Mesocosm Studies on the Efficacy of Bioamended Activated Carbon

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Mesocosm Studies on the Efficacy of Bioamended Activated Carbon for Treating PCB-Impacted Sediment Rayford B. Payne, Upal Ghosh, Harold Douglas May, Christopher W. Marshall, and Kevin R Sowers Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b01935 • Publication Date (Web): 15 Aug 2017 Downloaded from http://pubs.acs.org on August 16, 2017

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Mesocosm Studies on the Efficacy of Bioamended Activated Carbon

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for Treating PCB-Impacted Sediment

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Rayford B Payne†, Upal Ghosh‡, Harold D May§, Christopher W. Marshallǁ⊥, and Kevin R

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Sowers†*

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Department of Marine Biotechnology, Institute of Marine and Environmental Technology,

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University of Maryland Baltimore County, Baltimore MD ‡

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Department of Chemical, Biochemical, and Environmental Engineering, University of

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Maryland Baltimore County, Baltimore MD §

Marine Biomedicine and Environmental Science Center, Department of Microbiology and

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Immunology, Medical University of South Carolina, Charleston SC ǁ

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Biosciences Division, Argonne National Laboratory, Argonne, Illinois



Current Address: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA

* Corresponding author: Kevin Sowers, Department of Marine Biotechnology, Institute of Marine and Environmental Technology, 701 E. Pratt St., Baltimore, Maryland 21202 Telephone: (410) 234-8878/FAX: (410) 234-8896, e-mail: [email protected]

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ABSTRACT

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This report describes results of a bench-scale treatability study to evaluate the efficacy of

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bioaugmentation with bioamended activated carbon (AC) for in-situ treatment of polychlorinated

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biphenyl (PCB) impacted sediments.

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microorganisms to degrade and reduce the overall concentration of PCBs in sediment was

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determined in 2 L recirculating mesocosms designed to simulate conditions in Abraham’s Creek

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in Quantico, Virginia. Ten sediment mesocosms were tested for the effects of AC alone, AC

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with slow release electron donor (cellulose) and different concentrations and combinations of

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PCB dehalogenating and degrading microorganisms added as bioamendments. A 78% reduction

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of total PCBs was observed using a cell titer of 5 × 105 Dehalobium chlorocoercia and

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Paraburkholderia xenovorans cells g-1 sediment with 1.5% AC as a delivery system. Levels of

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both higher and lower chlorinated congeners were reduced throughout the sediment column

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indicating that both anaerobic reductive dechlorination and aerobic degradation occurred

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concurrently.

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bioaugmented treatments. Toxicity associated with co-planar PCBs was reduced by 90% after

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treatment based on toxic equivalency of dioxin-like congeners. These results suggest that an in

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situ treatment employing the simultaneous application of anaerobic and aerobic microorganisms

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on AC could be an effective, environmentally sustainable strategy to reduce PCB levels in

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contaminated sediment.

To this end, the ability of PCB transforming

Porewater concentrations of all PCB homologs were reduced 94-97% for

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INTRODUCTION

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Polychlorinated biphenyls (PCBs) were used widely in transformers, capacitors and other

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electrical applications for over five decades because of their superior flame resistance and

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insulating properties 1. Other common uses included fluorescent light ballasts, inks, paints,

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hydraulic oils, adhesives and carbonless paper. The manufacture of PCBs was banned in the

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United States in 1979 and worldwide in 2001.

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environment during their widespread use, PCBs continue to pose a health risk because their

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propensity for bioaccumulation in food chains and potential toxicity as a result of consumption

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by animals and humans 1.

However, as a result of release into the

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The commonly accepted methods for treatment of sediments impacted with PCBs are

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dredging and disposal or capping. Dredging is effective for reducing PCB levels in sediments,

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but it is costly, disruptive to the ecosystems and increases the potential risk if PCBs are released

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into the water 2. Capping with passive materials such as sand is an effective treatment approach

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for containment of PCBs in sediment, but because it is subject to potential abiotic and biotic

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disruption it does not completely eliminate the risk of later exposure and can be disruptive to the

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existing ecosystem 3. A recently developed method that is gaining acceptance is the application

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of amendments such as activated carbon (AC) to PCB impacted sediments to sequester PCBs 4.

