Plant Soil (2007) 291:81–92 DOI 10.1007/s11104-006-9176-2
ORIGINAL PAPER
Leaching of isothiocyanates through intact soil following simulated biofumigation Mette Laegdsmand Æ Anne Louise Gimsing Æ Bjarne W. Strobel Æ Jens Christian Sørensen Æ Ole Hørbye Jacobsen Æ Hans Christian Bruun Hansen
Received: 21 August 2006 / Accepted: 28 November 2006 / Published online: 30 December 2006 Springer Science+Business Media B.V. 2006
Abstract Biofumigation can be used as an alternative to conventional soil fumigation to control soil-borne pests. With biofumigation, plant tissue with a natural content of glucosinolates (cruciferous plants) is damaged and incorporated into the topsoil. When the plant tissue is damaged, the glucosinolates come into contact with the endogenous enzyme myrosinase, which catalyse the hydrolysis of glucosinolates into various products depending on the reaction conditions. Isothiocyanates are among the potential products formed from these reactions. We investigated if the isothiocyanates from rape plant material were leached through the soil to drain depth when a heavy rainstorm followed the biofumigation. We applied isothiocyanates from rape plant material (1,480 lmol m–2) to four large (0.6 m diameter, 1.0 m long) intact soil monoliths from a loamy and a sandy soil and conducted a leaching experiment under semi-
field conditions. The soil monoliths were irrigated with 70–90 mm (10 mm h–1) and the concentrations of three isothiocyanates (3-butenyl, 4-pentenyl and 2-phenethyl) were monitored in the leachate. Between 0 and 14.8 mmol isothiocyanates were leached for each mol of isothiocyanates applied during application of 70–90 mm irrigation. The distribution coefficient estimated from leached concentrations was 0.04–1.19 for 3butenyl, 0.04–1.15 for 4-pentenyl isothiocyanate and 0.037–0.97 for 2-phenethyl isothiocyanate. The concentration of total isothiocyanates in the leachate was in the same order of magnitude as the LD50 of isothiocyanates for sensitive aquatic organisms. Keywords Attenuation Biofumigation Glucosinolate Isothiocyanate Soil Transport
Introduction M. Laegdsmand (&) O. H. Jacobsen Department of Agroecology, Danish Institute of Agricultural Sciences, Research Centre Foulum, PO Box 50, 8830 Tjele, Denmark e-mail:
[email protected] A. L. Gimsing B. W. Strobel J. C. Sørensen H. C. B. Hansen Department of Natural Sciences, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark
Soil-borne pathogens like nematodes and fungi have traditionally been controlled by soil fumigation with the synthetic compounds methyl bromide and methyl isothiocyanate (Kirkegaard and Matthiessen 2004; Matthiessen and Shackleton 2005). Substituting these synthetic compounds with naturally produced compounds has recently gained much interest, especially because from 2006 the use of methylbromide has been
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living organisms including bacteria, fungi, nematodes and wireworms (Lazzeri et al. 1993; Elberson et al. 1996; Brown and Morra 1997; Sarwar et al. 1998; Manici et al. 2000; Lazzeri and Manici 2001; Smith and Kirkegaard 2002; Rumberger and Marschner 2003) and the isothiocyanates also have herbicidal activity (Petersen et al. 2001; Haramoto and Gallandt 2004; Norsworthy and Meehan 2005). Different isothiocyanates show dose-dependent reactivity towards different organisms, and it is therefore likely that nontarget organisms may also be affected by the isothiocyanates. A few studies have confirmed the toxicity of the isothiocyanates to non-target organisms both in soil (Bending and Lincoln 2000; Rumberger and Marschner 2003; Ibekwe 2004) and in the aquatic environment (Schultz et al. 2005). This means that isothiocyanates originating from biofumigation may affect soil functioning. Futhermore, if the isothiocyanates are transported to rivers or streams by overland flow, by leaching through the soil profile or by draining, they will potentially cause harmful effects on non-target organisms living in the aquatic environments. Isothiocyanates are lipophilic compounds with low water solubility and they seem to be both sorbed and degraded quickly in soil (Rumberger and Marschner 2003; Warton