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Organic carbon amendments for enhanced biological attenuation of trace organic contaminants in biochar-amended stormwater biofilters Bridget A. Ulrich, Marta Vignola, Katelynn Edgehouse, David Werner, and Christopher P. Higgins Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 19 Jun 2017 Downloaded from http://pubs.acs.org on June 19, 2017
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Organic carbon amendments for enhanced biological attenuation of trace organic contaminants in biochar-amended stormwater biofilters Bridget A. Ulrich,† Marta Vignola,§ Katelynn Edgehouse,‡ David Werner,§ and Christopher P. Higgins†,* †
Department of Civil and Environmental Engineering Colorado School of Mines, Golden, CO §
School of Civil Engineering and Geosciences Newcastle University, Newcastle upon Tyne, United Kingdom ‡
Department of Chemistry Cleveland State University, Cleveland, Ohio Submitted to Environmental Science & Technology *To whom correspondence should be addressed. E-mail:
[email protected] Phone: (720)-984-2116 Fax: (303) 273-3413
Number of pages: Number of figures: Number of tables: Number of words:
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ABSTRACT
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This study sought to evaluate how dissolved organic carbon (DOC) affects attenuation of
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trace organic contaminants (TOrCs) in biochar-amended stormwater biofilters. It was
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hypothesized that (1) DOC-augmented runoff would demonstrate enhanced TOrC
40
biodegradation, and (2) biochar-amended sand bearing DOC-cultivated biofilms would
41
achieve enhanced TOrC attenuation due to sorptive retention and biodegradation.
42
Microcosm and column experiments were conducted; utilizing actual runoff, DOC from
43
straw and compost, and a suite of TOrCs. Biodegradation of TOrCs in runoff was more
44
enhanced by compost DOC than straw DOC (particularly for atrazine, prometon,
45
benzotriazole, and fipronil). 16S rRNA gene quantification and sequencing revealed that
46
growth-induced microbial community changes were, amongst replicates, most consistent
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for compost-augmented microcosms, and least consistent for raw runoff microcosms.
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Compost DOC most robustly enhanced utilization of TOrCs as carbon substrates,
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possibly due to higher residual nutrient levels upon TOrC exposure. Sand columns
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containing just 0.5 wt% biochar maintained sorptive TOrC retention in the presence of
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compost-DOC-cultivated biofilms, and TOrC removal was further enhanced by
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biological activity. Overall, these results suggest that co-amendment with biochar and
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compost may robustly enhance TOrC attenuation in stormwater biofilters; a finding of
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significance for efforts to mitigate the impacts of runoff on water quality.
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INTRODUCTION
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Stormwater runoff has degraded urban water quality by transporting contaminants
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to receiving waters.1 Low Impact Development (LID) systems have gained popularity
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because they remove many conventional contaminants during infiltration (e.g., metals
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and petroleum hydrocarbons).2,3 However, removal of trace organic contaminants
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(TOrCs) in LID systems is poorly understood, despite their broad presence in urban
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runoff.4 In particular, many toxic stormwater TOrCs, such as urban-use pesticides (e.g.,
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phenylurea and triazine herbicides, phenylpyrazole insecticides),5–7 flame retardants (e.g.,
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organophosphates),8 and chemicals from vehicle fluids (e.g., benzotriazoles),9 are also
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relatively mobile; allowing them to pass through LID systems.10
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Amendment of LID systems with biochar has shown promise for improved
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removal of metals, bacteria, nutrients, and TOrCs.11–15 In particular, a recent study
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revealed remarkable TOrC removal in intermittently dosed, biochar-amended infiltration
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columns: >99% TOrC removal from spiked natural water was maintained throughout
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cumulative treatment of an annual treatment volume estimated for the Denver area.16
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However, it remains unclear if sorptive TOrC retention can be maintained throughout an
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entire system lifetime (i.e., more than 15 years, as limited by accumulation of non-
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degradable metals).17 It would therefore be desirable to stimulate biodegradation to
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reduce potential accumulation, as has been demonstrated previously for conventional
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stormwater biofilters.18 These systems contain an upper layer amended with organic
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carbon (e.g., compost or straw), stimulating biological activity by slowly releasing
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growth substrates (e.g., dissolved organic carbon, DOC; and nutrients such as nitrogen,
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N, and phosphorus, P). As this may also cause undesirable sorbent fouling and reduced
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TOrC sorptive retention,19 it is of interest to determine if DOC can allow enhanced
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biological TOrC attenuation despite this potential tradeoff.
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While several studies have evaluated the effects of DOC on microbial
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communities and TOrC biodegradation in wastewaters and surface waters,20–22 it remains
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unclear how the underlying phenomena will manifest in stormwater biofilters. TOrC
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biodegradation may be affected by increased overall microbial abundances, as well as
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growth-induced changes in microbial communities. For example, broader TOrC
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biodegradation potential has been demonstrated in wastewater communities with greater
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biodiversity (i.e., community richness and evenness),20 which has been linked to
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availability of ambient carbon and nitrogen.22 Further, DOC can affect TOrC-degrading
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activity by influencing biofilm formation and structure.23 DOC quality and composition
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may be drivers behind these phenomena. For example, easily utilizable carbon substrates
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may allow rapid microbial growth, while growth on more complex substrates may lead to
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broader substrate utilization potential.24 The carbon-to-nitrogen ratio (C:N) of leached
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DOC can also affect growth (and in turn TOrC utilization) by establishing either carbon-
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limited or nutrient-limited growth conditions.25,26 As DOC quality and composition can
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be controlled in biofilters through selection of organic carbon amendments, biofilters
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could potentially be designed to achieve optimal TOrC biodegradation.
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The objective of this study was to assess how DOC from organic carbon
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amendments affects TOrC attenuation in biochar-amended biofilters. Efforts were
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focused on biological processes, as relevant abiotic processes (i.e., sorptive TOrC
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retention in presence of biochar and DOC) were assessed in a previous study.14 It was
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hypothesized that (1) augmenting runoff with DOC would enhance TOrC biodegradation
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due to increased microbial growth, activity, and potentially growth-stimulated changes in
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community composition; and (2) biochar-amended media bearing DOC-cultivated
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biofilms would demonstrate enhanced TOrC attenuation due to a combination of TOrC
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sorptive retention and biodegradation. The first hypothesis was assessed in microcosm
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experiments, and the second in column experiments. To maintain environmental
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relevance, these experiments utilized actual runoff to obtain representative microbial
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consortia, straw and woody plant-based compost to obtain DOC of varying quality, and a
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suite of TOrCs and transformation products (TPs) to represent compound classes that are
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likely to impact water quality (i.e., both toxic and mobile). The results from this study
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provided useful insight for the design of infiltration systems to mitigate impacts of runoff
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on receiving water quality.
