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Defluorination of sodium monofluoroacetate by soil microorganisms from central Australia
Soil Biology & Biochemistry 33 (2001) 227±234
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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 fungus Fusarium 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. q 2001 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 invertebrates except where individual species have had evolutionary 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 of Gastrolobium), 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 * 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. E-mail address: [email protected] (L.E. Twigg).
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-bearing vegetation in southwestern Western Australia (Twigg et al., 1996). In fact several genera of soil fungi (e.g. Fusarium, 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 ubiquitous 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 matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 0038-071 7(00)00134-6
228
L.E. Twigg, L.V. Socha / Soil Biology & Biochemistry 33 (2001) 227±234
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 (248 10 0 S; 1328 50 0 E) 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, n 3: 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 examining 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 undisturbed 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 l 21 KH2PO4, and 1 g l 21 (NH4)2SO4 adjusted 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 l 21) and FeSO47H2O (10 mg l 21), 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 polycarbonate 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. min 21). There was one bottle per replicate with three replicates per soil collection period. After 12 d incubation, the concentration of F 2 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 timecourse 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 F 2 concentration measured using the F 2 electrode (see above). Background levels of F 2 in 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 F 2 was then measured using the F 2 electrode. Background levels for the 1080 solution were also determined using the F 2 electrode. Because ¯uoride ions can bind to soil particles (Barrow and Shaw, 1977), the recovery of added F 2 from both 1 g and 5 g sterile soil samples was also determined. The amount of F 2 binding to the soil was measured by adding a known amount of F 2 to 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 F 2 determined using the F 2 electrode. This was compared to the measurement of F 2 in solutions with identical F 2 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 l 21 KH2PO4, and 1 g l 21 (NH4)2SO4 at pH 6.8; fungi: 0.2 mg l 21 CaCl2 and 10 mg l 21 FeSO47H2O adjusted to pH 5.6), and with and without 5 g of sterile soil. Bacterial suspensions (1.5 £ 10 9 cells ml 21) 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 scraping off aerial mycelium from 48 h-old cultures into 5 ml sterile 0.85% NaCl w/v and sterile 20 mM 1080. As appropriate, 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 determined 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 n 3 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 for Streptomyctes sp. 1 (Actinomycetes) were presented separately, and were exclude from the `bacteria' category during the analyses, because this species had marked differences in their isolation and growth requirements (e.g. aerial mycelium) compared to that of the other bacteria. 3. Results 3.1. Recovery of added F 2 Little F 2 was found to bind to the soil. Mean recovery rates for 1.25±5.0 and 10.0±40.0 mg of added F 2 were 95.5 ^ 0.8 % (SEM, n 3; and 98.5 ^ 4.4% (SEM, n 4; respectively. Little free F 2 occurred naturally in the soil
229
