with chlorinated 3 ethenes at an industrial area in Barcelona (Spain) 4 Natàlia Blázquez-Pallía,b, Mònica Rosellc, Joan Variasb, Marçal Boschb, Albert Solerc, 5 Teresa Vicenta, Ernest Marco-Urreaa,* 6 a Departament d’Enginyeria Química, Biològica i Ambiental, Universitat Autònoma de 7 Barcelona (UAB), c/ de les Sitges s/n, 08193 Cerdanyola del Vallès, Spain. b 8 Litoclean, S.L., c/ Numància 36, 08029 Barcelona, Spain. c 9 Grup MAiMA, SGR Mineralogia Aplicada, Geoquímica i Geomicrobiologia, 10 Departament de Mineralogia, Petrologia i Geologia Aplicada, Facultat de Ciències de la 11 Terra, Universitat de Barcelona (UB), c/ Martí Franquès s/n, 08028 Barcelona, Spain. 12 * 13 Corresponding author: 14 Departament d’Enginyeria Química, Biològica i Ambiental, Universitat Autònoma de 15 Barcelona (UAB), c/ de les Sitges s/n, 08193 Cerdanyola del Vallès, Spain. E-mail: 16 ernest.marco@uab.cat, Phone: +34 935812694. 17 18 Environmental Pollution 19 Total number of pages (including cover): 32 20 Figures: 3 21 Tables: 1 1 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 22 Abstract 23 The bioremediation potential of an aquifer contaminated with tetrachloroethene (PCE) 24 was assessed by combining hydrogeochemical data of the site, microcosm studies, 25 metabolites concentrations, compound specific-stable carbon isotope analysis and the 26 identification of selected reductive dechlorination biomarker genes. The characterization 27 of the site through 10 monitoring wells evidenced that leaked PCE was transformed to 28 TCE and cis-DCE via hydrogenolysis. Carbon isotopic mass balance of chlorinated 29 ethenes pointed to two distinct sources of contamination and discarded relevant alternate 30 degradation pathways in the aquifer. Application of specific-genus primers targeting 31 Dehalococcoides mccartyi species and the vinyl chloride-to-ethene reductive 32 dehalogenase vcrA indicated the presence of autochthonous bacteria capable of the 33 complete dechlorination of PCE. The observed cis-DCE stall was consistent with the 34 aquifer geochemistry (positive redox potentials; presence of dissolved oxygen, nitrate, 35 and sulphate; absence of ferrous iron), which was thermodynamically favourable to 36 dechlorinate highly chlorinated ethenes but required lower redox potentials to evolve 37 beyond cis-DCE to the innocuous end product ethene. Accordingly, the addition of lactate 38 or a mixture of ethanol plus methanol as electron donor sources in parallel field-derived 39 anoxic microcosms accelerated dechlorination of PCE and passed cis-DCE up to ethene, 40 unlike the controls (without amendments, representative of field natural attenuation). 41 Lactate fermentation produced acetate at near-stoichiometric amounts. The array of 42 techniques used in this study provided complementary lines of evidence to suggest that 43 enhanced anaerobic bioremediation using lactate as electron donor source is a feasible 44 strategy to successfully decontaminate this site. 45 46 Capsule 47 The combination of complementary diagnostic techniques provides different lines of 48 evidence for in situ bioremediation potential of a tetrachloroethene-contaminated aquifer. 49 50 Keywords 51 Stable isotope; Dehalococcoides mccartyi; biostimulation; DCE stall; reductive 52 dehalogenase. 2 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 53 1. Introduction 54 Tetrachloroethene (PCE) and trichloroethene (TCE) are widely used as degreasing agents 55 in industry and are frequently detected in subsurface waters due to improper disposal or 56 accidental spills. The lesser chlorinated ethenes, dichloroethene (DCE) and vinyl chloride 57 (VC), are usually detected in groundwater because of incomplete reductive dechlorination 58 reactions of PCE and TCE, in a common phenomenon referred to as “DCE or VC stall” 59 (Bradley, 2000; Stroo and Ward, 2010). In this situation, PCE, TCE and their daughter 60 products coexist in groundwater forming a hazardous chemical mixture. These 61 compounds are all regulated and considered priority substances by the Agency for Toxic 62 Substances and Disease Registry of the United States (ATSDR, 2016) and by the 63 2008/105/EC European Directive (European Commission, 2008), which set maximum 64 contaminant levels (MCL) for all of them. 65 Remediation strategies for contaminated aquifers have traditionally included 66 physicochemical treatments such as pump and treat, thermal desorption, in situ chemical 67 oxidation and soil vapor extraction, among others. However, in the last decade, in situ 68 bioremediation, which uses the metabolism of microorganisms to degrade pollutants, has 69 emerged as one of the most preferable technologies used to clean up the subsoil (Pandey 70 et al., 2009). This shift towards in situ biological treatments is likely due to their cost- 71 effectiveness and less invasive features compared with the conventional physicochemical 72 methods (Lemming et al., 2010; Lyon and Vogel, 2013). 