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EXPERIMENTAL SECTION
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TOrC Analysis. The TOrC suite was selected in a previous study,4 and included atrazine,
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diuron, tris(3-chloro-2-propyl)phosphate (TCPP), tris(3-chloro-ethyl)phosphate (TCEP),
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prometon, benzotriazoles (1H-benzotriazole in microcosm experiments, 5-methyl-
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benzotriazole in column experiments), fipronil, oryzalin, and 2,4-dichlorophenoxyacetic
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acid (2,4-D results are reported in the Supporting Information, because it behaved
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unusually relative to the other analyzed TOrCs for reasons that remain unclear).
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Transformation
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desethylatrazine, DEA; deisopropylatrazine, DIA), diuron (1-(3,4-dichlorophenyl)-3-
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methylurea, DCPMU; 3,4-dichloroaniline, DCA), and fipronil (fipronil sulfone and
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sulfide). TOrCs were measured by liquid chromatography tandem mass spectrometry
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(LC-MS/MS), and ionized by electrospray ionization (ESI) in the positive or negative
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mode (0.025 μg/L limit of quantitation, details in Method S1). Optima™-grade methanol
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and water (Fischer Scientific) were used unless noted otherwise.
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DOC Extracts. Concentrated DOC extracts (used to augment DOC levels in
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microcosms) were prepared in runoff that was collected following three separate rain
products
(TPs)
were
analyzed
for
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events, such that it was stored for less than 24 hours prior to use in microcosm, column,
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and DOC characterization experiments. Runoff was collected in sterilized glass jugs from
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a pipe discharging runoff from a residential neighborhood in Golden, CO. Approximately
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500 mL of sediment (leaves, sticks, and silt) was added to each jug to introduce bacteria
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and DOC. The runoff was equilibrated at room temperature on a shaker table overnight
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and centrifuged for 5 minutes at 800 RCF to remove large particles. To enable rapid
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production of concentrated DOC extracts (to allow augmentation of runoff with DOC
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simulating leachate from rain events on the order of hours), runoff (2 L) containing either
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a woody plant-based compost (EcoGrow® compost, obtained from A1 Organics; Eaton,
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CO, 500 mL) or wheat straw (Longmont, CO; 2L, packed) was incubated at 30 °C on a
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shaker table overnight in the dark (such that the leached DOC was minimally affected by
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exposure to UV radiation).27 Extracts were filtered to 0.45 μm (such that minimal residual
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bacteria remained prior to subsequent sterilization by autoclave), analyzed with a
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Shimadzu Total Organic Carbon Analyzer to determine DOC and total nitrogen (TN),
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then diluted with filtered runoff to adjust the concentration as necessary.
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Fresh DOC extracts were analyzed by ultra-violet visible (UV-vis) spectroscopy
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(Figure S1) from which specific UV absorption was determined (SUVA, absorption at
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254 nm; listed in Table S4 with additionally calculated absorption parameters). SUVA is
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an indicator of DOC aromaticity,28 and negative associations between SUVA and DOC
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biodegradability have been previously reported for soil leachates.29 SUVA was higher for
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the compost DOC (3.0 L·mgC-1·m-1 for compost DOC, versus 0.7 L·mgC-1·m-1 for straw
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DOC), likely because the compost was derived from lignin-rich, woody material.30 The
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C:N ratios for fresh compost and straw extracts were 8.3 and 15.8, suggesting that they
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provided carbon-limited and nutrient-limited growth conditions, respectively (considering
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an optimal C:N of 10).31 Higher residual (post-incubation) DOC levels in compost
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microcosms suggested that the compost DOC was less biodegradable (52% and 89%
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DOC biodegraded in compost and straw microcosms, respectively; Table S5). Lower
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residual levels of P (Table S6) and N (Table S5) in straw microcosms indicated nutrient-
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limited growth conditions.
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Microcosm Experiments. Three types of aqueous microcosm media (raw runoff, and
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runoff augmented with DOC from compost or straw) were used to prepare eight biotic
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microcosms (five monitored for TOrCs, three sacrificed for additional analyses) and five
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biologically inhibited controls. Erlenmeyer flasks (150 mL) were filled with 5 g sand
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(Sigma Aldrich) and 10 mL DOC extract or filtered raw runoff, then autoclaved. The
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microbial consortium was introduced with 40 mL of collected runoff, such that the DOC
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prior to incubation (and subsequent dilution) was 205 mg/L in straw and compost
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microcosms (6 mg/L DOC in raw runoff). Microcosms were aerobically incubated for 14
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days in the dark at 30 °C on a shaker table to allow for microbial growth. Reduced
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dissolved oxygen (DO, Hach® DO probe) consumption in sacrificed microcosms
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indicated stabilized biological activity after this duration. Microcosms were then diluted
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to 125 mL with autoclaved synthetic stormwater (composition in Table S3), and a sodium
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azide (NaN3) solution (5 mL of 4 g/L in DI water, 154 mg/L final concentration) was
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added to control microcosms to inhibit biological activity (biotic microcosms diluted with
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5 mL autoclaved DI water). These dilutions allowed sufficient volume for numerous
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sampling events, and reduced the DOC to more representative levels (i.e., less than 50
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mg/L, Table S5).
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TOrCs were introduced by spiking microcosms with 52 μL of a methanol carrier
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solution (time 0), such that initial individual TOrC concentrations (40 μg/L) permitted
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small sample volumes without exceeding environmentally relevant levels.4 Spiked
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microcosms were shaken on a shaker table at room temperature and sampled over 76
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days (daily to weekly sampling). Prior to each sampling event, autoclaved DI water was
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added to correct for evaporation losses (individual volumes determined gravimetrically to
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avoid dilution). Aliquots (250 μL) were transferred with sterile pipette tips to 2 mL
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microcentrifuge tubes containing 200 μL internal standard solution (4 μg/L isotope
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surrogates in methanol) and 1550 μL of Milli-Q™ water containing 500 mg/L sodium
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azide to prevent biodegradation during storage (4°C, less than 30 days). Solid phase
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extraction (SPE) was carried out immediately prior to LC-MS/MS analysis using
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Phenomenex Strata®-X 33u Polymeric Reversed Phase cartridges (30mg/1mL) following
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the manufacturer’s recommended protocol. Nitrate (Nitrate TNT Plus Hach Kit), optical
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density (OD, absorbance at 600 nm), and DO were also measured on 3 – 4 occasions
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(Figure S2).
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Column Experiments. Columns (Kimble Chase Economy FLEX-COLUMN® 6 cm
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length, 2.5 cm I.D.) were dry-packed with sand (Sigma Aldrich, 210 – 297 μm particles,
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column diameter: particle size greater than 50 to prevent boundary effects),32 or sand with
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0.5 wt% biochar (53 – 250 μM particle sizes, Milli-Q™-rinsed), and porosity was
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estimated gravimetrically after saturation with water (0.35 ± 0.1 for biochar columns,
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0.32 ± 0.1 for sand columns). Mountain Crest Gardens biochar (MCG-biochar, produced
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by gasification of pinewood by Gropro, Inc., in Etna, CA; material properties in Table
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S7) was selected for its high surface area (317 m2/g) and proven TOrC retention
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performance.14 While higher biochar doses may be desirable in full-scale systems (e.g.,
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greater than 5 wt%),16 a low dose was necessary to allow TOrC breakthrough within a
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reasonable experimental time frame. Batch sorption experiments with the DOC extract
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and biochar revealed that the biochar had similar sorption affinities for both types of
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DOC (Method S2, log Kd,DOC [L/kg] = 3.2).