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By sequestering soluble PCBs, AC effectively decreases the bioavailability of PCBs thereby

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minimizing the risk of exposure to the food chain 5. However, none of these methods has the

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potential to accelerate the degradation of PCBs beyond the rates observed for natural attenuation.

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Biological degradation of PCBs in the environment occurs by anaerobic dechlorination of

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highly chlorinated congeners followed by the aerobic degradation of the dechlorination products.

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The primary rate-limiting factor is the low native abundance of PCB dechlorinators in sediments

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PCB impacted sediment. For example, sequential treatment of PCB impacted sediment in an

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anaerobic PCB halorespiring enrichment followed by transfer in an aerobic culture containing

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Paraburkholderia xenovorans LB400 was reported to degrade weathered Aroclor 1248 and

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Aroclor 1260 by as much a 70% and 67%, respectively

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demonstrated the complementary role of anaerobic dechlorination and aerobic degradation in

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PCB degradation, they were conducted in closed microcosms with controlled redox conditions.

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In addition, delivery of microorganisms through a water column into sediments at field scale is a

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challenge. A promising approach is to deliver the microorganisms on solid particles that could

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integrate into the sediment environment.

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extensive dechlorination in Aroclor 1260 enriched sediment microcosms amended with AC.

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Payne et al. 10-11 applied granular activated carbon in the form of SediMite™ 4, a pelletized form

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of AC, inoculated with anaerobic halorespiring and aerobic degrading bacteria in static estuarine

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Baltimore Harbor sediment mesocosms and observed over 75% reduction in weathered Aroclor

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1260 levels in only 120 days.

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bioamendments, the report demonstrated that AC in principle could be used as a delivery system

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for PCB degrading microorganisms that would form a solid substrate for active microbial

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transformation. However, the study only tested two microorganisms at a single cell titer.

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. Thus, enhancing this natural process with bioaugmentation is a potential treatment strategy for

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. However, although both studies

Kjellerup et al.

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reported a shift towards more

In addition to enhanced degradation of PCB levels by the

A key aspect of planning a bioremediation field application is determining the optimal

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loading rate of treatment material into the sediment.

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parameters for optimal efficacy of bioaugmentation using pelletized AC as a potential carrier to

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deliver PCB degrading microorganisms to PCB impacted sediments.

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conducted in mesocosms containing PCB contaminated sediment from a watershed drainage

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In this study, we test and identify

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Experiments were

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pond adjacent to Chopawamsic Creek, a tributary of the Potomac River. The study employs

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static sediment mesocosms with recirculating overlying water to simulate in situ redox

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conditions by maintaining a dissolved oxygen concentration in the water column similar to that

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in the water column at the site. Parameters tested include effects of organic carbon,

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bioamendment titer, and different combinations of microorganisms on the total and porewater

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concentration of PCBs in sediments over the course of 375 days.

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MATERIALS AND METHODS

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Sediment collection and homogenization. PCB impacted sediment (25 L) was collected on 18

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October, 2012 from Abraham’s Creek in Quantico, VA with a grab sampler at 39°16.8 N,

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76°36.2 W and transported to the lab in four sealed 20 L polypropylene buckets. Water for the

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treatability study was collected from the site in a 20 L carboy. Sediment collected from the site

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was transferred to a 100 L basin in an anaerobic glove bag under a nitrogen atmosphere and the

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pooled sediment was thoroughly mixed with a 10 cm mud mixer (TBC Inc., Long Beach, CA)

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mounted on a power drill. Sediment was stored in the dark at 4°C prior to use.

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Preparation of bioamended AC. Halorespiring (PCB dehalogenating) bacteria were grown

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anaerobically in estuarine mineral medium (ECl)

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with the following electron donors and acceptors, respectively: “Dehalobium chlorocoercia”

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DF-1 12 with sodium formate (10 mM) and PCB 61 (2,3,4,5-tetrachlorobiphenyl; 173 µM), strain

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o-17

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Chloroflexus phylotypes SF1 and DEH10 14 with a fatty acid mixture containing sodium salts of

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acetate, propionate and butyrate (2.5 mM each) and Aroclor 1260 (100 mg L-1). Cultures were

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grown in 160-ml serum bottles containing 50 ml of medium sealed under N2-CO2 (4:1) with 20-

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mm Teflon-coated butyl stoppers (West Pharmaceutical, Inc., Exton, PA) and incubated

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statically at 30°C in the dark. For preparation of the bioamendment, halorespiring bacteria were

