DEGRADATION RATE OF SODIUM FLUOROACETATE IN THREE NEW ZEALAND SOILS GRANT NORTHCOTT,y DWAYNE JENSEN,y LUCIA YING,y and PENNY FISHER*z yPlant & Food Research Ruakura, Hamilton, New Zealand zLandcare Research, Lincoln, New Zealand (Submitted 20 October 2013; Returned for Revision 20 November 2013; Accepted 23 January 2014) Abstract: The degradation rate of sodium fluoroacetate (SFA) was assessed in a laboratory microcosm study incorporating 3 New Zealand soil types under different temperature (5 8C, 10 8C, or 20 8C) and soil moisture (35% or 60% water holding capacity) conditions using guideline 307 from the Organisation for Economic Co-operation and Development. A combination of nonlabeled and radiolabeled 14 C- SFA was added to soil microcosms, with sampling and analysis protocols for soil, soil extracts, and evolved CO 2 established using liquid scintillation counting and liquid chromatography–mass spectrometry. Degradation products of SFA and their rates of formation were similar in the 3 soil types. The major degradation pathway for SFA was through microbial degradation to the hydroxyl metabolite, hydroxyacetic acid, and microbial mineralization to CO 2 , which constituted the major transformation product. Temperature, rather than soil type or moisture content, was the dominant factor affecting the rate of degradation. Soil treatments incubated at 20 8C displayed a more rapid loss of 14 C-SFA residues than lower temperature treatments. The transformation half-life (DT50) of SFA in the 3 soils increased with decreasing temperature, varying from 6 d to 8 d at 20 8C, 10 d to 21 d at 10 8C, and 22 d to 43 d at 5 8C. Environ Toxicol Chem 2014;33:1048–1058. # 2014 SETAC Keywords: 1080 Degradation time Hydroxyacetic acid Pesticide Sodium fluoroacetate Soil INTRODUCTION Sodium fluoroacetate (SFA; FCH 2 CO 2 Na) is registered as a pesticide in New Zealand and Australia, for delivery in bait to control pest mammals, and is commonly referred to as 1080. In New Zealand, aerial or bait station application of cereal pellet or chopped carrot bait containing up to 0.15% SFA by weight is used for broad-scale management of introduced brushtail possums (Trichosurus vulpecula), rodents (Rattus spp.) and, to a lesser extent, rabbits (Oryctolagus cuniculus). After application, bait remaining uneaten on the ground is subject to dissipation and degradation through exposure to rainfall [1]. Exposure to environmental moisture results in water-soluble SFA leaching from bait into litter and soil [2]. Thus, the degradation of SFA and the rate of formation of degradation products in soil are of interest for environmental risk assessment. Potential degradation pathways of SFA in soil include chemical hydrolysis in solution and biotic mechanisms of metabolism or mineralization. Degradation in solution begins with dissociation of the sodium ion followed by hydrolysis of fluoroacetate to produce fluoromethane and bicarbonate. Metabolism of SFA following ingestion by soil organisms occurs via conversion to fluorocitrate within mitochondria [3] or by root uptake of fluoroacetate in solution followed by metabolism by plants. Mineralization of SFA through microbial degradation produces fluoride ion and hydroxyacetic acid (HAA; also termed glycolate/glycolic acid). Previous studies have identified soil bacteria that could utilize fluoroacetate as a sole carbon source through enzymatic cleavage of the carbon– fluorine bond [4–6] as well as the enzymes responsible and their gene coding in specific bacteria [7]. Previous investigations [5,6,8–11] have identified a signifi- cant role of some bacteria and fungi in the degradation of SFA in natural soils. Temperature and moisture content, as regulators of microbial activity, were expected to affect the rate of SFA degradation in New Zealand soils [12]. In 2007, a regulatory reassessment of SFA [13] by the Environmental Risk Manage- ment Authority New Zealand (now Environmental Protection Agency, New Zealand) determined that existing data on the degradation pathways and rates of SFA in soil were limited in scope and applicability, and were not conducted in accordance with international test methods. The present study addressed a recommendation [13] to generate additional data using guideline 307 of the Organisation for Economic Co-operation and Development (OECD) [14] under aerobic test conditions. In addition to the test temperatures of 20 8C and 10 8C stipulated in the test guideline, an additional treatment of 5 8C was included to simulate the New Zealand winter conditions when SFA is most commonly applied. An additional test treatment simulating relatively dry soil conditions was also included. Test soils used needed to represent regions of New Zealand subject to aerial application of SFA bait, and encompass variability in soil organic matter and clay content. MATERIALS AND METHODS Within the protocol described by OECD guideline 307 [14], we used a combination of nonlabeled and radiolabeled SFA to determine the rate of degradation and the nature and rates of formation and decline of transformation products. The guideline recommends that test soil moisture content should be 40% to 60% water-holding capacity (WHC). In the present study we used 60% WHC as 1 treatment variable and also included 35% WHC as another, to address the question of degradation of SFA under drier soil conditions. In addition to the test temperatures of 10 8C and 20 8C specified by OECD guideline 307 [14], an additional 5 8C treatment was incorporated into the experiment to better represent the range of temperatures prevalent in New Zealand, particularly as SFA baits are often applied during winter when soil temperatures are lowest. The assessment was * Address correspondence to [email protected]. Published online 29 January 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/etc.2536 Environmental Toxicology and Chemistry, Vol. 33, No. 5, pp. 1048–1058, 2014 # 2014 SETAC Printed in the USA 1048
Transcript
DEGRADATION RATE OF SODIUM FLUOROACETATE IN THREE NEW ZEALAND SOILS
GRANT NORTHCOTT,y DWAYNE JENSEN,y LUCIA YING,y and PENNY FISHER*zyPlant & Food Research Ruakura, Hamilton, New Zealand
zLandcare Research, Lincoln, New Zealand
(Submitted 20 October 2013; Returned for Revision 20 November 2013; Accepted 23 January 2014)
Abstract: The degradation rate of sodium fluoroacetate (SFA)was assessed in a laboratorymicrocosm study incorporating 3NewZealandsoil types under different temperature (5 8C, 10 8C, or 20 8C) and soil moisture (35% or 60% water holding capacity) conditions usingguideline 307 from the Organisation for Economic Co-operation and Development. A combination of nonlabeled and radiolabeled 14C-SFA was added to soil microcosms, with sampling and analysis protocols for soil, soil extracts, and evolved CO2 established using liquidscintillation counting and liquid chromatography–mass spectrometry. Degradation products of SFA and their rates of formation weresimilar in the 3 soil types. The major degradation pathway for SFA was through microbial degradation to the hydroxyl metabolite,hydroxyacetic acid, and microbial mineralization to CO2, which constituted the major transformation product. Temperature, rather thansoil type or moisture content, was the dominant factor affecting the rate of degradation. Soil treatments incubated at 20 8C displayed amore rapid loss of 14C-SFA residues than lower temperature treatments. The transformation half-life (DT50) of SFA in the 3 soilsincreased with decreasing temperature, varying from 6 d to 8 d at 20 8C, 10 d to 21 d at 10 8C, and 22 d to 43 d at 5 8C. Environ ToxicolChem 2014;33:1048–1058. # 2014 SETAC
Keywords: 1080 Degradation time Hydroxyacetic acid Pesticide Sodium fluoroacetate Soil
INTRODUCTION
Sodium fluoroacetate (SFA; FCH2CO2Na) is registered as apesticide in New Zealand and Australia, for delivery in bait tocontrol pest mammals, and is commonly referred to as 1080. InNew Zealand, aerial or bait station application of cereal pellet orchopped carrot bait containing up to 0.15% SFA by weight isused for broad-scale management of introduced brushtailpossums (Trichosurus vulpecula), rodents (Rattus spp.) and,to a lesser extent, rabbits (Oryctolagus cuniculus). Afterapplication, bait remaining uneaten on the ground is subjectto dissipation and degradation through exposure to rainfall [1].Exposure to environmental moisture results in water-solubleSFA leaching from bait into litter and soil [2]. Thus, thedegradation of SFA and the rate of formation of degradationproducts in soil are of interest for environmental risk assessment.