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Biofilms were seeded on columns with concentrated solutions prepared similarly
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to the microcosm media. Over three cycles, undiluted, autoclaved straw and compost
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DOC extracts (>1000 mg/L DOC) were inoculated (either with runoff or the previously
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incubated culture) and incubated until increases in OD indicated exponential growth
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(Method S3, growth curves in Figure S4). After flushing with CO2 to ensure saturation,
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columns were injected with one pore volume (PV, approximately 10 mL) of seeding
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solution at 1 mL/min in upflow, and then slowly cycled for 24 hours (0.05 mL/min, 0.6
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cm/hr linear velocity) to allow microbial attachment. Columns were then equilibrated
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with synthetic stormwater containing 10 mg/L DOC at 0.21 mL/min (2.6 cm/hr linear
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velocity) for 10 days (influent reservoirs refreshed every 2-3 days) until effluent DO
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levels stabilized (Figure S5). Tracer tests using KBr (10 g/L) as a conservative tracer and
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a Hach® conductivity meter showed that hydraulic retention times were minimally
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affected by biofilm formation (i.e., breakthrough less than 10 minutes before the
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predicted 2.3 hour hydraulic retention time).
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Columns were then injected with synthetic stormwater containing 10 mg/L DOC
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and 10 μg/L TOrCs (methanol from TOrC carrier solution evaporated under N2) for 23
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days (675 PVs) and 18 days (528 PVs), respectively (injection times differed due to
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slower microbial growth in the compost DOC seeding solution, Figure S3). Influents
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were refreshed every 24 hours to minimize TOrC biodegradation within the reservoir.
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NaN3 (400 mg/L) was added to influents for control sand-biochar columns (one control
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and three biotic replicates per DOC type). Following TOrC injection, columns were
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flushed with TOrC-free synthetic stormwater for the remainder of the experiment.
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Effluent samples (collected every 1 – 100 PVs over the total 1100 PVs) were preserved
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by diluting 10% with 4 g/L NaN3 (biotic columns only) and stored at 4°C in the dark.
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Isotope dilution was carried out immediately prior to LC-MS/MS analysis (ESI positive
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mode only due to constraints on sample analysis).
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Microbial Analyses. Microbial community composition and structure were investigated
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by high-throughput sequencing (pyrosequencing). Due to constraints on sample analysis,
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DNA extraction and sequencing was carried out for 3 representative biotic microcosms
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from each condition (9 total), and the column seeding solutions (Figure S4). The subset
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of microcosms chosen for microbial sequencing analysis included, from each condition,
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two TOrC-monitored microcosms (Figure S9; samples C1, C5, S1, S2, R1, and R5) and
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one unmonitored microcosm (samples C6, S6, and R6), such that community differences
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due to the TOrC sampling process could be identified if present. Immediately following
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the final sampling event, the sand and remaining aqueous media were centrifuged in
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sterile 50 mL Falcon tubes at 3220 RCF for 60 min. After decanting the liquid, the top
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500 mg of sand (wet weight) was collected for DNA extraction. DNA was extracted in
239
duplicate using a PowerSoil DNA Isolation Kit (MO Bio Laboratories, Inc.), according to
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the manufacturer’s instructions (stored at -80 °C until subsequent analysis).
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Polymerase Chain Reaction (PCR) amplification of the V4-V5 region of bacterial
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16S rRNA genes for each DNA extract was conducted using fusion primers containing a
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PGM sequencing adaptor, a ‘GT’ spacer, and a unique 12 base pair Golay barcode to
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allow multiplex analyses (primers 515F: 5’-GTGNCAGCMGCCGCGGTAA-3’, and
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926R: 5’-CCGYCAATTYMTTTRAGTTT-3’).33 PCR reactions were conducted using
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the FastStart High Fidelity PCR System and the PCR Nucleotide Mix (Roche Diagnostics
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GmbH), with the following thermocycle program: (i) 2 minutes denaturation at 95°C, (ii)
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30 cycles of 30 minutes denaturation at 95°C, (iii) 30 second annealing at 56°C, (iv) 45
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minutes elongation at 72°C, and (v) 7 minutes elongation at 72°C. PCR products were
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purified using Agencourt AMPure XP reagent (Beckman Coulter) and quantified using a
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Qubit dsDNA HS Assay kit (Invitrogen) on a Qubit® 2.0 Fluorometer. The single PCR
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libraries were pooled in equimolar amounts, further purified using a Pippin Prep System
253
(Life Technologies) following the manufacturer’s protocol. Quantitative PCR (qPCR) of
254
overall bacterial 16S rRNA genes was also conducted to qualitatively compare gene copy
255
numbers among samples (Method S4).
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Sequencing was carried out with an Ion Torrent Personal Genome Machine®
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(PGM) System (Life Technology) at Newcastle University. A total of 1,863,582
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sequences were obtained and processed in QIIME (v 1.7.0) using the bioinformatics
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pipeline. The sequences were trimmed to remove primers and barcodes, quality filtered
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(considering a minimum quality score of 20) and chimera checked (ChimeraSlayer)
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resulting in 739,973 total sequences. Sequences were then clustered into Operational
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Taxonomic Unit (OTU) at 97% sequence similarity level by the uclust algorithm, and a
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representative sequence from each OTU was selected and taxonomically identified using
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the Greengenes database34 and classified using the RDP naïve Bayesian rRNA
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classifier.35–37 Representative sequences and correspondent taxonomic assignment were
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used to build a table of OTU abundances at different levels of taxonomy. Microbial
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community diversity within and across the samples was determined using the QIIME
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(v.1.7.0) pipeline and Primer v6 software.38,39 The Bray Curtis similarity metric was
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generated and represented by a 2-dimensional non-metric multidimensional scaling
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(nMDS) plot.39 Additionally, an analysis of similarities (ANOSIM) was conducted on the
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Bray Curtis dissimilarity matrix (square root transformed) using PRIMER v6. Statistical
272
analysis was performed in R on the alpha diversity indices (Shannon index).
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RESULTS AND DISCUSSION
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TOrC biodegradation in microcosms. Biodegradation of most monitored TOrCs was
275
observed under at least one experimental condition, and generally occurred consistently
276
across replicates (Figures 1 and S6). Oryzalin and benzotriazole were biodegraded the
277
most quickly; while atrazine, prometon, fipronil, and diuron were biodegraded more
278
slowly. TCPP and TCEP were generally resistant to biodegradation, with the exception
279
that a single biotic raw runoff microcosm showed sudden and rapid degradation of both
280
compounds after 30 days (henceforth referenced as the ‘outlier’ microcosm).
