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BIODEGRADATION AND UPTAKE OF THE PESTICIDE SULFLURAMID IN A SOIL/CARROT MESOCOSM Itsaso Zabaleta, Ekhine Bizkarguenaga, Deborah B. O. Nunoo, Lara Schultes, Juliana Leonel, Ailette Prieto, Olatz Zuloaga, and Jonathan P. Benskin Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03876 • Publication Date (Web): 08 Feb 2018 Downloaded from http://pubs.acs.org on February 9, 2018

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Environmental Science & Technology

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BIODEGRADATION AND UPTAKE OF THE PESTICIDE SULFLURAMID IN A SOIL/CARROT MESOCOSM

3 Itsaso Zabaleta1,2*, Ekhine Bizkarguenaga1,2*, Deborah B. O. Nunoo2, Lara Schultes2, Juliana Leonel3, Ailette Prieto1,4, Olatz Zuloaga1,4, Jonathan P. Benskin2

4 5 6 7 8

1

Department of Analytical Chemistry, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), Bilbao, Spain. 2

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Department of Environmental Science and Analytical Chemistry (ACES), Stockholm University, Stockholm, Sweden.

11 12

3

Department of Geoscience, Universidade Federal de Santa Catarina, Florianópolis, Brazil.

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4

Research Centre for Experimental Marine Biology and Biotechnology (PIE), University of the Basque Country (UPV/EHU), Plentzia, Spain.

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

* Corresponding authors: [email protected] Tel.: 0034 946015551 Fax: 0034 946013500 [email protected] Tel.: 0034 946015551 Fax: 0034 946013500

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ACS Paragon Plus Environment


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ABSTRACT

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N-ethyl perfluorooctane sulfonamide (EtFOSA) is the active ingredient of Sulfluramid, a

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pesticide which is used extensively in South America for control of leaf-cutting ants. Despite

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being a known precursor to perfluorooctane sulfonate (PFOS), the importance of EtFOSA as a

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source of environmental PFOS remains unclear. In the present work, uptake, leaching, and

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biodegradation of EtFOSA and its transformation products were assessed over 81 days in

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soil/carrot (Daucus carota ssp sativus) mesocosms for the first time. Experiments performed in

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the presence of carrot produced PFOS yields of up to 34 % using a technical EtFOSA standard,

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and up to 277 % using Grão Forte, a commercial Sulfluramid bait formulation containing

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0.0024 %

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sulfonamide (FOSA), and perfluorooctanoic acid (PFOA) also formed over the course of the

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experiments. Leachate contained low levels of transformation products and a high FOSA:PFOS

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ratio, consistent with recent observations in Brazilian surface water. In carrot, the more

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hydrophilic transformation products (e.g. PFOS) occurred primarily in the leaves, while the

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more hydrophobic products (e.g. FOSA, FOSAA and EtFOSA) occurred in peel and core.

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Remarkably, isomer-specific analysis revealed that the linear EtFOSA isomer biodegraded

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significantly faster than branched isomers. These data collectively show that the application of

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Sulfluramid baits can lead to the occurrence of PFOS in crops and in the surrounding

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environment, in considerably higher yields than previously thought.

EtFOSA.

Perfluorooctane

sulfonamido

acetate

(FOSAA),

perfluorooctane

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KEYWORDS: EtFOSA, Sulfluramid, soil, biodegradation, carrot, PFAS isomers

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Supporting Information: Materials and reagents, sample extraction, and supporting figures

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and tables.

