De¯uorination of sodium mono¯uoroacetate by soil microorganisms from
central Australia
L.E. Twigg*, L.V. Socha
Scienti®c Services Division, Parks & Wildlife Commission, Northern Territory, P.O. Box 1046, Alice Springs, NT 0871, Australia Received 13 October 1999; received in revised form 11 April 2000; accepted 22 June 2000
Abstract
Sodium mono¯uoroacetate (1080) is a commonly used vertebrate pesticide throughout Australia and New Zealand. However, little is known about the persistence of 1080 in arid environments, or whether soil microorganisms capable of de¯uorinating 1080 are present in soils from arid Australia. Soil samples (3 replicates) from central Australia were collected on seven occasions over an 8-month period, and the microorganisms capable of de¯uorinating 1080 were isolated. When grown in an inorganic medium containing 20 mM 1080 as the sole C source, 24 species were able to de¯uorinate 1080: 13 bacteria and 11 fungi. The abundance of these microorganisms appeared to be in¯uenced by climatic conditions with the relative abundance of many species increasing after rain. The fungusFusarium oxysporum had by far the greatest de¯uorinating ability, and de¯uorinated approximately 45% of added 1080 within 12 d. De¯uorination of 1080 added to soil was signi®cantly greater at pH 5.6 compared to pH 6.8, suggesting that the fungal species were important de¯uorinators in these soils. In a 28-d time course trial, de¯uorination of added 1080 by soil microorganisms appeared to asymptote after 21±28 d. The presence of these microorganisms in soil from central Australia indicates that 1080 can be used safely even in arid environments. 1080 is unlikely to persist in these soils, or to contaminate ground water. The implications of these ®ndings with respect to the environmental safety of 1080 in other regions where 1080 baits are used are also discussed.q2001 Elsevier Science Ltd. All rights reserved.
Keywords: Microbial de¯uorination; Pest control; Sodium mono¯uoroacetate; Soil persistence
1. Introduction
Sodium mono¯uoroacetate (Compound 1080) is highly toxic to most endothermic vertebrates and many inverte-brates except where individual species have had evolution-ary exposure to naturally occurring ¯uoroacetate-bearing vegetation (Twigg and King, 1991). Most of the plants in Australia which produce ¯uoroacetate belong to a single
genus (34 species ofGastrolobium), and most are con®ned
to the southwest of Western Australia although three species do occur in parts of northern and central Australia (two
species of Gastrolobium plus Acacia georginae; Aplin,
1971; Oelrichs and McEwan, 1961; Twigg and King, 1991). 1080 poison is also an important vertebrate pesticide
in Australia where, under strict guidelines, 1080
impregnated baits are commonly used for controlling
rabbits (Oryctolagus cuniculus), foxes (Vulpes vulpes) and
dingoes (Canis familiaris dingo) (Thomson, 1986; McIlroy
et al., 1988; Saunders et al., 1995; Williams et al., 1995). Because 1080 is highly water soluble and readily leached from baits (Wheeler and Oliver, 1978; McIlroy et al., 1988), there has been some concern regarding the persistence of the ¯uoroacetate entering the environment both from the toxic baits, and from ¯uoroacetate-bearing plants (Par®t et al., 1994; Walker 1994; Twigg et al., 1996). However, this concern has not been realised as 1080 does not persist in soil or waterways at least, in areas with ¯uoroacetate-bear-ing vegetation in southwestern Western Australia (Twigg et
al., 1996). In fact several genera of soil fungi (e.g.
Fusar-ium,Penicillium) and bacteria (e.g.Pseudomonas,Bacillus) from these soils are now known to degrade 1080 (Wong et al., 1992). While some of these microorganisms are ubiqui-tous and commonly occur in a variety of moist soils (Kelly, 1965; Bong et al., 1979), little is known about the ability of soil microorganisms from arid and semi-arid regions to degrade 1080.
Here we report on the ability of soil microorganisms from arid central Australia to de¯uorinate 1080. We also examine the relative abundance of these microorganisms both before and after rainfall events, and make some comments as to the likely persistence of 1080 in this environment.
0038-0717/01/$ - see front matterq2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 1 3 4 - 6
www.elsevier.com/locate/soilbio
* Corresponding author. Present Address: Vertebrate Pest Research Services, Agriculture Western Australia, Bougainvillea Avenue, Forrest-®eld, WA 6058 Australia. Tel.:161-8-9366-2330; fax:161-8-9366-2342.
