Article pubs.acs.org/est
Fate of Polyfluoroalkyl Phosphate Diesters and Their Metabolites in Biosolids-Applied Soil: Biodegradation and Plant Uptake in Greenhouse and Field Experiments Holly Lee, Alex G. Tevlin, Scotia A. Mabury, and Scott A. Mabury* Department of Chemistry, University of Toronto, 80 St. George St., Toronto, Ontario, Canada, M5S 3H6 S Supporting Information *
ABSTRACT: Significant contamination of perfluoroalkyl acids (PFAAs) in wastewater treatment plant (WWTP) sludge implicates the practice of applying treated sludge or biosolids as a potential source of these chemicals onto agricultural farmlands. Recent efforts to characterize the sources of PFAAs in the environment have unveiled a number of fluorotelomer-based materials that are capable of degrading to the perfluoroalkyl carboxylates (PFCAs), such as the polyfluoroalkyl phosphate diesters (diPAPs), which have been detected in WWTP and paper fiber biosolids. Here, a greenhouse microcosm was used to investigate the fate of endogenous diPAPs and PFCAs present in WWTP and paper fiber biosolids upon amendment of these materials with soil that had been sown with Medicago truncatula plants. Biodegradation pathways and plant uptake were further elucidated in a separate greenhouse microcosm supplemented with high concentrations of 6:2 diPAP. Biosolid-amended soil exhibited increased concentrations of diPAPs (4−83 ng/g dry weight (dw)) and PFCAs (0.1−19 ng/g dw), as compared to control soils (nd−1.4 ng/g dw). Both plant uptake and biotransformation contributed to the observed decline in diPAP soil concentrations over time. Biotransformation was further evidenced by the degradation of 6:2 diPAP to its corresponding fluorotelomer intermediates and C4−C7 PFCAs. Substantial plant accumulation of endogenous PFCAs present in the biosolids (0.1−138 ng/g wet weight (ww)) and those produced from 6:2 diPAP degradation (100−58 000 ng/g ww) were observed within 1.5 months of application, with the congener profile dominated by the short-chain PFCAs (C4−C6). This pattern was corroborated by the inverse relationship observed between the plant−soil accumulation factor (PSAF, Cplant/Csoil) and carbon chain length (p < 0.05, r = 0.90−0.97). These results were complemented by a field study in which the fate of diPAPs and PFCAs was investigated upon application of compost and paper fiber biosolids to two farm fields. Together, these studies provide the first evidence of soil biodegradation of diPAPs and the subsequent uptake of these chemicals and their metabolites into plants.
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INTRODUCTION The high concentrations (ng/g) of perfluoroalkyl and polyfluoroalkyl substances (PFASs) often reported in wastewater treatment plant (WWTP) sludge1−5 and their demonstrated capacity to sorb to sludge6 suggest this matrix may be a significant reservoir for these chemicals in the environment. This is of concern because a significant fraction (40%) of treated WWTP sludge or biosolids is directed toward land application in Ontario (ON), Canada.7 Of the 140 000 tons of biosolids generated at the largest WWTP in Toronto, ON in 2012, 38% was applied directly on land, while 41% was further pelletized and chemically treated for soil amendment.8 Paper fiber biosolids are generated from the wastewater treatment process of recycled paper products in pulp and paper mills, and like WWTP biosolids, they are largely applied (20%) as a soil conditioner in Ontario.7 Composting of household organic waste, such as food scraps, garden waste, and soiled food packaging, and the subsequent use of these materials as a soil conditioner represents an additional method to divert waste from landfills. Analysis of WWTP sludge and paper fiber biosolids collected across Ontario from 2002 to 2008 reported © 2013 American Chemical Society