(1.14 mg g 21, n 4; deionised water (0.16 mg ml21, n 5 or the 20 mM 1080 solution (2.5 mg ml 21, n 5: Because of the high recovery rates of added F 2, our data are presented as unadjusted values. The recovery of F 2 ion from known amounts of 1080 as determined by the amount of inorganic ¯uoride subsequently released following degradation 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 380 mg of F 2. 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 F 2 d 21 g soil 21 A 1 B1 £ time 1 B2 £ time 2, where time is in days, and A (0.928), B1 (1.050) and B2 (20.017) are nonlinear regression constants n 105; r 0:654 However, soil samples appeared to have greater de¯uorinating 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 F 19:27; df 1, 26, P 0:0002: The ability of soil to de¯uorinate 1080 was also in¯uenced by time of year F 3:42; df 6, 26, P 0:013 with the highest de¯uorination occurring in early November after 54 mm of rainfall (Table 1). The interaction was also significant F 2:78; df 6, 26, P 0:032 indicating the effects of pH varied between time periods. The pH of the soil at the ®eld sites was 6.6 n 3: 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 temperatures 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 Australian soil. Microorganisms were least abundant during, or following, periods of low rainfall (Table 1). Fusarium was 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 consecutive collection periods. Two species of bacteria were not identi®ed (Table 1). 3.4. De¯uorination by microbial isolates All 24 isolates were capable of de¯uorinating 1080 when
230
L.E. Twigg, L.V. Socha / Soil Biology & Biochemistry 33 (2001) 227±234 20
9.1 mg F 2 g sterile soil 21) and 19.5 mg F 2 ml 21 inoculant n 11 £ 2; and Streptomycessp.1, 58.5 mg F 2 ml 21 inoculant (or 6.3 mg F 2 g sterile soil 21) and 2 21 15.4 mg F ml inoculant n 1 £ 2:
(a)
Fluoride released (µg F-/g soil)
15
10
4. Discussion 5
0 0 30
7
14
21
28
14
21
28
(b)
25 20 15 10 5 0 0
7
Days of incubation Fig. 1. Mean de¯uorination of added 1080 (i.e. 380 mg F 2) by 5 g soil samples incubated at 288C (day) and 158C (night) over 28 d. (a) The mean of repeated incubations of soil from each site n 3 for each collection period n 7; and the curvilinear line ®t for all sites is shown r 0:99; n 21: Symbols represent the different sites. (b) Mean of the three sites for soils collected in March (A), April (P), May (W), July (X), September (K) and November (V). Except for April n 6 n 3:
1080 was their sole source of carbon (Fig. 3a). In the absence of soil, the fungal isolates had greater de¯uorinating ability than the bacteria F 5:09; df 1, 44, P 0:03: Within the fungi, de¯uorination by F. oxysporum was greater than for any other species F 9:30; df 10, 11, P , 0:001; but de¯uorination by bacterial species was similar F 5:09; df 11, 12, P 0:10: 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 F 10:03; df 1, 44, P , 0:003; with F. oxysporum better than all other fungal species F 21:48; df 10, 11, P , 0:001: However, de¯uorination by bacteria now differed between species with B. megaterium and C. albidus having the highest rates (Fig. 3b; F 7:76; df 11, 12, P 0:001: Fungal de¯uorination in the presence of sterile soil increased approximately four-fold with F. oxysporum having 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, 32.7 mg F 2 ml 21 inoculant (or 3.5 mg F 2 g sterile soil 21) and 15.1 mg F 2 ml 21 inoculant n 12 £ 2; fungi, 82.6 mg F 2 ml 21 inoculant (or
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. subtilis are widespread occurring in both countries. F. oxysporum is the most ef®cient and proli®c de¯uorinator of 1080 of all the microorganisms 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 Weeksella to 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 moisture (Par®t et al., 1994). The more likely cause of Wong et al. (1992) ®ndings is that the initial isolation of soil microorganisms 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 microorganism 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 F 2 binding 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 F 2 to 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 F 2 was low, ranging from 2±5%, depending upon the
a
9.0 30.3 20.7
1.5 28.8 17.3
1.0 0.7 0 0.7 0.3 5.7 2.0 4.7 2.0 2.0 0
267
800
3 2 0 2 1 17 6 14 6 6 0
0 33 0 533 133 167 300 0 500 367 167 0
Mn
0 100 0 1600 400 500 900 0 1500 1100 500 0
Tot
Total unique species for April 18.
Total Rainfall (mm) Mean Max Temperature (8C) Mean Min Temperature (8C)
Weather (two week periods)?