2. Materials and methods
2.1. Study area and collection of soil samples
Soil samples were collected from Palm Paddock within
the Finke Gorge National Park (248100S; 1328500E) which
is approximately 150 km west of Alice Springs. To the best of our knowledge, none of the soils tested had any known previous exposure to 1080. Soil substrates in this region are generally calcerous stony rises with some red clays/sandy loam's in the low lying areas, but they also include areas of
sandstone. The dominant vegetation is hummock (Triodia)/
Acacia grasslands. Landforms include undulating plains,
steep hillsides, dissected plateaus, watercourses, and the Finke river (dominant feature). The ®ne red sandy loam soil within Palm Paddock has a 20% crust of cryptogam
(micro¯ora). Mean soil pH was 6.6 (SEM 0.12, n3:
Average annual rainfall, which can be highly variable, is 260 mm (monthly maxima range from 0±496 mm). Temperatures can be extreme with recorded maxima of
108C in July (mean, 208C) to over 448C in January
(mean, 378C) (Bureau of Meteorology Records, Northern
Territory).
Our current study was part of a larger investigation exam-ining the longevity of 1080 meat baits in central Australia (Twigg et al., 2000). To examine what micro¯ora were present in the soil in Palm Paddock where these trials were carried out, and to determine temporal variation in the relative abundance of these microorganisms, 30±40 g soil samples (1±8 cm depth) were collected from undis-turbed soil at permanently marked locations over a 32 week period commencing in March 1998. There were 7 sampling periods: Day 0, then 0.5, 1, 2, 4, 6 and 8 months after the 1080 baits were placed into the predator-proof cages. The ®ve cages used in the longevity trials were in a circular pattern with approximately 20 m between each cage (i.e. soil collection site). The soil samples were collected within 1 m of three of these cages. Thus there were three replicate soil collections for each sample period. All soil samples were placed into individual resealable plastic
containers and kept at 78C until analysis. Rainfall, and
ambient and soil temperature (depth 5 cm), were monitored daily at the site using a ENVIRODATA AUSTRALIA EASIDATA data logger.
2.2. De¯uorinating activity of soil microorganisms
Methods used for determining the de¯uorinating activity of soil microorganisms were similar to those described by Wong et al. (1992). All water was deionised and autoclaved
at 1218C and 15 kPa for 15 min. To avoid heat degradation,
the 1080 solution was sterilised using a 0.22 um Millipore ®lter membrane. An enriched, autoclaved broth containing
2 g l21KH2PO4, and 1 g l21(NH4)2SO4adjusted to pH 6.8
with a few drops of 0.1 M NaOH was used for the bacterial
incubations. For fungi, the broth contained traces of CaCl2
(0.2 mg l21) and FeSO47H2O (10 mg l21), and was adjusted
to pH 5.6 with a few drops of 0.1 M NaOH. Ten ml aliquots of these broths were dispensed into sterile 120 ml
polycar-bonate bottles. After cooling to 508C, 20 mM of 1080 and
1 g of air-dried soil were added to each bottle. The bottles
were incubated at 278C on an orbital shaker (180 rev.
min21). There was one bottle per replicate with three
repli-cates per soil collection period. After 12 d incubation, the
concentration of F2 in the culture broths was determined
using an Orion ¯uoride electrode (model 94-09-00), an Orion EA 940 expandable ion analyser, an Orion single junction reference electrode 90-01 and an Orion automatic temperature compensation probe.
A time-course experiment was used to determine the de¯uorination activity of the microorganisms present in the soil samples. One ml of 20 mM 1080 was added to 5 g of soil (®nal moisture content about 15% w/w) in 120 ml sterile polycarbonate bottles, and the bottles incubated at
288C. (12 h day) and 158C (12 h night). Five bottles were
established for the soil from each collection period for each site and the amount of de¯uorination of 1080 was measured at 7 d intervals from 0 to 28 d (i.e. 5
time-course periods£3 sites£7 soil collection periods, n
105:At each time- course collection period, one of the
bottles was removed from the incubator, 10 ml of sterile
water was added, and after 30 min, the F2concentration
measured using the F2electrode (see above).