et al. 2003; Matthiessen et al. 2004b; Gimsing et al. 2006). Studies on the fate of isothiocyanates in soil show that in non-sterile soil they degraded within a few days (Rumberger and Marschner 2003; Matthiessen et al. 2004b; Gimsing et al. 2006) with most of the reaction taking place within a few hours. Results show that the reaction is faster in topsoil than in subsoil (Gimsing et al. 2006). In sterile soil, a fast reaction typically takes place within an hour, which reduces the extractable concentration of isothiocyanate in soil to approximately 50% of the initially added amount, whereafter the concentration slowly reduces (Rumberger and Mars-
banned in the EU (according to the Montreal protocol) due to its negative impact on the ozone layer. Control of soil-borne pests based on naturally produced compounds has been coined biofumigation (Kirkegaard and Matthiessen 2004) and the compounds which have been most extensively investigated are the glucosinolates and some of their hydrolysis products—the isothiocyanates. Glucosinolates are compounds produced by cruciferous plants such as rape, mustard, canola, cabbage and broccoli (Kjaer 1976). The glucosinolate molecule (1’-Thio-b-D-glucopyranosyl-alkyl-Z-N-hydroximin sulphate esters) contains a thioglucose moiety, a sulphonated oxime and a variable side chain derived from an amino acid. More than 120 different side chains have been described (Manici et al. 2000; Mithen 2001). When the plant tissue is ruptured the glucosinolates come into contact with the endogenous enzyme myrosinase, which hydrolyses the glucosinolates to biologically active isothiocyanates, thiocyanates, nitriles or oxazolidine2-ethione, see Fig. 1 (Sørensen 1990; Sarwar et al. 1998; Mithen 2001). The isothiocyanates are generally considered as the most toxic of the hydrolysis products (Brown and Morra 1997) and biofumigation has so far been concentrated on maximising the formation of isothiocyanates (Warton et al. 2001; Morra and Kirkegaard 2002; Matthiessen et al. 2004b). Among the cruciferous plants, the brassicas seem the most promising biofumigant crops. Most research has been done on rape (Brassica napus) and Indian mustard (Brassica juncea) (Kirkegaard and Matthiessen 2004; Gimsing and Kirkegaard 2005), because these plants contain several isothiocyanate-liberating glucosinolates (Kirkegaard and Sarwar 1998). Biofumigation is performed by incorporating the green mulch of Brassica plants in the upper part of the soil in which the isothiocyanates can be released. Several studies have confirmed that the isothiocyanates are toxic to a range of pathogenic soilFig. 1 General structure of glucosinolates and their hydrolysis products. Only the most commonly encountered hydrolysis products are shown
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Glucose
R
S
SH
C
C NOSO 3 -
Glucosinolate
R
+ Glucose
R
N
C
R
C
N
R
S
C
S
Isothiocyanate Nitrile
NOSO 3 -
Unstable intermediate product
N
Thiocyanate
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chner 2003; Matthiessen et al. 2004b; Gimsing et al. 2006). This reduction is probably due to a fast sorption reaction and volatilisation. These results point to the importance of microbial degradation for the attanuation of isothiocyanates in soil. Enhanced biodegradation of isothiocyanates has been observed for soils frequently grown with Brassicas, indicating that some microorganisms are capable of fast degradation of isothiocyanates (Gimsing et al. 2006; Warton et al. 2003; Matthiessen et al. 2004a; Warton and Matthiessen 2005). Even though isothiocyanates are quickly sorbed and degraded in soil, it has been shown that the synthetic methyl isothiocyanate can leach through soil (Guo et al. 2003). In this study we investigated the leaching of isothiocyanates following simulated biofumigation with blended rape plant material and extracts from rape plant in 1-m deep intact soil monoliths. We followed the transport of isothiocyanates during biofumigation to investigate if the isothiocyanates could be leached to 1 m depth on a sandy and a loamy soil when a heavy rainfall (70– 90 mm, 10 mm/h) follows immediately after the application of the plant material and extracts.