Total number of species All species
Aspergillus ¯avus Aspergillus fumigatus Aspergillus sp. 1 Fusarium avenaceum Fusarium compactum Fusarium equiseti Fusarium oxysporum Fusarium proliferatum Fusarium semitectum? Fusarium solani Penicillium spinulosum
Fungi
Total number of species
Acinetobacter sp. 1 Arthrobacter sp. 1 Arthrobacter sp. 2 Aureobacterium sp. 1 Aureobacterium sp. 2 Bacillus megaterium Bacillus subtilis Cryptococcus albidus Pseudomonas alcaligenes Weeksella virosa Unknown, gram 1 bacillus Unknown, gram 1 coccus Actinomycetes Streptomyces sp. 1
Bacteria
Microorganism
March
9 18
1 2 0 1 1 2 2 2 1 2 0
9
2
0 1 0 3 2 2 1 0 1 3 2 0
NS
59.7 33.1 14.7
10 0 9 0 10 1 0 0 4 3 0
0
0 1300 0 200 0 0 100 0 1000 0 200 0
Tot
April #1
3.3 0 3.0 0 3.3 0.3 0 0 0.7 1.0 0
0
0 433 0 67 0 0 33 0 333 0 67 0
Mn
6 11 a
1 0 1 0 1 1 0 0 2 1 0
5
0
0 1 0 1 0 0 1 0 1 0 1 0
NS
15 3 0 4 9 0 0 0 0 22 0
600
1200 1100 0 0 0 3800 1900 0 2300 2600 2600 800
Tot
0.1 24.4 13.4
5.0 1.0 0 1.3 3.0 0 0 0 0 7.3 0
200
400 367 0 0 0 1267 633 0 767 867 867 267
Mn
April #2
5 14 a
2 1 0 1 3 0 0 0 0 2 0
9
1
1 1 0 0 0 3 1 0 1 2 3 1
NS
0.0 26.2 10.4
0 0 0 3 2 4 5 5 12 3 5
2200
1200 0 0 600 500 0 0 500 0 900 1100 1100
Tot
May
0.0 24.0 8.4
0 0 0 1.0 0.7 1.3 1.7 1.7 4.0 1.0 1.7
733
400 0 0 200 167 0 0 167 0 300 367 367
Mn
8 16
0 0 0 2 1 2 3 2 3 1 1
8
3
2 0 0 1 1 0 0 2 0 1 2 1
NS
0.0 24.4 6.6
0 1 2 2 1 0 1 1 4 0 0
1100
0 300 100 400 0 0 300 0 200 1200 900 0
Tot
July
35.6 25.0 7.2
0 0.3 0.7 0.7 0.3 0 0.3 0.3 1.3 0 0
367
0 100 33 133 0 0 100 0 67 400 300 0
Mn
7 15
0 1 1 2 1 0 1 1 1 0 0
8
2
0 1 1 1 0 0 1 0 1 3 1 0
NS
0.0 28.3 10.0
0 0 5 7 11 3 11 0 4 3 0
600
500 0 2300 0 800 0 0 200 0 0 1300 600
Tot
21.0 29.1 12.8
0 0 1.7 2.3 3.7 1.0 3.7 0 1.3 1.0 0
200
167 0 767 0 267 0 0 67 0 0 433 200
Mn
September
7 14
0 0 2 3 2 2 3 0 1 1 0
7
1
1 0 3 0 1 0 0 1 0 0 2 1
NS
53.8 37.4 16.8
0 4 3 13 9 33 22 1 16 6 10
700
2500 0 2900 1200 200 200 0 0 0 0 1000 0
Tot
ND ND ND
0 1.3 1.0 4.3 3.0 11.0 7.3 0.3 5.3 2.0 3.3
233
833 0 967 400 67 67 0 0 0 0 333 0
Mn
November
10 17
0 2 1 2 2 3 3 1 3 2 2
7
2
2 0 2 2 1 1 0 0 0 0 2 0
NS
Table 1 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 n 3 containing that isolate for that collection period, ND, data not collected
L.E. Twigg, L.V. Socha / Soil Biology & Biochemistry 33 (2001) 227±234 231
232
L.E. Twigg, L.V. Socha / Soil Biology & Biochemistry 33 (2001) 227±234 pH 5.6
Fluoride released (µgF-/g soil)
800
pH 6.8
600
400
200
0 Mar
Apr
Apr
May
Ju l
Sep
N ov
Month Fig. 2. The effect of pH on the mean (^SEM, n 3 de¯uorination of added 1080 (i.e. 3800 mg F 2) by 1 g soil samples grown in 10 ml of 20 mM 1080 solution at 278C for 12 d. Soil was collected from each site for each collection period n 7:
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¯uorination, the low levels of organic matter in our soil samples suggest that alternative sources of carbon, or available nitrogen, may be limited in arid zone soils. Such a response is supported by our time course experiment, where de¯uorination 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¯uorinating 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
Fluoride released (µgF-/ml)
500
(a) Broth only
11
400 Bacteria
9 Fungi
300 7 200 5
FA c Degraded (%)
13
100 3 Acin sp1 Ar th sp1 Ar th sp2 Aure sp1 Aure sp2 Baci meg Baci sub Cryp alb Pseu alc Week vir Unkn bac Unkn coc Asp flav Asp fum Asp sp1 F u s av e Fus com Fus equ Fus oxy Fus pro Fus sem Fus sol Pen spi Str sp1 All Bac All Fungi
0
50
35 30 20 Bacteria
Fungi
25 20 15
10
10 5 0
0 Acin sp1 Ar th sp1 Ar th sp2 Aure sp1 Aure sp2 Baci meg Baci sub Cryp alb Pseu alc Week vir Unkn bac Unkn coc Asp flav Asp fum Asp sp1 F u s av e Fus com Fus equ Fus oxy Fus pro Fus sem Fus sol Pen spi Str sp1 All Bac All Fungi
Fluoride released (µgF-/g soil)
40
FA c Degraded (%)
45
(b) Broth & sterile soil
30
Fig. 3. Mean (^SEM, n 2) de¯uorination of added 1080 by soil microorganisms from central Australia after 12 d incubation at 278C in enriched media (see methods). (a) By microbial isolates grown in 10 ml of 20 mM 1080 solution (sole carbon source, with 3800 mg added F 2), and (b) microbial isolates grown in 1 ml of 20 mM 1080 solution (i.e. 380 mg F 2) plus 5 g of sterile soil at 278C for 12 d.