3



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INTRODUCTION

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Perfluorooctane sulfonate (PFOS; C8F17SO3-) has attracted considerable international regulatory

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and scientific attention due to its widespread occurrence and links to adverse health effects in

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humans and wildlife.1 On account of these risks, PFOS and its precursors were added to

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Annex B of the United Nations Stockholm Convention on Persistent Organic Pollutants in

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2009,2 and to the list of priority hazardous substances in the EU water policy Directive

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2013/39/EU in 2013.3 Presently, manufacturing of PFOS and PFOS-precursors continues in

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some countries under Stockholm Convention production and use exemptions. These

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contemporary sources of PFOS are poorly characterized and may pose a considerable ongoing

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risk to humans and wildlife.4–6

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Brazil is currently among the main global producers of the PFOS-precursor N-ethyl

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perfluorooctane sulfonamide (EtFOSA; C8F17SO2NHC2H5), which is the active ingredient in

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Sulfluramid, a commercial pesticide. EtFOSA is produced from the starting material

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perfluorooctane sulfonyl fluoride (POSF; C8F17SO2F), which is imported into Brazil from

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China. Brazil holds an exemption under Annex B to manufacture and use Sulfluramid to manage

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leaf-cutting ants from the genus Atta ssp. and Acromyrmex spp., which jeopardize agricultural

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activities in parts of Latin America.7 Alternatives to Sulfluramid are not currently available; and

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while the country is phasing out production and use of baits for domestic use, commercial

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applications in agriculture are expected to continue into the foreseeable future.8

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The manufacture and use of Sulfluramid in Brazil from 2004 to 2015 is expected to produce

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between 167 and 603 tonnes of PFOS.9,10 However, there are considerable uncertainties

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surrounding these estimates, owing to an absence of manufacturing data but also a lack of

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information surrounding PFOS yields in the environment. For example, the only study to

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investigate soil biodegradation of EtFOSA reported very low (4 mol%) yields of PFOS

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following incubation of a pure standard of EtFOSA over 182 days.11 Studies involving other

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perfluorooctane sulfonamides have demonstrated considerably higher PFOS yields (and in some

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cases formation of perfluoroalkyl carboxylic acids) under biological12–15 and abiotic16,17

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conditions (reviewed elsewhere18). Among these studies, a soil-vegetable mesocosm study

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involving perfluorooctane sulfonamide (FOSA), the N-dealkylation product of EtFOSA,

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demonstrated that FOSA was totally degraded to PFOS in presence of carrot while no

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degradation was observed in absence of vegetable.12 Collectively these data suggest that in the

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natural environment (and in particular in the presence of a vegetable crop), yields of PFOS from

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EtFOSA may be considerably higher than 4%. However, to date there are no soil-vegetable

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mesocosm studies involving EtFOSA or commercial sulfluramid formulations. 4


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Data on the environmental occurrence of EtFOSA in South and Central America are also

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scarce.9 One study reported low but detectable levels of EtFOSA in air samples from Costa

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Rica19 while others have observed elevated concentrations of potential EtFOSA transformation

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products in both South American surface waters9,20,21 and biota.22 The unusually high ratio of

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FOSA:PFOS in Brazilian surface water is hypothesized to be a marker of Sulfluramid use, but

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this requires further investigation. To date, there are no studies which have examined the

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occurrence of EtFOSA or its transformation products around agricultural regions where

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Sulfluramid is deployed. Such data, together with improved estimates of EtFOSA production

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and PFOS degradation yields, are clearly needed in order to determine the importance of

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Sulfluramid as a source of environmental PFOS.

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Despite some recent work involving leaching and plant uptake12,23–25 of PFAAs, only two

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studies have investigated the fate of PFOS-precursors in a soil-vegetable mesocosm.12,26 There

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are no peer-reviewed studies investigating the fate and behavior of commercial Sulfluramid

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baits in soil-vegetable mesocosm. Considering the use pattern of Sulfluramid, this information

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is urgently needed in order to characterize the likelihood of environmental contamination

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arising from the use of this commercial pesticide. The purpose of this study was to investigate

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biodegradation, leaching, plant uptake, and distribution of EtFOSA and its transformation

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products in soil-carrot mesocosms. Experiments were performed with both technical standards

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and a commercially available, characterized Sulfluramid bait, providing new estimates for

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EtFOSA-derived PFOS formation under environmentally-relevant conditions. Furthermore,

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since commercial EtFOSA is manufactured as an isomeric mixture, we studied the fate and

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behavior of individual isomers using isomer-specific analytical methodologies. To our

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knowledge this is the first isomer-specific study of any PFAS in a soil and/or soil-vegetable

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mesocosm. Collectively, these data provide valuable new insight on the importance of EtFOSA

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as a contemporary source of PFOS.