Background levels of F2in each soil type, and water used
during the trials was determined by mixing 5 g soil in 10 ml deionised water. This mixture was allowed to stand for 30 min in polycarbonate bottles and the concentration of
F2was then measured using the F2electrode. Background
levels for the 1080 solution were also determined using the
F2electrode. Because ¯uoride ions can bind to soil particles
(Barrow and Shaw, 1977), the recovery of added F2from
both 1 g and 5 g sterile soil samples was also determined.
The amount of F2 binding to the soil was measured by
adding a known amount of F2to 5 or 1 g of sterile soil in
a known amount of the standard fungi culture broth without 1080. This was then allowed to stand for 24 h. Deionised water (10 ml) was added to the 5 g sample containers, the containers allowed to stand for 30 min, and the recovery rate
of added F2 determined using the F2electrode. This was
compared to the measurement of F2 in solutions with
identical F2 concentrations without soil. There were two
replicates for each soil type. This approach simulated the two main incubation methods used. All trials were corrected for background levels before determining de¯uorination rates.
2.3. Isolation of 1080 microorganisms
After 12 d incubation, for bacteria, a 100-fold dilution using deionised water was made from each of the enriched culture broths and then plated onto nutrient agar (NA). Enriched culture broths for fungi were plated undiluted L.E. Twigg, L.V. Socha / Soil Biology & Biochemistry 33 (2001) 227±234
onto potato-dextrose agar (PDA). NA plates were incubated
at 308C and PDA plates at 258C. Single colonies of different
microbial species were subcultured onto NA or PDA and subsequently identi®ed. Bacterial colonies were identi®ed to genera or species using the Analytical Pro®le Index (API) strips (bioMerieus sa) and associated software (APILAB). Fungal colonies were identi®ed using known morphological characteristics described by Raper and Fennell (1965), Booth (1971), Pitt (1979), and Burgess et al. (1988).
2.4. De¯uorinating activity of microbial isolates
The de¯uorinating ability of each isolate was determined in the presence of 20 mM 1080 with trace elements
(bacteria: 2 g l21 KH2PO4, and 1 g l2
1
(NH4)2SO4 at pH
6.8; fungi: 0.2 mg l21 CaCl2 and 10 mg l21 FeSO47H2O
adjusted to pH 5.6), and with and without 5 g of sterile
soil. Bacterial suspensions (1.5£109cells ml21) were
prepared in sterile 0.85% NaCl w/v for the 1080 only inoculum, and in a 20 mM 1080 solution for the sterile soil inoculum. Fungal suspensions were prepared by scrap-ing off aerial mycelium from 48 h-old cultures into 5 ml sterile 0.85% NaCl w/v and sterile 20 mM 1080. As appro-priate, the samples were inoculated with either 1 ml of inoculum to 10 ml of sterile 20 mM 1080 solution or 1 ml inoculum to 5 g of sterile soil. There were two independent broth cultures for each isolate for each soil treatment.
The broths were kept at 278C for 12 d in sterile 120 ml
polycarbonate bottles.
2.5. Statistical analysis
Statistical analyses were undertaken using Statistica (StatSoft 1994). The decay curve for added 1080 was deter-mined using nonlinear regression. The effect of pH and time on the de¯uorination of 1080 by soil was assessed using a ®xed effects ANOVA with the individual cage locations
n3 acting as a blocking factor (Winer et al., 1991).
Differences in the de¯uorination ability between microbial isolates were tested using log-transformed de¯uorination rates, and a ®xed effects ANOVA with the Tukey HSD
post-hoc test (Winer et al., 1991). Data forStreptomyctes
sp. 1 (Actinomycetes) were presented separately, and were
exclude from the `bacteria' category during the analyses, because this species had marked differences in their isola-tion and growth requirements (e.g. aerial mycelium) compared to that of the other bacteria.
3. Results
3.1. Recovery of added F2
Little F2was found to bind to the soil. Mean recovery rates
for 1.25±5.0 and 10.0±40.0mg of added F2were 95.5^0.8
% (SEM, n3; and 98.5^4.4% (SEM, n4;
respectively. Little free F2 occurred naturally in the soil
(1.14mg g21,n4;deionised water (0.16mg ml
21
,n5
or the 20 mM 1080 solution (2.5mg ml21,n5:Because
of the high recovery rates of added F2, our data are
presented as unadjusted values. The recovery of F2 ion
from known amounts of 1080 as determined by the amount of inorganic ¯uoride subsequently released following degra-dation by oxygen combustion can range from 90±97.5% (Peters and Baxter, 1974). However, for our purposes, we assumed that all added 1080 could be de¯uorinated such
that 1 ml of 20 mM 1080 would yield 380mg of F2. This
value was used for all the percentage calculations of the amount of added 1080 de¯uorinated.