73 Organohalide-respiring bacteria (OHRB) are the key microorganisms in the 74 bioremediation of chlorinated ethenes because they can use them as terminal electron 75 acceptors during microbial respiration, resulting in the formation of lesser-chlorinated 76 compounds (Leys et al., 2013). OHRB tend to harbour several distinct reductive 3 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 77 dehalogenases (RDase), which are the enzymes driving the organohalide respiration 78 process. The identification of their function allows for the exploitation of the genes 79 encoding the catalytic subunit, reductive dehalogenase homologous A subunit (rdhA), as 80 biomarkers to assess the intrinsic bioremediation potential of contaminated sites 81 (Cupples, 2008; Hug et al., 2013; Hug and Edwards, 2013; Pöritz et al., 2013). OHRB are 82 strictly anaerobic and when redox potential or nutritional requirements (i.e. acetate and 83 hydrogen as carbon source and electron donor, respectively) are not adequate, the aquifer 84 can be conditioned by supplying organic fermentable substrates such as lactate, butyrate 85 or benzoate in a process referred to as biostimulation (Adrian and Löffler, 2016; Leeson 86 et al., 2004). Some OHRB can partially dechlorinate PCE to TCE or cis-DCE, such as 87 Clostridium sp., Dehalobacter sp., Desulfitobacterium sp., Desulfuromonas sp., 88 Geobacter sp., or Sulfurospirillum sp. (Löffler et al., 2013), but Dehalococcoides 89 mccartyi sp. (Dhc) has been considered, for a long time, the unique genus capable of fully 90 dechlorinating PCE to innocuous ethene (Cupples et al., 2003; He et al., 2005; Sung et 91 al., 2006). However, it may be possible that other bacteria, still unknown and not 92 described yet, are able to perform such reaction, as recent studies have shown that a 93 Dehalogenimonas species can dechlorinate TCE to ethene (Yang et al., 2017b), and that 94 a bacterial consortium not containing Dhc was able to completely dechlorinate PCE as 95 well (Yu et al., 2016). Likewise, Lu et al. (2006) observed that VC disappeared at 96 contaminated sites where no Dhc markers were present, and Da Silva and Alvarez (2008) 97 and He et al. (2015) demonstrated that dechlorination activity and formation of ethene is 98 not always correlated with the presence of Dhc genes. 99 Different approaches allow for the monitoring and characterization of in situ 100 biodegradation of organic contaminants and organohalides in particular (Bombach et al., 101 2010; Nijenhuis and Kuntze, 2016). Monitoring of contaminants’ concentrations can 4 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 102 provide information on the removal of certain contaminants in the polluted site, but the 103 main drawback is that physical attenuation processes such as dilution, sorption or 104 dispersion cannot be distinguished from biological processes and, therefore, these 105 measurements can sometimes be misinterpreted. Likewise, co-contamination and off-site 106 sources of the contaminants make difficult site degradation assessments. To overcome 107 this bottleneck, a more reliable method that is increasingly used to monitor in situ 108 chlorinated ethenes transformation is the compound specific isotope analysis (CSIA) 109 (Hunkeler et al., 1999; Nijenhuis et al., 2007; Palau et al., 2014; Wanner et al., 2016). 110 This technique measures the abundance ratio of specific stable isotopes (i.e. 13C/12C) in 111 targeted molecules relative to an international standard. In principle, lighter isotope 112 molecules react at a higher rate than heavier isotope molecules during biochemical 113 transformations. This results in a progressive enrichment of the heavier isotope that can 114 be used to confirm and quantify in situ biodegradation and elucidate degradation 115 pathways (Elsner et al., 2010). CSIA can also give information regarding source 116 apportionment of the involved pollutants in field studies (Filippini et al., 2018; Hunkeler 117 et al., 2008; Nijenhuis et al., 2013). 118 The detection of Dhc and specific rdhA genes based on polymerase chain reaction 119 (PCR) is a qualitative indication of the reductive dechlorination potential at contaminated 120 sites, but it does not provide information about the physiological activity of Dhc. 121 Likewise, stable carbon isotope fractionation of PCE or TCE can serve as an indicator of 122 in situ reductive dechlorination of these contaminants, but it does not confirm that 123 biodegradation beyond cis-DCE is feasible. Therefore, it is necessary an integrated 124 approach to provide different and complementary lines of evidence for the assessment of 125 the potential of contaminated sites to fully dechlorinate chlorinated solvents (Badin et al., 126 2016; Courbet et al., 2011; Stelzer et al., 2009). 5 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 127 Consequently, the aim of this work was to assess the bioremediation potential of a 128 chlorinated ethenes contaminated site from an industrial area in Barcelona (Spain) using 129 several techniques, including (1) the assessment of the hydrogeochemical conditions of 130 the aquifer, (2) the analysis of the carbon isotopic composition of chlorinated ethenes, (3) 131 the establishment of microcosms to evaluate the effect of different biostimulants to 132 detoxify groundwater samples, and (4) the use of PCR primers targeting specific 133 functional genes. 134 135 2. Materials and methods 136 2.1. Chemicals 137 PCE (99.9% purity) and TCE (≥98% purity) were purchased from Panreac, and ethene 138 (≥99.95% purity) and sodium lactate (≥98% purity) from Sigma-Aldrich. Methanol and 139 ethanol were purchased from Scharlab at the highest purity available. Other chemicals 140 and reagents used for the present study were purchased from Sigma-Aldrich, 141 Thermofisher and Bio-Rad at scientific grade or higher. 142 2.2. Hydrogeochemical description of the aquifer 143 The studied aquifer is located in the province of Barcelona (Spain). This site was 144 significantly contaminated with PCE due to improper disposal practices after its former 145 use as degreasing agent at an industrial plant. The aquifer is an unconfined bedrock 146 mainly consisting of Tertiary sediments. 