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EXPERIMENTAL METHODS

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Standards and Reagents

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A full list of chemicals and reagents is provided in the Supporting Information (SI). Technical

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EtFOSA (95%) originated from Lancaster Synthesis (Wyndham, NH).27 Isomeric purity could

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not be determined due to a lack of purified branched isomer standard. Grão Forte, a commercial

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Sulfluramid formulation (determined to contain 0.0024 % EtFOSA (∑branched+linear

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isomers)) was obtained from Insetimax Industrial Chemicals (Brazil). L-EtFOSA, L-FOSA,

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perfluorooctane sulfonamido acetate (L-FOSAA), perfluorodecanoate (PFDA), characterized

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isomeric mixtures of PFOS and perfluorooctanoate (PFOA), and the isotopically labeled 5



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standards of EtFOSA, FOSA, PFOS, PFOA and PFDA were purchased from Wellington

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Laboratories (Guelph, ON, Canada) (Table S1).

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Experimental Design and Soil Fortification

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A total of six, 81-day mesocosm experiments were carried out concurrently (Table 1).

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Experiments 1 and 2 were conducted in duplicate and involved incubation of technical EtFOSA

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(3.8 mg kg-1; ∑branched+linear isomers) in microbially-active soil (referred to herein as `active´

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soil), with and without carrot (Daucus carota ssp sativus), respectively. Experiments 3, 4, and 5,

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were designed as control incubations: Experiment 3 was conducted in duplicate and involved

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fortifying soil autoclaved at 112 °C under vacuum for 4 hours (referred to herein as `inactive´

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soil) with technical EtFOSA (3.8 mg kg-1, ∑branched+linear isomers) to monitor leaching and

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abiotic losses. Experiments 4 and 5 were single blank experiments which contained unfortified

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active soils without and with carrot, respectively, to monitor contamination introduced from

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water and air. Finally, Experiment 6 was carried out in duplicate and involved fortifying active

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soil containing carrot with the commercial Sulfluramid formulation Grão Forte (0.0024 %

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EtFOSA, ∑branched+linear isomers). All experiments contained PFDA (100 ng g-1) which

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functioned as an internal negative control, as previously described.15

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An acidic sandy loam soil (pH = 5.7 ± 0.2), which is common to regions of Brazil28 was used in

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the present work. Soil chemistry parameters are provided in Table S2. In Experiments 1-3

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(Table 1), soils were weighed, covered with acetone and fortified with technical EtFOSA in

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order to achieve a 3.8 mg kg-1 nominal concentration. After stirring for 24 hours, the soil-

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acetone mixture was placed under a fume hood in order to let the solvent evaporate. Soil was

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then aged for one week. For Experiment 6, 10 g of Grão Forte was added to the surface of each

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pot containing 2 kg of soil.

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Plant Cultivation and Sampling

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All experiments were performed in a climate-controlled greenhouse with interior conditions set

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to 25 °C / 50 % humidity during the day (14 h) and 18 °C / 60 % humidity at night (10 h). Prior

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to germination, seeds were soaked in Milli-Q water. The washed seeds were distributed

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randomly on dampened filter paper in a Petri dish and covered with moistened filter paper.