Potential degradation pathways of SFA in soil includechemical hydrolysis in solution and biotic mechanisms ofmetabolism or mineralization. Degradation in solution beginswith dissociation of the sodium ion followed by hydrolysis offluoroacetate to produce fluoromethane and bicarbonate.Metabolism of SFA following ingestion by soil organismsoccurs via conversion to fluorocitrate within mitochondria [3] orby root uptake of fluoroacetate in solution followed bymetabolism by plants. Mineralization of SFA through microbialdegradation produces fluoride ion and hydroxyacetic acid(HAA; also termed glycolate/glycolic acid). Previous studieshave identified soil bacteria that could utilize fluoroacetate as asole carbon source through enzymatic cleavage of the carbon–fluorine bond [4–6] as well as the enzymes responsible and theirgene coding in specific bacteria [7].
Previous investigations [5,6,8–11] have identified a signifi-cant role of some bacteria and fungi in the degradation of SFA in
natural soils. Temperature and moisture content, as regulators ofmicrobial activity, were expected to affect the rate of SFAdegradation in New Zealand soils [12]. In 2007, a regulatoryreassessment of SFA [13] by the Environmental Risk Manage-ment Authority New Zealand (now Environmental ProtectionAgency, New Zealand) determined that existing data on thedegradation pathways and rates of SFA in soil were limited inscope and applicability, and were not conducted in accordancewith international test methods.
The present study addressed a recommendation [13] togenerate additional data using guideline 307 of the Organisationfor Economic Co-operation and Development (OECD) [14]under aerobic test conditions. In addition to the test temperaturesof 20 8C and 10 8C stipulated in the test guideline, an additionaltreatment of 5 8C was included to simulate the New Zealandwinter conditions when SFA is most commonly applied. Anadditional test treatment simulating relatively dry soil conditionswas also included. Test soils used needed to represent regions ofNew Zealand subject to aerial application of SFA bait, andencompass variability in soil organic matter and clay content.
MATERIALS AND METHODS
Within the protocol described by OECD guideline 307 [14],we used a combination of nonlabeled and radiolabeled SFA todetermine the rate of degradation and the nature and rates offormation and decline of transformation products. The guidelinerecommends that test soil moisture content should be 40% to60% water-holding capacity (WHC). In the present study weused 60% WHC as 1 treatment variable and also included 35%WHC as another, to address the question of degradation of SFAunder drier soil conditions. In addition to the test temperatures of10 8C and 20 8C specified by OECD guideline 307 [14], anadditional 5 8C treatment was incorporated into the experimentto better represent the range of temperatures prevalent in NewZealand, particularly as SFA baits are often applied duringwinter when soil temperatures are lowest. The assessment was
* Address correspondence to [email protected] online 29 January 2014 in Wiley Online Library
(wileyonlinelibrary.com).DOI: 10.1002/etc.2536
Environmental Toxicology and Chemistry, Vol. 33, No. 5, pp. 1048–1058, 2014# 2014 SETAC
Printed in the USA
1048
completed to Good Laboratory Practice standard at Plant & FoodResearch’s Food and Biological Chemistry laboratory betweenMay 2009 and December 2010. More detailed technicaldescription of the test system and analytical methods used inthe assessment are provided by Northcott [15].
Test soil selection, sampling, and preparation
Three test soils were selected using spatial data to identifyrepresentative areas of New Zealand over which SFA bait hasbeen applied aerially and is likely to be applied in future. Thegeographic information system (GIS) data were sourced fromthe VectorNet database (Animal Health Board,Wellington, NewZealand) for aerial bait applications for possum control duringJuly 2007 to February 2008, and from the Department ofConservation (Research Development and Improvement Divi-sion, Christchurch, New Zealand) for aerial application forpossum and rodent control over areas of conservation estate inthe period 2006 to 2007. These GIS data were matched to areasof the dominant New Zealand Soil Classification Soil Orders(National Soil Database, Landcare Research) to produce aranking of soil order by area exposed to aerial bait application.Three orders (brown soils, podzols, and pumice soils)represented the greatest combined area exposed to SFA baitsafter aerial application and themost diversity in terms of regionallocation and variation of physical, chemical, and biologicalcharacteristics. Collection sites (Table 1) were selected using theNational Soils Database (Landcare Research) to identify existingreference sites and provide descriptive information.
Soil from the 3 reference sites was sampled to a maximumdepth of 20 cm. At each site, soil was taken from at least 5locations approximately 10m apart. Large stones, vegetationand leaf litter, visible earthworms, and the upper layer of grassand root material were removed by hand. The collected soil fromeach location was transferred into large plastic bags and looselytied to allow exchange of air. The soil samples were transportedchilled (<8 8C) to the testing laboratory. Clods >3 cm werebroken apart manually, and for each soil type the contents of allsample bags were mixed together by hand, with visible pieces ofplant material and stone removed during mixing. The fullymixed composite soil for each sampling site and soil order weretransferred into loosely tied, labeled plastic bags and stored at5 8C.
Approximately 10 kg of each composite soil sample wassieved to 2mm, and soil dry weight and gravimetric watercontent (GWC) were determined by drying triplicate subsamplesof sieved soil fractions to constant weight at 105 8C. Water-holding capacity was measured using the method of Hoper [16].Maximum WHC was calculated as the corresponding GWCusing the soil dry weight data. The GWC of sieved field-moistsoils was either close to or greater than that required to achieve60% or 35% WHC, necessitating partial air-drying of the soilsbefore readjusting to the required experimental WHC, asdescribed by Northcott [15].
Sterile soil for control treatments was prepared by autoclavingpreweighed portions of soil 3 times. The soil was autoclaved at121 8C (1 bar) for 30min and maintained within the sealed
Table 1. Details of test soil sampling and descriptions
OrderLocation and reference
from database
Global positioning systemcoordinate and date of
sample collection Existing dataa
Physical andchemical
characteristicsb
Podzol Orikaka Sandy Loam, West Coast,South Island
GR L29 2425373 593 4737 59834071.400N Beech–podocarp forest, Profiledescription 0-B3
Medium sand 11%
2 January 2009 Fine sand 9%Silt 13%Clay 54%pH 4.6
Total C 12.6%Total N 0.50%MBC 1362
Brown Soil Matiri, West Coast, South Island 24810075.600E BLA Acid allophanic brown Coarse sand 1%GR L30 2410730 5909418 59809039.200N Upland yellow-brown earth, lowland
podocarp forestMedium sand 6%
8 January 2009 Fine sand 30%Silt 39%Clay 24%pH 3.7
GR U18 2795100 6257100 62857010.700N Mānuka exotic forest, sphagnum Medium sand 9%12 January 2009 Fine sand 18%
Silt 43%Clay 27%pH 5.4
Total C 7.68%Total N 0.33%MBC1014
aHorizon descriptions accessible on New Zealand soils database; search by series name or identity number: http://soils.landcareresearch.co.nz/contents/SoilData_NSD_ReportsFlat.aspx?currentPage¼SoilData_NSD_ReportsFlat&menuItem¼SoilDatab Particle Size Distribution testing of Fine Earth Fraction (New Zealand Classification), andmeasurements of pH, total carbon and nitrogen, andmicrobial biomasscarbon (MBC; mg/kg) undertaken by Landcare Research, Hamilton, New Zealand.
