Article pubs.acs.org/est

Bioaccumulation of Perfluoroalkyl Acids by Earthworms (Eisenia fetida) Exposed to Contaminated Soils Courtney D. Rich,† Andrea C. Blaine,† Lakhwinder Hundal,‡ and Christopher P. Higgins*,† †

NSF Engineering Research Center ReNUWIt, Department of Civil and Environmental Engineering, Colorado School of Mines, Golden, Colorado 80401, United States ‡ Metropolitan Water Reclamation District of Greater Chicago, Chicago, Illinois 60611, United States S Supporting Information *

ABSTRACT: The presence of perfluoroalkyl acids (PFAAs) in biosolidsamended and aqueous film-forming foam (AFFF)-impacted soils results in two potential pathways for movement of these environmental contaminants into terrestrial foodwebs. Uptake of PFAAs by earthworms (Eisenia fetida) exposed to unspiked soils with varying levels of PFAAs (a control soil, an industrially impacted biosolids-amended soil, a municipal biosolidsamended soil, and two AFFF-impacted soils) was measured. Standard 28 day exposure experiments were conducted in each soil, and measurements taken at additional time points in the municipal soil were used to model the kinetics of uptake. Uptake and elimination rates and modeling suggested that steady state bioaccumulation was reached within 28 days of exposure for all PFAAs. The highest concentrations in the earthworms were for perfluorooctane sulfonate (PFOS) in the AFFF-impacted Soil A (2160 ng/g) and perfluorododecanoate (PFDoA) in the industrially impacted soil (737 ng/g). Wet-weight (ww) and organic carbon (OC)-based biota soil accumulation factors (BSAFs) for the earthworms were calculated after 28 days of exposure for all five soils. The highest BSAF in the industrially impacted soil was for PFDoA (0.42 goc/gww,worm). Bioaccumulation factors (BAFs, dry-weight-basis, dw) were also calculated at 28 days for each of the soils. With the exception of the control soil and perfluorodecanoate (PFDA) in the industrially impacted soil, all BAF values were above unity, with the highest being for perfluorohexanesulfonate (PFHxS) in the AFFF-impacted Soil A (139 gdw,soil/ gdw,worm). BSAFs and BAFs increased with increasing chain length for the perfluorocarboxylates (PFCAs) and decreased with increasing chain length for the perfluoroalkyl sulfonates (PFSAs). The results indicate that PFAA bioaccumulation into earthworms depends on soil concentrations, soil characteristics, analyte, and duration of exposure, and that accumulation into earthworms may be a potential route of entry of PFAAs into terrestrial foodwebs.

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INTRODUCTION Recently, much attention has been focused on the bioaccumulation of perfluoroalkyl acids (PFAAs) in plants, aquatic organisms, and higher trophic level terrestrial animals.1−4 PFAAs and their precursors are used for many industrial and consumer products including fire-fighting foams, nonstick food packaging, and stain repellents.5 PFAAs are known to be ubiquitous and persistent, and longer-chain PFAAs can bioaccumulate, resulting in the presence of PFAAs in wildlife, humans, water, sediment, air, and house dust.6−8 Some of the long-chain PFAAs (perfluorooctanoate (PFOA) or longer for perfluorocarboxylates (PFCAs) and perfluorohexane sulfonate (PFHxS) or longer for perfluoroalkyl sulfates (PFSAs)9) have half-lives in humans lasting up to eight years, and in general are not rapidly eliminated from other higher trophic level organisms.10 After collection in conventional wastewater treatment plants (WWTPs), mass flows of PFAAs are maintained or increased via production of PFAAs from precursors during treatment processes.11 They are subsequently released into the environment through biosolids and effluents.12 Biosolids may contain © 2014 American Chemical Society

elevated levels of PFAAs and are commonly land-applied in the United States as an important nutrient supplement for crops.1,11,13−15 The issue of PFAAs in biosolids was highlighted in several studies that evaluated the environmental fate of PFAAs in Decatur, Alabama (U.S.), where PFAA-contaminated sewage sludge was unintentionally applied to pasture land.16−20 These studies reported high concentrations of PFAAs in soils16−18 and water samples,19 and there were detectable levels of PFAAs in cow’s milk20 because of high concentrations in the industrially contaminated sludge. However, it is unclear as to whether land applied municipal biosolids that contain low levels of PFAAs could become a potential source of PFAA exposure for earthworms and hence terrestrial foodwebs. The use of PFAAs and PFAA precursors in aqueous filmforming foam (AFFF) presents another route for high concentrations of PFAAs to be introduced to the environment. Received: Revised: Accepted: Published: 881