L.E. Twigg, L.V. Socha / Soil Biology & Biochemistry 33 (2001) 227±234
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 ^ 0:05 mg F2 ml21 ; n 2 occurred in sterile soil incubations in the absence of microorganisms. Thus we are con®dent that the de¯uorination 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 of Fusarium and the exceptional ability of F. 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 contamination. 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 bait 21 (or approximately 25 mg 1080 g 21). 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. Pseudomonas spp. and F. oxysporum are also capable of degrading factory waste products containing 1080 (Kirkpatrick, 1999). The bacteria, P. cepacia has been isolated from the seed of ¯uoroacetateproducing 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 bait 21) can remain toxic for over 12 months (Twigg et al., 2000).
233
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 AravenaRoman 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 genera Gastrolobium and Oxylobium. Western Australian Department 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 for Fusarium Research. 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 of Pseudomonas 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 in Acacia 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 Workshop 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, Eupenicillium and Talaromyces. 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 Vertebrate Pests: Foxes. Bureau of Resource Sciences. Australian Government Printing Services, Canberra (141pp). StatSoft, 1994. Statistica for the Macintosh. StatSoft Inc, Tulsa, USA (1064pp). Thomson, P.C., 1986. The effectiveness of aerial baiting for the control of
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dingoes in north-western Australia. Australian Wildlife Research 13, 165±176. Twigg, L.E., King, D.R., 1991. The impact of ¯uoroacetate-bearing vegetation 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.). Proceedings 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.
www.elsevier.com/locate/soilbio
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 fungus Fusarium 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. q 2001 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 invertebrates except where individual species have had evolutionary 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 of Gastrolobium), 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 * 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. E-mail address: [email protected] (L.E. Twigg).
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-bearing vegetation in southwestern Western Australia (Twigg et al., 1996). In fact several genera of soil fungi (e.g. Fusarium, 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 ubiquitous 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 matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 0038-071 7(00)00134-6
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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 (248 10 0 S; 1328 50 0 E) 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, n 3: 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 examining 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 undisturbed 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 l 21 KH2PO4, and 1 g l 21 (NH4)2SO4 adjusted 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 l 21) and FeSO47H2O (10 mg l 21), 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 polycarbonate 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. min 21). There was one bottle per replicate with three replicates per soil collection period. After 12 d incubation, the concentration of F 2 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 timecourse 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 F 2 concentration measured using the F 2 electrode (see above). Background levels of F 2 in 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 F 2 was then measured using the F 2 electrode. Background levels for the 1080 solution were also determined using the F 2 electrode. Because ¯uoride ions can bind to soil particles (Barrow and Shaw, 1977), the recovery of added F 2 from both 1 g and 5 g sterile soil samples was also determined. The amount of F 2 binding to the soil was measured by adding a known amount of F 2 to 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 F 2 determined using the F 2 electrode. This was compared to the measurement of F 2 in solutions with identical F 2 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 l 21 KH2PO4, and 1 g l 21 (NH4)2SO4 at pH 6.8; fungi: 0.2 mg l 21 CaCl2 and 10 mg l 21 FeSO47H2O adjusted to pH 5.6), and with and without 5 g of sterile soil. Bacterial suspensions (1.5 £ 10 9 cells ml 21) 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 scraping off aerial mycelium from 48 h-old cultures into 5 ml sterile 0.85% NaCl w/v and sterile 20 mM 1080. As appropriate, 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 determined 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 n 3 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 for Streptomyctes sp. 1 (Actinomycetes) were presented separately, and were exclude from the `bacteria' category during the analyses, because this species had marked differences in their isolation and growth requirements (e.g. aerial mycelium) compared to that of the other bacteria. 3. Results 3.1. Recovery of added F 2 Little F 2 was found to bind to the soil. Mean recovery rates for 1.25±5.0 and 10.0±40.0 mg of added F 2 were 95.5 ^ 0.8 % (SEM, n 3; and 98.5 ^ 4.4% (SEM, n 4; respectively. Little free F 2 occurred naturally in the soil