Degradation of sodium fluoroacetate in soil Environ Toxicol Chem 33, 2014 1049
autoclave until it had cooled to 30 8C (�5–6 h). The autoclavecycle was repeated, and the soils were then left to incubate atambient temperature for 24 h to allow surviving bacterial spores togerminate. Following this period of incubation, the soils wereautoclaved at 122 8C (1.35 bar) for 35min. All subsequentmanipulations of the sterilized soils were conducted underlaminar-flow and sterile conditions. The 5 8C, 10 8C, and 20 8Ctreatments were housed in separate temperature-controlled roomsmonitored by calibrated electronic monitors and minimum–
maximum thermometers. Pre-equilibration of all test soils at 5 8C,10 8C, and 20 8Cwas undertaken for aminimumof 4wk,with anygerminating seedlings removed by hand.
Soil spiking procedure
An initial moisture adjustment was used to introduce SFA tosoil as an aqueous spike using a modification of the methoddescribed by Brinch et al. [17], thereby avoiding the potentialalteration of the sorption behavior of SFA in the test soils [18].Soil within each flask was spiked with a mixture of nonlabeledand radiolabeled SFA (fluoroacetic acid, [1-14C] sodium salt)equivalent to 18mg of SFA, which was the nominal quantity in asingle 12-g cereal pellet bait containing 0.15% (w/w) SFA. Theaqueous spike was evenly distributed over quarter portions ofsoil that were subsequently combined with the remainingvolume of soil. Sterile soil treatments were spiked with the sameprocedure but using soil that had been autoclaved within theprevious 2 d and a solution of SFA prepared in sterile MilliQwater (Millipore).
Spiking of soil in the 3 temperature treatments was staggeredto accommodate the sampling schedule. Immediately afterspiking, subsamples (0.5–0.9 g) of soil from each treatment wereweighed into individual paper combustion cones, with 6replicates and 10 replicates taken from each spiked sterile andnonsterile treatment, respectively. These replicate subsampleswere stored at –50 8C to 80 8C until analysis by sample oxidationto confirm the activity and homogeneity of the [14C]-SFAportion of the spikes.
Flow-through incubation flask system
The test system was based on the design specified in OECDguideline 307 [14], and by the Soil Science Society of America[19]. Spiked soil treatments in 250-mL glass Schott bottles wereincubated in flow-through, dark conditions under 3 temperature(5 8C, 10 8C, or 20 8C) and 2 soil moisture (35% or 60% WHC)treatments. Eighteen flasks were incubated at each temperature(n¼ 6 for each soil type). Each group of 6 flasks comprisedduplicate nonsterile and a sterile spiked soil for each of the 35%and 60% WHC treatments. Each flask contained an equivalentdry weight of 105 g of soil, but the total final weight of soilprepared for each varied depending on the adjusted GWCrequired to attain the treatment WHC. Flasks were sealed with ascrew cap containing a Teflon plug and Viton O-ring insert,through which a controlled air flow (5mL/min) was deliveredand then directed through a trap containing a polyurethane foam(PUF) plug, and bubbled into glass scintillation vials containingalkali solution (5mL of 1M NaOH) to trap CO2/
14CO2.
Sample types and sampling intervals
The 10 8C treatments were spiked first, followed by the 20 8Ctreatments 4 d later and the 5 8C treatments 2 d later. The OECDguideline 307 [14] specifies that the study should normally notexceed 120 d, but we used a longer incubation period to ensure acomplete characterization of the decline of SFA concentrationsover time. Sampling and analyses included measurement of
evolved 14CO2 from alkali trap solutions; extraction of volatilePUF plug traps; analysis of soil subsamples for extractableresidual radioactivity, parent SFA, and transformation products;and total residual radioactivity by total oxidation. Air flow to theincubation flasks was temporarily suspended during samplingprocedures.
Subsamples of soil (�10 g) taken during the incubationperiod were stored in sealed capped vials at�20 8C to 50 8C untilanalysis. At soil sampling, each incubation flask was weighed,and any reduction resulting frommoisture loss was compensatedfor by the addition of an appropriate volume of sterile water,which wasmixed into the soil tomaintain the requiredWHC. Forthe 5 8C and 20 8C treatments, incubation time was 135 d, andsoil sampled at 0 d, 3 d, 4 d, 7 d, 10 d, 21 d, 22 d, 35 d, 64 d, and135 d. The 10 8C treatments were incubated for 136 d, and soilwas sampled at 0 d, 8 d, 14 d, 21 d, 28 d, 42 d, 63 d, and 136 d.
Alkali trap solutions were sampled and changed at 1 d, 2 d,3 d, 4 d, 5 d, 7 d, 10 d, 14 d, 21 d, 28 d, 35 d, 42 d, 56 d, 63 d, 70 d,84 d, 98 d, 112 d, and 126 d after spiking, with some minorvariations later in the incubation period (see Results section).The PUF plugs were sampled and changed on days 10, 21, 49,77, and 112 of incubation, and stored in sealed glass vials at–20 8C until analysis.
Sample extraction and analysis
Total oxidation of soil subsamples to determine the totalresidual activity of 14C was carried out using a PerkinElmer 307oxidizer. Quality assurance measures included a control soilcombustion blank, a control soil spiked with a solution of [14C]-toluene (0.02mL of 305Bq/mL) to assess combustion efficien-cy, combustion blanks to assess the carryover of radioactivitybetween samples, and a comparative standard prepared from the[14C]-toluene Spec-Chec solution (PerkinElmer). Soil (0.25–1 g) was weighed into paper combustion cones, 0.1mL ofCombustaid solution (PerkinElmer) was added, and the contentswere capped with a combustion pad. Samples were combustedfor 2.5min. Evolved 14CO2 was trapped and eluted in 10mL ofCarbosorb E (PerkinElmer) and mixed with 10ml of PermafluorE scintillation cocktail (PerkinElmer) in 23-mL vials; then 14Cradioactivity was measured by liquid scintillation counting.
Residues of SFA were extracted from soil subsamples (2.5 g),using amodification of the extractionmethoddescribedbyWrightet al. [20]. Quality assurance samples for each batch of samplesincluded an extraction solution blank; a control soil blank; acontrol soil spiked with the internal standard chloroacetic acid,SFA, and HAA; and a comparative standard prepared bydispensing the same spike compound solutions into a 10-mLvolumetric flask. Approximately 2.5 g of soil was spiked with theinternal standard (chloroacetic acid) and extracted with 5mL ofmagnesium carbonate–saturated aqueous solution by sonicationfor 20min (Bandelin Sonorex Digital 10P), followed by shakingfor 20min at 300 rpm (IKA KS 501). The soil slurry wascentrifuged (Hettich Rotanta 460R) to separate the soil andaqueous extract,whichwasdecanted into a plastic centrifuge tube.The extraction of the soilwas repeated, and the combined aqueousextract was filtered through a glass fiber filter (Labserve, 25-mmdiameter). The concentration of SFA,HAA, and chloroacetic acidin the soil extracts was determined by liquid chromatographymass–spectrometry (LCMS), and total extractable [14]C residuesby liquid scintillation counting.