3.2. De¯uorination ability of soil
Soil samples from central Australia de¯uorinated 23% of added 1080 within 28 d; however, de¯uorinating activity of these soils appeared to asymptote after 21±28 d (Fig. 1a).
The model decay curve used was: mg F2d21g
soil21A1B1£time1B2£time2, where time is in
days, and A (0.928), B1 (1.050) and B2 (20.017) are
nonlinear regression constants n105; r0:654
However, soil samples appeared to have greater de¯uorinat-ing ability after signi®cant rainfall events, as the highest rates were observed for the November, May and March soil samples and these collection periods were preceded by moderate rainfall (Fig. 1b; Table 1).
De¯uorination by soil samples (1 g) incubated in the enriched medium with added 1080 (Fig. 2) was greater at
pH 5.6 than at pH 6.8 F19:27;df1, 26,P0:0002:
The ability of soil to de¯uorinate 1080 was also in¯uenced
by time of year F3:42;df6, 26,P0:013with the
highest de¯uorination occurring in early November after 54 mm of rainfall (Table 1). The interaction was also
signif-icant F2:78; df6, 26, P0:032 indicating the
effects of pH varied between time periods. The pH of the
soil at the ®eld sites was 6.6 n3: Approximately
223 mm of rainfall occurred during the trial, with a weekly mean of 6.95 mm (range 0±60 mm). Rainfall was greatest in early April and early November, and the highest tempera-tures occurred during November to March (Table 1).
3.3. Relative abundance of soil microorganisms
Twenty-four species of microorganisms capable of de¯uorinating 1080 were isolated from the central Austra-lian soil. Microorganisms were least abundant during, or
following, periods of low rainfall (Table 1).Fusariumwas
the most abundant fungi, with species of this genus present in most months. The presence and abundance of bacteria was more varied with some species totally absent for conse-cutive collection periods. Two species of bacteria were not identi®ed (Table 1).
3.4. De¯uorination by microbial isolates
1080 was their sole source of carbon (Fig. 3a). In the absence of soil, the fungal isolates had greater
de¯uorinat-ing ability than the bacteria F5:09; df1, 44, P
0:03: Within the fungi, de¯uorination by F. oxysporum
was greater than for any other species F9:30;df10,
11,P,0:001;but de¯uorination by bacterial species was
similar F5:09; df11, 12, P0:10: However, the
amount of 1080 de¯uorinated increased considerably when isolates were provided with an additional carbon source in the form of added sterile soil (Fig. 3b). The fungal isolates again had greater de¯uorinating ability than the
bacteria F10:03; df1, 44, P,0:003; with F.
oxysporumbetter than all other fungal species F21:48;
df10, 11, P,0:001: However, de¯uorination by
bacteria now differed between species with B. megaterium
andC. albidushaving the highest rates (Fig. 3b;F7:76;
df11, 12, P0:001: Fungal de¯uorination in the
presence of sterile soil increased approximately four-fold
withF. oxysporumhaving by far the greatest de¯uorinating
ability of any microbial isolate. Bacterial de¯uorination increased only two-fold (Fig. 3). Mean de¯uorination rates after 12 d incubation with 1080, and with and without
sterile soil, were: bacteria, 32.7mg F2ml21 inoculant (or
3.5mg F2g sterile soil21) and 15.1mg F2ml21 inoculant
Twenty-four species of microorganisms, which were capable of growing in the presence of 1080 were isolated from soil in arid Australia. Although not all microorganisms
could be identi®ed, species of Bacillus, Pseudomonas,
Aspergillus, Penicillium and Streptomyces capable of
de¯uorinating 1080 are known to occur in soil in temperate climates in Australia (Wong et al., 1991, 1992; Kirkpatrick, 1999) and New Zealand (Bong et al., 1979; Walker, 1994).
In fact, F. oxysporum, F. solani and B. subtilisare
wide-spread occurring in both countries.F. oxysporumis the most
ef®cient and proli®c de¯uorinator of 1080 of all the micro-organisms capable of detoxifying 1080 identi®ed to date (Wong et al., 1991, 1992; Walker, 1994; Kirkpatrick,
1999; our study). However, the ability of Acinetobacter,
Arthrobacter, Aureobacterium, Cryptococcus and
Week-sellato de¯uorinate 1080 has not been recorded previously.