August 23, 2014 December 16, 2014 December 17, 2014 December 17, 2014 DOI: 10.1021/es504152d Environ. Sci. Technol. 2015, 49, 881−888

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Table 1. Characteristics of the Five Field Soils Used in the Bioaccumulation Experiments and Experimental Durations pH organic carbon (%) soil texture worm exposure (days)

control soil 7.7 1.4 silt loam 28

industrially impacted soil 6.9 6.5 clay loam 28

municipal soil 6.5 6.0 loam 0−28

AFFF Soil A 7.9 1.7 clay 28

AFFF Soil B 7.9 1.3 clay 28

The objective of this study was to evaluate the bioaccumulation of PFAAs from PFAA-impacted soils in earthworms (Eisenia fetida). The earthworm was chosen as the test organism due to its critical role in the terrestrial food web and its constant contact and ingestion of soil.37 Bioaccumulation experiments were performed using 28 day exposures of earthworms to fieldcollected (unspiked) PFAA-impacted soils. Equilibrium-based bioaccumulation factors (BAFs) and BSAFs were calculated for earthworms after 28 days of exposure to each soil. The uptake kinetics of PFAAs in earthworms was also measured over the 28 days and modeled using a standard biokinetic model that allowed for the estimation of both uptake and elimination rates. To our knowledge, this is the first study to look at bioaccumulation of PFAAs in earthworms from unspiked biosolids-amended soils and AFFF-impacted soils.

High levels of PFAA contamination have been reported in groundwater and soils around fire fighter training facilities.21−26 While the primary risk driver at these sites may be the potential for human consumption of contaminated groundwater, the presence of these compounds and their potential precursors in the soils, as determined by the total oxidizable precursor (TOP) assay,22 may also result in unacceptable ecological risks if significant bioaccumulation within the terrestrial foodwebs occurs. Again, at many of these sites, earthworms may serve as the gateway for chemical movement from the contaminated soils into the terrestrial foodweb. Several recent studies have documented the toxicity of two PFAAs, perfluorooctanoate (PFOA) and perfluorooctane sulfate (PFOS), in wildlife and laboratory animals.27−29 Bioaccumulation, particularly of PFOS, has been widely observed in higher trophic level animals. Müller et al.4 found biomagnification of PFOS and all of the long-chain perfluorocarboxylates (PFCAs), with the exception of PFOA, in caribou and wolves. Vestergren et al.3 observed biomagnification in dairy cows from contaminated food and water sources for PFOS and the long-chain PFCAs, again with the exception of PFOA. Custer et al.30 found biomagnification of PFOS and long-chain PFCAs in tree swallows that primarily feed on aquatic insects. Although longer chain PFAAs have been shown to bioaccumulate and biomagnify in aquatic organisms2,31 and higher trophic level animals,3,4,30 there are much less data associated with the bioaccumulation of PFAAs in lower trophic level terrestrial organisms. One recent study did document bioaccumulation of PFOS (up to 2410 ng/ g) in earthworms collected near a fluorochemical manufacturing facility.32 In addition, Xu et al. found that exposure to soil concentrations of PFOS varying from 10 to 120 mg/kg could damage the DNA of the earthworms.33 Separately, Zareitalabad et al. found that at a soil concentration of 100 mg/kg of PFOA and PFOS, there was a decrease in earthworm survival.34 At levels of 500 mg/kg of PFOA and PFOS, complete earthworm mortality was observed.34 Most relevant to the study herein, Zhao et al. examined bioaccumulation of PFAAs into earthworms from spiked soils.35 The bioaccumulation was calculated as a biota soil accumulation factor (BSAF), which was normalized for the organic carbon content in the soil. An important finding was that bioaccumulation in earthworms increased with increasing chain length, though the highest BSAF reported (0.537 goc/gww) was for perfluorododecanoate (PFDoA) in soil spiked to 100 ng/ g.35 This study used spiked-soil systems to evaluate bioaccumulation of PFAAs, which may not accurately represent field conditions, as chemical bioavailability to earthworms from spiked soils is generally not representative of typical field conditions because bioavailability is modified due to aging effects.36 Earthworms are important terrestrial organisms that play a critical role at the bottom of the food chain. For this reason, it is imperative to have an accurate measure of bioaccumulation upon which to base terrestrial foodweb models, as bioaccumulation of PFAAs in earthworms could result in unacceptable risks for higher trophic level organisms via biomagnification.