The LCMS analysis was performed on a ThermoFisherScientific Surveyor liquid chromatography system coupled to anautosampler and heated column compartment. Separations of5-mL injections of aqueous soil extracts were carried out using a
1050 Environ Toxicol Chem 33, 2014 G. Northcott et al.
Zorbax Extend-C18 column (150mm� 2.1mm, 5mm, Agilent)with an isocraticmobile phase consisting of 10%methanol–waterwith tributylamine (5mM)and formicacid (5mM)at aflowrateof0.2mL/min. The column temperature was maintained at 35 8Cthroughout.
Mass spectrometric analysis was completed on a Thermo-Fisher Scientific LCQ Deca-ion trap mass spectrometer systemusing atmospheric pressure chemical ionization in the negativemode. Vaporizer temperature was 250 8C, sheath and auxiliarynitrogen gas flows were 35U and 10U, respectively, and theheated capillary was maintained at 200 8C. Mass spectral datawere acquired between 4min and 15min using selected ionmonitoring (SIM). The compound-specific SIM ions monitoredduring the analysis were 75m/z for HAA, 77m/z for SFA, and93.5m/z for chloroacetic acid.
The alkali trapping solution was mixed with 15mL of UltimaGoldXR scintillation cocktail (PerkinElmer), and the trapped andaccumulated radioactivity was measured by liquid scintillationcounting. Samples were counted for a period of 20min or athreshold of 0.5% uncertainty, whichever was reached sooner.Counting data (counts/min) were quench-corrected to provide thecorresponding activity in disintegrations/min (DPM).The activityof alkali trapping solutions and aqueous sample extracts wascorrected against a quench curve constructed using certified 14Cquench standardsprepared inUltimaGold liquid scintillationfluid(National Institute of Standards and Technology [NIST]).Theactivity of oxidized samples was corrected against a quench curveconstructed from a dilution series of Carbosorb E in Permafluor Escintillationfluid spikedwith a knownamountof 14C radioactivity([14C]-toluene). This in-house–prepared quench curve wasvalidated against certified NIST 14C quench standards.
The PUF plugs were transferred to empty 20-mL polypro-pylene solid-phase extraction tubes mounted in a vacuummanifold, and 5mL of MilliQ water was added. Each plug wassequentially compressed and extended to adsorb and expresswater. This process was repeated 4 times to ensure evensaturation; then the plug was fully compressed and vacuum-applied to collect the aqueous extracts in glass scintillation vials.The extraction process was repeated and combined with the firstextract, and the extracted radioactivity was measured by liquidscintillation counting. Quality assurance samples consisting of asolution blank, control blank PUF plug, PUF plug spiked with1.8 kBq of [14C]-1080, and [14C]-1080 spike comparative wereprepared and extracted with each batch of extracted PUF plugs.
Statistical analysis
Except for the 14CO2 mineralization profiles, all estimateswere calculated on a soil dry weight basis. Statistical analysiswas undertaken using Microsoft Excel 2003 and 2007. Thedecline of SFA concentration in soil with time was describedaccording to first-order kinetics
CðtÞ ¼ C0 expð�ktÞ ð1Þ
whereC0 is the initial concentration of SFA spiked in soil (mg/kgsoil), C(t) is the concentration of SFA in soil (mg/kg soil) at timet, t is the time (d), and k is the degradation rate constant (d). Therate constant k was estimated by fitting Equation 1 to theexperimental data with SigmaPlot for Windows Version 10.0(Release 10.0.1.2.) using the Levenberg–Marquardt algorithm todetermine parameter values. For nonlinear curve fitting, theinitial parameter value for C0 was set to the theoretically appliedamount of SFA initially spiked into the soil. SigmaPlot’sAutomatic Initial Parameter Estimate Functions were used to
estimate parameter k, and the initial concentration was optimizedto obtain best fit to the data, based on the assumption that theinitial concentration was subject to experimental error. Between10 iterations and 27 iterations were performed to derivesolutions. Parameter values together with their standard errorsand the adjusted r2 values of the best-fit solution were obtained.
Half-lives for the degradation of SFA were calculated from kusing the standard equation t1/2¼ ln(0.5)/k, or t1/2¼ 0.6932/k.The 50%, 75%, and 90% disappearance times of SFA (DT50,DT75,and DT90 values) were estimated by substituting the SFAconcentration at time zero (C0), defining C(t) as 0.5�C0 or0.1�C0 , and entering the estimated k for each soil treatmentinto Equation 1 and solving for the time in days (t). Totalrecovered radioactivity obtained from each flow-throughincubation flask was calculated as the sum of [14C]-radioactivityremaining in the soil within the flask at time x, the cumulative[14C]-radioactivity removed in each soil subsample at time x, andthe cumulative radioactivity measured as [14C]-CO2 at time x.Total recovered [14C]-radioactivity for each flask was expressedas a percentage of the total [14C]-SFA radioactivity spiked intothe soil and added to each flask at the initiation of the study andmeasured by total sample oxidation and liquid scintillationcounting of [14C]-radioactivity.
Quality assurance in soil spikes, temperature conditions, andanalytical methods
Mean and standard deviation 14C-radioactivity of sub-sampled spiked soils (DPM [14C]/g) were used to calculatethe percentage relative standard deviation (%RSD) as a measureof spike homogeneity. Mean (� 95% confidence interval [CI]),median, minimum, and maximum %RSD (n¼ 36) were4.8%� 1.2%, 3.8%, 0.6%, and 13.8%, respectively, demon-strating a homogeneous distribution of SFA spikes within andbetween individual soil treatments. Higher %RSD values wereobtained for spikes in the 60% WHC soil treatments andparticularly the Matiri soil type, which maintained a higher levelof clumping after shaking and mixing. Two outlier valuesobtained for spikes in Matiri soil at 60% WHC corresponded tominimum and maximum values of 26 500DPM/g and40 700DPM/g soil. The 95% confidence limit for meanradioactivity across all spiked soil treatments (n¼ 36) was34 600� 800DPM/g soil, further demonstrating the reproduc-ibility of the spiking procedure. The accuracy of the adoptedspiking procedure was determined by comparing the activity of[14C]-SFA in spiked soil (by total sample oxidation) against thatof comparative spike solutions prepared from the same [14C]-SFA solutions used to spike the soil treatments, and reported aspercentage spike recovery. The 95% confidence limit for themean recovery of [14C]-SFA radioactivity and correspondingmedian were 97%� 5% and 95%, respectively (n¼ 34),excluding the 2 outliers identified.
The acceptable temperature range specified by OECDguideline 307 [14] for 20 8C and 10 8C treatments is� 2 8C,and a similar level of precision was applied to the additional 5 8Csoil treatments. Temperatures in the 5 8C and 10 8C treatmentfacilities were maintained within the specified range for theduration of the study. In the 20 8C treatment facility, recordedtemperatures spiked above the accepted range of 20 8C� 2 8C ononly 42 out of a total of 9302 individual temperaturemeasurements when the facility was accessed during verywarm outside conditions. The temperatures in excess of 22 8Cwithin the facility occurred for a period of 10 h on the second tolast day of the study, ranging from 22.01 8C to 22.34 8C;however, the controlled environment was maintained within the
Degradation of sodium fluoroacetate in soil Environ Toxicol Chem 33, 2014 1051
range specified by the OECD guideline for the duration of thetransformation experiment. Mean radioactivity (� 95% CI)measured in blank control soils (n¼ 23) by total soil sampleoxidation was 62DPM� 6DPM, equivalent to the backgroundlevel of radioactivity. The 95% CI for combustion efficiency ofthe total sample oxidizer, as determined by the recovery of [14C]-toluene spiked onto control soil and combusted, was 99� 2%(n¼ 23), and the 95% CI for the mean level of radioactivitycarryover between consecutive combusted samples was0.1� 0.1% (n¼ 23).