147 For the geological and hydrogeological characterization of the studied area, 55 148 rotational probes between 10 to 20 m depth with continuous sample extraction were 149 carried out and habilitated as piezometers. According to the probes, from the bottom to 6 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 150 top, mainly three main lithological facies were differentiated: i) a lower layer of red marl 151 located at 10 to 3 m depth, ii) an intermediated brown silty mudstone, and iii) a higher 152 level represented by ochre silty mudstone that are located up to 3 to 6 m depth. An 153 intermediated layer of silty mudstone with sandstone beds is developed in the south part 154 of the site, and it locally evolves to a sandstone – microconglomerate strata that probably 155 corresponds to an old Tertiary sedimentary paleochannel. This most transmissive layer is 156 intersected at the piezometer MW-7. Such Tertiary formation is locally covered by 157 Quaternary deposits including sands, silts and clays with a variable vertical extension and 158 anthropogenic materials (concrete, etc.). 159 For site characterization and monitoring, 55 wells completely screened were 160 installed. The water table is located at depths ranging between 2.5 and 7.5 m below 161 ground surface (266 to 272 m.a.s.l.). Groundwater is mainly concentrated in the sandstone 162 or microconglomerate intervals that intercalate with the mudstone matrix forming a multi- 163 layered aquifer. Due to the limited continuity of the permeable stratum, the potential 164 hydrological exploitation of the area is low. Locally, when monitoring wells intersect 165 layers of sandstone, silty sandstones, silty mudstones and microconglomerates, the 166 hydraulic conductivity increases to medium–low. Pumping tests determined that the 167 hydraulic parameters of the aquifer are from 0.1 to 0.8 m2/day for transmissivity, and 168 from 0.02 to 0.32 m/day for hydraulic conductivity . 169 2.3. Collection of aquifer samples 170 The plume characterization monitoring campaign was carried out in May 2016 on 10 171 monitoring wells identified by numerical codes (Figure 1) using a peristaltic pump. First, 172 the following parameters were measured in situ: (1) the piezometric level; (2) the worker 173 short-term exposure to volatile organic compounds (VOCs) and other gases, with a 7 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 174 MiniRAE Lite direct-reading photoionization detector (RAE Systems, Spain), and (3) the 175 hydrogeochemical parameters of groundwater once they were stabilized using a flow- 176 through cell to avoid contact with the atmosphere (temperature, pH, electric conductivity, 177 and redox potential (Eh)), with a multiparameter probe 3430 WTW (Weilheim). For Eh 178 the redox sensor was a SenTix ORP 900 and the measurements were corrected to the 179 standard hydrogen electrode system (UH) by adding the reference electrode potential at 180 the groundwater temperature to the measured potential. Concentration of dissolved 181 oxygen was measured with a Dissolved Oxygen Meter HI 9147 (Hanna Instruments). 182 Then, samples from the aquifer were obtained. 183 Groundwater for stable carbon isotope analysis and chemical characterization was 184 collected at 1 m above the bottom of the 10 wells, to avoid sediments. The ones for CSIA 185 were killed with NaOH (pH>10) to prevent further biodegradation reactions. 186 For the establishment of microcosms, groundwater with fine sediments was collected 187 from the bottom of the monitoring well MW-2 in transparent autoclaved glass bottles, 188 which were previously filled with N2 gas to minimise bacterial contact with oxygen, and 189 sealed with PTFE caps to minimise VOCs’ adsorption. All groundwater samples were 190 kept in the dark at 4ºC until analysis. 191 2.4. Laboratory microcosms 192 To study whether organic fermentable substrates could enhance the biodegradation of 193 chlorinated ethenes and ethene formation, three different treatments were prepared in 194 triplicate: (1) control containing only groundwater, (2) groundwater with a mixture of 195 methanol plus ethanol (3 mM each), and (3) groundwater with sodium lactate (3 mM). 196 Each microcosm consisted of 100 mL glass serum sterile bottles sealed with Teflon- 197 coated butyl rubber septa and aluminium crimp caps, and contained 65 mL of sampled 8 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 198 groundwater and fine sediments. All microcosms were prepared in an anaerobic glovebox 199 and incubated at 25 °C in the dark under static conditions. After setting up the 200 microcosms, the initial concentration of chlorinated ethenes was analysed by headspace 201 gas chromatography as described in section 2.5 (Table S1) and afterwards it was 202 periodically monitored. 203 To determine the carbon isotopic fractionation (εC) during anaerobic reductive 204 dechlorination of PCE, six parallel cultures were simultaneously prepared as described 205 above, but using anaerobic defined media as reported elsewhere (Martín-González et al., 206 2015) and groundwater from MW-2 (1.5% v/v) as inoculum. Each microcosm was spiked 207 with PCE (160 µM), and sacrificed with NaOH (10 M) at different time points of PCE 208 degradation. In this experiment, three different controls were included, at least in 209 duplicate: (1) NaOH-killed controls containing inoculum and PCE; (2) NaOH-killed 210 controls with inoculum but without PCE, and (3) abiotic controls with PCE but without 211 inoculum, to account for abiotic transformations and control potential impurities from the 212 PCE stock solution. 