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twice sub-cultured 1:10 in 500 mL medium with 100 µM PCE substituted for PCB as the

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electron acceptor to remove any residual PCBs. The halorespiring cultures were harvested

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anaerobically under N2-CO2 (4:1) by centrifugation in 250 mL Oakridge bottles 15. Cell pellets

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. Halorespiring cultures were maintained

with sodium acetate (10 mM) and PCB 65 (2,3,5,6-tetrachlorobiphenyl; 173 µM) and

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were resuspended in 100 mL of ECl medium without PCE to a final concentration of 5 × 107

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cells mL-1. Paraburkholderia xenovorans LB400 16,17 was grown aerobically in M9 minimal medium

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with solid biphenyl crystals (5 mM; solubility in water, 2.89×10-2 mM) as the carbon source

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and electron donor as described previously

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were incubated at 30 °C with shaking at 100 rpm to an O.D.600 of 1.0 (ca. 4×108 cells mL-1),

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harvested by centrifugation, and the cell pellet was suspended in 100 mL of sterile M9 medium

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without biphenyl.

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. Cultures (100 ml) in 500 ml Erlenmeyer flasks

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Immediately before addition to mesocosms the anaerobe (DF-1, ο-17 and/or SF1-

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DEH10) and aerobe (LB400) were combined in a manual pump sprayer reservoir (1.75 L Flo-

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Master 56HD, Root-Lowell Mfc Co., Lowell, MI). For each mesocosm the concentrated cultures

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were sprayed onto 70 g of SediMite™ pellets (Sediment Solutions, Ellicott City MD), an

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aggregate of activated carbon (AC), sand, clay binder and where indicated 1% cellulose, at

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concentrations indicated below. Bioamended AC was immediately mixed into mesocosms as

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indicated below with a Teflon spoon and mesocosms were allowed to settle for 4 hours before

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baseline sampling. The final concentration of SediMite™ in sediment was 3% for a final

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concentration of 1.5% (dry wt) AC.

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Preparation of mesocosms. Aliquots of homogenized sediment (1.75 L) were transferred under

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an atmosphere of N2 in an anaerobic glove bag to two-liter thin layer chromatography tanks (ID

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in cm: 8W x 27H x 12D, sediment depth 6 cm, water depth 2 cm) modified as shown in Figure

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S1. After treatments shown in Table 1, the tanks were sealed with glass plates aerated water

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collected from the site was continuously circulated with a 12-channel peristaltic pump (Watson-

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Marlow) at a hydraulic retention time of one hour. The aerated water entered the tanks through a

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stainless steel manifold to create a linear flow over the surface of the sediment, thereby

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minimizing the risk of PCB loss due to volatilization and sediment turbation into the water

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column Aerated water was pumped from a sparging flask containing a fritted glass gassing tube

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connected to an air supply. The dissolved oxygen concentration in the water column, measured

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with a polarographic electrode (Mettler Toledo, Columbus OH), was maintained at 6.7±0.3 mg

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kg-1 at the mesocosm inlets and 6.5±0.3 mg kg-1 at the outlets, which is equivalent to 6.75 mg kg-

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1

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Amberlite XAD-2 (Rohm & Haas Co, Newark, DE) resin in the base of the sparging flask to

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remove soluble PCBs prior to aeration. Mesocosms were operated at a temperature of 22-24oC

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in the dark.

reported at the site in June 2012. Water flowing out of the mesocosm was passed through

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Sediment analysis. Sediment TOC analysis was performed as described by Grossman and

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Ghosh

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5000A) and non-dispersive infrared gas analyzer as recommended by the manufacturer.

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using a Shimadzu TOC analyzer with a solids sample module (TOC-5000A and SSM-

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PCB extraction and analysis of sediment. Sediment mesocosms were sampled in triplicate

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using a sterile 5 mL syringe barrel as a coring device. A numbered grid and random number

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generator were used to ensure that samples were collected from random locations.

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Approximately 5g wet weight sediment was dried with pelletized diatomaceous earth (Dionex,

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Sunnyvale, CA) in a desiccator at room temperature. The dried sediment (1 g) was extracted

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with an Accelerated Solvent Extractor (Dionex) following EPA method 3545 as described

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previously 10. PCB 166 (10 µl stock of 400 µg L-1 hexane) was added as a surrogate to correct

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for extraction efficiency. PCBs 30 and 204 (400 µg L-1 each in 10 µl acetone) were added as

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internal standards.