Materials and methods Soils The soils used for sampling the soil monoliths were a loamy soil (Sj. Odde) and a sandy soil (Jyndevad). The two soils represent the two most common soil types in Denmark. Both soils have been used for organic farming with application of manure, the loamy soil since 1951 and the sandy since 1990. The Sj. Odde soil is Table 1 Key parameters for the two soils at four depths: texture, content of organic carbon, measured intervals of saturated hydraulic conductivity (n = 9) and bulk density with mean values in brackets (n = 9) Ap,1 is plough layer and Ap,2 is plough pan
situated in the northeastern part of Denmark and is formed on calcareous glacial till and the Jyndevad is situated in the southwestern part of Denmark on a glacial outwash plain. According to Soil Taxonomy (Soil Survey Staff 1999), the Jyndevad soil is a Humic Psammentic Dystrudept and the Sj. Odde soil is a Typic Argiudoll. Samples for measurements of soil water retention, bulk density and saturated hydraulic conductivity were taken from four depths with nine replicate samples at each depth. Table 1 shows some physical and chemical properties of the two soils. We measured texture and organic carbon content on loose samples of soil; and saturated hydraulic conductivity by the constant head method (Klute and Dirksen 1986) and dry bulk density on 100 cm3 samples. Duplicate, undisturbed soil monoliths were sampled at the two sites in March 2004. A steel cylinder (diameter 0.6 m, length 1.0 m), suspended from a rack was hammered into the soil. Before each push of the cylinder into the soil, a roughly 10 cm deep soil layer was removed around the cylinder at a distance of 2 cm from its circumference to ease insertion whilst retaining the intactness of soil monoliths. When the cylinder was filled with soil a steel plate was hammered horizontally under the cylinder and bolted onto it to prevent the soil from falling out. The two replicate monoliths were taken approximately 3 m apart. During excavation at Jyndevad we observed that the boundary between layer B1 and layer B2 was at 70 cm depth for monolith I and 90 cm depth for monolith II. Hence, the coarse sandy layer B1 was approximately 20 cm thicker in monolith II than in monolith I. The soil monoliths were installed in a trench on a funnel base filled with glass spheres (600–
Sand:silt:clay %
Organic carbon %
Ksat cm h–1
Bulk density 106 g m–3
Sj. Odde
Ap,1 Ap,2 Bt Ck
67:12:21 – 41:22:37 33:28:39
2.2 – 0.4 0.1
0.05–203 0.31–203 7.9–725 0.00–234
(50) (74) (174) (53)
1.29–1.63 1.58–1.76 1.59–1.70 1.76–1.88
(1.49) (1.67) (1.64) (1.82)
Jyndevad
Ap,1 Ap,2 B1 B2
92:3:5 – 94:1:5 94:1:5
2.4 – 0.04 0.04
8–17 (12) 3–66 (21) 112–200 (152) 41–63 (52)
1.30–1.48 1.51–1.59 1.42–1.52 1.50–1.55
(1.41) (1.55) (1.49) (1.52)
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Fig. 2 Experimental setup for the soil monolith leaching
Wodden box with 4 cm Styrofoam Irrigation head Output TDR-probe Soil Monolith
Temperature sensor
Funnel
Soil
Artificial rainwater
Soil
Sample Concrete trench
800 lm diameter) (Fig. 2). The pressure head at the lower end of the monoliths at equilibrium was –15 hPa due to hanging water column in the glass spheres. A wooden box isolated with 40 mm Styrofoam covered the trench, except where the monoliths were placed to ensure a realistic temperature in the monoliths. Time domain reflectometry (TDR) probes (400 mm long, 100 mm spacing) were installed in the soil at two depths (30 and 80 cm) and temperature sensors were installed at three depths (5, 50 and 90 cm). The soil was grown with barley and fertilized with pig slurry prior to the simulated biofumigation. Simulated biofumigation Seeds from rape plants (Brassica napus) were sown in potting soil in boxes in a greenhouse and irrigated to water-holding capacity once a week. Light and heat was applied to the greenhouse for 12 h a day. After 3 weeks we harvested the rape plants. Freshly harvested plants (1,500 g per monolith) were cut in an industrial vegetable cutter (Robot Coupe SNC, France) and afterwards blended (Waring Commercial Laboratory blender, Waring Product Division, New Hartford, Conneticut, USA) with 1,300 ml deionized water
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for 2 min. About 5 g KBr and 22.0 g rape plant extract powder was mixed with 500 ml of deionized water. The rape extract was made as a semipurified rapeseed glucosinolate concentrate (25% W/W) in pilot scale. The glucosinolates were dominated by aliphatic type glucosinolates and the content as well as the profile was established based on analysis of both intact and desulfo glucosinolates (Michaelsen et al. 1992). The blended rape plants and the rape extract were mixed and left to hydrolyse for 1 h to allow for the hydrolysis of the glucosinolates. The mixture of plants and extract was spread homogenously on the surface of the soil monolith and the irrigation experiment was conducted. The total amount applied (see later) was approximately 1,480 lmol m–2 (3-Butenyl-isothiocyanate 1,080 lmol m–2, 4-Pentenyl-isothiocyanate 327 lmol m–2, 2-Phenethyl-isothiocyanate 77 lmol m–2). Irrigation Artificial rain water (NaCl 5.84 mg l–1, CaCl2(2H2O) 1.47 mg l–1, MgCl2(6H2O) 2.03 mg l–1) was applied to each column using a drip irrigation device. The irrigation intensity was 10 mm h–1. We chose to irrigate with around 80 mm since this
Plant Soil (2007) 291:81–92