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Less than 20% removal was observed in the biologically inhibited control
282
microcosms for most TOrCs (Figure 1), suggesting that the effects of dilution and
283
reaction with NaN3 were minor relative to biodegradation. Removal of oryzalin and
284
benzotriazole in controls was greater than the other TOrCs, though still less than their
285
removal in biotic microcosms. This was potentially due to incomplete activation of
286
biological activity by NaN3; considering that these TOrCs were the most strongly
287
biodegraded overall, and reduced DO levels in DOC-augmented controls relative to raw
288
runoff controls suggested that microbial activity persisted to some degree despite the
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presence of NaN3 (Figure S2). The extent of biodegradation at the end of the experiment
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was therefore assessed relative to the raw runoff controls (calculated extents of
291
biodegradation reported as fractions and μmols in Figures S8 and S9, respectively).
292
Interestingly, the outlier raw runoff microcosm demonstrated the greatest overall TOrC
293
biodegradation among the individual microcosms (i.e., highest sum of all monitored
294
TOrCs biodegraded, Figure S9), and is henceforth considered separately from the raw
295
runoff microcosms unless noted otherwise. Compost microcosms consistently
296
demonstrated the most overall TOrC biodegradation across experimental replicates, while
297
straw microcosms overall were not significantly different from raw runoff microcosms (p
298
values from the two-tailed student t test for comparison of raw runoff microcosms to
299
compost and straw microcosms were 0.0011 and 0.20, respectively; Table S8). The
300
compost microcosms demonstrated enhanced biodegradation of fipronil, benzotriazole,
301
atrazine, and prometon (p values = 0.0069, 0.0017, 0.0043, and 0.023, respectively, for
302
comparison to raw runoff); while straw microcosms demonstrated slightly enhanced
303
biodegradation of fipronil and benzotriazole (p = 0.0031 and 0.017, respectively, for
304
comparison to raw runoff).
305
In general, the most abundant monitored TPs for fipronil, diuron, and atrazine
306
were fipronil sulfone (Figure 1C), DCPMU (Figure 1D), and desethylatrazine (Figure
307
1E), respectively (additional TPs in Figure S7); except for the outlier microcosm, for
308
which the most abundant fipronil TP was fipronil sulfide. To determine if unmonitored
309
TPs were generated, mass balance errors were calculated according to differences in
310
concentration between (1) each parent TOrC in raw runoff controls, and (2) the sum of
311
each parent TOrC and their monitored TPs in biotic microcosms (Table S9). Atrazine and
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diuron showed low mass balance errors for the first 60 days, after which errors exceeded
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10%. This coincided with the peak concentrations of the measured primary TPs,
314
suggesting biodegradation of primary TPs to unmonitored secondary TPs. In contrast,
315
fipronil mass balance errors exceeded 10% after just 13 days, suggesting that
316
unmonitored primary TPs may have been generated.
317
Trends in lag time (delays before biodegradation) across experimental replicates
318
provided insight into biodegradation mechanisms. For example, oryzalin, benzotriazole,
319
and fipronil were biodegraded in all DOC-augmented microcosms without a lag time;
320
suggesting biodegradation by co-metabolic processes (i.e., degradation by constitutively
321
expressed, non-specific enzymes). Atrazine and prometon, in contrast, were only
322
biodegraded in compost microcosms following a 30-day lag time. Depletion of
323
preferential carbon substrates may have caused this lag time, subsequently allowing
324
TOrC biodegradation due to adaptive expression of TOrC-degrading enzymes40,41 or
325
reduced substrate-substrate inhibitions.42 The greater aromatic character of the compost
326
DOC may have contributed to this behavior, as these more complex carbon substrates
327
may have been more slowly utilized and hence still present upon TOrC exposure. TCPP
328
and TCEP biodegradation in the outlier microcosm was particularly abrupt after the 30-
329
day lag time, potentially due to the sudden activity of specialist bacteria. Decreased DO
330
(Figure S2A), increased OD (Figure S2B), and reduced DOC (Table S5) relative to raw
331
runoff microcosms indicate accelerated growth in this microcosm following TOrC
332
addition, potentially due to methanol exposure (discussed further below).
333
Microcosm microbial communities. The 2D nMDS analysis showed that the microbial
334
communities in all DOC-augmented microcosms (i.e., both straw and compost
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microcosms) clustered apart from those in raw runoff microcosms (Figure 2A), indicating
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DOC-stimulated, growth-induced changes in community structure. The communities of
337
the DOC-augmented microcosms were relatively similar to each other (58% Bray Curtis
338
similarity overall, see dendrogram in Figure S10), while compost DOC communities
339
showed the highest similarity among replicates (73%). As communities were more
340
similar among the DOC-augmented microcosms, the more significantly enhanced TOrC
341
biodegradation in compost microcosms relative to straw microcosms (as compared to the
342
raw runoff microcosms) was likely due to either subtle differences in communities (e.g.,
343
differences in relative abundances of specific OTUs), and/or other factors relating to
344
microbial abundance and activity. The outlier microcosm did not cluster with the other
345
microcosms, suggesting that microbial community differences caused its unique
346
biodegradation behavior. It is important to note that growth on methanol from the TOrC
347
carrier solution may have also affected communities. It is possible that this masked
348
community differences that had already developed during incubation, potentially
349
increasing the similarity among the DOC-augmented microcosm communities. For the
350
outlier microcosm, it is possible that growth of methanol-utilizing bacteria was stimulated
351
in the absence of other DOC sources, potentially contributing to its unique community
352
structure.
353
Gene copy numbers (GCNs) of overall bacterial 16S rRNA genes were
354
consistently high for compost microcosms, while they were lower and more variable for
355
raw runoff and straw microcosms (Figure 2B). Considering that low residual DOC levels
356
(Table S5) suggest that growth occurred in all DOC-augmented microcosms, the higher
357
GCNs in compost microcosms may have been due to greater abundances of active
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bacteria upon DNA extraction (at the end of the experiment). GCNs were higher in the
359
outlier microcosm than the raw runoff microcosms, further indicating accelerated growth
360
in this microcosm. Only small differences in the Shannon index were observed among the
361
microcosms (indicative of community richness and evenness, Table S10), therefore alpha
362
diversity likely had only minor effects on observed trends. This is unsurprising, as the
363
inoculum effectively fixed the species richness across microcosms (i.e., the same number
364
of species were introduced into all microcosms upon inoculation). While averaged
365
Shannon indices were higher for compost and straw microcosms than for raw runoff
366
microcosms (3.6 and 3.4, versus 3.2, respectively), differences were not statistically
367
significant (p = 0.072 and 0.66, respectively, for comparison to raw runoff microcosms).
368
Notably, an especially low Shannon index was observed for one straw microcosm (2.95,
369
microcosm S2, discussed below).
370
Figure 3 shows the most prevalent differences in OTU relative abundances that
371
were identified among the microcosms (family-level OTU bar plot in Figure S11).