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MATERIALS AND METHODS Chemicals. Stable isotope standards and native perfluorinated standards were obtained from Wellington Laboratories (Guelph, ON, Canada). Analytes include perfluorooctanoate(PFOA), perfluorononanoate (PFNA), perfluorodecanoate(PFDA), perfluoroundecanoate (PFUdA), PFDoA, PFHxS, PFOS, and perfluorodecane sulfonate (PFDS). All analytes had matched stable isotope standards except for PFDS, for which the PFOS mass-labeled standard was employed. A list of the standards can be found in Supporting Information (SI) Table S1. All chemicals, unless otherwise specified, were obtained from Sigma-Aldrich. Standards were prepared in a 70/30 (v/v) solution of methanol and water with 0.01% ammonium hydroxide. Extractions were completed using HPLC-grade acetonitrile, HPLC-grade methanol, and water from a Milli-Q system (Millipore, Billerica, MA). Supelclean ENVI-Carb was used for extraction cleanup. HPLC-grade water and methanol were employed for use in liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Soils. Five field-collected soils containing elevated levels of PFAAs were used for the bioaccumulation experiments. Three of the soils were characterized previously: an unamended control soil, a “municipal” soil that had received long-term field application of municipal biosolids (applied at reclamation rates for 20 years for a cumulative application of 1654 Mg/ha), and an “industrially impacted” soil consisting of PFAA-contaminated biosolids mixed with the control field soil.1 The other two soils were collected from an AFFF-impacted former fire fighter training area at Ellsworth Air Force Base, South Dakota. One soil was collected adjacent to the source zone (fire fighter training area burn pit; Soil A) and the other was collected approximately 180 m down gradient from the source zone (Soil B). The soils were collected from the surface to a depth of 0.3 m with a hand auger.25 Additional details as to the scope and extent of PFAA contamination at the fire fighter training facility at Ellsworth can be found elsewhere.22,25 All soil samples were sieved (6.3 mm) to remove large rocks and debris and stored at 4°C prior to use. The soils were also characterized for the fraction of organic carbon (Walkley-Black method), the soil texture, and the pH at a 882

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3000 rpm (1811 RCF). The extract was decanted into a 20 mL scintillation vial and evaporated to dryness under nitrogen. After extraction, the sample was reconstituted in 1 mL of acidic methanol (1% v/v acetic acid). From the 1 mL of reconstituted sample, 100 μL was removed and diluted 10-fold with acidic methanol (to 1 mL). This was added to a packed column containing 200 mg of Envicarb for cleanup. After cleanup, a second 10-fold dilution with water was completed using 150 μL of cleaned-up extract (to a final volume of 1.5 mL) prior to analysis via LC-MS/MS. The dry weight conversion factor for the worms was determined by drying a separate triplicate aliquot of depurated worms at 105°C overnight. A previously established protocol employing stable-isotope surrogate standards40 was used for soil extraction of all the soils at Day 28 as well as the Day 1 for the municipal soil. This soil method was also included in the National Institute of Standards and Technology (NIST)-led development of PFAA concentration values being added to the Certificates of Analysis for two Standard Reference Materials (sludge, house dust).41 In addition, the four soils (not including the control) were analyzed for PFAA precursors using the TOP assay as outlined in Houtz et al.22,42 Protocols for both the soil extraction and the TOP assay can be found in the SI. Analysis. All PFAAs were analyzed with isotope dilution via LC-MS/MS using previously described conditions.40 Briefly, chromatography was performed using a Shimadzu LC-20AD unit (Kyoto, Japan) with aqueous ammonium acetate (10 mM) and methanol (10 mM) delivered at a flow rate of 800 μL/min. A Shimadzu SIL-5000 Auto Injector loaded the sample (1 mL) onto a 50 mm × 4.6 mm Gemini C18 column with a 3-μm particle size (Phenomenex, Torrance, CA) coupled to a C18 Guard Cartridge. Initially, the eluent conditions were 50% methanol and 50% water; methanol was ramped up to 95% over 4 min, held at 95% for 4 min, ramped back down to 50% over 1.5 min, and finally re-equilibrated at 50% until 13 min. All analytes were monitored for two transitions using scheduled multiple reaction monitoring on an MDS Sciex Applied Biosystems API 3200 (MDS Sciex, Ontario) system operating in negative electrospray ionization mode. Quality Control. Analyst software was used for quantitation of all LC-MS/MS data. Triplicate experimental containers were used for all time points sampled. Means and standard errors of experimental replicates were reported (n = 3). The lowest calibration standard that was calculated to be within 30% of its actual value was used to define the limit of quantitation (LOQ); the injected calibration curves typically ranged from 1 to 200 000 ng/L. The LOQs in the worm tissues were analyte-, sample-, and run-dependent and ranged from 0.715 to 2.86 ng/g ww. Contamination was monitored through experimental blanks (worms collected at time zero; n = 6) and extraction blanks extracted with no added material (extracted with each batch of worm samples; n = 3), and injection blanks. Any losses during extraction and LC-MS/MS ionization were corrected for by internal surrogate standards used for each analyte. The average surrogate recovery for all worm samples and all analytes was 47%. Analyte-specific surrogate recovery values as well as the results of surrogate-corrected spike recovery experiments for worm tissues can be found in SI Table S3. Bioaccumulation Metrics. Bioaccumulation factors (BAFs) were calculated for worms (after 28 days of exposure) in each soil by dividing the concentration of chemical in the worm (converted to a dry weight basis) by the concentration in the soil (on a dry weight basis)(eq 1):