is the largest rain event occurring in Denmark in 100 years. The risk of leaching of isothiocyanates from biofumigation would be in the first day after application since the isothiocyanates are volatile and have high degradation rates. The temperature during the irrigation experiment was between 8 and 12C depending on soil depth. The effluent was sampled during the irrigation experiments and the last sample was taken approximately 12 h after irrigation was stopped. Samples were frozen immediately after sampling and kept at –20C until analysis. The effluent samples were analyzed for electrical conductivity (EC), turbidity and total organic carbon (TOC) and concentrations of isothiocyanates. Outflow, soil water content and temperature were monitored during the leaching experiments. EC was measured with a conductivity-meter (Radiometer, Copenhagen, Denmark). The TOC in all effluent samples was measured on a Shimadzu TOC-5000A (Shimadzu, Kyoto, Japan) equipped with a suspended particles kit ensuring that all carbon in suspended particles was included in the analyses. Based on the glucosinolate profile of the plant material and the rape extract used for the simulated biofumigation, it was chosen to limit this study to the investigation of the leaching of three of the most common isothiocyanate hydrolysis products, which were 3-butenyl, 4-pentenyl and 2-phenethyl isothiocyanates. For determination of the isothiocyanate content in the percolated water, 20.00 ml water was transferred to a 50 ml separating funnel and added 3 ml ethylacetate (EA) spiked with 30 lg l–1 propyl-isothiocyanate as internal standard (IS). The funnel was thoroughly rotated for 3 min before resting and separation of the phases with extraction efficiency >99.9%. The EA extract evaporated to approx. 0.5 ml and dried by passing 1.5 cm anhydrous Na2SO4 packed in a Pasteur pipette with a glass wool bed. The dried sample was collected in amber GC vials and closed with PTFE lined septa. The concentration of isothiocyanate was determined by gas chromatography with mass spectrometer (GC-MS) (PolarisGCQ, ThermoFinnigan, Austin). The GC column was 30 m · 0.25 mm id, 0.25 lm film thickness Rtx5MS (Restek, Bellefonte, PA, USA) with He carrier gas at constant flow of 1.2 ml min–1. The
85
temperature program was 40C (2 min), 15C min–1 to 285C (1.67 min), transfer line 275C, MS ion source 200C and scan range 50– 200 m/z. Quantification was made by selective ion monitoring (SIM) of the 72 m/z fragment for propyl-isothiocyanate (IS), 3-butenyl-isothiocyanate and 4-pentenyl-isothiocyanate, and 163 m/z fragment for 2-phenethyl-isothiocyanate. Injector temperature was 200C in splitless mode, and injection volume was 2 ll sample and 1 ll air with hot needle. The autosampler was programmed to do three solvent pre-washes of the syringe, three sample washes, three sample pull-ups and three solvent washes after injection. The limit of detection was approx. 25 nmol l–1 for the GCMS analysis, and thereby approx. 1 nmol l–1 for the water samples. Modelling The software CXTFIT (ver. 2.1) (Toride et al. 1999) was used to estimate the hydrological parameters, the mass of leachable organic carbon and the sorption affinity of isothiocyanates in the four soil monoliths. A two-region model is used when physical non-equilibrium affects the transport whereas a two-site model is used for simulating chemical non-equilibrium caused by kinetic sorption. Gimsing et al. (2006) found that sorption of the isothiocyanates to the two soils used in these experiments was a fast process and hence chemical non-equilibrium will only have minor influence on the transport. In an aggregated soil, such as the loamy Sj. Odde soil, physical nonequilibrium will dominate the transport. Therefore we used a two-region model for these simulations. The transport of solutes in the mobile domain was simulated by the convectiondispersion equation with equilibrium sorption in the mobile and immobile domain (Eq. 1). Degradation rates in the mobile and immobile domains were assumed to be the same and degradation in the sorbed fraction was assumed to be zero (Eq. 2). bR
@C1 1 @ 2 C1 @C1 ¼ xðC1 C2 Þ l1 C1 P @Z2 @T @Z ð1Þ
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86
ð1 bÞR
Plant Soil (2007) 291:81–92
@C2 ¼ xðC1 C2 Þ l2 C2 ; @T
a better understanding of the transport processes in the soil. The parameters estimated with the model should be evaluated in the light of this. The constant water content was calculated from the measured water content in the individual monolith and the constant flow was calculated from the irrigation intensity. We estimated D, b and x from bromide concentrations in the leachate for each monolith, assuming that the experimental water content and flow was constant and that the bromide was applied as a Dirac pulse. The isothiocyanates were applied with a high amount of organic material from the rape plants. We assumed that the majority of organic matter was transported as small organic particles and that the entire mobile flow domain was not accessible for the flow of the organic particles. Therefore, we estimated the actual amount of organic carbon that could be leached (MTOC) and a new ratio between mobile and immobile water contents available for flow of organic particles (bTOC) from the breakthrough of TOC, assuming that there was no degradation of organic matter during the experimental period. We also estimated the apparent retardation factors for the whole soil profile (R) of the three isothiocyanates, setting the first-order degradation rate constants (l) to 3.6 · 10–6 and 4.0 · 10–6 s–1 on the loamy and the sandy soil, respectively, for all isothiocyanates. This was based on laboratory experiments at 10C (data not yet published). Subsequently, the apparent sorption coefficients (K*d) were calculated from R. The estimation of parameters was done by using the inverse modelling option in CXTFIT (Toride et al. 1999).