372
Several OTUs that are known to facilitate nitrogen-cycling processes were enriched in
373
DOC-augmented microcosms relative to raw runoff; including those within the families
374
Rhodospirlliceae
375
Comamonadaceae (Comamonas; denitrification),45 and Rhodocyclaceae (Thauera;
376
denitrification).46 This reflects previous findings of coincidentally enhanced nitrogen-
377
cycling processes and TOrC biodegradation among wastewater communities.21,47 An
378
OTU from the genus Pseudomonas was enriched only in straw microcosms (particularly
379
microcosm S2, causing its low Shannon index; Figure S11). Though many Pseudomonas
380
species are known TOrC-degraders,48 the OTU's abundance was not positively associated
(N2-fixation),43
Nitrospirae
(Nitrospira;
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381
with individual or overall TOrC biodegradation (Spearman’s rank and Pearson
382
correlation tests described in Method S5, Tables S11 – S14). As Pseudomonas can
383
undergo metabolic regulation,49 this OTU may have initially out-competed other bacteria
384
for utilization of the straw-derived carbon substrates, potentially until residual levels of P
385
and N could no longer support growth (Tables S5 and S6). This potentially explains the
386
reduced TOrC biodegradation in straw microcosms relative to compost microcosms, as
387
microbial activity and hence TOrC biodegradation would presumably be diminished if
388
nutrients were depleted prior to TOrC introduction. Interestingly, the outlier microcosm
389
was enriched with OTUs within the family Sphingomonadaceae (markers in Figure 3),
390
which contains several genera of TOrC-degrading bacteria (e.g., Sphingomonas).50,51
391
While these OTUs could potentially be specialist TCEP and TCPP degrading bacteria,
392
they may also just covary with such bacteria.
393
Overall, while DOC-stimulated growth led to communities that were starkly
394
different from raw runoff communities, community differences among the DOC-
395
augmented microcosms were more subtle. However, the similarity in both growth and
396
community composition amongst replicates was the highest for the compost microcosms,
397
coinciding with the greatest overall TOrC biodegradation. One possible explanation for
398
this behavior is that the compost microcosms provided conditions that more robustly
399
promoted TOrC-degrading activity upon TOrC exposure. For example, higher residual N
400
and P levels may have facilitated the utilization of TOrCs as carbon sources, potentially
401
allowing sustained microbial activity throughout the experiment. These conditions may
402
have arisen due to several factors relating to DOC composition and quality, or a
403
combination thereof; including the initial C:N of the DOC (i.e., providing carbon-limited
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rather than nutrient-limited growth conditions), as well as effects of DOC aromaticity on
405
substrate utilization potential. In particular, the higher aromaticity of the compost DOC
406
may have led to adaptive expression of TOrC-degrading enzymes. This is further
407
supported by the biodegradation lag times observed for atrazine and prometon, which
408
also showed the most significantly enhanced biodegradation relative to straw microcosms
409
(p = 0.00077 and 0.00067, respectively, Table S8).
410
While the underlying phenomena behind the observed TOrC biodegradation
411
trends cannot be fully elucidated, these findings indicate that compost DOC may indeed
412
support TOrC-degrading microbial activity in stormwater biofilters more effectively than
413
straw DOC. However, the observed time-scale for TOrC biodegradation (weeks to
414
months), is much higher than hydraulic residence times for distributed infiltration
415
systems (hours to days). In this respect, biochar-amended biofilters may be especially
416
effective if sorptive retention is maintained, as increased TOrC retention times may allow
417
more time for biodegradation. The column experiments provided further insight into
418
these questions.
419
Column Experiments. Sand-only columns cultivated with compost DOC
420
demonstrated longer TOrC retention times than those cultivated with straw DOC (i.e.,
421
TOrC breakthrough was observed after approximately 100 PVs in sand-only compost
422
columns, versus less than 10 PVs for sand-only straw columns; Figures 4 and S12).
423
Compost columns also demonstrated greater microbial activity during TOrC injection and
424
flushing, as indicated by lower effluent DO levels (Figure S5). Notably, TOrC retention
425
times were enhanced by amendment of sand with just 0.5 wt% biochar (i.e., breakthrough
426
occurred approximately 100 PVs later in biochar-sand columns than in sand-only
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427
columns), despite the severe fouling expected to be caused by the concentrated seeding
428
solutions (with DOC concentrations potentially orders of magnitude above representative
429
levels). It is therefore likely that TOrC retention can be maintained in actual biofilters,
430
which presumably contain media with significantly higher biochar compositions (i.e.,
431
greater than 5 wt%).16
432
In addition to higher TOrC retention times, compost columns also demonstrated
433
greater percent removal of most TOrCs (i.e., lower recovery at the end of the experiment
434
as indicated by a Method of Moments analysis; Figure S13). This appeared to be at least
435
partially due to biological processes, as inhibition of biological activity with NaN3
436
reduced removal of atrazine by 20%, as well as TCEP and TCPP to a lesser degree
437
(though slowly receding concentrations during flushing may have slightly affected
438
recovery calculations). This could potentially indicate TOrC biodegradation within
439
columns (i.e., 20% greater atrazine removal in the absence of NaN3 potentially indicates
440
up to 20% biodegradation). This is an interesting result, as conventional porous media
441
transport theory suggests that only the dissolved TOrC fraction is bioavailable, and that
442
TOrCs are therefore biodegraded on the time-scale of the hydraulic retention time.52 Thus
443
biodegradation would be unlikely to amount to observable levels within the columns
444
unless biodegradation were particularly “rapid” (as the hydraulic retention time in
445
columns was approximately 2 hours).
446
Interestingly,
atrazine
retention
times
were
comparable
to
expected
447
biodegradation time-scales (i.e., 10-20 days) and appeared to be associated with atrazine
448
removal (i.e., biochar-sand columns demonstrated both atrazine retention times and
449
atrazine removal percentages that were nearly double those for sand-only columns).
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While a possible explanation for this behavior is the biodegradation of TOrCs sorbed to
451
biochar (which has been observed for some cases),53 this apparently contradicts
452
conventional transport theories. A more feasible explanation may be biodegradation of
453
TOrCs loosely associated with the immobilized DOC and the biofilm itself. Localized
454
biodegradation of these more readily desorbed TOrCs would continuously replenish
455
sorption sites, making it appear that sorbed TOrCs were biodegraded. Moreover, the
456
biofilm structure (potentially affected by DOC quality) could also affect TOrC
457
biodegradation (e.g., due to beneficial co-settlement of TOrC-degrading bacteria).23
458
Overall, the column experiments revealed that TOrC sorptive retention was
459
maintained by biochar-amended media bearing DOC-cultivated biofilms, and that overall
460
TOrC attenuation was improved in the presence of biological activity stimulated by
461
compost DOC. As it remains unclear if TOrCs were actually biodegraded within
462
columns, studies evaluating systems with longer TOrC retention times may provide
463
insight into whether TOrCs can be biodegraded significantly within biochar-amended
464
biofilters.