detection of varying chain lengths of perfluoroalkyl carboxylates (PFCAs, 7−10 perfluorinated carbons (CF’s)) and perfluorooctane sulfonate (PFOS) at ng/g concentrations and, for the first time, the polyfluoroalkyl phosphate diesters (diPAPs; up to 2100 ng/g in WWTP sludge and 3800 ng/g paper fiber biosolids).5 DiPAPs belong to a suite of commercial fluorotelomer-based materials, such as the acrylate-based polymers (FTACPs),9,10 sulfonates (FTSAs),11 and stearate monoesters (FTSs),12 all of which have a demonstrated capacity to biodegrade to PFCAs in either soil or WWTP-simulated environments.13 Detection of fluorotelomer saturated (FTCAs) and unsaturated (FTUCAs) carboxylates in WWTP sludge and effluents,4,14,15 both of which are intermediate metabolites of fluorotelomer-based precursor degradation to the PFCAs, also suggests WWTPs are exposed to contaminated influents containing diPAPs and other Received: Revised: Accepted: Published: 340
September 4, 2013 November 15, 2013 December 6, 2013 December 6, 2013 dx.doi.org/10.1021/es403949z | Environ. Sci. Technol. 2014, 48, 340−349
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fluorotelomer-based materials. In addition to direct PFCA emission sources to WWTPs, such as contaminated discharges from nearby fluorochemical industries16 and disposal of consumer products containing PFCAs,17−19 biodegradation of commercial fluorotelomer-based products represents an additional source of PFCAs in WWTP media. As such, one concern of biosolids application onto farmlands centers over its potential as an exposure route of PFASs to soil and its surrounding environment, as was observed in soil,20 tile drainage water,21 and assorted plant crops22 during laboratory and field experiments. These concerns were recently highlighted in farmlands in Decatur, Alabama that have received >10 years of biosolids application from a local WWTP known to have processed effluents from nearby fluorochemical manufacturers. Some of the highest soil concentrations of perfluoroalkyl acids (PFAAs) were reported in the biosolids-amended soils collected in 2007 (mean: 1500 ng/g dry weight (dw), perfluorooctanoate (PFOA); 940 ng/g dw PFOS), as compared to the lack of quantitation observed in background soils that have never received biosolids application.23 Similarly, elevated PFAA concentrations were observed in plants (10−200 ng/g dw PFOA; 1−20 ng/g dw PFOS)24 and surface and well water (up to 11 000 ng/L in surface water; up to 6400 ng/L in well water)25 sampled near the impacted fields. The fact that varying chain lengths of fluorotelomer alcohols (6:2 to 14:2 FTOHs) were also detected in these same plant samples24 and biosolidsamended soils26 further supports potential biodegradation of fluorotelomer-based materials that may be present in the biosolids-applied soil. A number of soil and WWTP-simulated biodegradation studies have reported FTOH as an intermediate metabolite of various fluorotelomer-based precursors, such as the diPAPs,13 FTACPs,9,10 and FTSs.12 However, it has not yet been demonstrated whether a similar transformation pathway may occur for these precursors in a soil-plant environment. Here, a 5.5-month greenhouse experiment was performed to investigate the fate of endogenous diPAPs and PFCAs in biosolids-amended soil sown with alfalfa plants, Medicago truncatula, which were chosen due to their ease of cultivation and common presence as a forage crop for livestock and to promote soil fertility in agricultural farmlands. As commercial greaseproofing formulations containing fluorinated phosphate surfactants like the diPAPs are typically composed of a mixture of varying perfluoroalkyl chain lengths (4−20 CF’s), as well as the monofluoroalkylated (monoPAP) and trifluoroalkylated (triPAP) phosphate esters,27−29 one diPAP congener (6:2) was specifically chosen for further elucidation of its biotransformation pathways in the soil and the plants. The influence of the perfluoroalkyl chain length on the plant uptake of the parent diPAPs and their corresponding metabolites was also examined. As diPAPs are marketed as greaseproofing agents in food contact papers27 and have been found in food packaging material,30,31 separate greenhouse and field experiments were performed to investigate the potential for endogenous diPAPs present in contaminated WWTP biosolids, paper fiber biosolids, and composts to carry over to amended soils and, subsequently, to plants grown on the same soils.