ð2Þ
3
Water content, m m
Fig. 3 Water content in the soil monoliths: closed symbols refer to monolith I and open symbols to monolith II, circles are for 30 cm depth and triangles for 80 cm depth
-3
where b is the fraction of mobile water (b = hm/h), q K R is the retardation factor ( R ¼ 1 þ bh d ), C1 is the dimensionless concentration of the mobile domain (C1 = cm/c0), C2 is the dimensionless concentration of the immobile domain (C2 = cim/c0), T is dimensionless time, Z is dimensionless length, P–1 is the dimensionless hydrodynamic dispersion coefficient (P = mL/D), D is hydrodynamic dispersion, x is the dimensionless rate of transfer between the mobile and the immobile domain (x = aL/hm), l1 and l2 are the dimensionless rates of degradation of the mobile domain and the immobile domain (l1 = Lhml/hm and l2 = Lhiml/hm). hm and him are the water contents of the mobile and the immobile domain, respectively; cm and cim are the concentrations in the mobile and the immobile domain, respectively; qb is the bulk density of the soil; K*d is the apparent distribution coefficient for isothiocyanate sorption for the entire soil monolith. Not all conditions for the model were fulfilled: The soil water content and flow varied with time and depth in the two soils during the experiment. The changes in the flow and water content on the Sj. Odde soil (Fig. 3) and hence the effect on model output was minor. On the Jyndevad soil water content rose during the first 4 h and was different in 30 and 80 cm depth. This may result in errors on the estimated K*d values on the Jyndevad soil. However, we used the model to characterize the soil monoliths and the different isothiocyanates from the observed leaching to get
0.5
Sj. Odde
Jyndevad
0.4 0.3 0.2 0.1 0.0 0
2
4
6
0
Time, h
123
2
4
6
8
Plant Soil (2007) 291:81–92
87
Results and discussion Table 1 shows that the saturated hydralic conductivity on Sj.Odde soil varied two orders of magnitude between the 100 cm3 samples. The minimum hydraulic conductivity measured in the topsoil was 0.5 mm h–1. This indicates that preferential flow will occur in the topsoil at irrigation intensities higher than 0.5 mm h–1. Therefore we will expect that preferential flow will occur in the Sj. Odde soil under the irrigation intensity (10 mm h–1) used in the present experiment. On the Jyndevad soil the measured saturated hydraulic conductivity of the topsoil samples are in the same order of magnitude and the minimum value is 80 mm h–1. Here the topsoil will not introduce preferential flow. However, the difference in hydraulic conductivity of the Ap,2 layer and the B1 layer may introduce fingering of flow below the interface of the Ap,2 layer and the B1 layer. The experiments of biofumigation followed by a large simulated precipitation event (70–90 mm) resulted in leaching of all three types of isothiocyanates (3-butenyl, 4-pentenyl and 2-phenethyl) from one of the monoliths of the loamy soil and from both monoliths of the sandy soil. No leaching of isothiocyanates was detected from monolith I of the loamy soil. The accumulated leaching during the irrigation varied between 0 and 21.9 lmol m–2 total isothiocyanate. During irrigation, between 0 and 14.8 mmol isothiocyanates was leached for each mol of isothiocyanates applied. The actual amount leached varied between replicates, soil type and isothiocyanate type (Table 2). The leaching of isothiocyanates had not declined to zero at the end of the irrigation period, so we expect that more isothi-
ocyanates would be leached with a longer irrigation period. The experiments show that isothiocyanates may be washed down to 1 m depth during a heavy rainstorm following biofumigation in the field. If the soil is drained or located near a surface water body, then the isothiocyanates can be transported into the water body; and waterliving organisms may be exposed to low concentrations of isothiocyanates. A study on the toxicity of isothiocyanates to larvae of the whitefringed weevil showed that the LD50 of methyl isothiocyanate was 0.04 mg l–1 at 5C, and the LD50 of 2-phenethyl isothiocyanate was 0.15 mg l–1 (Matthiessen and Shackleton 2005). The mean concentrations of isothiocyanates in the leachate varied between 0 and 0.05 mg l–1. Hence, the concentrations of isothiocyanates in the leachate were in the same order of magnitude as the LD50 for the isothiocyanates. The simulated and measured values of bromide are shown in Fig. 4. We observed that simulated and measured values of bromide concentrations were similar in all monoliths (R2 is between 0.96 and 1.00). The estimated values of hydrodynamic dispersion (D) from the four monoliths varied within the range 0.10–6.6 · 10–6 m2 s–1 (Table 3). Values of the fraction of mobile water (b) varied between 0.31 and 0.76, and exchange rates between the mobile and the immobile domain (x) varied between 1.0 and 5.3. Monolith II from Sjællands Odde had a four times higher hydrodynamic dispersion compared to monolith I. This shows that the distribution of porewater velocities is wider in monolith II. Leaching of 25% of the applied tracer occurs with outflow of 18 and 4.7 mm leachate on Sj. Odde replicate I and II