465
Environmental Implications. The findings of this study are of particular
466
significance for efforts to mitigate impacts of runoff on water quality through
467
optimization of contaminant removal in infiltration systems. In particular, in addition to
468
improving TOrC attenuation, co-amendment of biofilters with compost and biochar may
469
comprehensively enhance performance by mitigating undesirable leaching of metals and
470
nutrients from compost;54–56 making vegetated systems (which require compost for plant
471
growth) more attractive. Vegetated systems may be further advantageous for improving
472
TOrC attenuation: biological activity stimulated in the root zone may further improve
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TOrC biodegradation,57 while enhanced desorption in the presence of root exudates may
474
prevent TOrC accumulation on biochar.58,59 Remarkable TOrC removal has indeed been
475
observed in vegetated infiltration columns representing biochar-amended biofilters;16 yet
476
as no TOrC breakthrough was observed, it remains unclear whether TOrC removal is due
477
to sorption alone, or to a combination of sorption and biodegradation. Therefore,
478
additional studies seeking to identify and quantify TOrC biodegradation in vegetated,
479
biochar-amended biofilters are of interest to determine if TOrC accumulation can be
480
prevented throughout the system entire lifetime.
481
Supporting information. This material is available free of charge via the Internet at
482
http://pubs.acs.org, and contains additional figures and tables and methodological details,
483
as referenced throughout the text. The sequencing data has been made available to the
484
public through the EMBL European Bioinformatics Institute (Project PRJEB19822,
485
http://www.ebi.ac.uk/).
486
Author information
487
Corresponding author. Christopher P. Higgins.
488
E-mail:
[email protected] 489
Phone: (720)-984-2116
490
Fax: (303) 273-3413
491
Acknowledgements.
492
Science Foundation Graduate Research Fellowship under Grant No. DGE-1057607 and
493
the National Science Foundation Engineering Research Center Program under
494
Cooperative Agreements EEC-1028968 (ReNUWIt) and EEC-1262655. Collaboration
This material is based upon work supported by the National
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with Newcastle University was facilitated by EPSRC grant EP/K021737/1 and Marie
496
Skłodowska-Curie Initial Training Network (MERMAID) grant 607492.
497
REFERENCES
498
(1)
499 500
National Research Council. Urban Stormwater Management in the United States; Washington, DC, 2009.
(2)
Hunt, W. F.; Traver, R. G.; Davis, A. P.; Emerson, C. H.; Collins, K. A.; Stagge, J.
501
H. Low impact development practices: designing to infiltrate in urban
502
environments. In Effects of urbanization on groundwater: an engineering case-
503
based approach for sustainable development; American Society of Civil
504
Engineers: Reston, VA, 2010; pp. 308–343.
505
(3)
Dietz, M. E. Low Impact Development Practices: A Review of Current Research
506
and Recommendations for Future Directions. Water. Air. Soil Pollut. 2007, 186,
507
351–363.
508
(4)
Grebel, J. E.; Mohanty, S. K.; Torkelson, A. A.; Boehm, A. B.; Higgins, C. P.;
509
Maxwell, R. M.; Nelson, K. L.; Sedlak, D. L. Engineered Infiltration Systems for
510
Urban Stormwater Reclamation. Environ. Eng. Sci. 2013, 30, 437–454.
511
(5)
Bucheli, T. D.; Müller, S. R.; Heberle, S.; Schwarzenbach, R. P. Occurrence and
512
behavior of pesticides in rainwater, roof runoff, and artificial stormwater
513
infiltration. Environ. Sci. Technol. 1998, 32, 3457–3464.
514
(6)
Gan, J.; Bondarenko, S.; Oki, L.; Haver, D.; Li, J. X. Occurrence of fipronil and its
515
biologically active derivatives in urban residential runoff. Environ. Sci. Technol.
516
2012, 46, 1489–1495.
517
(7)
Huang, X.; Fong, S.; Deanovic, L.; Young, T. M. Toxicity of herbicides in
ACS Paragon Plus Environment
22
Environmental Science & Technology
518 519
Page 24 of 35
highway runoff. Environ. Toxicol. Chem. 2005, 24, 2336–2340. (8)
Regnery, J.; Püttmann, W. Occurrence and fate of organophosphorus flame
520
retardants and plasticizers in urban and remote surface waters in Germany. Water
521
Res. 2010, 44, 4097–4104.
522
(9)
Stachel, B.; Holthuis, J.-U.; Schulz, W.; Seitz, W.; Weber, W. H.; Tegge, K.-T.;
523
Dobner, I. Treatment Techniques and Analysis of Stormwater Run-off from Roads
524
in Hamburg, Germany. In Xenobiotics in the urban water cycle; 2010; Vol. 16, pp.
525
445–461.
526
(10)
Zhang, K.; Randelovic, A.; Page, D.; McCarthy, D. T.; Deletic, A. The validation
527
of stormwater biofilters for micropollutant removal using in situ challenge tests.
528
Ecol. Eng. 2014, 67, 1–10.
529
(11)
Beesley, L.; Moreno-Jiménez, E.; Gomez-Eyles, J. L. Effects of biochar and
530
greenwaste compost amendments on mobility, bioavailability and toxicity of
531
inorganic and organic contaminants in a multi-element polluted soil. Environ.
532
Pollut. 2010, 158, 2282–2287.
533
(12)
Mohanty, S. K.; Cantrell, K. B.; Nelson, K. L.; Boehm, A. B. Efficacy of biochar
534
to remove Escherichia coli from stormwater under steady and intermittent flow.
535
Water Res. 2014, 61C, 288–296.
536
(13)
537 538
Reddy, K. R.; Xie, T.; Dastgheibi, S. Nutrients Removal from Urban Stormwater by Different Filter Materials. Water, Air, Soil Pollut. 2013, 225, 1778.
(14)
Ulrich, B. A.; Im, E. A.; Werner, D.; Higgins, C. P. Biochar and activated carbon
539
for enhanced trace organic contaminant retention in stormwater infiltration
540
systems. Environ. Sci. Technol. 2015, 49, 6222–6230.
ACS Paragon Plus Environment
23
Page 25 of 35
541
Environmental Science & Technology
(15)
Shimabuku, K. K.; Kearns, J. P.; Martinez, J. E.; Mahoney, R. B.; Moreno-
542
Vasquez, L.; Summers, R. S. Biochar sorbents for sulfamethoxazole removal from
543
surface water, stormwater, and wastewater effluent. Water Res. 2016, 96, S60–
544
S65.
545
(16)
Ulrich, B. A.; Loehnert, M.; Higgins, C. P. Improved contaminant removal in
546
vegetated stormwater biofilters amended with biochar. Environmental Science:
547
Water Research & Technology, 2017, DOI: 10.1039/c7ew00070g.
548
(17)
Davis, A. P.; Shokouhian, M.; Sharma, H.; Minami, C.; Winogradoff, D. Water
549
quality improvement through bioretention: lead, copper, and zinc removal. Water
550
Environ. Res. 2003, 75, 73–82.
551
(18)
Lefevre, G. H.; Hozalski, R. M.; Novak, P. J. The role of biodegradation in
552
limiting the accumulation of petroleum hydrocarbons in raingarden soils. Water
553
Res. 2012, 46, 6753–6762.