commercial laboratory (Agvise Laboratories, Northwood, ND). Soil characteristics are reported in Table 1. Earthworms. Adult earthworms (E. fetida) were obtained from Carolina Biological Supply (Burlington, NC) and subsequently kept at room temperature in hydrated peat moss. The worms were fed three times a week with alfalfa that was hydrated in a 1:1 ratio of water to food and allowed to age for 2 days before being spread over the top of the worm culture. Prior to exposing the worms to the contaminated soils, the worms were depurated in sets of five in the dark for 24 h on wet filter paper and the initial weight was recorded after the depuration. Experimental Setup. The bioaccumulation experiment was conducted in polypropylene Nalgene bottles (1 L) with triplicate bottles prepared for each time point and soil. As preliminary tests with soil aliquots adjusted to 75% of the water holding capacity for each soil (as per standard protocols38) indicated significant worm avoidance of several of the soils, additional preliminary worm burrowing experiments were conducted to determine the minimal moisture needed for each soil to ensure the worms would not avoid the soil. Once the soil moisture content needed to ensure worm burrowing was determined, the moisture content for each soil was adjusted prior to the start of the experiment. These gravimetric moisture contents were: 11% for the control soil, 23% for the industrially impacted soil, 25% for the municipal soil, 15% for the AFFF Soil A, and 16% for the AFFF Soil B. After sieving the soils, 500 mL (calculated based on American Society for Testing and Materials protocol30) of soil was added to each container. Worms were added to the containers (five worms per container) and kept in an incubator at 25°C with constant light exposure. Six sets of worms were frozen at −80°C for a reference point at time zero and were not exposed to any of the soils. Each container was topped with cheese cloth held on by a rubber band and the starting weight of each container was recorded. The moisture content was held constant by adding distilled water to each container every other day to maintain the initial mass, and a water reservoir at the bottom of the incubator was employed to add humidity and minimize evaporative losses of water. Worms were collected after 28 days in all five soils to determine bioaccumulation. To ensure 28 days was sufficient to achieve steady state and to enable kinetic modeling, additional sets of triplicate containers using the municipal soil were prepared for sampling on days 1, 3, 5, 7, 9, 12, 16, and 21. At each time point, containers were removed from the incubator and the worms and soil were prepared for extraction. The worms were removed individually with a dental pick and placed in a Petri dish on wet filter paper to depurate for 24 h in the dark prior to a final weight being recorded and being frozen in a 50 mL polypropylene tube at −80°C until analysis. The mass of each worm sample at the beginning and the end of the exposure can be found in SI Table S2. No differences in burrowing habits were observed during the course of the exposure. After removal of the worms, 50 mL of the soil was placed in a 50 mL polypropylene tube and frozen (−20°C) until extraction. Extractions. The extraction protocol for the worms was modified from Malinsky et al.,39 primarily adjusting for a larger sample mass. Each tube of worms was thawed and subsequently spiked with 2 ng of isotopically labeled surrogate; the tube was then allowed to sit for 30 min before adding 20 mL of acetonitrile. All five worms within a tube were then homogenized into a composite sample using an Omni Prep (Omni International) hand-held homogenizer with the Omni TH motor and “hard tissue” plastic tips. The sample was frozen (−20°C) for at least 1 h and afterward centrifuged for 20 min at 883