213 2.5. Analytical methods 214 Chemical characterization of groundwater was determined through the analysis of major 215 anions and cations. Aliquots of samples were preserved with nitric acid to measure total 216 concentrations of Fe, Ca and Na by inductively coupled plasma-optic emission 217 spectrometry (ICP-OES, Optima 3200 RL) and by inductively coupled plasma mass 218 spectrometry (ICP-MS, Elan 6000) at the Centres Científics i Tecnològics de la 219 Universitat de Barcelona (CCiT-UB). HCO3- was determined by titration (METROHM 220 702SM Titrino). NO3-, Cl- and SO4-2 concentrations were analyzed by high-performance 221 liquid chromatography (HPLC) using a WATERS 515 HPLC pump with an IC-PAC 9 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 222 anion column and a WATERS detector (mod 432) at the CCiT-UB. 223 Headspace samples (500 µL) were collected from microcosms to quantify chlorinated 224 ethenes and ethene by gas chromatography (GC) coupled to a flame ionization detector 225 (FID) as described elsewhere (Martín-González et al., 2015). Lactate, pyruvate, acetate, 226 and formate were analysed from 1 mL filtered liquid samples (0.22 μm, Millex) by HPLC 227 as previously described (Mortan et al., 2017). 228 Stable carbon isotope analyses were performed with an Agilent 6890 GC equipped 229 with a split/splitless injector, coupled to a Delta Plus isotope ratio mass spectrometer 230 (IRMS) through a GC-Combustion III interface (Thermo Finnigan). The analyses were 231 carried out by headspace solid-phase microextraction (HS-SPME) following the same 232 methodology used in Martín-González et al. (2015), but the injector temperature was 233 250ºC and the initial oven temperature of 60ºC was kept for 5 min. Carbon isotopic 234 compositions of the contaminants are reported in delta notation (δ13C), relative to the 235 international standard VPDB (Vienna Pee Dee Belemnite), following 𝑅𝑠𝑎𝑚𝑝𝑙𝑒 236 𝛿 13 𝐶 = ( − 1) (Eq.1) 𝑅𝑠𝑡𝑑 237 where Rsample and Rstd refer to the isotope ratios (13C/12C) of the sample and the standard, 238 respectively (Elsner, 2010). δ13C is usually expressed in parts per mil (‰). All samples 239 were measured at least in duplicate and corrected for slight carbon isotopic fractionation 240 induced by the HS-SPME with respect to daily aqueous control standards of chlorinated 241 ethenes with known carbon isotope ratios. The δ13C of these pure in-house standards was 242 determined previously using a Flash EA1112 (Carlo-Erba) elemental analyzer (EA) 243 coupled to a Delta C Finnigan MAT IRMS (Thermo Finnigan) through a Conflo III 244 interface (Thermo Finnigan) using six international reference materials (NBS 19, IAEA- 10 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 245 CH-6, USGS40, IAEA-600, IAEACH-7, L-SVEC) with respect to the VPDB standard 246 according to (Coplen et al., 2006). All the chlorinated ethenes aqueous control standards 247 that were injected together with the experimental samples had a one standard deviation 248 (1σ) lower than 0.5‰ and their mean values were used to normalise the δ13C of the 249 samples, the uncertainties of which were calculated by error propagation. 250 Since PCE was the unique chlorinated precursor released in this aquifer and it is 251 transformed sequentially via reductive dechlorination to TCE, cis-DCE and minor 252 amounts of VC, the weighted average of the isotope signature of the chlorinated solvents 253 must remain constant if VC is not further degraded and PCE released over the years has 254 identical isotopic composition (Aeppli et al., 2010; Hunkeler et al., 1999; Palau et al., 255 2014) . Carbon isotopic mass balance for chlorinated ethenes at each well of the site was 256 calculated for the sequential reductive dechlorination, as follows 257 𝛿 13 𝐶𝑠𝑢𝑚 = 𝑥𝑃𝐶𝐸 · 𝛿 13 𝐶𝑃𝐶𝐸 + 𝑥𝑇𝐶𝐸 · 𝛿 13 𝐶𝑇𝐶𝐸 + 𝑥𝐷𝐶𝐸 · 𝛿 13 𝐶𝐷𝐶𝐸 + 𝑥𝑉𝐶 · 𝛿 13 𝐶𝑉𝐶 (Eq.2) 258 where x refers to the molar fraction of each compound with respect to the total molar mass 259 (sum of chlorinated ethenes) at the sampling event. Calculated 𝛿 13 𝐶𝑠𝑢𝑚 from wells at the 260 field site were compared to assess potential point sources of PCE in the aquifer. 261 A simplified version of the Rayleigh equation allows to quantify the carbon isotopic 262 fractionation, εC (Elsner, 2010), which defines the relationship between changes in carbon 263 isotopic composition (Rt/R0) and concentrations (f = Ct/C0) with time, as follows 𝑅𝑡 264 ln ( ) = 𝜀𝐶 · ln(𝑓) (Eq.3) 𝑅0 265 where Rt/R0 can be described as (δ13Ct + 1) / (δ13C0 + 1) according to δ13C definition. εC 266 can be used to quantify the extent of biodegradation of a target contaminant if a site- 267 specific εC-value can be obtained (Elsner, 2010). In addition, the εC value is characteristic 11 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 268 for a given degradation pathway and can provide information into the reactions taking 269 place in the field (Elsner, 2010). 270 2.6. DNA extraction and PCR 271 DNA was extracted from enriched cultures inoculated with aquifer samples from MW-2. 272 Cell harvesting was carried out via centrifugation of 65 mL-samples. Genomic DNA was 273 isolated using NucleoSpin Tissue DNA extraction kit following the instructions provided 274 by the manufacturer (Macherey-Nagel). Primer sets used to detect Dhc 16S rRNA gene 275 and Dhc reductive dehalogenase gene vcrA were previously described (Manchester et al., 276 2012; Ritalahti et al., 2006) (Table S2). Dehalococcoides mccartyi strain BTF08 (Pöritz 277 et al., 2013) was used as positive control. Each 10 µL reaction mixture contained 5 µL of 278 iQ Supermix (2x) (Bio-Rad), 250 nM of each primer (1 µL volume each) (Thermofisher) 279 and a concentration of template DNA ranging between 5-50 ng/µl (3 µL volume). The 280 thermal program used for PCR amplification of Dhc and vcrA genes was described 281 elsewhere (Martín-González et al., 2015; Ritalahti et al., 2006). 