Table 2 The amount of isothiocyanates leached relative to the applied amount 3-Butenyl-isothiocyanate (nmol leached) (lmol applied)–1 Sj.Odde 0.0 I Sj.Odde 12.6 II Jyndevad 1.6 I
4-Pentenyl-isothiocyanate (nmol leached) (lmol applied)–1
2-Phenethyl-isothiocyanate (nmol leached) (lmol applied)–1
Total (nmol leached) (lmol applied)–1
0.0
0.0
0.0
13.6
50.2
14.8
1.3
0.6
1.5
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Bromide concentration, mg L
-1
Fig. 4 Simulated and measured concentrations of the conservative tracer (bromide) in the leachate of the soil monoliths. Lines are simulated values and circles are measured values. Closed symbols refer to monolith I and open symbols to monolith II
80
Sj. Odde
Jyndevad
60
40
20
0 0
2
4
6
8
0
2
4
6
8
10
Table 3 Estimated hydraulic parameters
n R2 D, 10–6 m2 s–1 b, x
Sj. Odde I
Sj.Odde II
Jyndevad I
Jyndevad II
10 0.99 1.8 (0.2) 0.31 (0.2) 5.3 (0.3)
9 1.00 6.6 (0.5) 0.76 (0.03) 2.1 (0.4)
8 0.99 0.20 (0.14) 0.55 (0.01) 1.0 (0.5)
7 0.96 0.10 (0.06) 0.31 (0.03) 4.6 (0.3)
Number of observations (n), coefficient of determination (R2) of simulated versus measured leaching of bromide and values of parameters estimated from BTCs of bromide (hydrodynamic dispersion (D), fraction of mobile water (b) and dimensionless rate of transfer between the mobile and the immobile domain (x))
TOC leached originated from the rape material located on the soil surface; no degradation of TOC took place; the TOC consisted mainly of organic particles and macromolecules. The last assumption requires that transport of TOC was restricted to a smaller mobile domain than the one estimated from the tracer experiments, due to the size-exclusion effect. There was a good agreement between the model and the measured concentrations of TOC on both monoliths from Sjællands Odde and from the monolith II from Jyndevad (R2 values were between 0.89 and 0.98), but on monolith I from Jyndevad the initial leaching of TOC could not be captured by the model (R2 = 0.56) (Fig. 5). The initially high
TOC concentration, mg L
Fig. 5 Simulated and measured concentrations of the total organic carbon in the leachate of the soil monoliths. Lines are simulated values and circles are measured values. Closed symbols refer to monolith I and open symbols to monolith II
-1
respectively. This demonstrates that there is a more direct flowpath for the water in monolith II compared to monolith I. On Jyndevad soil 25% of the applied tracer was leached with 26 and 10.7 mm on monolith I and II respectively. On the Jyndevad soil monolith II shoved a more direct flowpath compared to monolith I. During the excavation procedure we also observed that the coarse B horizon of the Jyndevad soil was 20 cm thicker in monolith II compared to monolith I. This thick coarse layer of monolith II may have caused hydraulic instability leading to preferential (finger-) flow. Simulation of concentrations of TOC in the leachate was done under these assumptions: all
500
Sj. Odde
300 200 100 0 0
123
Jyndevad
400
2
4
6
8
0
2
4
6
8
10
Plant Soil (2007) 291:81–92
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leaching of organic carbon may be due to loss of native soil organic matter. The estimated parameters for the total mass of organic carbon leached from rape plant material and rape plant extracts (MTOC); and the reduction of the mobile water fraction (bTOC/b) are shown in Table 4. The applied amount of carbon from the rape plant material and the rape plant extracts was approximately 640 g TOC m–2. The estimate for MTOC was 254 and 270 g m–2 on the monoliths from Sjællands Odde and 41 and 456 g TOC m–2 for the monoliths from Jyndevad. Jyndevad I had the lowest leaching and Jyndevad II had the highest leaching of TOC. The reduction in the mobile domain (bTOC/b) was 0.76–0.87 in both monoliths from Sjællands Odde and Jyndevad I; no reduction in the mobile domain was observed in Table 4 Estimated parameters regarding transport of organic carbon Sj. Odde Sj Odde I II n 10 0.97 R2 MTOC g m–2 254 0.87 bTOC b–1
9 0.89 270 0.75
Jyndevad Jyndevad I II 8 0.56 41 0.76
7 0.98 456 1.00
Number of observations, coefficient of determination (R2) of simulated versus measured leaching of TOC and values of parameters estimated from leaching of TOC (Mass of leachable TOC added with rape plant material (MTOC) and reduction in fraction of mobile water (bTOC b–1))