described in the SI. The WWTP biosolids and compost were collected in 2009, while the paper fiber biosolids were collected in 2008. Greenhouse Microcosm Experiment. Using an Odjob concrete mixer (Scepter Corporation, Toronto, ON), WWTP biosolids were mixed with soil at a rate of 16 g of biosolids/kg of soil (≈ 8.7 metric dry tons/ha), which was slightly higher than the maximal 5-year application rate of 8 tons/ha permitted in Ontario.32 In a separate experiment, the same biosolids were mixed with paper fiber biosolids at a ratio of 1:4 that corresponded to application rates of 16 g of WWTP biosolids/kg of soil and 67 g of paper fiber biosolids/kg of soil (≈36 tons/ha,) respectively, the latter of which was similar to the maximal 5-year application rate of 30 tons/ha for the spreading of paper fiber biosolids in Ontario.33 These biosolidsamended soils were then transferred to pots (∼600 g/pot) into which 5−10 seeds of M. truncatula were planted per pot, followed by inoculation with a mixture of cultured rhizobia strains, known to form symbiosis with M. truncatula in nature. Preparation of the rhizobia mixture is described in the SI. As the pots contained holes at the bottom, a catch plate was placed under each pot to capture any analytes that may have leached from the soil during watering. The pots were grouped into four treatments, as shown in Figure S1: (1) soil without biosolids amendment (n = 1 per time point); (2) WWTP biosolids-amended soil sown with plant seeds (n = 3 per time point); (3) soil amended with 1:4 mixture of WWTP biosolids and paper fiber biosolids and sown with plant seeds (n = 3 per time point); and (4) WWTP biosolids-amended soil sown with plant seeds and premixed with 100 mg of 6:2 diPAP from an ethanol-based standard (n = 3 per time point). Treatment 1 served as the PFAS-free blank and plant-free control, while treatments 2 and 3 were included to investigate the fate of endogenous PFASs that may be present in the WWTP and paper fiber biosolids. In treatment 4, 6:2 diPAP was added as a parent chemical to monitor for its potential biodegradation and uptake into the plants. The use of 6:2 diPAP as the parent here was based on industry preference for the perfluorohexyl (6 CF’s) chain length in the manufacture of fluorinated surfactants,34 and the fact that the diester typically exhibits the highest product efficiency in oil repellency applications, as compared to the mono- and triesters.28 The pots were watered daily and kept in a greenhouse (Earth Sciences Centre, University of Toronto, ON) for 5.5 months under natural sunlight and supplementary illumination (200 μmol/m2/sec) and a temperature cycle of 25/21 °C day/night. Field Experiment. Using a tractor-drawn spreader, 600 tons of a mixture of 1 part compost and 4 parts paper fiber biosolids were applied to 2 farmfields (15-acre and 5-acre) in Northumberland County, ON in May 2009 (Figure S2). A 1acre section of the 15-acre field was planted with pumpkins (Cucurbita maxima). Sampling, Extraction, and Analysis. In the greenhouse experiment, initial concentrations of diPAPs and PFCAs in the soil were measured by sacrificing one pot (n = 1) from each of the four treatment groups. At 1.5, 3.5, and 5.5 months, triplicate pots (n = 3) were sacrificed for each treatment, while the blank soil in treatment 1 was sampled as one pot at each time point. During sampling, the entire plant, including the roots, was harvested, shaken gently to remove any adhering soil particles, and archived together in plastic bags. The soil was wholly removed from each pot and mixed with 100−300 mg of sodium azide (NaN3) in a plastic bag to inhibit further microbial
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MATERIALS AND METHODS Materials. A list of all standards and reagents used in this study is provided in the Supporting Information (SI). All target analytes are listed in Table S1. Collection of WWTP and paper fiber biosolids, compost, and alfalfa plant seeds is further 341
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Figure 1. Concentrations of diPAPs and PFCAs (ng/g dw) observed in WWTP biosolids-amended soil (A), WWTP biosolids- and paper fiber biosolids-amended soil (B), and compost- and paper fiber biosolids-amended soil (C) in the greenhouse and field experiments. Each data point represents the arithmetic mean concentration of the triplicate (n = 3) sampling. The error bar represents the standard error.