554
(19)
Kwon, S.; Pignatello, J. J. Effect of natural organic substances on the surface and
555
adsorptive properties of environmental black carbon (char): pseudo pore blockage
556
by model lipid components and its implications for N2-probed surface properties
557
of natural sorbents. Environ. Sci. Technol. 2005, 39, 7932–7939.
558
(20)
Johnson, D. R.; Helbling, D. E.; Lee, T. K.; Park, J.; Fenner, K.; Kohler, H. P. E.;
559
Ackermann, M. Association of biodiversity with the rates of micropollutant
560
biotransformations among full-scale wastewater treatment plant communities.
561
Appl. Environ. Microbiol. 2015, 81, 666–675.
562 563
(21)
Helbling, D. E.; Johnson, D. R.; Honti, M.; Fenner, K. Process Parameters and Microbial Community Characteristics. Environ. Sci. Technol. 2012, 46, 10579–
ACS Paragon Plus Environment
24
Environmental Science & Technology
564 565
10588. (22)
Johnson, D. R.; Lee, T. K.; Park, J.; Fenner, K.; Helbling, D. E. The functional and
566
taxonomic richness of wastewater treatment plant microbial communities are
567
associated with each other and with ambient nitrogen and carbon availability.
568
Environ. Microbiol. 2014, 17, 4851–4860.
569
Page 26 of 35
(23)
Horemans, B.; Breugelmans, P.; Hofkens, J.; Smolders, E.; Springael, D.
570
Environmental dissolved organic matter governs biofilm formation and subsequent
571
linuron degradation activity of a linuron-degrading bacterial consortium. Appl.
572
Environ. Microbiol. 2013, 79, 4534–4542.
573
(24)
Schutter, M.; Dick, R. Shifts in substrate utilization potential and structure of soil
574
microbial communities in response to carbon substrates. Soil Biol. Biochem. 2001,
575
33, 1481–1491.
576
(25)
577 578
substrate C:N ratio and concentration. Aquat. Microb. Ecol. 1999, 19, 105–118. (26)
579 580
Touratier, F.; Legendre, L.; Vezina, A. Model of bacterial growth influenced by
Bachand, P. A. M.; Horne, A. J. Denitrification in constructed free-water surface wetlands: II. Effects of vegetation and temperature. Ecol. Eng. 1999, 14, 17–32.
(27)
Wetzel, R. G.; Hatcher, P. G.; Bianchi, T. S. Natural photolysis by ultraviolet
581
irradiance of recalcitrant dissolved organic matter to simple substrates for rapid
582
bacterial metabolism. Limnol. Oceanogr. 1995, 40, 1369–1380.
583
(28)
Weishaar, J. L.; Aiken, G. R.; Bergamaschi, B. A.; Fram, M. S.; Fujii, R.; Mopper,
584
K. Evaluation of specific ultraviolet absorbance as an indicator of the chemical
585
composition and reactivity of dissolved organic carbon. Environ. Sci. Technol.
586
2003, 37, 4702–4708.
ACS Paragon Plus Environment
25
Page 27 of 35
587
Environmental Science & Technology
(29)
Olefeldt, D.; Turetsky, M. R.; Blodau, C. Altered Composition and Microbial
588
versus UV-Mediated Degradation of Dissolved Organic Matter in Boreal Soils
589
Following Wildfire. Ecosystems 2013, 16, 1396–1412.
590
(30)
591
McKendry, P. Energy production from biomass (part 1): overview of biomass. Bioresour. Technol 2002, 83, 37–46.
592
(31)
Alexander, M. Introduction to Soil Microbiology. Soil Sci. 1978, 125, 275–336.
593
(32)
Lang, J. S.; Giron, J. J.; Hansen, A. T.; Trussell, R. R.; Hodges, W. E.
594
Investigating filter performance as a function of the ratio of filter size to media
595
size. J. / Am. Water Work. Assoc. 1993, 85, 122–130.
596
(33)
Hamady, M.; Knight, R.; Hamady, M.; Knight, R. Tools , techniques , and
597
challenges Microbial community profiling for human microbiome projects: Tools,
598
techniques, and challenges. 2009, 19, 1141–1152.
599
(34)
600 601
LBL. Greengenes: 16S rDNA Data and Tools http://greengenes.lbl.gov/cgibin/nph-index.cgi.
(35)
McDonald, D.; Price, M. N.; Goodrich, J.; Nawrocki, E. P.; DeSantis, T. Z.;
602
Probst, A.; Andersen, G. L.; Knight, R.; Hugenholtz, P. An improved Greengenes
603
taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria
604
and archaea. Isme J 2012, 6, 610–618.
605
(36)
Werner, J. J.; Koren, O.; Hugenholtz, P.; DeSantis, T. Z.; Walters, W. a; Caporaso,
606
J. G.; Angenent, L. T.; Knight, R.; Ley, R. E. Impact of training sets on
607
classification of high-throughput bacterial 16s rRNA gene surveys. ISME J. 2012,
608
6, 94–103.
609
(37)
Wang, Q.; Garrity, G. M.; Tiedje, J. M.; Cole, J. R. Naive Bayesian classifier for
ACS Paragon Plus Environment
26
Environmental Science & Technology
610
rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl.
611
Environ. Microbiol. 2007, 73, 5261–5267.
612
(38)
Page 28 of 35
Caporaso, J. G.; Kuczynski, J.; Stombaugh, J.; Bittinger, K.; Bushman, F. D.;
613
Costello, E. K.; Fierer, N.; Peña, A. G.; Goodrich, K.; Gordon, J. I.; et al. QIIME
614
allows analysis of high-throughput community sequencing data. 2011, 7, 335–336.
615
(39)
Clarke, K. R.; Somerfield, P. J.; Chapman, M. G. On resemblance measures for
616
ecological studies, including taxonomic dissimilarities and a zero-adjusted Bray-
617
Curtis coefficient for denuded assemblages. J. Exp. Mar. Bio. Ecol. 2006, 330, 55–
618
80.
619
(40)
Dinamarca, M. A.; Ruiz-Manzano, A.; Rojo, F. Inactivation of cytochrome o
620
ubiquinol oxidase relieves catabolic repression of the Pseudomonas putida GPo1
621
alkane degradation pathway. J. Bacteriol. 2002, 184, 3785–3793.
622
(41)
Amador, C. I.; Canosa, I.; Govantes, F.; Santero, E. Lack of CbrB in Pseudomonas
623
putida affects not only amino acids metabolism but also different stress responses
624
and biofilm development. Environ. Microbiol. 2010, 12, 1748–1761.
625
(42)
Liu, L.; Helbling, D. E.; Kohler, H. E.; Smets, B. F. A Model Framework to
626
Describe Growth-Linked Biodegradation of Trace-Level Pollutants in the Presence
627
of Coincidental Carbon Substrates and Microbes. Environ. Sci. Technol. 2014, 48,
628
13358–13366.
629
(43)
Madigan, M.; Cox, S. S.; Stegeman, R. A. Nitrogen fixation and nitrogenase
630
activities in members of the family Rhodospirillaceae. J. Bacteriol. 1984, 157, 73–
631
78.