Our ®ndings are also in contrast with those of Wong et al. (1992) who were unable to isolate any soil microorganisms capable of de¯uorinating 1080 from four arid/semi-arid sites in Australia. There are several possible reasons for this. Their sandy soil from the Tanami Desert site in central Australia may not have had such organisms. However, in sandy loams in New Zealand, 50% of 1080 (6.1 mg added)
was detoxi®ed within 38 d at 218C with 9±20% soil
moist-ure (Par®t et al., 1994). The more likely cause of Wong et al. (1992) ®ndings is that the initial isolation of soil
microor-ganisms was undertaken at a temperature (45±508C) which
inhibited microbial growth. Our incubation and isolation procedures were undertaken using a temperature range of
15±308C. Furthermore, in New Zealand silt loams, 50% of
added 1080 was degraded within 10 d at 238C, 30 d at 108C,
and 80 d at 58C (Par®t et al., 1994). This is similar to the
rates we observed where about 10±50% of added 1080 was
de¯uorinated within 12 d at 278C.
The rate of de¯uorination for similar species of microor-ganism often differed between our study and studies by Wong et al. (1991, 1992). The latter found de¯uorination rates ranging from 4±78% of added 1080 within 12 d at
278C. The high level of F2binding to the soil may have
been a confounding factor during Wong et al.'s (1992) trials
(.60% by back calculation of data presented). The binding
of F2to soil in their trials was only determined for one soil
type and then extrapolated to all soils tested. The correction factor used to overcome this binding may have led to an over estimation of the amount of 1080 de¯uorinated in some instances. Although not measured, our soils contained little obvious organic matter and hence the amount of binding of
F2 was low, ranging from 2±5%, depending upon the
L.E.
Twigg,
L.V.
Socha
/
Soil
Biology
&
Biochemi
stry
33
(2001)
227
±
234
231
The relative abundance of microorganisms isolated from soil in central Australia over an eight month period and which were capable of growing in an enriched media containing 10 ml of 20 mM 1080. Weather variables for the corresponding periods are also shown over two week intervals. Tot, Total number of colonies for all three sites; Mn, Mean number of colonies from the three sites; NS, Number of sites n3
containing that isolate for that collection period, ND, data not collected
March April #1 April #2 May July September November
Microorganism Tot Mn NS Tot Mn NS Tot Mn NS Tot Mn NS Tot Mn NS Tot Mn NS Tot Mn NS
Bacteria
Acinetobactersp. 1 0 0 0 0 0 0 1200 400 1 1200 400 2 0 0 0 500 167 1 2500 833 2
Arthrobactersp. 1 100 33 1 1300 433 1 1100 367 1 0 0 0 300 100 1 0 0 0 0 0 0
Arthrobactersp. 2 0 0 0 0 0 0 0 0 0 0 0 0 100 33 1 2300 767 3 2900 967 2
Aureobacteriumsp. 1 1600 533 3 200 67 1 0 0 0 600 200 1 400 133 1 0 0 0 1200 400 2
Aureobacteriumsp. 2 400 133 2 0 0 0 0 0 0 500 167 1 0 0 0 800 267 1 200 67 1
Bacillus megaterium 500 167 2 0 0 0 3800 1267 3 0 0 0 0 0 0 0 0 0 200 67 1
Bacillus subtilis 900 300 1 100 33 1 1900 633 1 0 0 0 300 100 1 0 0 0 0 0 0
Cryptococcus albidus 0 0 0 0 0 0 0 0 0 500 167 2 0 0 0 200 67 1 0 0 0
Pseudomonas alcaligenes 1500 500 1 1000 333 1 2300 767 1 0 0 0 200 67 1 0 0 0 0 0 0