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Figure 1. Concentrations of PFAAs in earthworms over time for PFOS (a), PFOA, PFDA, PFDoA, and PFHxS (b) exposed to municipal soil for up to 28 days (672 h). The kinetics of accumulation of the additional PFAAs were omitted for clarity, but can be found in SI Figure S1.

Table 2. Fitted Kinetic Parameters for Uptake of PFAAs, Estimated Steady State BSAF and BAF Values, and Measured 28 Day BSAF and BAF Valuesa

a

analyte

ks (goc·gww−1·h−1) x 104

ke (h−1) x 104

estimated steady state BSAF (goc·gww−1)

measured BSAF (goc·gww−1)

estimated steady State BAF (gdw,soil·gdw,worm−1)

measured BAF (gdw,soil· gdw,worm−1)

PFOA PFNA PFDA PFUdA PFDoA PFHxS PFOS PFDS

10.3 ± 3.8 10.2 ± 3.1 7.0 ± 1.8 8.0 ± 3.0 4.2 ± 0.6 13.9 ± 1.9 15.6 ± 2.7 0.7 ± 0.2

512 ± 199 268 ± 85.5 143 ± 42.6 230 ± 93.1 40.1 ± 9.5 63.2 ± 11.8 93.0 ± 19.9 41.1 ± 19.1

0.020 ± 0.011 0.038 ± 0.017 0.049 ± 0.019 0.035 ± 0.019 0.105 ± 0.029 0.220 ± 0.051 0.168 ± 0.046 0.017 ± 0.009

0.019 ± 0.0024 0.034 ± 0.0055 0.048 ± 0.010 0.033 ± 0.0056 0.092 ± 0.019 0.23 ± 0.040 0.17 ± 0.032 0.015 ± 0.0035

2.15 ± 1.16 4.08 ± 1.79 5.27 ± 2.08 3.71 ± 2.04 11.3 ± 3.11 23.6 ± 5.44 18.0 ± 4.95 1.82 ± 0.99

2.07 ± 0.26 3.64 ± 0.59 5.17 ± 1.08 3.50 ± 0.60 9.82 ± 2.07 24.6 ± 4.27 18.1 ± 3.44 1.59 ± 0.37

The estimated steady state BSAF was calculated as ks/ke and the estimated steady state BAF was calculated as ks/ke·(wet weight of worm/foc).

determined from separate depuration experiments), we have previously observed very good agreement between ke values determined for PFAAs in worms using the two approaches.2 For the present study, analysis of the Day 1 and Day 28 municipal soil levels indicated that the concentration of PFAAs in the soil did not statistically vary over time (α = 0.05), and thus λ was set to zero.

Cworm(ng/gdw)

BAF =

Csoil(ng/gdw)

(1)

Biota soil accumulation factors (BSAFs) were also calculated for worms (after 28 days of exposure) to normalize for the organic carbon content of the different soils (eq 2): BSAF =

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Cworm(ng/g ww) Csoil(ng/gdw)/foc

RESULTS AND DISCUSSION Kinetics of PFAA Bioaccumulation in Earthworms. Data from the municipal soil treatment was used to develop a kinetic model of bioaccumulation of each PFAA (not differentiating between linear and branched isomers) in the earthworm. The municipal soil, which had received biosolids at reclamation rates for 20 years, had the highest concentrations of PFOS (243 ng/ gdw) and PFDS (113 ng/gdw) and much lower concentrations of PFOA (14.8 ng/gdw), PFHxS (3.03 ng/gdw) and PFUdA (5.32 ng/gdw). Similarly, worm concentrations were also highest for PFOS (683 ng/gww) and PFDS (28.1 ng/gww) with lower concentrations of PFOA (4.76 ng/gww) and PFNA (3.98 ng/ gww). All PFAA concentrations for soils and worms can be found in SI Tables S4−S7. PFAA concentrations measured in the worms from the municipal soil were plotted versus time (Figure 1) and fit with the kinetic model (eq 3). Additional analytes are shown in SI Figure S1. The selected model resulted in ks values that ranged from 4.22 to 10.3 (×10−4 goc·gww−1·h−1) and ke values that ranged from