months, a soil core was taken and separated into three segments of 5 in. each (0−5, 5−10, 10−15 in.). At 3.5 months, three pumpkins were harvested (2 months after planting) from the 1-acre field, each dissected into the leaves, flower, stalk, roots, and fruit to be stored separately. Sample analysis was performed with high pressure liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), using an Agilent 1100 HPLC coupled to an API4000 triple quadrupole MS (Applied Biosystems/MDS Sciex) operating under negative electrospray ionization mode. Further instrumental parameters are provided in the SI. Quality Assurance of Data. Quantitation of the PFCAs, FTCAs, and FTUCAs was performed using mass-labeled internal standards, with the exception of those analytes for
activity. The plates placed under each pot were also archived in plastic bags. All samples were stored at 4 °C until analysis. Soil, plants, and catch plates were extracted by methods described in the SI. Prior to their application on the farmfield, the compost and paper fiber biosolids were extracted as described in the SI. Soil samples were collected from nine sites across the two fields 3 days prior to application and subsequently at 1.5, 4, and 6.5 months after application (Figure S2). At each site, 20 spadefuls of surface soil were collected from a 9 m2 area and mixed in a 5gallon bucket, from which ∼500 g was sampled and stored as described above. This procedure was repeated three times at one site as a measure of site heterogeneity. Background soil from an adjacent untreated field was also collected. At 2 342
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for the control soil used in treatment 1. The C4−C14 PFCAs were present at concentrations ranging from nd to 1.8 ng/g dw in the soil. The polyfluoroalkyl PFCA precursors (i.e., diPAPs, FTCAs, and FTUCAs) were detected occasionally in the control soil (nd−0.6 ng/g dw). Upon initial amendment (0 month) of the soil with WWTP biosolids (treatment 2) and paper fiber biosolids (treatment 3), total diPAP (up to 300 ng/ g dw) and total PFCA (up to 50 ng/g dw) concentrations were observed in the soil (Figures 1A,B, S3, and S4). As was previously observed in Ontario WWTP sludge,5 a suite of 6:2 to 10:2/12:2 diPAPs were detected in the WWTP biosolidsamended soil here at concentrations ranging from 3.9 ± 0.7 ng/ g dw for 6:2/8:2 diPAP to 51.1 ± 5.7 ng/g dw for 10:2 diPAP (Figures 1A and S3). These diPAPs were also observed at concentrations ranging from 23.7 ± 4.5 ng/g dw for 6:2 diPAP to 82.5 ± 10.0 ng/g dw for 10:2 diPAP in soil amended at a 1:4 ratio of WWTP biosolids and paper fiber solids, respectively (Figures 1B and S4). Together with the significant diPAP concentrations (28−1070 ng/g, mean) previously reported in these same paper fiber solids,5 the high diPAP concentrations observed here in the paper fiber biosolids-amended soil are consistent with the prevalent use of these chemicals in food contact paper applications.27 An increasing prevalence of the longer chain diPAPs was observed in both types of treated soil, which corroborates the sorption dependency on chain lengths that has been previously reported for PFCAs and PFSAs in sediments35 and soils.36 The observation of different perfluoroalkyl chain lengths of diPAPs in the amended soil here and previously in WWTP sludge and paper fiber biosolids5 is also consistent with environmental exposure to commercial fluorotelomer-based products. A decline was observed in the concentrations of diPAPs in both types of treated soil over time (Figures 1A,B, S3, and S4), which may be due to sorption to the pots, soil, and/or biosolids; leaching to the catch plates during watering; biodegradation; and translocation into plants. Accumulations of 104 ± 13 ng and 107 ± 20 ng of total diPAPs (ΣdiPAPs) were observed over time in the catch plates placed under the WWTP biosolids-amended pots and WWTP- and paper fiber biosolids-amended pots, respectively. These masses corresponded