The limits of detection for SFA, HAA, and chloroacetic acidin soil extracts analyzed by LCMS were 0.025mg/mL, 0.1mg/mL, and 0.5mg/mL, respectively, equating to limits of detectionof 0.1mg/kg, 0.4mg/kg, and 2.0mg/kg, respectively, on a soildry-weight basis. The relatively higher limits of detection forchloroacetic acid was inconsequential because it was used as theinternal standard at a nominal concentration of 60mg/kg.Nominal concentrations of the SFA and HAA quality assurancerecovery spikes added to control soil were equivalent to 37mg/kg and 50mg/kg. Mean recoveries (� 95% CI) fell within the70% to 110% range of acceptance specified by OECD guideline307 [13], being 104� 1% (n¼ 459) for chloroacetic acid,103� 3% (n¼ 22) for SFA, and 75� 7% (n¼ 22) for HAA.
RESULTS
Mineralization of [14C]-SFA
At 20 8C the fastest rates of SFA mineralization occurredwithin the first 30 d in all soil types. At 133 d the meancumulative percentages of original [14C]-SFA mineralized were77% (Kaingaroa at 60%WHC), 70% (Kaingaroa at 35%WHC),84% (Matiri at 60% WHC), 63% (Matiri at 35% WHC), 60%(Orikaka at 60% WHC), and 70% (Orikaka at 35% WHC).Mineralization occurred in 20 8C sterile controls at relativelyslow rates, reaching cumulative percentage mineralized end-points ranging from 21.8% to 44.4%. At 10 8C the fastest rates ofmineralization occurred within the first 40 d, and by 143 d themean cumulative percentages of original [14C]-SFA mineralizedwere 77% (Kaingaroa at 60% WHC), 64% (Kaingaroa at 35%WHC), 84% (Matiri at 60%WHC), 59% (Matiri at 35%WHC),73% (Orikaka at 60%WHC), and 64% (Orikaka at 35%WHC).Some mineralization occurred in 10 8C sterile controls after thefirst 40 d to total cumulative percentage endpoints ranging from3.2% to 21%. At 5 8C the fastest mineralization rates of SFAoccurred over the first 70 d, and at 135 d the mean cumulativepercentages of original [14C]-SFA mineralized were 66%(Kaingaroa at 60% WHC), 67% (Kaingaroa at 35% WHC),75% (Matiri at 60% WHC), 74% (Matiri at 35% WHC), 83%(Orikaka at 60%WHC), and 80% (Orikaka at 35%WHC). Somemineralization occurred in 5 8C sterile controls after the first40 d, reaching cumulative percentage endpoints ranging from1% to 30%. As an example, Figure 1 shows the percentage ofmineralized [14C]-SFA produced during the incubation ofOrikaka soil at 35% WHC.
[14C]-SFA residues in PUF plugs, soil, and soil extracts andradioactivity mass balance
Analysis of PUF plugs indicated that negligible volatilizationof [14C]-SFA or [14C]-HAA occurred over the course of thestudy. The positioning of the 14C atom at carbon 1 within theSFA molecule corresponds to the carbon atom within thecarboxylic acid functional group. Therefore the absence of 14Cradioactivity in the PUF plugs demonstrates that volatilization of[14C]-SFA or [14C]-HAA did not occur, or was negligible. The
PUF plugs from 20 8C treatments sampled at 10 d of incubation(the period at which greatest mineralization of SFA occurred)showed radioactivity to a maximum of 1300DPM, whichrepresented<1% of the radioactivitymineralized and released as14CO2 over the same 10-d period and <0.03% of the totalradioactivity initially spiked. Consequently, PUF plugs sampledlater in the study were not analyzed for residual radioactivity.
Total [14C]-SFA residues determined by total oxidation of soilsamples declined more rapidly over the first 22 d of incubation at20 8C than at 10 8C or 5 8C (Figure 2). Overall, the steriletreatments retained high percentages of radioactivity over the first20 d of incubation, with some loss becoming apparent at day 20(20 8C treatments) and day 40 (10 8C and 5 8C treatments). Theamount of extractable [14C]-SFA residues from soil exhibited asimilar profile to that of the bound residual soil activity. However,the percentage of extractable radioactivity was less than thecorresponding total [14C]-SFA residues for every soil, moisturecontent, and incubation temperature. This reduction was correlat-ed with the observed decrease in total soil [14C]-SFA residues.Extractability of 14C soil residues was temperature dependent, sothat as temperature decreased, the percentage of extractableresidues remaining at later sampling time points increased. Thiswas demonstrated in the time it took until less than 10% of theoriginal [14C]-SFA radioactivity spiked into the soil remainedextractable, namely, day22 for the 20 8C treatments, day 42 for the10 8C treatments, and day 135 for the 5 8C treatments.
Total [14C]-radioactivity mass balances were calculated forindividual flasks on each occasion they were sampled (totaln¼ 378; Table 2). The mean (95% CI) total recovered 14Cradioactivity was 99%� 1%, with 91% of the 378 mass balanceresults within the range of 90% to 100% specified by Henry [10].Twenty-seven of the remaining 9% (34 results) of individualmass balance calculations provided recoveries within the rangeof 80% to 120%, and 7 (2% of the total number) fell outside thisrange (76%, 132%, 121%, 124%, 128%, 124%, and 74%).While these recoveries fall outside the range recommended inOECD guideline 307 [14], they remain within the range of 60%to 140% recovery considered acceptable for analysis of pesticideresidues by the European Commission [21].
LCMS analysis of soil samples
Figure 2 shows the soil concentrations of SFA measured byLCMS in duplicate nonsterile biometer flasks, with the
Figure 1. Percentage of mineralized sodium fluoroacetate ([14C]-SFA)produced during the incubation of Orikaka soil at 35% water-holdingcapacity (WHC). Maximum and minimum values are marked by smallerversion of symbols corresponding to ^¼ 20 8C treatment, *¼ 10 8Ctreatment, ~¼ 5 8C treatment, ^¼ 20 8C sterile treatment, �¼ 10 8Csterile treatment, and D¼ 5 8C sterile treatment.
1052 Environ Toxicol Chem 33, 2014 G. Northcott et al.
percentage difference in the measured concentration betweenreplicate flasks ranging from 0.06% to 27%, with a mean of5.93%. Higher percentage differences were obtained over timeas the concentration of SFA decreased and the absolutedifference in measured concentration between replicates becamemore significant. The rate of SFA disappearance across all 3 soiltypes and both moisture treatments decreased with decreasingtemperature (Figure 3). There was little apparent difference inthe rate of SFA disappearance rate between all treatmentsincubated at 20 8C. At 10 8C, and further at 5 8C, the rate ofdisappearance of SFA in the Kaingaroa soil at 35% WHC wasless than that at 60% WHC. This difference did not occur in theMatiri and Orikaka soils at 10 8C or 5 8C.
All sterile treatments demonstrated some loss of SFA withincreasing incubation time, the extent of which increased withtemperature. There was an initial period during which SFAconcentration remained relatively constant, and this lag periodincreased with decreasing temperature. The decline in SFAconcentration following the lag period showed a general increasein rate and extent with increasing temperature. The relative
importance of abiotic degradation observed in sterile soiltreatments was assessed by comparing the concentration of SFAbetween corresponding sterile and nonsterile treatments (Ta-ble 3). This showed significantly greater concentrations of SFAin sterile treatments compared with the corresponding nonsteriletreatments over the respective lag periods. At 20 8C and 10 8C,the concentration of SFA remaining in nonsterile treatments afterthe lag period was close to 0 or less than the limits of detection. Incomparison, SFA concentrations in sterile treatments decreasedby an average of 12% over the same period. The percentage ofSFA remaining in the 5 8C nonsterile soils over the lag periodrelative to that in the 5 8C sterile soil treatments (Table 3) variedfrom 0.2% for Kaingaroa soil at 60% WHC to 19% for Matirisoil at 60% WHC, with an average of 13%.