632
(44)
Hovanec, T. a; Taylor, L. T.; Blakis, A.; Delong, F. Nitrospira -Like Bacteria
ACS Paragon Plus Environment
27
Page 29 of 35
Environmental Science & Technology
633
Associated with Nitrite Oxidation in Freshwater Aquaria Nitrospira -Like Bacteria
634
Associated with Nitrite Oxidation in Freshwater Aquaria. Appl. Environ.
635
Microbiol. 1998, 64, 258.
636
(45)
Willems, A.; Vos, P. D. E. Comamonas. Prokaryotes 2006, 723–736.
637
(46)
Waterbury, J. B. The Prokaryotes. Physiology 2006, 1053–1073.
638
(47)
Koh, Y. K. K.; Chiu, T. Y.; Boobis, A. R.; Scrimshaw, M. D.; Bagnall, J. P.;
639
Soares, A.; Pollard, S.; Cartmell, E.; Lester, J. N. Influence of operating
640
parameters on the biodegradation of steroid estrogens and nonylphenolic
641
compounds during biological wastewater treatment processes. Environ. Sci.
642
Technol. 2009, 43, 6646–6654.
643
(48)
644 645
Assaf, N. a; Turco, R. F. Accelerated biodegradation of atrazine by a microbial consortium is possible in culture and soil. Biodegradation 1994, 5, 29–35.
(49)
Rojo, F. Carbon catabolite repression in Pseudomonas: Optimizing metabolic
646
versatility and interactions with the environment. FEMS Microbiol. Rev. 2010, 34,
647
658–684.
648
(50)
Waterbury, J. B. The Family Spingomonadaceae, In The Prokaryotes; 2006.
649
(51)
Gabriel, F. L. P.; Heidlberger, A.; Rentsch, D.; Giger, W.; Guenther, K.; Kohler,
650
H.-P. E. A novel metabolic pathway for degradation of 4-nonylphenol
651
environmental contaminants by Sphingomonas xenophaga Bayram: ipso-
652
hydroxylation and intramolecular rearrangement. J. Biol. Chem. 2005, 280, 15526–
653
15533.
654 655
(52)
Hemond, H. F.; Fetcher, E. J. Section 3.6: Biodegradation in the Subsurface Environment. In Chemical Fate and Transport in the Environment; Elsvier Inc.:
ACS Paragon Plus Environment
28
Environmental Science & Technology
656 657
San Diego, CA, 2015; pp. 279–285. (53)
Tong, H.; Hu, M.; Li, F. B.; Liu, C. S.; Chen, M. J. Biochar enhances the microbial
658
and chemical transformation of pentachlorophenol in paddy soil. Soil Biol.
659
Biochem. 2014, 70, 142–150.
660
Page 30 of 35
(54)
Mullane, J. M.; Flury, M.; Iqbal, H.; Freeze, P. M.; Hinman, C.; Cogger, C. G.;
661
Shi, Z. Intermittent rainstorms cause pulses of nitrogen, phosphorus, and copper in
662
leachate from compost in bioretention systems. Sci. Total Environ. 2015, 537,
663
294–303.
664
(55)
Reddy, K. R.; Xie, T.; Dastgheibi, S. Evaluation of Biochar as a Potential Filter
665
Media for the Removal of Mixed Contaminants from Urban Storm Water Runoff.
666
J. Environ. Eng. 2014, 140, 4014043.
667
(56)
Beesley, L.; Moreno-Jiménez, E.; Gomez-Eyles, J. L. Effects of biochar and
668
greenwaste compost amendments on mobility, bioavailability and toxicity of
669
inorganic and organic contaminants in a multi-element polluted soil. Environ.
670
Pollut. 2010, 158, 2282–2287.
671
(57)
Joner, E. J.; Johansen, A.; Loibner, A. P.; dela Cruz, M. A.; Szolar, O. H. J.;
672
Portal, J.-M.; Leyval, C. Rhizosphere Effects on Microbial Community Structure
673
and Dissipation and Toxicity of Polycyclic Aromatic Hydrocarbons (PAHs) in
674
Spiked Soil. Environ. Sci. Technol. 2001, 35, 2773–2777.
675
(58)
Lefevre, G. H.; Hozalski, R. M.; Novak, P. J. Root Exudate Enhanced
676
Contaminant Desorption: An Abiotic Contribution to the Rhizosphere Effect.
677
Environ. Sci. Technol. 2013, 47, 11545–11553.
678
(59)
Lefevre, G. H.; Hozalski, R. M.; Novak, P. J. The role of biodegradation in
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limiting the accumulation of petroleum hydrocarbons in raingarden soils. Water
680
Res. 2012, 46, 6753–6762.
681
(60)
Cornelissen, G.; Haftka, J.; Parsons, J.; Gustafsson, O. Sorption to black carbon of
682
organic compounds with varying polarity and planarity. Environ. Sci. Technol.
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2005, 39, 3688–3694.
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FIGURES
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Figure 1. Concentrations of (A) oryzalin, (B) benzotriazole, (C) fipronil, (D) diuron, (E) atrazine, and (F) TCEP in microcosms (prometon and TCPP are shown in Figure S6). Error bars indicate standard deviations for replicate microcosms, and significant differences between inhibited controls and biotic microcosms are assumed to be due to biodegradation. Select transformation products (TPs) are also shown for atrazine (desethylatrazine), fipronil (fipronil sulfone and fipronil sulfide), and diuron (3,4dichloromethylphenyl urea, DCPMU). Additional TPs are shown in Figure S7. ACS Paragon Plus Environment
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Figure 2. (A) Two-dimensional non-metric multidimensional scaling (2D nMDS) plot of sequenced microcosm microbial communities (markers) and Bray Curtis similarities among samples (lines). Similarities are also shown in the dendrogram in Figure S11 (similarity analysis conducted using PRIMER v6). (B) Gene copy numbers per mass of DNA-extracted sand for each sequenced sample, determined by qPCR of total bacterial 16S rRNA genes (flocculation was observed in sample S1, likely causing its elevated gene copy numbers). Error bars indicate the standard deviation of triplicate measurements.
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Figure 3. Relative abundances of OTUs that were (i) identified at the genus level, and (ii) observed to be enriched by at least one order of magnitude relative to raw runoff (note the log-scale of the y axis). Bars show averages for replicate microcosms, with error bars showing the standard deviations. Markers show relative abundances for the outlier microcosm (not included in the averages for the raw runoff microcosms). Comparisons were made between averaged relative abundances, with the exception of the outlier microcosm, for which its individual values were compared to the averaged values for the other raw runoff microcosms.
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Figure 4. Breakthrough of (A, B) atrazine, (C, D) methylbenzotriazole, and (E, F) TCPP in sand-only and sand-biochar columns (0.5 wt% biochar), with biofilms cultivated from compost DOC (green) or straw DOC (yellow). Results for biologically inhibited control sand-biochar columns are shown by dotted lines. Columns were injected with 10 μg/L TOrCs, then flushed with TOrC-free synthetic stormwater. Diuron was exceptionally well removed overall, as has been observed previously.14,60
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