to odd carbon chain pattern is consistent with the biological production of PFCAs from fluorotelomer-based materials.37−40 Despite the observed decline in diPAP concentrations in the soil and plant samples over time, no consistent evolution of PFCAs was observed in either of these compartments. However, the occasional detection of various FTCAs and FTUCAs in both the plants and soil is evidence of biotransformation of some fluorotelomer-based precursor materials present in the system. Identifying these specific precursors is complicated by the diverse functionalities incorporated in the manufacture of commercial fluorotelomer-based chemicals (e.g., phosphates, sulfonates, ethoxylates, polymers)41 and the fact that these products may contain mixtures of different chain lengths and other fluorotelomer-based residuals, like FTOHs.42 In the interest of elucidating transformation pathways, plant uptake, and metabolite profiles from potential soil and/or plant degradation
of diPAPs, 6:2 diPAP was added at high concentrations (μg/g), at least an order of magnitude higher than those observed endogenously in WWTP biosolids. Biotransformation of 6:2 diPAP in the Greenhouse Microcosm. The decline in 6:2 diPAP soil concentrations followed first order kinetics with a disappearance half-life of ∼2 months (kdisappearance = 0.342 ± 0.002/month; r = 1.00; p < 0.0001; Figure 2). As described above, the dissipation of diPAPs in soil may occur through multiple pathways. Leaching was a minor loss pathway for 6:2 diPAP, as only 712 ± 252 ng was observed to accumulate over time in the catch plates, which corresponded to C7) which preferred to reside in the soil, as shown in Figure S5. The PSAFs calculated here from plants sown in WWTP biosolids-amended soil (1.46 ± 0.49; PFOA) were typically higher than those measured from carrots, potatoes, and cucumbers grown in biosolids-amended soil in the laboratory (0.01−0.05 from edible portions; 0.38−0.99 from leaves and stalks; PFOA)22 and grasses collected from the contaminated fields in Decatur, AL (0.09−0.65; PFOA).24 These differences may be due to variable uptake abilities across different plant species and may also depend on the extent of the 346
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Environmental Implications. This study provides the first evidence of biotransformation of diPAPs and the subsequent plant uptake of these chemicals and their metabolites in a soilplant environment. This has important implications for diPAPs and other commercial fluorotelomer-based chemicals that have been discovered in the WWTP environment,2,5 as land application of biosolids may represent a significant source of these commercial materials and their biodegradation metabolites to soils. Analysis of soil amended with WWTP biosolids, paper fiber biosolids, and compost revealed significant diPAP and PFCA concentrations both in the greenhouse and field experiments. Translocation of PFCAs into the plants was observed to favour the short-chain congeners, such as PFBA, PFPeA, and PFHxA, although longer PFCAs (C7−C12) were also detected. Published data for PFASs in edible plants are sparse, but the plant−soil accumulation observed here and by others22,24,48 suggests plant uptake may be an important entryway for PFASs into the food chain through consumption of crops and meat raised on contaminated fields. The discovery of elevated PFAS concentrations in the biosolids-applied fields in Decatur, AL23−26 has triggered concern over potential contamination of beef cattle that have been grazing on these fields for 12 years.54 Analytical data on these animals have not been published, except for one raw milk sample, obtained from a bulk tank supplied by these cattle, which reported a detectable concentration of PFOS at 0.16 ng/g, but no other PFAAs monitored.55 Nevertheless, recent work by Kowalczyk et al. observed significant accumulation of PFOS (168−240 μg/L, plasma; 885−1172 ng/g, liver) and to a lesser extent, PFOA (