Jyndevad II. Fast transport (due to finger-flow) of tracer dominated Jyndevad II and this may be the reason why no further reduction of the mobile domain was observed. The estimated values of the apparent distribution coefficients (K*d) for 3-butenyl, 4-pentenyl and 2-phenethyl varied between 0.04 and 1.2 l kg– 1 (Table 5). K*d of the aliphatic isothiocyanates (3butenyl-isothiocyanate and 4-pentenyl-isothiocyanate) estimated from the monoliths was 1.2 on Sjællands Odde soil and 0.08 on Jyndevad soil. Gimsing et al. (2006) found that the Kd values in batch experiments for the aliphatic prop-2-enyl was 0.54 and 0.51 l kg–1 at the Sjællands Odde soil for the A and B horizons, respectively, and 0.95 and 0.35 l kg–1 on the Jyndevad soil. For the aromatic 2-phenethyl isothiocyanate K*d was 0.97 on Sjællands Odde and 0.66 on Jyndevad. Gimsing et al. (2006) found that the Kd value determined by batch experiments for benzyl isothiocyanate was 2.95 and 0.16 l kg–1 for A and B horizons, respectively, on the Sjællands Odde soil and 6.75 and 2.44 l kg–1 for the Jyndevad soil. The apparent K*d values for the Sjællans Odde soil was approximately the same as the Kd values measured by Gimsing et al. (2006), but the apparent K*d values of the monoliths with Jyndevad soil was lower than the Kd values measured by Gimsing et al. (2006). Slow kinetic sorption on the sandy Jyndevad soil may explain these differences, since the model used for estimation
Table 5 Estimated parameters regarding transport of isothiocyanate
Sj. Odde II MSE R K*d (l kg–1) Jyndevad I MSE R K*d (l kg–1) Jyndevad II MSE R K*d (l kg–1)
3-Butenyl isothiocyanate
4-Pentenyl isothiocyanate
2-Phenethyl isothiocyanate
4181 6.9 (0.5) a 1.19
572 6.7 (0.6) a 1.15
950 5.8 (1.1) a 0.97
98 1.29 (0.01) a 0.040
11 1.29 (0.09) a 0.040
0.15 1.27 (0.01) a 0.037
34 1.72 (0.03) a 0.11
46 1.95 (0.03) b 0.14
5.2 1.61 (0.03) a 0.094
Mean square error (MSE) of the estimation, estimated retardation factor (R) of the isothiocyanates in the entire monolith estimated with the mobile–immobile model and calculated distribution coefficient (K*d). Values in brackets are mean square error of the estimated retardation factor. Estimated values of R on the different isothiocyanates with different letters are significantly different
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of the sorption parameters did not include sorption kinetics. Other studies have shown that isothiocyanates are mainly sorbed to the organic matter in the soil, and the more hydrophobic the soil, the stronger the isothiocyanate is sorbed to the organic matter (Gimsing et al. 2006). Violation of the assumption in CXTFIT of constant flow and water content on the Jyndevad monoliths may also have led to errors in the estimates of K*d. From the structure of the different isothiocyanates we would expect that the hydrophobicity and hence the K*d values of the three isothiocyanates for a certain soil monolith was in the order: K*d(2-phenethyl-isothiocyanate) > K*d(4-pentenyl isothiocya* nate) > Kd(3-butenyl-isothiocyanate) The estimated value of K*d for 2-phenethylisothiocyanate was the lower of the three isothiocyanates in all monoliths where leaching occured. This is contrary to what was expected and only significantly different from 4-pentenylisothiocyanate in Jyndevad II. This indicates that the reactive convective-dispersive transport in solution was not the only process involved in the transport, but that colloid-facilitated transport may have enhanced the leaching of the isothiocyanates. The lower apparent K*d of the 0.5
Sj. Odde replicate I Jyndevad replicate I
0.4
-1
0.3
ITC concentration, µmol L
Fig. 6 Simulated and measured concentrations of the three isothiocyanates in the leachate of the soil monoliths. Lines are simulated values and symbols are measured values
2-phenethyl-isothiocyanate could also have been the result of a too high degradation rate coefficient used in the simulations, since simulations had the same degradation rate coefficient for all three isothiocyanates. However, if we estimated the K*d of 2-phenethyl-isothiocyanate under the assumption of no degradation, the difference of the new K*d relative to the original was less than 1%. In the model only transport in the water phase is considered but the isothiocyanates are volatile and part of the isothiocyanates may have evaporated. If the volatilisation rate is lower for 2-phenethyl-isothiocyanate compared to the aliphatic isothiocyanates we would estimate a lower distribution coefficient of 2-phenethyl-isothiocyanate. On monolith II of the Sjællands Odde soil leaching patterns of isothiocyanates could not be simulated correctly, due to the simplicity of the model (Fig. 6). For this monolith we observed an initial peak in the concentrations of all three isothiocyanates after 2 h of irrigation (at just 5 mm outflow). The mobile–immobile model did not simulate this peak. TOC concentrations started to rise at the same time as the initial peak in concentrations of isothiocyanates. This suggests that the initial peak in isothiocyanate concentra-