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PCB congeners were analyzed using a Hewlett-Packard 6890 series II gas chromatograph

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(GC) with a DB-1 capillary column (60 m × 0.25 mm × 0.25 µm; JW Scientific, Folsom, CA)

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and a

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congeners resolved in 130 individual peaks

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resolved and analyzed using a Hewlett-Packard 5890 series II gas chromatograph (GC) with a

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HT8 8% phenyl polycarborane-siloxane capillary column (60 m × 0.22 mm × 0.25 µm; SGE)

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and a 63Ni electron capture detector as described previously 21. PCB congeners 77, 81, 105, 114,

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118, 123, 126, 169, 156, 157, 167 and 189 were quantified with a 10-point calibration curve (2-

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800 µg L-1) using PCBs 30 and 204 as internal standards.

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Ni electron capture detector by a modified method of EPA 8082, which detected 173 10

. Co-planar dioxin-like PCB congeners were

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PCB extraction and analysis of passive samplers and XAD resin. Polyoxymethylene (POM,

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77 µM; CS Hyde Co., Lake Villa, IL) membranes cut into 1 x 7 cm strips were pre-cleaned with

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hexane followed by methanol, then secured in stainless steel screens with aluminum staples.

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Three POM samplers were inserted vertically in the sediment column of each mesocosm. The

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samplers were removed from the mesocosms 120 and 375 days after treatments, rinsed with

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water to remove sediment, and analyzed for PCBs as described in Beckingham et al.

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concentrations in POM were converted to estimated PCB concentrations in the porewater phase

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based on equilibrium partitioning constants 23. Performance reference compounds were not used.

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Amberlite XAD-2 resin (Rhom & Haas, Philadelphia, PA) was pre-cleaned with hexane

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followed by methanol prior to use and extracted for PCB analysis after 375 days as described

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above for the passive samplers.

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. PCB

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DNA extraction and enumeration of bacterial bioamendments. DNA was extracted by

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adding 0.25 g sediment (wet wt.) from each sample core to a PowerBead microcentrifuge tube

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(Power Soil DNA Isolation Kit, MOBIO Laboratories, Inc., Carlsbad, CA) as previously

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described 10. Extracted DNA samples had an A260/280 ratio of ≥ 1.6 and an A260/230 ratio of ≥

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2.0. Enumeration of microorganisms in each subcore was performed by real-time quantitative

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PCR (qPCR) using iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA). Primers

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included CIOP0/CIOP1 specific for the bphA gene operon of Paraburkholderia xenovorans

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LB400 and SKFPat9F/SKFPat9R specific for a putative reductive dehalogenase of “Dehalobium

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chlorocoercia” DF-1 as previously described

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enumerated by qPCR using primers Chl348F/Dehal844R specific for genes encoding 16S rRNA

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in the halorespiring chloroflexi

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with R2 = 0.999.

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(CIOP0/CIOP1), 1.3 (SKFPat9F/SKFPat9R) and 2.9 (Chl348F/Dehal844R).

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10-11, 24

.

All other halorespiring strains were

. Amplification efficiencies of standards were >89.0±9.0%

The linear range was 0.1 to 1x10-6 ng and the y-intercept was 1.2

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Analysis of the microbial community. DNA was prepared for amplification using the 5PRIME

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MasterMix (5 PRIME, Inc, Gaithersburg, MD) and 0.2 µM 515F/806R primers

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amplified using the following conditions: 3 minutes at 94°C, 35 cycles of 94°C for 45 seconds,

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50°C for 1 minute, and 72°C for 1.5 minutes, with a final extension for 10 min at 72°C.

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Amplicons were then pooled to 70 ng DNA per sample, and a clean pool was then generated

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using the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). DNA was prepared for

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amplification using the 5PRIME MasterMix (5 PRIME, Inc.) and 0.2 µM of 515F/806R primers

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targeting the V4 region of the 16S rRNA gene. Clean pools were sequenced on an Illumina

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. DNA was

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MiSeq using V3 chemistry to obtain 2x150 base pair reads. The average sequencing coverage

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was over 17,000 sequences per sample.

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Materials.

Community analyses are described in Supplemental

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Data availability. Study: PRJNA355587 (SRP095095), sample: PCB mesocosms

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(SRS1859570), experiment: mesocosm_seqs (SRX2422444), run: mesocosm_seqs.fastq.gz

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(SRR5110056).