229
(1.14 mg g 21, n 4; deionised water (0.16 mg ml21, n 5 or the 20 mM 1080 solution (2.5 mg ml 21, n 5: Because of the high recovery rates of added F 2, our data are presented as unadjusted values. The recovery of F 2 ion from known amounts of 1080 as determined by the amount of inorganic ¯uoride subsequently released following degradation 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 380 mg of F 2. 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 F 2 d 21 g soil 21 A 1 B1 £ time 1 B2 £ time 2, where time is in days, and A (0.928), B1 (1.050) and B2 (20.017) are nonlinear regression constants n 105; r 0:654 However, soil samples appeared to have greater de¯uorinating 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 F 19:27; df 1, 26, P 0:0002: The ability of soil to de¯uorinate 1080 was also in¯uenced by time of year F 3:42; df 6, 26, P 0:013 with the highest de¯uorination occurring in early November after 54 mm of rainfall (Table 1). The interaction was also significant F 2:78; df 6, 26, P 0:032 indicating the effects of pH varied between time periods. The pH of the soil at the ®eld sites was 6.6 n 3: 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 temperatures 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 Australian soil. Microorganisms were least abundant during, or following, periods of low rainfall (Table 1). Fusarium was 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 consecutive collection periods. Two species of bacteria were not identi®ed (Table 1). 3.4. De¯uorination by microbial isolates All 24 isolates were capable of de¯uorinating 1080 when
230
L.E. Twigg, L.V. Socha / Soil Biology & Biochemistry 33 (2001) 227±234 20
9.1 mg F 2 g sterile soil 21) and 19.5 mg F 2 ml 21 inoculant n 11 £ 2; and Streptomycessp.1, 58.5 mg F 2 ml 21 inoculant (or 6.3 mg F 2 g sterile soil 21) and 2 21 15.4 mg F ml inoculant n 1 £ 2:
(a)
Fluoride released (µg F-/g soil)
15
10
4. Discussion 5
0 0 30
7
14
21
28
14
21
28
(b)
25 20 15 10 5 0 0
7
Days of incubation Fig. 1. Mean de¯uorination of added 1080 (i.e. 380 mg F 2) by 5 g soil samples incubated at 288C (day) and 158C (night) over 28 d. (a) The mean of repeated incubations of soil from each site n 3 for each collection period n 7; and the curvilinear line ®t for all sites is shown r 0:99; n 21: Symbols represent the different sites. (b) Mean of the three sites for soils collected in March (A), April (P), May (W), July (X), September (K) and November (V). Except for April n 6 n 3:
1080 was their sole source of carbon (Fig. 3a). In the absence of soil, the fungal isolates had greater de¯uorinating ability than the bacteria F 5:09; df 1, 44, P 0:03: Within the fungi, de¯uorination by F. oxysporum was greater than for any other species F 9:30; df 10, 11, P , 0:001; but de¯uorination by bacterial species was similar F 5:09; df 11, 12, P 0:10: 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 F 10:03; df 1, 44, P , 0:003; with F. oxysporum better than all other fungal species F 21:48; df 10, 11, P , 0:001: However, de¯uorination by bacteria now differed between species with B. megaterium and C. albidus having the highest rates (Fig. 3b; F 7:76; df 11, 12, P 0:001: Fungal de¯uorination in the presence of sterile soil increased approximately four-fold with F. oxysporum having 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, 32.7 mg F 2 ml 21 inoculant (or 3.5 mg F 2 g sterile soil 21) and 15.1 mg F 2 ml 21 inoculant n 12 £ 2; fungi, 82.6 mg F 2 ml 21 inoculant (or
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. subtilis are widespread occurring in both countries. F. oxysporum is the most ef®cient and proli®c de¯uorinator of 1080 of all the microorganisms 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 Weeksella to 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 moisture (Par®t et al., 1994). The more likely cause of Wong et al. (1992) ®ndings is that the initial isolation of soil microorganisms 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 microorganism 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 F 2 binding 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 F 2 to 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 F 2 was low, ranging from 2±5%, depending upon the
a
9.0 30.3 20.7
1.5 28.8 17.3
1.0 0.7 0 0.7 0.3 5.7 2.0 4.7 2.0 2.0 0
267
800
3 2 0 2 1 17 6 14 6 6 0
0 33 0 533 133 167 300 0 500 367 167 0
Mn
0 100 0 1600 400 500 900 0 1500 1100 500 0
Tot
Total unique species for April 18.