Degradation times for SFA and formation of HAA in soiltreatments
In sterile treatments, SFA degradation did not follow a first-or second-order kinetic decay model; thus half-life and otherdegradation parameters could not be determined with any
Table 2. Summary of [14C]-radioactivity mass balancesa
a Statistical results of the [14C]-radioactivity mass balance for the 18 individual microcosms incubated at specified temperature. Data are expressed as a percentageof the initial [14C]-SFA radioactivity spiked into each soil microcosm, except for the 95% confidence interval, which represents the mean [14C]-SFA radioactivitymass balance calculated for the 18 individual microcosms.SFA¼ sodium fluoroacetate; CI¼ confidence interval.
Figure 2. Residual radioactivity remaining in Orikaka soil 35%water-holding capacity (WHC) treatment as a function of temperature for nonsterile soil and sterilesoil. Sampling occasions for 5 8C and 20 8C treatments correspond to 0 d, 3 d, 7 d, 10 d, 22 d, 35 d, 64 d, and 135 d and sampling occasions for 10 8C treatmentscorrespond to 0 d, 8 d, 14 d, 21 d, 28 d, 42 d, 63 d, and 135 d.
Degradation of sodium fluoroacetate in soil Environ Toxicol Chem 33, 2014 1053
certainty. Fitting the first-order exponential model to SFAdegradation profiles in all nonsterile treatments provided r2
values (Table 4) considerably higher than the minimum requiredvalue of 0.7 specified in OECD guideline 307 [14]. Exponential
decay curves for the 3 soil types are shown in Figure 3. Estimatesof DT50, DT75, and DT90 values for SFA degradation anddisappearance in the soil treatments (Table 4) increased as theincubation temperature decreased from 20 8C to 10 8C, andfurther to 5 8C. There was no overlap between the degradation-time parameters calculated for the 3 incubation temperatures,demonstrating a trend of reducing rate of SFA degradation withdecreasing temperature.
An effect of soil type or moisture content on SFA degradationparameters was not as clear. Half-life and DT50 values in alltreatments incubated at 20 8C were statistically indistinguish-able, with the exception of DT50 values for the Orikaka 60%WHC and Kaingaroa 35% WHC treatments. Similarly, DT90values for the 20 8C treatments were also statistically indistin-guishable with the exception of Orikaka soil incubated at 35 8Cand 60% WHC. However, these differences were very small,ranging from 0.1 d in the DT50 values to 1 d for the DT90 values.At 10 8C, degradation parameters began to exhibit differencesbetween the 3 soil types at the same moisture content, andbetween the 2 moisture levels for some of the same soils(Table 4). This was most obvious in Kaingaroa soil, where thehalf-life, DT50, and DT90 values increased from 12.4 d, 13.6 d,and 42 d in 65% WHC treatments to 18.2 d, 21 d, and 63 d in35% WHC treatments. Matiri soil followed an opposite trend,however, with half-life, DT50 and DT90 values decreasing assoil moisture decreased from 65% to 35%WHC. In Orikaka soil,the half-life, DT50, and DT90 values at 65% and 35% WHCwere statistically indistinguishable from each other, suggestingthat soil moisture content was not a critical factor for degradationof SFA in this soil type. At 5 8C, Kaingaroa soil DT50 and DT90values followed the same trend observed at 10 8C, with half-lifeincreasing as soil moisture decreased. In comparison, the half-life, DT50 and DT90 values in Matiri and Orikaka treatments at5 8C in both 65% WHC and 35% WHC treatments were
Figure 3. Residual concentration of sodium fluoroacetate and first-order decay curve fitting in Kaingaroa soil (first column), Matiri soil (second column), andOrikaka soil (third column) at 35% water-holding capacity (WHC; first row) and 60% WHC (second row).
Table 3. Residual concentrations of sodium fluoroacetate (SFA) in sterileand nonsterile soil treatmentsa
aValues for residual SFA are inmg/kg dryweight of soil. The lag period is thetime over which SFA concentrations remained relatively constant in thesterile soil treatment.ND¼ not detected, below limit of detection; WHC¼water-holdingcapacity.
1054 Environ Toxicol Chem 33, 2014 G. Northcott et al.
statistically indistinguishable. Overall, moisture content wasonly a critical parameter in SFA degradation in the Kaingaroasoil (but not the other 2 soils).
Relatively low, uniform concentrations of HAAwere presentin nonsterile treatments throughout the study (Table 5). The plots(Figure 4) for the formation of HAA residues and theirsubsequent disappearance did not follow a first- or second-order kinetic decay model, so degradation parameters could notbe determined with any certainty. Differences in HAAconcentrations between treatments were not statistically signifi-cant, and there was no consistent trend in the concentration ofHAA within the temperature and moisture treatments of a soiltype, nor between the 3 soil types. Maximum HAA concen-trations (Figure 4) were 7.46mg/kg, 11.48mg/kg, and 15.76mg/kg respectively for the Kaingaroa, Matiri, and Orikaka soil typesat 35% WHC, corresponding to 4.4%, 6.7%, and 9.2% of theoriginal quantity of SFA spiked into the treatments.
DISCUSSION
Measured decreases in SFA concentrations in nonsterile soiltreatments over the same periods indicate that the degradationwas dominated by biological activity, and that abiotic degrada-tion was a minor loss mechanism. We assumed that thesubsequent appearance of mineralized SFA (14CO2) innominally sterile soil samples was the result of incompletesterilization by the autoclaving procedures used.While completesterilization of soil can be achieved by gamma-irradiation, thisprocedure was not available in New Zealand. Other means tominimize bacterial regrowth such as the use of the growthinhibitors NaN3 or HgCl2 were considered but dismissed: NaN3
is toxic, is recognized as a respiratory irritant, and is potentiallyexplosive. The latter risk alone precluded the use of NaN3 in ourexperiments as subsamples of soil were subjected to total sampleoxidation in an oxygen-rich high-temperature atmosphere. Theconcentration of HgCl2 recommended to inhibit microbialactivity in soil (500mg/kg dry soil [22]) would have resulted in
the addition of 130 ppm Cl– to sterile soil treatments.Uncertainties regarding the potential for this mass of Cl– toalter the properties of sterile soil and introduce unforeseenartefacts were such that HgCl2 was not used. The lower rates ofmineralization measured in the 5 8C and 10 8C sterile controlswere consistent with reduced biological activity at lowertemperatures, and an increasing lag period for residual bacterialand fungal spores to re-establish sufficient activity to metabolize
Table 4. Model-estimated parameters for the degradation of sodium fluoroacetate (SFA) across all soil treatmentsa
a Values are the mean� the absolute standard error calculated from the standard error of estimated values for k, the degradation rate constant derived from the first-order kinetic model C(t)¼C0 exp(�kt).WHC¼water-holding capacity.
Table 5. Concentration of hydroxyacetic acid in nonsterile soil treatmentsa
aMinimum, maximum, median, and mean concentrations of hydroxyaceticacid in mg/kg dry weight of soil calculated from data obtained from bothtreatment replicates and all sampling times.ND¼ not detected, below limit of detection; WHC¼water-holdingcapacity.