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RESULTS and DISCUSSION

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Effect of treatments on reductive dechlorination and degradation of PCBs. Water content of

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the homogenized sediment was 74% ± 6 and total organic carbon (TOC) was 6.7% ± 0.5. Total

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PCB concentration in the pooled sediment samples was 3.40 ± 0.50 mg kg-1 with a mean of 3.5 ±

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0.1 chlorines per biphenyl. Additional characterization of sediment is provided in Supplemental

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Materials. The effect of treatments on PCB levels in Abraham’s Creek sediment is shown in

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Figure 1 and Table 2. The results in non-bioamended treatments 1, 2 and 3 indicate that there

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was no significant (ρ>0.05) biostimulation of indigenous bacterial populations or abiotic loss as

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a result of mixing, addition of AC, or addition of cellulose as a carbon source. This observation

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indicates that biostimulation of indigenous halorespiring and degrading PCB microorganisms

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would not be effective for reduction of PCB levels in Abraham’s Creek sediment at rates greater

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than those observed for natural attenuation.

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All bioamended treatments showed significant degradation with the exception of

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treatments 4 and 9. A titer of 103 cells g-1 showed no significant effect on PCB concentration

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over the course of 375 days, whereas bioamended treatments 5 and 6 showed significant

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degradation, activity, 58 and 78%, respectively. In treatment 6 most of the degradation, 55%,

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was observed in the first 30 days and maximum degradation of 78% was observed in treatment 6

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after 120 days. This value is similar to the maximum value of 80% total PCB reduction observed

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365 days after bioamendment of weathered Baltimore Harbor sediment mesocosms reported by

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Payne et al. 11. In contrast, degradation in treatment 5, 7, 8, 9 and 10 continued after 120 days.

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The addition of cellulose as a slow release carbon source in combination with

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bioamendments appeared to increase the extent of PCB reduction. However, the difference in

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PCB levels after 376 days in treatment 6, which contained 105 cells g-1 of DF-1 and LB400 with

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cellulose, and treatment 7, which contained the same bioamendment titer without cellulose was

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not significant (ρ>0.05). The results suggest that digestible organic carbon was sufficiently

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available in the sediments.

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Nona- through hexa-chlorobiphenyls were reduced by 80% in treatment 6 to a final total

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concentration of 0.12 mg kg-1 after 375 days (Figure S2). Since these homolog groups are not

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attacked or poorly attacked by LB400 27, most of the reduction in concentration is attributable to

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reductive dechlorination by DF-1 and possibly by indigenous halorespirers

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homologs accounted for only 22% of the total weathered PCBs in Abrahams Creek sediment,

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bioaugmentation with a halorespirer such as DF-1 would have a critical role in less weathered

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sediment where nona- to hexa-chlorobiphenyls can account for an average 88% of the total

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congener mass in Aroclor 1260 28. Pentachlorobiphenyls 146, 151 and 153, which accounted for

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5.2% of the total mean PCB concentration in the sediment, were degraded by 86, 87 and 89%,

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respectively, in treatment 6. However, these congeners are not dechlorinated by DF-1

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poorly degraded by LB400

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bioamendment with DF-1, a phenomenon that has been reported previously, 10,11,12 indicates that

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indigenous halorespiring or degrading microorganisms were active in the mesocosm, possibly

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stimulated by dechlorination products of DF-1.

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.

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. Although these

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and

The transformation of non-substrate congeners after

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Halorespiring bacteria with different substrate specificities were added to mesocosms 8

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through 10 to test their effectiveness for reducing total PCB concentrations as co-amendments

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with LB400. The extent of degradation with addition of o-17 in treatment 8, which preferentially

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attacks congeners in single flanked ortho substituted and double flanked meta chlorine

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substituted positions, and with DEH10 and SF-1 in treatment 9, which preferentially attack

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congeners in single and double flanked meta chlorine substituted positions, was less than that

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observed with a similar cell concentration of DF-1 in treatment 6. Although DF-1 is limited to

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reduction of doubly flanked chlorines, the dechlorination patterns were similar between the

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amendments.