282 283 3. Results 284 3.1.Physicochemical characterization of the site 285 The hydrogeochemical data collected from the 10 monitoring wells of the aquifer is 286 summarized in Table 1. MW-4 was never impacted by chlorinated ethenes contamination 287 and therefore is considered as the natural background of the area, which belongs to 288 magnesium-calcium-bicarbonate facies. The distribution of native potential electron 289 acceptors in the aquifer varied among different monitoring wells. Dissolved oxygen 290 concentration ranged from 0.2 to 2.0 mg/L, total Fe was insignificant (<0.1 mg/L), nitrate 12 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 291 concentration exceeded 25 mg/L in all piezometers except in PZ-1 and Prof A, and 292 sulphate was detected at elevated levels in most of the wells (values ranging from 94 to 293 1435 mg/L). Elevated levels of chloride were detected in most of the wells (up to 1053 294 mg/L at the most contaminated well PZ-5), whereas MW-4 showed the lowest value (30.5 295 mg/L), considered the natural background. The averaged concentration of bicarbonate, 296 which serves as an indicator of the natural buffering capacity of the aquifer, was 459 ± 297 146 mg/L. The pH and temperature values were on average 7.4 ± 0.2 and 19 ± 2 ºC, 298 respectively. 299 The concentration analyses of chlorinated ethenes across the site showed that PCE was 300 the main VOC in the aquifer (concentrations ranging from 2.1 to 77 µM), but it was 301 always accompanied by minor amounts of TCE and cis-DCE (Figure 2, Table S1). Traces 302 of trans-DCE and VC were detected in MW-2 and, as expected, no chlorinated ethenes 303 were detected in MW-4 (Table S1). 304 3.2. Carbon stable isotope analysis of chlorinated ethenes 305 The carbon isotopic signatures (δ13C) of PCE and its dechlorination products were 306 analysed at the different monitoring wells to investigate the relevance of biodegradation 307 processes at the contaminated site. They were found from -32.6 to -26.4‰, -37.7 to - 308 29.7‰, and -33.0 to -26.0‰ for PCE, TCE, and cis-DCE, respectively (Figure 2, Table 309 S1 for more details). In the case of PCE, which is the source of the contamination plume, 310 all values were within the range of commercial solvents (-37.2 to -23.2‰) (Jendrzejewski 311 et al., 2001; van Warmerdam et al., 1995). The isotopic mass balance based on the 312 concentration-weighted δ13C signatures of the chlorinated ethenes and assuming 313 hydrogenolysis (δ13Csum, Eq. 2) was established for each well (Figure 2, Table S1). Except 314 for wells MW-7 and Prof A, which had similar isotopic balances of -26.4 ± 0.6‰ and - 13 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 315 26.0 ± 0.7‰, respectively, the δ13Csum-values of the rest of the wells were within the same 316 average value of -31 ± 1‰. 317 In an attempt to determine the site-specific εC for PCE dechlorination in this aquifer, 318 six parallel cultures inoculated with 1.5% v/v MW-2 groundwater were killed after 319 approximately 0, 7, 21, 46, 79, 81 and 82% of PCE degradation and the δ13C of 320 chlorinated ethenes was analysed. Abiotic and NaOH-killed controls showed no PCE 321 degradation as its concentration remained constant throughout the whole experiment (163 322 ± 5 µM, n=7, Figure S1A). In the active microcosms, although the δ13C of PCE shifted 323 1.6‰ after 82% degradation (see Figure S1B) and the one standard deviation (1σ) for 324 duplicate measurements were, for all samples, below total instrumental uncertainty of 325 0.5‰ (Sherwood Lollar et al., 2007), PCE dechlorination to TCE did not fit the Rayleigh 326 model. The data exhibited poor linearity (R2 = 0.46) when plotting according to Eq.3 and 327 the calculated εC would be -0.6 ± 0.8‰, showing a 95% confidence interval bigger than 328 the value itself and, therefore, expressed as not significant. In contrast, TCE showed a 329 stronger enrichment when degrading to cis-DCE. 330 3.3. Laboratory microcosms amended with different biostimulants 331 Three different microcosm treatments were prepared with groundwater material from 332 well MW-2 to test whether different fermentable organic compounds could enhance the 333 complete reductive dechlorination of PCE to ethene. This well was chosen for the 334 microcosms experiment because it was a candidate for the injection of amendments in a 335 foreseeable future in situ biostimulation pilot test. 336 The unamended microcosms used as natural biodegradation controls fully converted 337 PCE to cis-DCE by day 150, but the reaction remained stalled at this stage without ethene 338 formation (Figure S2A). 14 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 339 In the microcosms amended with a mixture of methanol plus ethanol, PCE was 340 degraded within the first week to cis-DCE and it accumulated in the medium for 85 d 341 (Figure S3A). At day 125, cis-DCE and VC disappeared from the medium. At day 139, 342 the microcosm was spiked with 75 µM of TCE and, after 60 days, TCE, cis-DCE and VC 343 were sequentially transformed, and ethene was detected (Figure S3A). 344 Microcosms amended with lactate also transformed PCE to cis-DCE within the first 345 week and the latter accumulated in the medium. At day 63, VC was detected and at day 346 75 ethene was observed for the first time. After 125 d, ethene was the only remaining 347 compound in the microcosms (Figure 3A). 