Weeksella virosa 1100 367 3 0 0 0 2600 867 2 900 300 1 1200 400 3 0 0 0 0 0 0
Unknown, gram1bacillus 500 167 2 200 67 1 2600 867 3 1100 367 2 900 300 1 1300 433 2 1000 333 2
Unknown, gram1coccus 0 0 0 0 0 0 800 267 1 1100 367 1 0 0 0 600 200 1 0 0 0
Actinomycetes
Streptomycessp. 1 800 267 2 0 0 0 600 200 1 2200 733 3 1100 367 2 600 200 1 700 233 2
Total number of species 9 5 9 8 8 7 7
Fungi
Aspergillus ¯avus 3 1.0 1 10 3.3 1 15 5.0 2 0 0 0 0 0 0 0 0 0 0 0 0
Aspergillus fumigatus 2 0.7 2 0 0 0 3 1.0 1 0 0 0 1 0.3 1 0 0 0 4 1.3 2
Aspergillussp. 1 0 0 0 9 3.0 1 0 0 0 0 0 0 2 0.7 1 5 1.7 2 3 1.0 1
Fusarium avenaceum 2 0.7 1 0 0 0 4 1.3 1 3 1.0 2 2 0.7 2 7 2.3 3 13 4.3 2
Fusarium compactum 1 0.3 1 10 3.3 1 9 3.0 3 2 0.7 1 1 0.3 1 11 3.7 2 9 3.0 2
Fusarium equiseti 17 5.7 2 1 0.3 1 0 0 0 4 1.3 2 0 0 0 3 1.0 2 33 11.0 3
Fusarium oxysporum 6 2.0 2 0 0 0 0 0 0 5 1.7 3 1 0.3 1 11 3.7 3 22 7.3 3
Fusarium proliferatum 14 4.7 2 0 0 0 0 0 0 5 1.7 2 1 0.3 1 0 0 0 1 0.3 1
Fusarium semitectum? 6 2.0 1 4 0.7 2 0 0 0 12 4.0 3 4 1.3 1 4 1.3 1 16 5.3 3
Fusarium solani 6 2.0 2 3 1.0 1 22 7.3 2 3 1.0 1 0 0 0 3 1.0 1 6 2.0 2
Penicillium spinulosum 0 0 0 0 0 0 0 0 0 5 1.7 1 0 0 0 0 0 0 10 3.3 2
Total number of species 9 6 5 8 7 7 10
All species 18 11a 14a 16 15 14 17
Weather (two week periods)?
Total Rainfall (mm) 9.0 1.5 59.7 0.1 0.0 0.0 0.0 35.6 0.0 21.0 53.8 ND
Mean Max Temperature (8C) 30.3 28.8 33.1 24.4 26.2 24.0 24.4 25.0 28.3 29.1 37.4 ND
Mean Min Temperature (8C) 20.7 17.3 14.7 13.4 10.4 8.4 6.6 7.2 10.0 12.8 16.8 ND
amount added. Kirkpatrick (1999) also reported rapid
de¯uorination of 1080 in factory waste by F. oxysporum
and species of Pseudomonas, with de¯uorination rates
ranging from 10 to 100% of added 1080 within 5 d at
308C. While Wong et al. (1991, 1992) also provided an
additional nitrogen source (peptone) to many of their broths, which could account for their greater levels of de¯uorina-tion, the low levels of organic matter in our soil samples suggest that alternative sources of carbon, or available nitro-gen, may be limited in arid zone soils. Such a response is supported by our time course experiment, where de¯uorina-tion of added 1080 appeared to asymptote after 21±28 d. Wong et al. (1992) also used 12 g of soil (more carbon?) compared to the 5 g of soil used in our trials; the amount of added 1080 in the two studies was the same.
The abundance of microorganisms capable of de¯uorinat-ing 1080 in central Australian soils generally increased following periods of rain. Adequate soil moisture is required to enable metabolism of substrates for vegetative and reproductive growth. The existence of a complex soil L.E. Twigg, L.V. Socha / Soil Biology & Biochemistry 33 (2001) 227±234
232 20 mM 1080 solution at 278C for 12 d. Soil was collected from each site for each collection period n7:
0
(b) Broth & sterile soil
B a c t e r i a F u n g i
micro¯ora is also dependent upon the presence of adequate food and energy sources (Gray and Williams, 1971). Similar to our ®ndings, the degradation of 1080 in sandy loams in New Zealand appears to be slower when soil moisture is
reduced (,9%; Par®t et al., 1994), suggesting the longevity
of 1080 in soil may be increased in some arid environments.