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Upon germination (12 - 14 days), 4 seedlings were transplanted to each pot containing 2 kg of

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soil (fortified or non-fortified). Each pot represented a single time point, and a total of 5 time

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points were sampled over the course of the experiment. Pots were arranged randomly and

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regularly watered with distilled water and Hoagland nutritive solution (Hoagland reagents and

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preparation procedure are included in SI). Leachate from each pot was collected at the same 6


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time as soil sampling on days 14, 28, 56 and 81, resulting in 14-day, 28-day, 56-day, and 81-day

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composite leachates samples. Exact volumes collected for each pot are provided in Table S3

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(SI). Blanks, consisting of Milli-Q water stored in the same PE bottles, were also analyzed in

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parallel to assess background contamination. On the last two time points, carrots were collected

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and divided into peel, core and leaf compartments. Soil was air-dried and carrots were freeze-

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dried. All samples were stored at -80 °C prior to extraction and analysis.

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Extraction and Cleanup

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The extraction procedure for determination of EtFOSA and its transformation products in Grão

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Forte bait is described in the SI. Validation of this method is described elsewhere.29 Soil and

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carrot extractions were performed according to Avendaño and Liu,11 with slight modifications.

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Briefly, 0.5 g of dried sample was fortified with 2 ng of isotopically-labeled standards and 8 mL

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acetonitrile (ACN), and then sonicated for 20 min. After sonication, the mixture was placed in

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an angular shaker for 40 min, centrifuged at 2900 rpm for 20 min and the ACN was transferred

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into a clean 15 mL polypropylene test tube. The procedure was repeated using ACN with

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25 mM sodium hydroxide and the supernatants were combined. The extracts were placed in a

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Turvobap LV evaporator and reduced to dryness (soil extracts) or to approx. 1 mL (carrot

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extracts). Soil extracts were reconstituted in 400 µL methanol (MeOH):Milli-Q water (1:1, v/v)

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with 20 mM formic acid and 20 mM ammonium formate, while a portion (200 µL) of the carrot

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extract was mixed with 200 µL of Milli-Q water containing 20 mM formic acid and 20 mM

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ammonium formate. Extracts were transferred to microvials prior to instrumental analysis.

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Leachate was filtered through borosilicate glass fiber filters prior to extraction using the

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procedure described by Gilljam et al.9 (see SI for details). The filters were extracted separately

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to assess potential sorption of target analytes, as previously described for solid matrices.

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Instrumental Analysis

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Quantitative analysis of EtFOSA and its transformation products was carried out by ultra

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performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) using a Waters

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Acquity UPLC coupled to a Xevo TQ-S triple quadrupole mass spectrometer (Waters). The

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method, which has been previously described,30 facilitates chromatographic separation and

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quantification of individual PFAS isomers (see example chromatograms in Figures S1-S4, SI).

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Extracts (10 µL) were injected onto an Ascentis Express F5 guard column (2.7 µm, 2.1 mm ×

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0.5 cm) coupled to an Ascentis Express F5 (2.7 µm, 2.1 mm × 10 cm) analytical column

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maintained at 30 °C. The mobile phase consisted of 20 mM formic acid and 20 mM ammonium

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formate in Milli-Q water (mobile phase A) and 100 % MeOH (mobile phase B). The flow rate

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was maintained at 0.25 mL min-1. The gradient profile started at 90 % A (hold time 1 min), 7



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followed by a linear decrease to 40 % A by 3 min, then to 12 % A by 14 min and finally 0 % by

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14.5 min (hold time 1 min). The mobile phase composition was returned to initial conditions by

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16.5 min and then equilibrated by 21.5 min. The mass spectrometer was operated under selected

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reaction monitoring (SRM) mode, with 2 to 5 transitions per analyte (see Table S1, SI).

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Quality Control

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Prior to analysis of samples, spike/recovery experiments were performed in soil (n = 4), carrot

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(n = 4) and glass filters (n = 4) at a fortification level of 30 ng g-1, and in water (n = 4) at a

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fortification level of 10 ng mL-1. Limits of detection (LODs) and quantification (LOQs) were

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estimated as the concentration producing a signal-to-noise ratio of 3 and 10, respectively (Table

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S4). We also evaluated potential losses from freeze-drying by analyzing soil fortified with target

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analytes with and without a freeze-drying step. Following method validation, ongoing

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assessment of method performance was carried out through the inclusion of blanks and spiked

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samples in every batch.