Total Rainfall (mm) Mean Max Temperature (8C) Mean Min Temperature (8C)
Weather (two week periods)?
Total number of species All species
Aspergillus ¯avus Aspergillus fumigatus Aspergillus sp. 1 Fusarium avenaceum Fusarium compactum Fusarium equiseti Fusarium oxysporum Fusarium proliferatum Fusarium semitectum? Fusarium solani Penicillium spinulosum
Fungi
Total number of species
Acinetobacter sp. 1 Arthrobacter sp. 1 Arthrobacter sp. 2 Aureobacterium sp. 1 Aureobacterium sp. 2 Bacillus megaterium Bacillus subtilis Cryptococcus albidus Pseudomonas alcaligenes Weeksella virosa Unknown, gram 1 bacillus Unknown, gram 1 coccus Actinomycetes Streptomyces sp. 1
Bacteria
Microorganism
March
9 18
1 2 0 1 1 2 2 2 1 2 0
9
2
0 1 0 3 2 2 1 0 1 3 2 0
NS
59.7 33.1 14.7
10 0 9 0 10 1 0 0 4 3 0
0
0 1300 0 200 0 0 100 0 1000 0 200 0
Tot
April #1
3.3 0 3.0 0 3.3 0.3 0 0 0.7 1.0 0
0
0 433 0 67 0 0 33 0 333 0 67 0
Mn
6 11 a
1 0 1 0 1 1 0 0 2 1 0
5
0
0 1 0 1 0 0 1 0 1 0 1 0
NS
15 3 0 4 9 0 0 0 0 22 0
600
1200 1100 0 0 0 3800 1900 0 2300 2600 2600 800
Tot
0.1 24.4 13.4
5.0 1.0 0 1.3 3.0 0 0 0 0 7.3 0
200
400 367 0 0 0 1267 633 0 767 867 867 267
Mn
April #2
5 14 a
2 1 0 1 3 0 0 0 0 2 0
9
1
1 1 0 0 0 3 1 0 1 2 3 1
NS
0.0 26.2 10.4
0 0 0 3 2 4 5 5 12 3 5
2200
1200 0 0 600 500 0 0 500 0 900 1100 1100
Tot
May
0.0 24.0 8.4
0 0 0 1.0 0.7 1.3 1.7 1.7 4.0 1.0 1.7
733
400 0 0 200 167 0 0 167 0 300 367 367
Mn
8 16
0 0 0 2 1 2 3 2 3 1 1
8
3
2 0 0 1 1 0 0 2 0 1 2 1
NS
0.0 24.4 6.6
0 1 2 2 1 0 1 1 4 0 0
1100
0 300 100 400 0 0 300 0 200 1200 900 0
Tot
July
35.6 25.0 7.2
0 0.3 0.7 0.7 0.3 0 0.3 0.3 1.3 0 0
367
0 100 33 133 0 0 100 0 67 400 300 0
Mn
7 15
0 1 1 2 1 0 1 1 1 0 0
8
2
0 1 1 1 0 0 1 0 1 3 1 0
NS
0.0 28.3 10.0
0 0 5 7 11 3 11 0 4 3 0
600
500 0 2300 0 800 0 0 200 0 0 1300 600
Tot
21.0 29.1 12.8
0 0 1.7 2.3 3.7 1.0 3.7 0 1.3 1.0 0
200
167 0 767 0 267 0 0 67 0 0 433 200
Mn
September
7 14
0 0 2 3 2 2 3 0 1 1 0
7
1
1 0 3 0 1 0 0 1 0 0 2 1
NS
53.8 37.4 16.8
0 4 3 13 9 33 22 1 16 6 10
700
2500 0 2900 1200 200 200 0 0 0 0 1000 0
Tot
ND ND ND
0 1.3 1.0 4.3 3.0 11.0 7.3 0.3 5.3 2.0 3.3
233
833 0 967 400 67 67 0 0 0 0 333 0
Mn
November
10 17
0 2 1 2 2 3 3 1 3 2 2
7
2
2 0 2 2 1 1 0 0 0 0 2 0
NS
Table 1 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 n 3 containing that isolate for that collection period, ND, data not collected
L.E. Twigg, L.V. Socha / Soil Biology & Biochemistry 33 (2001) 227±234 231
232
L.E. Twigg, L.V. Socha / Soil Biology & Biochemistry 33 (2001) 227±234 pH 5.6
Fluoride released (µgF-/g soil)
800
pH 6.8
600
400
200
0 Mar
Apr
Apr
May
Ju l
Sep
N ov
Month Fig. 2. The effect of pH on the mean (^SEM, n 3 de¯uorination of added 1080 (i.e. 3800 mg F 2) by 1 g soil samples grown in 10 ml of 20 mM 1080 solution at 278C for 12 d. Soil was collected from each site for each collection period n 7:
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¯uorination, the low levels of organic matter in our soil samples suggest that alternative sources of carbon, or available nitrogen, may be limited in arid zone soils. Such a response is supported by our time course experiment, where de¯uorination 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¯uorinating 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