Degradation of sodium fluoroacetate in soil Environ Toxicol Chem 33, 2014 1055
SFA. Other studies of the degradation of SFA in soil [6,11] havealso measured defluorination in soil treatments that weresterilized by autoclaving, concluding that this occurred in theabsence of microorganisms. In any future similar studies, itwould be useful to confirm whether microorganisms werepresent in nominally sterile soil treatments, for example, byposthoc culturing, providing a carbon source and measuringoxygen consumption in the soil or by measuring the effect ofadding nitrogen or phosphorus on the rate of SFA loss in sterilesoils.
While identification of the specific soil microorganismsresponsible for the degradation of SFA was beyond the scope ofthe present study, previous research has identified various soilbacteria and fungi, including Penicillium, Pseudomonas,Fusarium, and Micromonospora species, which are capable ofdefluorinating SFA [8,11]. In the New Zealand context,Pseudomonas and Fusarium species with defluorinating abilityhave been described [23], and Henry [10] described a species ofPenicillium isolated from leaves of broadleaf (Griselinialittoralis) that degraded fluoroacetate. In a study of NewZealand soils, Walker [24] described microbes capable ofutilizing fluoroacetate as a sole source of carbon, and others thatdisplayed defluorinating activity given a supplementary carbonsource for growth. Our data demonstrate that microorganismspresent in the 3 New Zealand soil types tested were able totransform and degrade residues of SFA under a range of climaticconditions.
Our data clearly show a primary influence of soil temperatureon the rate of SFA degradation, with reduced degradation rates atlower temperatures in all soil types. It is assumed that activitiesof similar microbial populations at increasing temperatures wereresponsible for differential degradation of SFA, rather than aneffect of temperature influencing the bioavailability of SFA. Ourfindings are in agreement with previously reported [12] DT50
estimates of 10 d at 23 8C, 30 d at 10 8C, and 80 d at 5 8C for SFAin a New Zealand sandy loam soil. Our experimental assess-ments yielded shorter degradation-time estimates for SFA in soilover a similar temperature range, withDT50 values ranging from6 d to 8 d at 20 8C, 10 d to 21 d at 10 8C, and 22 d to 43 d at 5 8C. Itis difficult to identify reasons for the differences in DT50estimates from the present study and those identified in Parfittet al. [12] because of the brevity of supporting informationprovided in Parfitt et al. [12]. Possible explanations includedifferences in the chemical and physical properties of the testsoils, the means by which the soils were treated and prepared,and stability of conditions during incubation of soils. Oneparticular difference between the 2 studies is that of moisturecontent/WHC, as described below.
The effect of soil moisture varied for the 3 New Zealand soilstested in the present study. In Kaingaroa pumice soil, thedegradation rate decreased as soil moisture decreased from 60%to 35%WHC. In Matiri brown soil, the rate of SFA degradationat 10 8C increased as soil moisture decreased from 60% to 35%WHC, but at 5 8C there was no statistical difference in thedegradation rate at both moisture levels. In the Orikaka podzolsoil, the degradation rate was independent of soil moisture at alltemperatures. The degradation parameters derived for SFA inNew Zealand soils in our experiment are lower (i.e., indicatefaster rates of degradation) than those reported by Parfitt et al.[12], where there were significant differences between the decaycurves obtained for SFA in a Conroy sandy loam incubated at21 8C as the gravimetric moisture content increased from 9%, to20%, and 36%. At the 36% moisture content, SFA was notdetectable in the soil at approximately 60 d, and had decreasedby approximately one-third at 35 d. By 35 d the SFAconcentration in soil with 20% moisture content had decreasedby approximately half, but at the much drier moisture content of9% there was no appreciable decrease in the SFA concentration
Figure 4. Formation of hydroxyacetic acid (HAA) in Kaingaroa soil (first column), Matiri soil (second column), and Orikaka soil (third column) at 35% water-holding capacity (WHC; first row) and 60%WHC (second row).
1056 Environ Toxicol Chem 33, 2014 G. Northcott et al.
at 35 d. It is not valid to directly compare the %WHC conditionsused in our experiment with the gravimetric moisture contenttreatments used by Parfitt et al. [12], which were at overall driertest soil conditions; our estimate is that the 9% gravimetricmoisture content used by Parfitt et al. [12] approximated 9% to17%WHC, and the 36% gravimetric content approximated 30%to 40% WHC.
The LCMS data confirmed that the disappearance of SFA inthe soil treatments was through transformation to HAA. Therelatively small quantities of HAA retained by the soils by theend of the incubation period indicated that this degradationproduct was itself readily utilized by soil microbial metabolismand was transformed and/or degraded further. This wasevidenced by data for total residual soil radioactivity andextractable radioactivity, which demonstrated persistent residualradioactivity in the incubated soils that LCMS analysesconfirmed was neither SFA or HAA. A proportion of thisresidual radioactivity therefore represented some furtherdegradation product(s) of HAA incorporated into the soilmicrobial cellular biomass and humic components. The OECDguideline 307 [14] defines a major transformation product as anyproduct representing�10% of the applied dose of the test articleat any time during the study. While HAA was identified as aprimary degradation product of SFA in our experiment, it did notconstitute a major transformation product under the conditionsof the present study.
Use of the procedures outlined by OECD guideline 307meant that larger organisms were excluded from test soils; thusour estimated degradation rates did not account for theirpotential contribution to degradation of SFA through metabo-lism to fluorocitrate. This transformation pathway may havebeen occurring through microorganisms present in the test soilsbut was not measured. This may have contributed to the smallproportions of extracted but unidentified, or unextracted 14Cresidues in the experimental soils. If fluoromethane was a majordegradation product of SFA in our test system, it would not havebeen fully retained by the volatile PUF trap or alkali trappingsolution, and this would have been reflected in a shortfall of theradioactivity mass balance. If the role of these other SFAdegradation pathways in soil was of concern, additional researchwould be required to establish the rate and extent of theformation of smaller fractions of other potential degradationproducts.
The data reported here are pertinent to regulatory environ-mental risk assessments where bait containing SFA is used forvertebrate pest management. In this context we note that SFA ishighly soluble in water [25], where the sodium–oxygen bonddissociates, leaving fluoroacetate in solution. In natural environ-ments, the role of dilution and movement with water in theenvironmental distribution and persistence of SFA is highlightedby other studies on leaching in soil [26], fate in groundwater[27], and toxicity to soil organisms [28]. Our findings haverelevance to Australian contexts where SFA is used as avertebrate pesticide and where fluoroacetate occurs naturally insome native vegetation [29] but are particularly applicable toNew Zealand as the world’s major user of SFA and wheresignificant community concerns exist about the environmentaleffects of broadcast bait application [30]. These data are ofpotential utility in modeling approaches to describing theenvironmental fate of SFA in New Zealand soils and climaticconditions [2]. While the SFA degradation data fitted a first-order decay model as required by the OECD guideline 307 [14],some of the mineralization kinetic data did not conform well tostandard first-order model functions. Further analysis will
explore second-order model fitting and the derivation of kineticparameters, particularly the rate constants for mineralization ofSFA and possible correlations with soil type and moisturecontent.
CONCLUSION
Degradation products of SFA and their rates of formationwere similar in the 3 New Zealand soils tested. In all 3 soils themajor degradation pathway was microbial metabolism, princi-pally through transformation to HAA. The relatively smallquantities of HAA retained by the soils during the course of andat the conclusion of the incubation period indicate that thisdegradation product is itself readily utilized by soil microbialmetabolism. Temperature, rather than soil type or moisturecontent, was the most important factor influencing the rate ofSFA degradation, with the rate of degradation slowing astemperature declined, but still occurring at 5 8C. In some soiltypes, soil moisture content may also influence the rate of SFAdegradation, with overall rates being slower under drier andcolder conditions.