(2)

BSAF values were not normalized to lipids since PFAAs do not readily accumulate in lipids,43 and worm concentrations were reported on a wet weight (ww) basis to allow for comparisons to earlier studies.2,31,35 Kinetic data were modeled using OriginPro 9.0 with a model that allows for possible contaminant concentration decline in the exposure media44 (eq 3): Corg =

ksCsoil,0 ke − λ

(e−λt − e−ket )

(3)

where Corg (ng/gww) is the concentration in the organism on a wet weight basis, Csoil,0 (ng/goc) is the initial concentration in the soil normalized to organic carbon content, ks (goc gww−1 h−1) is the uptake rate constant, ke (h−1) is the fitted elimination rate constant, and λ (h−1) is the first-order soil loss rate constant. Though both uptake (ka) and elimination (ke) rate constants were fitted from the same data (as opposed to ke values being 884

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Figure 2. BSAF (a) and BAF (b) values for each analyte for the biosolids-amended soils. Bars represented by < had worm concentrations that were < LOQ.

4.01 to 51.2 (×10−3 h−1) for the PFCAs. For the PFSAs, the ks values ranged from 0.699 to 15.6 (×10−4 goc·gww−1·h−1) and the ke values ranged from 4.11 to 9.30 (×10−3 h−1). The ks and ke parameters for each analyte are shown in Table 2. The uptake and elimination rates tend to decrease with an increase in chain length for both the PFCAs and PFSAs. The decrease in elimination rate with increasing chain length has been seen in other organisms as well.43,45 The estimated steady state BSAF values from the model (i.e., ks/ke) ranged from 0.017 to 0.220 goc/gww and generally increased with increasing chain length for the PFCAs. Interestingly, they decreased with increasing chain length for the PFSAs. In addition, the BSAF values were, in general, higher for the PFSAs than PFCAs of equal perfluorocarbon chain length. Previous studies have shown greater bioaccumulation in rainbow trout for PFSAs than PFCAs.43 The larger sulfonate headgroup compared to the carboxylate headgroup could explain why the bioaccumulation decreases with increasing chain length for PFSAs due to increased sorption to the soils as seen in Higgins et al.46 The increased sorption of the long chain PFSAs would decrease the bioavailability of the analytes to the earthworms. Zhao et al.35 also reported BSAF values for uptake into earthworms with a measured BSAF value for PFOS from a 200 ng/gdw spiked soil of 0.13 goc/gww, which is in good agreement with the estimated steady state BSAF value reported in Table 2. BSAF values reported by Zhao et al.35 for other analytes were generally on the same order of magnitude as those from the present study; however, they tended to be higher for PFCAs and lower for PFSAs. The estimated steady state BAF values from the model (ks/ke converted to a dry weight basis using the measured conversion factor of 6.42 gww/gdw but not organic carbon normalized) ranged from 1.82 to 23.6 gdw,soil/gdw,worm. The BAF values followed the same trends as the BSAF values. Both the estimated steady state BSAF and BAF values correspond well to the measured values calculated for the 28 day concentrations of PFAAs in the worms; all modeled values lie within the error range of the measured values. This agreement between model and experimental values indicates that the bioaccumulation of PFAAs in the worms had reached steady state by the end of 28 days.