Fluoride released (µgF-/ml)
500
(a) Broth only
11
400 Bacteria
9 Fungi
300 7 200 5
FA c Degraded (%)
13
100 3 Acin sp1 Ar th sp1 Ar th sp2 Aure sp1 Aure sp2 Baci meg Baci sub Cryp alb Pseu alc Week vir Unkn bac Unkn coc Asp flav Asp fum Asp sp1 F u s av e Fus com Fus equ Fus oxy Fus pro Fus sem Fus sol Pen spi Str sp1 All Bac All Fungi
0
50
35 30 20 Bacteria
Fungi
25 20 15
10
10 5 0
0 Acin sp1 Ar th sp1 Ar th sp2 Aure sp1 Aure sp2 Baci meg Baci sub Cryp alb Pseu alc Week vir Unkn bac Unkn coc Asp flav Asp fum Asp sp1 F u s av e Fus com Fus equ Fus oxy Fus pro Fus sem Fus sol Pen spi Str sp1 All Bac All Fungi
Fluoride released (µgF-/g soil)
40
FA c Degraded (%)
45
(b) Broth & sterile soil
30
Fig. 3. Mean (^SEM, n 2) de¯uorination of added 1080 by soil microorganisms from central Australia after 12 d incubation at 278C in enriched media (see methods). (a) By microbial isolates grown in 10 ml of 20 mM 1080 solution (sole carbon source, with 3800 mg added F 2), and (b) microbial isolates grown in 1 ml of 20 mM 1080 solution (i.e. 380 mg F 2) plus 5 g of sterile soil at 278C for 12 d.
L.E. Twigg, L.V. Socha / Soil Biology & Biochemistry 33 (2001) 227±234
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 ^ 0:05 mg F2 ml21 ; n 2 occurred in sterile soil incubations in the absence of microorganisms. Thus we are con®dent that the de¯uorination 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 of Fusarium and the exceptional ability of F. 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 contamination. 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 bait 21 (or approximately 25 mg 1080 g 21). 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. Pseudomonas spp. and F. oxysporum are also capable of degrading factory waste products containing 1080 (Kirkpatrick, 1999). The bacteria, P. cepacia has been isolated from the seed of ¯uoroacetateproducing 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 bait 21) can remain toxic for over 12 months (Twigg et al., 2000).
233
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 AravenaRoman 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 genera Gastrolobium and Oxylobium. Western Australian Department 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 for Fusarium Research. 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 of Pseudomonas 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 in Acacia 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 Workshop 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, Eupenicillium and Talaromyces. 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 Vertebrate Pests: Foxes. Bureau of Resource Sciences. Australian Government Printing Services, Canberra (141pp). StatSoft, 1994. Statistica for the Macintosh. StatSoft Inc, Tulsa, USA (1064pp). Thomson, P.C., 1986. The effectiveness of aerial baiting for the control of
234
L.E. Twigg, L.V. Socha / Soil Biology & Biochemistry 33 (2001) 227±234
dingoes in north-western Australia. Australian Wildlife Research 13, 165±176. Twigg, L.E., King, D.R., 1991. The impact of ¯uoroacetate-bearing vegetation 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.). Proceedings 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.