Acknowledgment—The research conducted in the present study wascontracted by the Animal Health Board and Department of Conservation,New Zealand. Thanks to D. McNaughton of Plant & Food Research forassistance preparing, monitoring, and auditing the Good Laboratory Practicestudy, and K. Mueller for modeling with Sigma Plot. Thanks to LandcareResearch staff J. Barringer, I. Lynn, S. Brown, and D. Thornburrow forscientific and technical support in soil identification and sampling. C. Bezarassisted with editing, and comments from P. Cowan and P. Livingstoneimproved earlier drafts of this manuscript. The authors have no conflicts ofinterest to declare with respect to this research.
2. SrinivasanMS, Suren A,Wech J, Schmidt J. 2011. Investigating the fateof sodium monofluoroacetate during rain events using modelling andfield studies. New Zeal J Mar Freshw Res 46:167–178.
3. Savarie PJ. 1984. Toxic characteristics of fluorocitrate, the toxicmetabolite of compound 1080. Proceedings, Eleventh Vertebrate PestConference, Sacramento, CA, USA, March 6-8, 1984. University ofCalifornia, Davis, CA, USA, pp 132–137.
4. Goldman P, Milne GW. 1966. Carbon–fluorine bond cleavage II.Studies on the mechanism of the defluorination of fluoroacetate. J BiolChem 23:5557–5559.
5. Horiuchi N. 1962. Breakage of the C–F bond in monofluoroacetate bysoil microbes. Seikagagu 34:92–98.
6. Twigg LE, Socha LV. 2001. Defluorination of sodium monofluor-oacetate by soil microorganisms from central Australia. Soil BiolBiochem 33:227–234.
7. Kurihara T, Yamauchi T, Ichiyama S, Takahata H, Esaki N. 2003.Purification, characterization, and gene cloning of a novel fluoroacetatedehalogenase from Burkholderia sp FA1. J Mol Catal B-Enzym 23:347–355.
8. KingDR, KirkpatrickWE,WongDH,Kinnear JE. 1994. Degradation of1080 in Australian soils. In Seawright AA, Eason CT, eds, Proceedingsof the Science Workshop on 1080. Miscellaneous Series 28. The RoyalSociety of New Zealand, Christchurch, New Zealand, pp 45–49.
9. Walker JRL, Bong CL. 1981. Metabolism of fluoroacetate by a soilPseudomonas sp. and Fusarium solani. Soil Biol Biochem 13:231–235.
10. Henry SJ. 1984. Biodegradation of sodium monofluoroacetate (“Com-pound 1080”). BSc Hons thesis. Botany Department, University ofCanterbury, Canterbury, New Zealand.
11. Gentle M, Cother E. 2014. Biodegradation of 1080: Testing soils insouth-eastern Australia for sodium fluoroacetate-degrading micro-organisms. Ecol Manage Restor. 15:52–57.
12. Parfitt RL, Eason CT, Morgan AJ, Wright GR, Burke CM. 1994. Thefate of sodium monofluoroacetate (1080) in soil and water. In SeawrightAA, Eason CT, eds, Proceedings of the Science Workshop on 1080.Miscellaneous Series 28. The Royal Society of New Zealand,Christchurch, New Zealand, pp 59–66.
Degradation of sodium fluoroacetate in soil Environ Toxicol Chem 33, 2014 1057
13. Environmental Risk Management Authority. 2007. Environmental RiskManagement Authority decision. Application for the reassessment of ahazardous substance under section 63 of the Hazardous Substances andNew Organisms Act 1996. Application HRE05002. [cited 2009September 22]. Available from: http://www.ermanz.govt.nz/newse-vents/1080/Decision%20_2007.08.10_%20FINAL.pdf.
14. Organisation for Economic Co-operation and Development. 2002. TestNo. 307: Aerobic and anaerobic transformation in soil. OECDGuidelines for the Testing of Chemicals. Paris, France.
15. Northcott GL. 2010. Aerobic transformation of monosodium fluoroa-cetate in soil: A GLP study. SPTS 4937, PFR client report 30865, PFRcontract 22439. The New Zealand Institute for Plant & Food Research,Ruakura, New Zealand.
16. Hoper H. 2006. Substrate-induced respiration. In Bloem J, Hopkins DW,Benedetti A, eds, Microbiological Methods for Assessing Soil Quality.CAB International, Wallingford, UK, pp 84–92.
17. Brinch UC, Ekelund F, Jacobsen CS. 2002. Method for spiking soilsamples with organic compounds. Appl Environ Microb 68:1808–1816.
18. Northcott GL, Jones KC. 2000. Spiking hydrophobic organiccompounds into soil and sediment: A review and critique of adoptedprocedures. Environ Toxicol Chem 19:2418–2430.
19. Wolf DC, Legg JO, Boutton TW. 1994. Isotopicmethods for the study ofsoil organic matter dynamics. In Bottomley PS, Angle JS, Weaver RW,eds,Methods of Soil Analysis Part 2: Microbiological and BiochemicalProperties. Soil Science Society of America, Madison, WI, USA, pp865–906.
20. Wright GR, Booth LH, Morriss GA, Potts MD, Brown L, Eason CT.2002. Assessing potential environmental contamination from compound1080 (sodium monofluoroacetate) in bait dust during possum controloperations. New Zeal J Agr Res 45:57–65.
21. European Commission. 1997. Quality control procedures for pesticideresidue analysis. InGuidelines for Residues Monitoring in the EuropeanUnion, Document 7826/VI/97. Brussels, Belgium, pp 1–25.
22. Wolf DC, Skipper HD. 1994. Soil sterilization. In Bottomley PS, AngleJS, Weaver RW, eds,Methods of Soil Analysis Part 2: Microbiologicaland Biochemical Properties. Soil Science Society of America, Madison,WI, USA, pp 41–51.
23. Bong CL, Cole ALJ, Walker JRL, Peters JA. 1979. Effect of sodiumfluoroacetate (“Compound 1080”) on the soil microflora. Soil BiolBiochem 11:13–18.
24. Walker JRL. 1994. Degradation of sodium monofluoroacetate by soilmicro-organisms. In Seawright AA, Eason CT, eds, Proceedings of theScienceWorkshop on 1080. Miscellaneous Series 28. The Royal Societyof New Zealand, Christchurch, New Zealand, pp 50–53.
25. Pelfrene AF. 2001. Rodenticides. In Krieger R, ed, Handbook ofPesticide Toxicology, 2nd ed, Vol II—Agents. Academic, San Diego,CA, USA, pp 1794–1836.
26. Parfitt RL, Eason CT, Hoff H, Heng LK. 1995. Sodium monofluor-oacetate (1080) leaching through soils. Bull Environ Contam Toxicol55:162–169.
27. Bowman RG. 1999. Fate of sodium monofluroacetate (1080) followingdisposal of pest bait to a landfill. New Zeal J Ecol 23:193–197.
28. O’Halloran K, Jones D, Booth L, Fisher P. 2005. Ecotoxicity of 1080 tosoil organisms. Environ Toxicol Chem 24:1211–1218.
29. Twigg LE, King DR, Bowen LH, Wright GR, Eason CT. 1996.Fluoroacetate content of some species of toxic Australian plantgenus, Gastrolobium, and its environmental persistence. Nat Toxins4:122–127.
30. Green W, Rohan M. 2011. Opposition to aerial 1080 poisoning forcontrol of invasive mammals in New Zealand: Risk perceptions andagency responses. J Roy Soc N Z 41:1–29.
1058 Environ Toxicol Chem 33, 2014 G. Northcott et al.