In our trials, little de¯uorination 1:28^0:05mg F
2ml21 ;
n2occurred in sterile soil incubations in the absence of
microorganisms. Thus we are con®dent that the de¯uorina-tion of 1080 in the presence of non-sterile soil was the result of microbial isolate activity. However, as both the spatial distribution and the abundance of these microbial isolates appeared to change over time, we recommend that soil samples need to be collected from a wide area in both space and time to be sure whether an individual species is present or not Ð or at least, soil moisture conditions need to be recorded. De¯uorination of 1080 was greater in our soil samples at pH 5.6 (Fig. 2), which is the preferred pH for many fungi. Although the pH of our soil was 6.6, we believe
that, because of the regular occurrence ofFusariumand the
exceptional ability ofF. oxysporum to de¯uorinate 1080,
fungi are probably the most important de¯uorinators of 1080 in central Australian soils. However, de¯uorinating activity seems to be dependent upon both the type and number of microorganisms present (Table 1).
The presence of numerous species of microorganisms, some with considerable ability to detoxify 1080, suggests that the half-life of 1080 in soils in many arid regions may be less than 40 d, particularly after signi®cant rainfall events. However, this will depend upon the species of microorganisms present, their abundance, their ability to de¯uorinate 1080, and the soil moisture conditions. 1080 can bind to cellulose (Hilton et al., 1969), and is readily leached through the soil pro®le (Par®t et al., 1994), thus pest control operations which utilise 1080 are extremely unlikely to result in any long term environmental contam-ination. 1080 is also readily degraded in waterways (Par®t et al., 1994; Twigg et al., 1996). The 1080-baits used in pest
control operations can contain up to 6 mg bait21 (or
approximately 25mg 1080 g21). This amount is well within
the observed de¯uorinating ability of the soil micro¯ora in Australia and New Zealand (Wong et al., 1991,1992; Par®t et al., 1994; our study). Furthermore, baits containing these
high concentrations are usually well spaced (.200 m apart),
which also helps with biosafety.Pseudomonasspp. andF.
oxysporum are also capable of degrading factory waste
products containing 1080 (Kirkpatrick, 1999). The bacteria,
P. cepaciahas been isolated from the seed of
¯uoroacetate-producing plants in South Africa, and this bacteria is capable of substantial de¯uorination of 1080 (Meyer, 1994). Consequently, both the target speci®city and rapid biodegradation of 1080, ensure that 1080 can be safely used in pest control programs in most areas of Australia and New Zealand. Despite this, some caution is required in arid
Australia because dried meat baits (6 mg 1080 bait21) can
remain toxic for over 12 months (Twigg et al., 2000).
Acknowledgements
We thank Dennis Matthews, Steve Eldridge, Lester Burgess and Win Kirkpatrick for their advice and help with parts of this project. Ian Arthur, and Max Aravena-Roman helped identify the bacteria. Dennis King and Glenn Edwards commented on earlier drafts.
References
Aplin, T.E.H., 1971. Poison plants of Western Australia: The toxic species of the generaGastrolobiumandOxylobium. Western Australian Depart-ment of Agriculture Bulletin No. 3772, pp. 1±64.
Barrow, N.J., Shaw, T.C., 1977. The slow reactions between soil and anions: 6 Effect of time and temperature of control on ¯uoride. Soil Science 124, 265±278.
Bong, C.L., Cole, A.L., Walker, J.R., Peters, J.A., 1979. Effect of sodium ¯uoroacetate (compound 1080) on the soil micro¯ora. Soil Biology & Biochemistry 11, 13±18.
Booth, C., 1971. The Genus Fusarium. Commonwealth Mycological Institute, Kew, Surrey, UK (237pp).
Burgess, L.W., Liddell, C.M., Summerell, B.A., 1988. Laboratory Manual forFusariumResearch. Department of Plant Pathology & Agricultural Entomology, University of Sydney, Sydney, Australia, 156pp. Gray, T.R., Williams, S.T., 1971. Soil Micro-organisms. Oliver & Boyd,
Edinburgh, (240pp).
Hilton, H.N., Yuen, R.H., Normura, N.S., 1969. Absorption of mono¯uoroacetate 2-14C ion and its translocation in sugarcane. Journal of Agriculture and Food Chemistry 17, 131±134.
Kelly, M., 1965. Isolation of bacteria able to metabolise ¯uoroacetate or ¯uoroacetamide. Nature 208, 809±810.
Kirkpatrick, W.E., 1999. Assessment of Sodium Fluoroacetate (1080) in Baits and Its Biodegradation by Microorganisms. MSc Dissertion, Curtin University of Technology, Bentley, Western Australia, Australia, 113pp.