348 To get an insight into the predominant fermentation pathways used by native microbial 349 populations for the organic substrates selected, low-molecular-weight fatty acids (lactate, 350 pyruvate, acetate, and formate) were monitored during the time-course experiments. 351 Organic acids were not detected in the unamended microcosms (Figure S2B). In the 352 microcosms amended with ethanol and methanol, approximately 3 mM of acetate was 353 produced after 15 d and it remained in the medium without a significant decrease for 354 approximately two months (Figure S3B). In the case of lactate-amended microcosms, 355 lactate was fermented to near-stoichiometric amounts of acetate, which was slowly 356 consumed in the microcosms and after 75 d it was completely depleted (Figure 3B). 357 A remarkable difference observed between the microcosms amended with lactate and 358 the mixture of ethanol plus methanol was the vigorous generation of methane in the latter 359 treatment. As depicted in Figure S4, methane was not detected in the control and it was 360 barely produced in the lactate-amended microcosms but, in the treatment with alcohols, 361 methane concentration remarkably increased after approximately 50 d without reaching 362 a plateau in the monitored period. 15 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 363 3.4. Identification of native OHRB 364 Dhc are keystone bacteria for the detoxification of chlorinated ethenes to nontoxic ethene. 365 PCR amplifications with Dhc 16S rRNA and the vcrA gene-targeted primers yielded 366 diagnostic amplicons indicating that the enrichment culture inoculated with groundwater 367 from MW-2 contained Dhc species implicated in the VC-to-ethene dechlorination (Figure 368 S5). 369 370 4. Discussion 371 The presence of the degradation products of the hydrogenolysis pathway of PCE in the 372 monitoring wells is a qualitative evidence for intrinsic anaerobic reductive dechlorination 373 because PCE was the only degreasing solvent used on site (Table S1). The values of 374 δ13Csum in 7 of 9 wells (Table S1) were within the same value (-31 ± 1‰) indicating that 375 they all share the same source of contamination and that PCE biodegradation beyond cis- 376 DCE was not significant in these seven wells (the balance would have become heavier as 377 the production of lighter VC was not taken into account). In the same way, it discarded 378 the existence of important alternate degradation pathways or production of unidentified 379 by-products. On the other hand, the δ13Csum observed in the other two distal wells (MW- 380 7 and Prof A), with an average value of δ13Csum of -26.2 ± 0.5‰ obtained from different 381 chlorinated ethenes in each well, suggest that two different sources of PCE could have 382 been leaked in this industrial area and they are statistically different from the rest 383 (ANOVA, p < 0.0009). An additional indicator of in situ PCE biodegradation is provided 384 by the concentration of chloride ions detected in the aquifer, which is between 3 and 30 385 times higher in the monitoring wells of the impacted aquifer compared to a non-impacted 386 well (MW-4), which served as a background control (Table 1). Furthermore, measured 16 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 387 temperature and pH showed neutral and temperate shallow groundwater conditions which 388 are quite optimal for bioremediation purposes. 389 The extent of in situ PCE biodegradation could not be quantified using the site-specific 390 εC of PCE because the variation of δ13C values obtained from the stablished microcosms 391 at different degradation stages of PCE was not linear. This fact leads us to the conclusion 392 that probably the degrading bacterial community was evolving differently in the 393 microcosm bottles which were sacrificed at different PCE degradation points. Several εC 394 values have been reported for the biodegradation of PCE under anoxic conditions in the 395 literature. They range from strong isotope fractionation (e.g. -16.7‰ by 396 Desulfitobacterium sp.) to very weak (-0.4 to -1.7‰) or even not significant isotope 397 fractionation (e.g. Sulfurospirillum, Desulfuromonas or Geobacter species all belonging 398 to ε-Proteobacteria, Table S3), and the reported εC of PCE during anaerobic reductive 399 dechlorination by Dhc isolates or Dhc-containing cultures ranges from -1.6 to -6.0‰ 400 (Table S3). The non-linear low fractionation (<2‰) obtained in our enrichment is likely 401 a combination of the degradation of several bacterial species present in the aquifer with a 402 major contribution of non-fractionating species. These results differ a bit from the higher 403 and significant, according to the EPA guide (Hunkeler et al., 2008), isotopic shift for PCE 404 of 3.6‰ observed on-site among the different monitoring wells with equal isotopic 405 balance (Figure 2, excluding MW-7 and Prof A), suggesting that such shift could be due 406 to a higher extent of degradation than the one measured in the microcosm (>82%) or a 407 major activity of higher fractionating species depending on specific well conditions. 408 Moreover, the remarkable isotopic enrichments observed in the same wells for TCE 409 (8.1‰) and cis-DCE (5.3‰) point clearly to biodegradation processes (Figure 2, 410 excluding MW-7 and Prof A). 