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chlorobiphenyls contained one or more doubly flanked chlorines

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attacked prior to singly flanked chlorines by both SF1/DEH10 and o-17

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in few observed differences between dechlorination patterns of higher chlorinated congeners by

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DF-1 and the other halorespiring bacteria. Differences in the dehalogenation patterns by the

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different halorespiring strains would also be masked by the relatively rapid degradation rates of

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aerobic degradation

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differences would likely have been observed as distinct singly flanked chlorines would have

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been available and targeted as PCBs were dechlorinated to congeners with six or less chlorines.

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Interestingly, total PCB degradation by the combination of all three halorespiring

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bioamendments appeared to be slightly less than bioaugmentation with only DF-1 and LB400 in

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treatments 5 and 6, although this difference was not significant (ρ>0.05). The results indicate

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that addition of 105 cells g-1 each of DF-1 and LB400 was the most robust treatment for reducing

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total PCB levels. This observation along with the fact that expected dechlorinated intermediates

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did not accumulate confirm that the total reduction in PCB concentration in the mesocosms was

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the result of anaerobic halorespiration and aerobic degradation occurring concurrently.

One explanation for this observation is that most of the hexa- to nona-

27, 30

, compared with halorespiration

14,10

.

28

, which are preferentially 14,29

. This would result

In the absence of LB400,

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PCB degradation throughout depth profile. The PCB concentrations were not significantly

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different (ρ>0.05) for the upper and lower 3 cm of the sediment column in any of the mesocosms

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(Figure S3). The results indicate that bioaugmentation was effective throughout the 6 cm

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sediment column. PCB degradation by LB400 requires oxygen for dioxygenase mediated ring

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cleavage

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water column, discoloration of the sediment by oxidation was only obvious in the top 0.3-0.5 cm.

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Below the oxidized layer the sediments remained black, likely due to reduced conditions

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maintained by anaerobic decay of native and added organic matter. Benthic activity due to

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worms was observed throughout the entire sediment depth in treatments 5 and 6 for the first four

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months, which also had the greatest extent of degradation. Based on these results it is not

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possible to draw any conclusion on the role of bioturbation for oxygenation of the lower

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sediment profile. However, the results indicate that even in mesocosms where benthic activity

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was not observed there was sufficient diffusion of oxygen through the porewater to support

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aerobic degradation. The similarity of the homolog patterns between the top and bottom cores

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after 120 days further confirms that both anaerobic dechlorination and aerobic degradation

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occurred throughout the sediment column in treatments 5 to 10.

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. Although dissolved oxygen levels were maintained at 6.7 mg L-1 throughout the

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Effects of treatments on toxicity of PCBs. Most of the toxic effects of PCBs for humans are

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mediated through the aryl hydrocarbon receptor (AhR), a cytosolic receptor protein present in

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most vertebrate tissues with high affinity for 2,3,7,8-substituted PCDD/Fs and some coplanar

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PCB congeners

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2,3,4,4’5-pentachlorobiphenyl (PCB 114), 2,3,3’,4,4’,5-hexachlorobiphenyl (PCB156) and

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2,3,3’,4,4’,5’-hexachlorobiphenyl (PCB 157) (Figure 2). The total concentration of these three

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congeners on day 0, 7.40 ng g-1 dw, was reduced 90% to 0.75 ng g-1 dw 375 days after treatment.

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Factoring in the toxic equivalency factor (TEF) for each coplanar congener relative to 2,3,7,8-

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tetra dibenzo-p-dioxin 33, the total toxic equivalency (TEQ) was reduced from 0.22 to 0.02 ρg g-

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. Three coplanar congeners were detected in Abraham’s Creek sediment:

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1

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estimating relative changes in potential exposure to dioxin-like chemicals from consumption of

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aquatic food products as this approach does not take into account a number of factors such as

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inhibition of TCDD toxic effects by other congeners in PCB mixtures

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bioaccumulation within the food chain such as partitioning coefficient 35.

. TEQ methodology in human risk assessment in the context of this study is only intended for

34

and factors affecting

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Effect of treatments on PCBs in porewater. Changes in PCB concentrations in porewater

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were measured by passive equilibrium sampling 375 days after treatments Figure 3. There was

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a 30-35% reduction in PCB porewater concentrations after treatment with non-bioamended AC

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in treatments 2 and 3 compared with untreated sediment.

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concentration of PCBs with abiotic AC is less compared with previous reports of >95%

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reduction of porewater PCBs with AC amendments 36. The difference can be largely attributed

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to the low AC dose in the present study (1.5%) compared to much higher doses of AC in

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previous work (>2.5%). The AC dose was kept low in the current study to focus primarily on

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the effect of bioamendment.