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Isomer Nomenclature and Identification

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In all cases, isomers were denoted by either ‘L-’ (linear isomer), ‘Br-’ (∑branched isomers), or

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a number denoting the location of the perfluoromethyl branching point (1-, 2-, 3-, etc.).

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Individual PFOS and PFOA isomers could be identified in chromatograms (Figure S1-S2) by

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matching their relative retention times and MS/MS product ions to those reported

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previously.30,31 In the case of EtFOSA (Figure S3), tentative structural assignments were made

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by comparing MS/MS product ions and retention times to that of PFOS. For example, 6-

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EtFOSA and 6-PFOS both produced m/z 169 product ion and eluted closest to their respective

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linear isomers, while 1-EtFOSA and 1-PFOS both eluted between 5- and 6-isomers and

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produced a unique m/z 419 product ion.

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Data Handling and Statistical Analysis

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Concentrations for a single time point in each experimental replicate were based on analysis of

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n = 3 soil or carrot samples or n = 1 sample of composite leachate. Quantification of target

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analytes was performed using an isotope dilution approach, with the exception of FOSAA,

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where matrix-matched calibration approach was performed due to the lack of a homologous

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isotopically labeled standard. Calibration curves (1/x weighting) were prepared from around the

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limit of quantification (LOQ) to 250 ng mL-1 and determination coefficients, R2, were always in

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the range of 0.994-0.998. Individual PFOS and PFOA isomers were determined using isomer-

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specific calibration curves prepared from characterized technical standards (see standards and

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reagents section). For targets where characterized isomeric mixtures were unavailable (i.e. 8


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EtFOSA, FOSA and FOSAA), Σ branched isomers were quantified separately from the linear

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isomer using a linear isomer calibration curve. In this case, the concentration of branched

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isomers should be considered semi-quantitative, owing to differences in response factors

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between branched and linear isomers.

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EtFOSA rate constants were determined by fitting soil concentrations (Csoil) to the equation

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ln(Csoil)= a - kdt, where kd is the apparent depletion rate constant, t is time, and a is a constant

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(See Figure S5). The apparent half-life (t1/2) was calculated by dividing ln(2) by the kd. Since the

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concentrations in replicate pots for a given experiment were not significantly different (p >

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0.05; Analysis of Variance (ANOVA) test), t1/2 was determined for each of the replicates, and

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these were used to calculate an average half-life and pooled standard deviation. Losses not

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accounted for by the internal negative control (e.g. from volatilization or irreversible sorption)

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as well as the potential for novel product formation were monitored by calculating the total

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number of moles in the system at each time point and comparing this to the total number of

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moles at t = 0. Finally, bioaccumulation factors (BAFs) were determined in carrot peel, core and

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leaf as a ratio between the concentration determined in each of the carrot compartment (d.w.)

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and the concentration detected in soil (d.w.).

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RESULTS AND DISCUSSION

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Quality Control

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Spike/recovery experiments involving L-FOSAA, L-FOSA, and isomeric mixtures of EtFOSA,

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PFOS, and PFOA resulted in internal standard-corrected percent recoveries ranging from 76-

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109 % for soil, 49-120 % for carrot, 76-130 % for leaching water and 65-130 % for filters (see

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Table S4), indicating good accuracy of the method. No significant differences (p > 0.05) were

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observed between freeze-dried and non-freeze dried soils, indicating that losses during the

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freeze-drying step were negligible (Figure S6). The internal negative control PFDA, which was

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incubated in all the experiments to monitor losses in situ, was recovered quantitatively from all

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pots and displayed no significant change in concentration over the course of the experiments

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(Figure 1). Monitoring of unfortified soil (Experiment 5) and soil-carrot (Experiment 4)

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mesocosms revealed the occurrence of PFOS PFOA, and FOSA in both soils and leachate. For

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PFOS and FOSA, soil and leachate concentrations in unfortified experiments were always

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