17 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 411 The detection of the biomarker genes implicated in the VC-to-ethene transformation 412 (Figure S5) indicated that the aquifer contained Dhc with the potential to detoxify PCE, 413 however, groundwater geochemistry exerted a primary control over anaerobic 414 dechlorination reactions. The presence of low concentrations of dissolved oxygen, the 415 relatively high concentrations of nitrate and sulphate, and the non-detection of iron (Table 416 1) indicate that the aquifer has mainly hypoxic to iron-reducing conditions, which is 417 thermodynamically appropriate to reduce highly chlorinated compounds such as PCE or 418 TCE to cis-DCE, but not to fully dechlorinate to harmless ethene (Bouwer, 2017). The 419 optimum redox potential for a complete reductive dechlorination is less than -100 mV 420 (Elsner and Hofstetter, 2011) but, as indicated in Table 1, no negative redox potentials 421 were measured in the monitored wells, although higher reducing microenvironments in 422 the aquifer cannot be discarded. As oxygen, nitrate, and sulphate are consumed, the redox 423 potential is expected to fall, but reduction of these electron acceptors can be hampered by 424 the lack of electron donor in groundwater (Yu et al., 2018). According to this hypothesis, 425 the addition of easily fermentable organic substrates (lactate and the mixture of ethanol 426 plus methanol) to the microcosms enhanced the dechlorination of PCE with respect to the 427 control by shortening the lag phase of PCE dechlorination and overcoming the “DCE 428 stall”, which permitted the full dechlorination to ethene (Figure 3A and S3A). 429 Lactate, ethanol, and methanol can be potentially transformed by native bacteria using 430 different pathways (Fennell et al., 1997). Production of acetate and hydrogen from 431 fermentation reactions is preferred to stimulate growth of OHRB because they can serve 432 as carbon source and electron donor, respectively. The production of ~ 3 mM of acetate 433 in the microcosms with a mixture of ethanol and methanol was consistent with the 434 stoichiometric transformation of ethanol (3 mM) to acetate and hydrogen (C2H6O + H2O 435 → C2H3O2- + H+ + 2H2) and the fermentation of methanol to carbon dioxide and hydrogen 18 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 436 (CH4O + 2H2O → CO2 + H2O + 4H2) (Figure S3B). Similarly, the near stoichiometric 437 conversion of lactate to acetate observed in Figure 3B agrees with the fermentation 438 reaction C3H5O3- + 2H2O → C2H3O-2 + HCO- + H+ + 2H2. The absence of short-chain 439 fatty acids in the microcosms used as controls corroborate that acetate was produced from 440 the organic amendments. 441 Acidity generated from fermentation reactions of the organic acids and dechlorination 442 reactions (i.e. HCl) can affect the success of the biodegradation of lesser chlorinated 443 ethenes (Christ et al., 2004). The successful reductive dechlorination of PCE to ethene 444 observed in the amended MW-2 microcosms shows that the aquifer was naturally well- 445 buffered (Table 1) and it can be assumed that pH was maintained within the range of 6- 446 8, which is described as optimal for dechlorinators (Yang et al., 2017a). 447 Similarly to our results, previous studies showed that methane production also 448 developed more slowly in lactate rather than ethanol-amended microcosms (Fennell et 449 al., 1997). Such pattern may be correlated with the amount of hydrogen released per mole 450 of ethanol and methanol, which is larger than that produced from lactate (Fennell et al., 451 1997). In the present work, since the concentration of fermentable organic substrates was 452 higher in the microcosms with ethanol and methanol (6 mM) than in the lactate ones (3 453 mM), the high amount of hydrogen released in the microcosms amended with the alcohols 454 could have caused a rapid shift to methanogenic conditions and stimulate the activity of 455 hydrogenotrophic methanogens with the subsequent methane production. 456 457 5. Conclusions 458 The use of an integrated approach that combined different complementary techniques 459 provided insights into the intrinsic biodegradation potential of a site contaminated with 19 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 460 chlorinated ethenes. The application of carbon stable isotopic balances and a statistical 461 analysis of the results suggested that two sources of PCE were responsible for the 462 contamination plume in this industrial area, but they have not been mixed, and that PCE 463 has been transformed, in any case, via the hydrogenolysis pathway to cis-DCE. The 464 identification of Dhc 16S rRNA and vcrA genes provided evidence of the aquifer potential 465 to detoxify PCE to ethene. The geochemistry of the aquifer suggested that activity of Dhc 466 in the cis-DCE stalled aquifer was impeded by the lack of sufficient electron donors to 467 lower the redox potential, and it was further corroborated with the establishment of 468 microcosms amended with fermentable substrates. The results obtained in this study 469 discourage natural attenuation as a remediation strategy in this contaminated site due to 470 the cis-DCE stall observed in microcosms miming the natural conditions of the aquifer 471 (unamended controls). The two treatments with organic amendments (lactate and the 472 mixture of ethanol plus methanol) accelerated the dechlorination of PCE and produced 473 ethene, but methane was vigorously produced in the microcosm containing methanol and 474 ethanol. In light of these results, an enhanced anaerobic bioremediation injecting lactate 475 as electron donor is recommended to detoxify this particular contaminated site. 476 477 6. Acknowledgements 478 This research has been supported by the