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bioamended with 103 (treatment 4), but the difference was not significant (ρ> 0.05) from the

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abiotic AC treatments 2 and 3. All of the remaining bioamended treatments showed a significant

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reduction of PCBs in the porewater compared with untreated sediment (ρ< 0.05) ranging from

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94% in treatment 10 to the greatest reduction, 97%, in treatment 6. Thus, the overall reductions

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in the freely dissolved porewater concentrations were larger than the corresponding reductions in

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the total PCBs in sediments indicating that the more soluble dechlorination products were not

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accumulating, but were further degraded by the aerobes. Presence of 1.5% AC was also

The reductions in porewater

Porewater PCB was reduced 40% after treatment with AC

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contributing to some of the reductions in porewater concentration beyond mass reduction by

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degradation.

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PCBs were not detected in the XAD resin indicating the reduction in PCB levels was not

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attributable to abiotic loss in the water column. Coplanar PCB congeners levels in the overlying

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water phase were below the detection limit of 0.01 ng L-1 in all of the mesocosms, including the

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untreated mesocosm. The results indicate that the addition of bioamendment was effective at

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reducing freely dissolved PCBs levels in sediment porewater, thereby reducing the potential for

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PCB flux into the overlying water and bioaccumulation in the aquatic food web including fish.

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Sustainability of the bioamendment. DF-1 and LB400 in treatment 6 were detected throughout

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the experiment, but their titer decreased approximately 2-3 orders of magnitude after 375 days

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(Figure 4), whereas the number of DF-1 and LB400 gene copies g-1 in treatments in non-

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bioaugmented treatments was below the theoretical detection limit of 102 gene copies g-1

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sediment. During the first sixty days when the cell titer was highest, there was a decrease in

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PCBs, but no significant increase in intermediate dechlorination products. Some dechlorination

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products were observed to accumulate on days 120 and 375, which coincides with two orders of

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magnitude decrease in the titer of LB400 (Figure S4). Overall, the results suggest that the

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aerobic degradation rate by LB400 was greater than the halorespiration rate of DF-1 in the first

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two months, but as the titer of LB400 decreased the net rate of degradation no longer exceeded

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that of anaerobic dechlorination, resulting in accumulation of some dechlorinated congeners.

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This observation is not surprising as degradation of halogenated biphenyls is co-metabolic and

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will not support growth 16. The titer of DF-1 also decreased over the course of 375 days, but at a

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slower rate than LB400. Although halorespiration of PCBs supports growth,14, 12 Lombard et al.

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relevant soluble PCB concentrations is too low to maintain a large population of bacteria. In the

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present study this limitation was overcome by introducing a high cell titer sufficient to reduce the

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bioavailable portion of PCBs without growth.

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calculated that the thermodynamic cell yield of halorespiring bacteria at environmentally

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In a prior study, activity in Baltimore Harbor sediment mesocosms bioaugmented with

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DF-1 and LB400 degradation was not detected after 80% of total PCBs were removed, although

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the remaining congeners were potential substrates for PCB dehalogenation or degradation. The

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report suggested that activity stopped because PCBs were no longer bioavailable and/or oxygen

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became limiting in the static mesocosms. However, in this study the dissolved oxygen in the

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water column was maintained at above 6% throughout the 375 days incubation period and there

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were no significant difference in the degradation patterns in the sediment column, which

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suggests that oxygen was likely not the limiting factor. Another possibility for the observed limit

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of degradation at 78% of the original PCB concentration in treatment 6 was the reduction in cell

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titer over time. However, repeating the treatment of mesocosm 6 after 375 days (105 cells g-1

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each of DF-1 and LB400 applied with AC and 0.03% cellulose) did not stimulate further

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degradation of total PCBs after 79 days, which indicates that the decline in activity was not due

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to a decrease in the titer of an active PCB degrading population or TOC. This observation

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combined with the low concentration of soluble PCBs (97% reduction) suggests that activity was

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inhibited by low bioavailability of the remaining PCBs. The total PCBs remaining are likely

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tightly bound to the AC and organic fraction in the sediment, and are below the threshold for

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uptake by the bioamendments.

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Effect of treatments on the indigenous microbial population. Overall, the microbial diversity

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significantly decreased over the course of the experiment (paired t-test, ρ