By comparison, the kinetic uptake and elimination rate constants reported in Zhao et al.35 were smaller (i.e., slower rates) than those reported here for all but one analyte (the ks value for PFDoA); these comparisons are shown in SI Figure S2. In both Zhao et al.35 and the current study, the earthworms were the same type, the soil type is similar (loamy surface soil35 vs. loam herein), and the organic carbon content was similar, making it unclear as to why significant differences were apparent. One possible explanation for a difference in uptake rates is that spiked soils that were used in Zhao et al.,35 which may have resulted in nonequilibrium conditions. In their study, spiked soils were shaken for 6 days and then incubated for 4 days prior to exposure. If equilibrium had not been reached, the PFAAs might not have been appropriately associated with the organic material in the soil upon which earthworms typically feed,47 resulting in an effective decreased exposure and slower uptake kinetics.35 Bioaccumulation from Field Soils. Worm Health. There was a low mortality rate of the worms after 28 days of exposure to the four field soils (5.0%) when compared to the control (13.3%). The worms did lose weight over the 28 day exposure, with an average weight loss of 24.3 ± 3.62% when all five worms were still alive. There was not a clear trend for which soil exposure resulted in more weight loss, suggesting that neither the soil type nor the PFAA concentrations (absolute or relative) affected the weight loss of the worms. Biosolids-Amended Soils. The biosolids-amended soils that were used for the 28 day exposure consisted of an industrially impacted soil and a municipal soil (plus a control soil), all of which were the same soils used in Blaine et al.1 For the industrially impacted soil, the highest BSAF value was for PFDoA (0.42 goc/gww) while the lowest reportable BSAF was for PFOA (0.040 goc/gww). The maximum BSAF for the municipal soil was for PFHxS (0.23 goc/gww) and the minimum BSAF was for PFDS (0.015 goc/gww). The control soil only resulted in a reportable BSAF value for PFNA (0.047 goc/gww). Figure 2a shows the BSAF values calculated (when possible) for each analyte. If either the concentration in the soil or worm was below the LOQ for that analyte, no BSAF or BAF values were calculated. All calculated BSAF values can be found in SI Table S8. For PFCAs, as the chain length increased, the BSAF also increased. This trend was also seen in Zhao et al.35 As can be seen 885

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Figure 3. BSAF (a) and BAF (b) values for AFFF-impacted soils.

formed using a fresh biosolids-based compost1 that potentially allowed for higher bioavailability. The differences could also be due to higher concentrations of PFCAs from the industrially impacted soil (as compared to the municipal soil), leading to higher bioaccumulation of those analytes in the industrially impacted soil. BAF values from the municipal and industrially impacted soils, when measurable, were greater than one for all of the analytes (Figure 2b). BAF values are used to assess risk from exposure,48 and BAF values greater than one indicate a higher concentration in the worms than the exposure media. The maximum BAF value was for PFDoA in the industrially impacted soil (41.4 gdw,soil/ gdw,worm). All BAF values are reported in SI Table S9. AFFF Impacted Soils. The BSAF values for earthworms from the AFFF-impacted Soil A were highest for PFHxS (0.37 goc/ gww), PFDoA (0.24 goc/gww), and PFOS (0.20 goc/gww); for earthworms from the AFFF-impacted Soil B, BSAF values were highest for PFHxS (0.20 goc/gww), PFOS (0.11 goc/gww), and PFDoA (0.10 goc/gww). The earthworms from the AFFFimpacted soils displayed BSAF values that were generally on the same order of magnitude of those seen in the biosolidsamended soils, further supporting the results. Figure 3a shows a trend of increasing BSAF values with increasing chain length for PFCAs and decreasing BSAF values with increasing chain length for PFSAs; this trend was also observed with the biosolidsamended soils. Similar to the BSAF values, the maximum BAFs for earthworms from the AFFF-impacted soils were for PFHxS, 139 gdw,soil/gdw,worm in Soil A and 99.6 gdw,soil/gdw,worm in Soil B. All of the BAF values for each analyte were greater than one, indicating accumulation from soil to worm. The trends with respect to chain length were also similar to those observed with BSAF values in the AFFF soils. Potential Role of Precursors. In an effort to discern the potential role of PFAA precursors in PFAA accumulation in the worms, the four soils (not including the control soil) were analyzed for PFAA precursors as per the TOP assay,22,42 with statistical difference of means between the oxidized and unoxidized soil extracts established with an analysis of variance (ANOVA) with Tukey’s Test (α = 0.05). Previous studies have suggested exposure to PFAA precursors can lead to PFAA body