Spanish Ministry of Economy and 479 Competitiveness, MINECO (CTM2013-48545-C2-1-R, CTM2016-75587-C2-1-R, 480 CGL2014-57215-C4-1-R and CGL2017-87216-C4-1-R projects) co-financed by the 481 European Union through the European Regional Development Fund (ERDF). This work 482 was also partly supported by the Generalitat de Catalunya through the consolidate 483 research groups (2017-SGR-14 and 2017SGR-1733) and N. Blázquez-Pallí’s Industrial 20 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 484 Doctorate grant (2015-DI-064). M. Rosell acknowledges a Ramón y Cajal contract 485 (RYC-2012-11920) from MINECO. The Departament d’Enginyeria Química, Biològica 486 i Ambiental of the Universitat Autònoma de Barcelona is a member of the Xarxa de 487 Referència en Biotecnologia de la Generalitat de Catalunya. We thank Dr. Ivonne 488 Nijenhuis for providing Dehalococcoides mccartyi BTF08 and CCiT-UB and Dr. Roger 489 Puig for excellent technical assistance. 490 491 492 21 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). 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Hydrol. 153, 92–105. 631 https://doi.org/10.1016/j.jconhyd.2013.06.004 27 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 632 Palau, J., Marchesi, M., Chambon, J.C.C., Aravena, R., Canals, À., Binning, P.J., Bjerg, 633 P.L., Otero, N., Soler, A., 2014. Multi-isotope (carbon and chlorine) analysis for 634 fingerprinting and site characterization at a fractured bedrock aquifer contaminated 635 by chlorinated ethenes. Sci. 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ISME 678 J. 11, 2767–2780. https://doi.org/10.1038/ismej.2017.127 29 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 679 Yu, J., Park, Y., Nguyen, V.K., Lee, T., 2016. PCE dechlorination by non- 680 Dehalococcoides in a microbial electrochemical system. J. Ind. Microbiol. 681 Biotechnol. 43, 1095–1103. https://doi.org/10.1007/s10295-016-1791-1 682 Yu, R., Andrachek, R.G., Lehmicke, L.G., Freedman, D.L., 2018. Remediation of 683 chlorinated ethenes in fractured sandstone by natural and enhanced biotic and abiotic 684 processes: a crushed rock microcosm study. Sci. Total Environ. 626, 497–506. 685 https://doi.org/10.1016/j.scitotenv.2018.01.064 686 687 30 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 688 Table 1. Hydrogeological and physicochemical parameters of the studied fully screened boreholes and groundwater samples. Numerical codes correspond 689 to monitoring wells depicted in Figure 1. MW-2 MW-3 MW-4 MW-6 MW-7 PZ-1 PZ-3 PZ-5 PZ-13 Prof A Borehole depth (m) 7.0 6.0 8.0 7.2 8.0 6.8 6.0 11.2 8.0 15.0 WT (m.a.s.l.) 268.2 269.3 270.4 269.1 265.5 272.2 271.8 n.a. 268.1 271.1 T (ºC) 17.6 24.5 16.5 19.1 18.8 20.7 20.3 18.3 18.4 18.9 pH 7.4 7.8 7.5 7.2 7.7 7.2 7.5 7.3 7.4 7.3 EC (mS/cm) 2.8 1.5 1.1 1.5 2.0 3.4 1.4 4.3 2.2 3.4 DO (mg/L) 0.2 n.m. n.m. 1.8 n.m. 1.2 1.0 2.0 1.5 n.m. Eh (mV) 247 337 392 288 266 469 401 361 273 94 SO4-2 (mg/L) 301.9 199.2 105.0 94.2 159.6 1435.1 182.4 179.2 181.7 144.3 NO3- (mg/L) 49.3 60.8 24.9 31.2 72.7 <20 130.5 41.3 116.8 <20 Fe (mg/L) <0.1 <0.02 <0.02 <0.02 <0.02 <0.1 <0.02 <0.1 <0.02 <0.1 Cl- (mg/L) 382.2 205.8 30.5 125.0 372.9 96.0 114.3 1053.0 368.6 711.3 HCO3- (mg/L) 605.3 251.9 585.3 652.3 260.1 543.4 351.3 369.1 364.5 605.3 Na (mg/L) 412.7 149.2 32.9 58.2 167.4 336.7 57.8 383.2 186.1 266.0 K (mg/L) 0.7 1.3 0.6 1.8 1.9 0.5 0.3 0.5 1.8 1.9 Ca (mg/L) 121.1 125.0 77.9 122.0 166.9 287.0 118.6 275.3 173.3 135.6 Mg (mg/L) 27.3 29.0 74.9 88.4 58.2 138.9 69.6 110.9 61.6 166.5 WT = water table, m.a.s.l. = meters above sea level, T = temperature, EC = electrical conductivity, DO = dissolved oxygen, Eh = redox potential, n.m. = not measured 690 31 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 691 692 Figure 1. Head contour lines, groundwater flow direction, location of boreholes and 693 suspected source points at the industrial contaminated area. 32 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 694 695 Figure 2. Molar concentrations distribution and carbon isotopic signatures (δ13C) of 696 chlorinated ethenes at the industrial contaminated area. The pie charts are proportionally 697 sized according to the total concentration of chlorinated ethenes in each well (from 2.8 to 698 82 µM). The carbon isotopic mass balance (δ13Csum) included all chlorinated ethenes 699 detected and it was calculated using Eq. 2. The numerical codes in the black rectangle 700 indicate the name of the well. Detailed information about the concentration and δ13C of 701 chlorinated ethenes in all monitoring wells can be found in Table S1. 33 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 702 34 This is the accepted version of the following article: Natàlia Blázquez-Pallí,Mònica Rosell, Joan Varias, Marçal Bosch, Albert Soler, Teresa Vicent, et al. Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain). Environmental Pollution, 244:2019, p.165-173, which has been published in final form at https://doi.org/10.1016/j.envpol.2018.10.013. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 703 Figure 3. Time-course of reductive dechlorination of chlorinated ethenes (●: PCE, ○: 704 TCE, ▲: cis-DCE, : trans-DCE, ■: VC, □: ethene and : sum of chlorinated ethenes 705 plus ethene, Panel A) and fermentation of lactate (●: lactate, □: pyruvate, ∆: acetate, : 706 formate, Panel B) in a lactate-amended microcosm constructed with aquifer materials 707 from well MW-2. Concentrations of chlorinated solvents and ethene are presented as 708 nominal concentrations. Data presented is from an individual microcosm, but it is 709 representative of triplicate microcosms. 710 35