McIlroy, J.C., Gifford, E.J., Carpenter, S.M., 1988. The effect of rainfall and blow¯y larvae on the toxicity of 1080-treated meat baits used in poisoning campaigns against wild dogs. Australian Wildlife Research 15, 473±483.
Meyer, M.J., 1994. Fluoroacetate metabolism ofPseudomonas cepacia. In: Seawright, A.A., Eason, C.T. (Eds.). Proceedings of the Science Workshop on 1080, Royal Society of New Zealand. Miscellaneous Series 28 SIR Publishing, Wellington, New Zealand, pp. 54±58. Oelrichs, P.B., McEwan, T., 1961. Isolation of the toxic principle inAcacia
georginae. Nature 190, 808±809.
Par®t, R.L., Eason, C.T., Morgan, A.J., Wright, G.R., Burke, C.M., 1994. The fate of sodium mono¯uoroacetate (1080) in soil and water. In: Seawright, A.A., Eason, C.T. (Eds.). Proceedings of the Science Work-shop on 1080, Royal Society of New Zealand. Miscellaneous Series 28 SIR Publishing, Wellington, New Zealand, pp. 59±66.
Peters, J.A., Baxter, K.L., 1974. Analytical determination of Compound 1080 (sodium ¯uoroacetate) residues in biological materials. Bulletin of Environmental Contamination & Toxicology 11, 177±183. Pitt, J.I., 1979. The Genus Penicillium and its Teleomorphic states,
EupenicilliumandTalaromyces. Academic Press, London, UK (634pp). Raper, K.B., Fennell, D.I., 1965. The Genus Aspergillus. Williams &
Wilkins, Baltimore, USA (686pp).
Saunders, G., Coman, B., Kinnear, J., Braysher, M., 1995. Managing Verte-brate Pests: Foxes. Bureau of Resource Sciences. Australian Government Printing Services, Canberra (141pp).
StatSoft, 1994. Statistica for the Macintosh. StatSoft Inc, Tulsa, USA (1064pp).
dingoes in north-western Australia. Australian Wildlife Research 13, 165±176.
Twigg, L.E., King, D.R., 1991. The impact of ¯uoroacetate-bearing vegeta-tion on native Australian fauna. Oikos 61, 412±430.
Twigg, L.E., King, D.R., Bowen, L.H., Wright, G.E., Eason, C.T., 1996. The ¯uoroacetate content of some species of the toxic plant genus Gastrolobium, and its environmental persistence. Natural Toxins 4, 122±127.
Twigg, L.E., Eldridge, S.R., Edwards, G.P., Shakeshaft, B.J., dePreu, N.D., Adams, N., 2000. The ef®cacy and longevity of 1080 meat baits used for dingo control in central Australia. Wildlife Research 27 (in Press).
Walker, J.R., 1994. Degradation of sodium mono¯uoroacetate by soil microorganisms. In: Seawright, A.A., Eason, C.T. (Eds.). Proceed-ings of the Science Workshop on 1080, Royal Society of New Zealand. Miscellaneous Series 28 SIR Publishing, Wellington, New Zealand, pp. 50±53.
Wheeler, S.H., Oliver, A.J., 1978. The effect of rainfall and moisture on the 1080 and pindone content of vacuum-impregnated oats used for the control of rabbits,Oryctolagus cuniculus. Australian Wildlife Research 5, 143±149.
Williams, C.K., Parer, I., Coman, B.J., Burley, J., Braysher, M.L., 1995. Managing Vertebrate Pests: Rabbits. Bureau of Resource Sciences. Australian Government Printing Services, Canberra (284pp). Winer, B.J., Brown, D.R., Michels, K.M., 1991. Statistical Principles in
Experimental Design. McGraw-Hill, New York, USA (1057pp). Wong, D.H., Kirkpatrick, W.E., Kinnear, J.E., King, D.R., 1991.
De¯uorination of sodium mono¯uoroacetate (1080) by microorganisms found in bait materials. Wildlife Research 18, 539±545.
Wong, D.H., Kirkpatrick, W.E., King, D.R., Kinnear, J.E., 1992. De¯uorination of sodium mono¯uoroacetate (1080) by microorganisms isolated from Western Australian soils. Soil Biology & Biochemistry 24, 833±838.