in SI Figure S3, the BSAF values increased approximately 0.13− 0.26 log units for each additional CF2 group added to the length of the perfluorocarboxylate. However, the opposite trend was observed for the PFSAs and does not correspond to what was observed by Zhao et al.35 Though the data are more limited than the PFCA data, in general, the log BSAF values decreased slightly with increasing chain length (SI Figure S3). It is unclear whether this difference may be an artifact of differing analyte suites, as Zhao et al.35 reported data for shorter chain PFSAs and did not report data for PFDS. Additionally, there could be decreased bioavailability of PFDS from the soil, which would explain the decrease in bioaccumulation for PFDS compared to the shorter chain PFSAs and would explain the decreasing trend seen in the present study compared to the increasing trend reported in Zhao et al.35 Overall, the BSAF values were on the same order of magnitude as those reported in Zhao et al.35 for both the municipal and industrially impacted soil, suggesting that the spiked systems used by Zhao et al. did not significantly increase the bioavailability of the PFAAs as compared to actual biosolidsamended field soils. This is important, as others36 have noted decreased bioavailability of organic chemicals in biosolidsamended soils. Moreover, given the differences in the measured uptake and elimination rate constants (SI Figure S2), the overall agreement of these data to those presented in Zhao et al.35 was somewhat surprising: clearly, the slower uptake rates were generally balanced by slower elimination rates, resulting in relatively good agreement in the steady-state BSAF values. When comparing the results of bioaccumulation in earthworms to that in sediment-dwelling worms (Lumbriculus variegatus), the BSAF values from the present study were generally an order of magnitude lower than those reported by Higgins et al.2 but were typically on the same order of magnitude as reported by Lasier et al.31 Even though the BSAF values are normalized to the organic carbon in the soil, there is generally more accumulation from the industrially impacted soil than from the municipal soil. These differences in accumulation between the industrially impacted soil and the municipal soil could be due to differences in soil properties, especially the composition of the organic carbon.1 The bioavailability of PFAAs may have been influenced by the nature of the organic carbon, as the municipal soil was an aged soil while the industrially impacted soil was 886

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burdens.2,49 In this study, these data were evaluated as to whether they could help explain the differences in bioaccumulation observed from the soils. AFFF Soil A and the municipal soil had the greatest number of increases in PFAA levels upon oxidation (SI Table S10), with only additional PFNA being generated upon oxidation in the industrially impacted soil and no PFAAs generated upon oxidation in AFFF Soil B. As accumulation of the PFAAs tended to be higher in the industrially impacted soil (as compared to the municipal soil; Figure 2) and AFFF Soil A (as compared to AFFF Soil B; Figure 3), these data suggest that differences in accumulation between the soils cannot be explained by the levels of precursors alone, at least as measured by the TOP assay. Additional analysis as to the specific identities of the precursors in these soils may be warranted, as it is possible (and even likely) that the biosolids-derived precursors may be very different (and exhibit different bioavailability and/or potential for biologically mediated conversion to PFAAs) than the AFFF-derived precursors. In other words, it is possible that the effects of precursors on the differing extents of PFAA accumulation observed herein are simply not discernible with the TOP assay. Implications. While there are some data available from the literature about uptake of PFAAs into earthworms from spiked soils, this is the first study to examine PFAA uptake into earthworms exposed to field-collected biosolids-amended soils and AFFF-impacted soils. From the reported data, it is clear that the earthworms are able to take up PFAAs from contaminated soil and that the uptake is chain length dependent. Preferential accumulation is observed for long-chain PFCAs and short-chain PFSAs. This would suggest that long-chain PFCAs and shortchain PFSAs have a higher potential to accumulate further up in the food chain as well. With the exception of PFDS in the industrially impacted soil, all BAFs for earthworms from PFAAimpacted soils were greater than one. This implies that PFAA contamination in the soil will not only enter the terrestrial food chain, but also bioaccumulate to much greater concentrations in the earthworm. With respect to risk assessment, this is important because typical BAF default (conservative) values of one may greatly underestimate the risk.48 In light of the potential for PFAA bioaccumulation in earthworms presented herein as well as the potential for higher trophic transport, the effects and extent of PFAA biomagnification in terrestrial food chains, particularly those associated with AFFF-impacted sites or industrially impacted biosolids applications, should be further evaluated.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Kate Percival and Evan Gray from the Colorado School of Mines for help with the worm assays as well as R. Hunter Anderson for insightful comments on an early draft of the manuscript.

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ASSOCIATED CONTENT

S Supporting Information *

Additional details are available regarding analytical methods, worm weight changes and mortality rates, soil extraction procedures, total oxidizable precursor assay procedure, worm spike recovery results, soil concentrations, earthworm concentrations, kinetic exposures, kinetic parameters comparison to Zhao et al.,35 BSAF values, BAF values, and changes in analyte concentration in the soils due to oxidation of precursors. This material is available free of charge via the Internet at http://pubs. acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: (720) 984-2116; fax: (303) 273-3413; e-mail: [email protected]. 887

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