3-butenyl isothiocyanate 4-pentenyl isothiocyanate 2-phenethyl isothiocyanate sim. 3-butenyl isothiocyanate sim. 4-pentenyl isothiocyanate sim. 2-phenethyl isothiocyanate
0.2 0.1 0.0
Jyndevad replicate II
Sj. Odde replicate II 0.4 0.3 0.2 0.1 0.0 0
2
4
6
8
0
Time, hours
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4
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8
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tion is a result of co-transport of isothiocyanates sorbed to mobile organic particles released from the rape material. If isothiocyanates were transported sorbed on organic particles, it may have been transported faster than dissolved isothiocyanates and the conservative tracer (bromide). The simulations of TOC also suggested that organic particles were leached at this time. A second peak in isothiocyanate concentrations was observed after approximately 5–7 h of irrigation (20– 45 mm outflow). The mobile–immobile model could not simulate this peak either. The second peak in isothiocyanate concentrations coincides with the peak in bromide. The second peak is the result of non-equilibrium transport of dissolved isothiocyanate, where the isothiocyanates bypass sorption sites of the soil leading to non-equilibrium in the soil and high concentrations of isothiocyanates in the leachate. This is either due to slow kinetic sorption (chemical non-equilibrium) or to preferential flow. Sorption in the mobile–immobile model is simulated by equilibrium sorption and the model does not account for chemical non-equilibrium in the soil. However Gimsing et al. 2006 showed that the sorption of isothiocyanates to soil is a fast process. We also simulated the concentrations of isothiocyanates in the leachate from monolith I with Sjællands Odde soil (not shown) using the hydraulic parameters for this monolith (Table 3) and the retardation factor estimated from monolith II. We found that the concentrations of isothiocyanates only exceeded the detection value in the last sample and only for 3-butenyl isothiocyanate. This explains why we did not detect any isothiocyanates in the leachate from monolith I. The simulated concentrations of isothiocyanates were in better agreement with measurements for the sandy monoliths. However, there were higher concentrations of 3butenyl isothiocyanate compared to the simulations 6 h after irrigation started on both sandy monoliths. 4-pentenyl and 2-phenethyl isothiocyanate did not show the same difference between measured and simulated values. The higher concentrations of leached TOC from 4 to 6 h after irrigation in Jyndevad I did not lead to measurable concentrations of isothiocyanates—hence we conclude that the higher levels of TOC leached between 4 and 6 h did not
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originate from the rape plant material, but is native soil organic matter. •
•
•
Isothiocyanates from rape plant material could be leached to 1 m depth on both loamy and sandy soil when a heavy storm followed simulated biofumigation. This shows that even though isothiocyanates have been demonstrated to sorb and degrade quickly in soil, it is possible that they may leach to drain depth in small amounts. Hence, there is a risk that aquatic organisms in nearby surface waters may become exposed to isothiocyanates. We measured three isothiocyanates (3-butenyl, 4-pentenyl and 2-phenethyl) in the leachate. We found that between 0 and 21.9 mmol was leached for each mol of isothiocyanate applied. We observed that the mean concentration of isothiocyanates in the leachate was in the same order of magnitude as the LD50 for sensitive aquatic organisms exposed to isothiocyanates. The estimated distribution coefficient for 2phenethyl isothiocyanate was lower than the estimated distribution coefficient for 3-butenyl isothiocyanate and 4-pentenyl isothiocyanate, even though the molecular structure of the 2phenethyl isothiocyanate suggests a higher hydrophobicity compared to 3-butenyl isothiocyanate and 4-pentenyl isothiocyanate. This reversed order was likely attributed to transport facilitated by colloidal organic particles of 2-phenethyl isothiocyanate.
Acknowledgements This work was funded by the Danish Research Councils (Contract no. 23-02-0152). We thank Stig T. Rasmussen, Bodil B. Christensen and Jørgen M. Nielsen for excellent technical assistance.
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